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    <title>Evolution of C4 photosynthesis predicted by constraint-based modelling</title>
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      <article itemscope="" itemtype="http://schema.org/Article" data-itemscope="root">
        <h1 itemprop="headline">Evolution of C4 photosynthesis predicted by constraint-based
          modelling</h1>
        <meta itemprop="image"
          content="https://via.placeholder.com/1200x714/dbdbdb/4a4a4a.png?text=Evolution%20of%20C4%20photosynthesis%20predicted%20by%20constraint-based%20modelling">
        <ol data-itemprop="authors">
          <li itemscope="" itemtype="http://schema.org/Person" itemprop="author">
            <meta itemprop="name" content="Mary-Ann Blätke"><span data-itemprop="givenNames"><span
                itemprop="givenName">Mary-Ann</span></span><span data-itemprop="familyNames"><span
                itemprop="familyName">Blätke</span></span><span data-itemprop="emails"><a
                itemprop="email"
                href="mailto:blaetke@ipk-gatersleben.de">blaetke@ipk-gatersleben.de</a></span><span
              data-itemprop="affiliations"><a itemprop="affiliation"
                href="#author-organization-1">1</a></span>
          </li>
          <li itemscope="" itemtype="http://schema.org/Person" itemprop="author">
            <meta itemprop="name" content="Andrea Bräutigam"><span data-itemprop="givenNames"><span
                itemprop="givenName">Andrea</span></span><span data-itemprop="familyNames"><span
                itemprop="familyName">Bräutigam</span></span><span data-itemprop="affiliations"><a
                itemprop="affiliation" href="#author-organization-1">1</a><a itemprop="affiliation"
                href="#author-organization-2">2</a></span>
          </li>
        </ol>
        <ol data-itemprop="affiliations">
          <li itemscope="" itemtype="http://schema.org/Organization" itemid="#author-organization-1"
            id="author-organization-1"><span itemprop="name">Leibniz Institute of Plant Genetics and
              Crop Plant Research (IPK)</span><address itemscope=""
              itemtype="http://schema.org/PostalAddress" itemprop="address"><span
                itemprop="addressLocality">Tübingen</span><span
                itemprop="addressCountry">Germany</span></address></li>
          <li itemscope="" itemtype="http://schema.org/Organization" itemid="#author-organization-2"
            id="author-organization-2"><span itemprop="name">Computational Biology, Faculty of
              Biology, Bielefeld University, Universitätsstraße</span><address itemscope=""
              itemtype="http://schema.org/PostalAddress" itemprop="address"><span
                itemprop="addressLocality">Bielefeld</span><span
                itemprop="addressCountry">Germany</span></address></li>
        </ol><span itemscope="" itemtype="http://schema.org/Organization" itemprop="publisher">
          <meta itemprop="name" content="Unknown"><span itemscope=""
            itemtype="http://schema.org/ImageObject" itemprop="logo">
            <meta itemprop="url"
              content="https://via.placeholder.com/600x60/dbdbdb/4a4a4a.png?text=Unknown">
          </span>
        </span><time itemprop="datePublished" datetime="2019-12-04">2019-12-04</time>
        <ul data-itemprop="genre">
          <li itemprop="genre">Research Article</li>
        </ul>
        <ul data-itemprop="about">
          <li itemscope="" itemtype="http://schema.org/DefinedTerm" itemprop="about"><span
              itemprop="name">Computational and Systems Biology</span></li>
          <li itemscope="" itemtype="http://schema.org/DefinedTerm" itemprop="about"><span
              itemprop="name">Plant Biology</span></li>
        </ul>
        <ul data-itemprop="keywords">
          <li itemprop="keywords">metabolic networks</li>
          <li itemprop="keywords">constraint-based model</li>
          <li itemprop="keywords">C4 photosynthesis</li>
          <li itemprop="keywords">model evolution</li>
          <li itemprop="keywords">flux balance analysis</li>
          <li itemprop="keywords">None</li>
        </ul>
        <ul data-itemprop="identifiers">
          <li itemscope="" itemtype="http://schema.org/PropertyValue" itemprop="identifier">
            <meta itemprop="propertyID"
              content="https://registry.identifiers.org/registry/publisher-id"><span
              itemprop="name">publisher-id</span><span itemprop="value"
              data-itemtype="http://schema.org/Number">49305</span>
          </li>
          <li itemscope="" itemtype="http://schema.org/PropertyValue" itemprop="identifier">
            <meta itemprop="propertyID" content="https://registry.identifiers.org/registry/doi">
            <span itemprop="name">doi</span><span itemprop="value">10.7554/eLife.49305</span>
          </li>
          <li itemscope="" itemtype="http://schema.org/PropertyValue" itemprop="identifier">
            <meta itemprop="propertyID"
              content="https://registry.identifiers.org/registry/elocation-id"><span
              itemprop="name">elocation-id</span><span itemprop="value">e49305</span>
          </li>
        </ul>
        <section data-itemprop="description">
          <h2 data-itemtype="http://schema.stenci.la/Heading">Abstract</h2>
          <meta itemprop="description"
            content="Constraint-based modelling (CBM) is a powerful tool for the analysis of evolutionary trajectories. Evolution, especially evolution in the distant past, is not easily accessible to laboratory experimentation. Modelling can provide a window into evolutionary processes by allowing the examination of selective pressures which lead to particular optimal solutions in the model. To study the evolution of C4 photosynthesis from a ground state of C3 photosynthesis, we initially construct a C3 model. After duplication into two cells to reflect typical C4 leaf architecture, we allow the model to predict the optimal metabolic solution under various conditions. The model thus identifies resource limitation in conjunction with high photorespiratory flux as a selective pressure relevant to the evolution of C4. It also predicts that light availability and distribution play a role in guiding the evolutionary choice of possible decarboxylation enzymes. The data shows evolutionary CBM in eukaryotes predicts molecular evolution with precision.">
          <p itemscope="" itemtype="http://schema.stenci.la/Paragraph">Constraint-based modelling
            (CBM) is a powerful tool for the analysis of evolutionary trajectories. Evolution,
            especially evolution in the distant past, is not easily accessible to laboratory
            experimentation. Modelling can provide a window into evolutionary processes by allowing
            the examination of selective pressures which lead to particular optimal solutions in the
            model. To study the evolution of C4 photosynthesis from a ground state of C3
            photosynthesis, we initially construct a C3 model. After duplication into two cells to
            reflect typical C4 leaf architecture, we allow the model to predict the optimal
            metabolic solution under various conditions. The model thus identifies resource
            limitation in conjunction with high photorespiratory flux as a selective pressure
            relevant to the evolution of C4. It also predicts that light availability and
            distribution play a role in guiding the evolutionary choice of possible decarboxylation
            enzymes. The data shows evolutionary CBM in eukaryotes predicts molecular evolution with
            precision.</p>
        </section>
        <h2 itemscope="" itemtype="http://schema.stenci.la/Heading" id="introduction">Introduction
        </h2>
        <p itemscope="" itemtype="http://schema.stenci.la/Paragraph">Identifying specific
          evolutionary trajectories and modelling the outcome of adaptive strategies at the
          molecular levels is a major challenge in evolutionary systems biology <cite itemscope=""
            itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a
              href="#bib55"><span>55</span><span>Papp et al.</span><span>2011</span></a></cite>. The
          evolution of novel metabolic pathways from existing parts may be predicted using
          constraint-based modelling (CBM) <cite itemscope=""
            itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a
              href="#bib52"><span>52</span><span>Orth et al.</span><span>2010</span></a></cite>. In
          CBM, selective pressures are coded via the objective functions for which the model is
          optimised. The factors which constrain evolution are integrated into the models via
          changes in model inputs or outputs and via flux constraints. We hypothesised that the
          evolution of the agriculturally important trait of C4 photosynthesis is accessible to CBM.
        </p>
        <p itemscope="" itemtype="http://schema.stenci.la/Paragraph">C4 photosynthesis evolved
          independently in at least 67 independent origins in the plant kingdom <cite itemscope=""
            itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a
              href="#bib65"><span>65</span><span>Scheben et al.</span><span>2017</span></a></cite>
          and it allows colonisation of marginal habitats <cite itemscope=""
            itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a
              href="#bib63"><span>63</span><span>Sage et al.</span><span>2012</span></a></cite> and
          high biomass production in annuals such as crops <span itemscope=""
            itemtype="http://schema.stenci.la/CiteGroup"><cite itemscope=""
              itemtype="http://schema.stenci.la/Cite"><a
                href="#bib62"><span>62</span><span>Sage</span><span>2004</span></a></cite><cite
              itemscope="" itemtype="http://schema.stenci.la/Cite"><a
                href="#bib23"><span>23</span><span>Edwards et
                  al.</span><span>2010</span></a></cite></span>. The C4 cycle acts as a biochemical
          pump which enriches the CO<sub itemscope=""
            itemtype="http://schema.stenci.la/Subscript"><span
              data-itemtype="http://schema.org/Number">2</span></sub> concentration at the site of
          Rubisco to overcome a major limitation of carbon fixation <cite itemscope=""
            itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a
              href="#bib62"><span>62</span><span>Sage</span><span>2004</span></a></cite>. Enrichment
          is beneficial because Rubisco, the carbon fixation enzyme, can react productively with
          CO<sub itemscope="" itemtype="http://schema.stenci.la/Subscript"><span
              data-itemtype="http://schema.org/Number">2</span></sub> and form two molecules of
          3-PGA, but it also reacts with O<sub itemscope=""
            itemtype="http://schema.stenci.la/Subscript"><span
              data-itemtype="http://schema.org/Number">2</span></sub> and produces
          2-phosphoglycolate which requires detoxification by photorespiration <cite itemscope=""
            itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a
              href="#bib51"><span>51</span><span>Ogren and Bowes</span><span>1971</span></a></cite>.
          The ratio between both reactions is determined by the enzyme specificity towards CO<sub
            itemscope="" itemtype="http://schema.stenci.la/Subscript"><span
              data-itemtype="http://schema.org/Number">2</span></sub>, by the temperature, and the
          concentrations of both reactants, which in turn is modulated by stresses such as drought
          and pathogen load. Evolution of Rubisco itself is constrained since any increase in
          specificity is paid for by a reduction in speed <cite itemscope=""
            itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a
              href="#bib73"><span>73</span><span>Spreitzer and
                Salvucci</span><span>2002</span></a></cite>. Lower speeds most likely cause
          maladaptivity since Rubisco is a comparatively slow enzyme and can comprise up to 50% of
          the total leaf protein <cite itemscope="" itemtype="http://schema.stenci.la/Cite"
            data-citationmode="Narrative"><a
              href="#bib24"><span>24</span><span>Ellis</span><span>1979</span></a></cite>. In the C4
          cycle, phosphoenolpyruvate carboxylase affixes CO<sub itemscope=""
            itemtype="http://schema.stenci.la/Subscript"><span
              data-itemtype="http://schema.org/Number">2</span></sub> to a C3 acid,
          phosphoenolpyruvate (PEP), forming a C4 acid, oxaloacetate (OAA). After stabilisation of
          the resulting C4 acid by reduction to malate or transamination to aspartate, it is
          transferred to the site of Rubisco and decarboxylated by one of three possible
          decarboxylation enzymes, NADP-dependent malic enzyme (NADP-ME), NAD-dependent malic enzyme
          (NAD-ME), or PEP carboxykinase (PEP-CK) <span itemscope=""
            itemtype="http://schema.stenci.la/CiteGroup"><cite itemscope=""
              itemtype="http://schema.stenci.la/Cite"><a
                href="#bib30"><span>30</span><span>Hatch</span><span>1987</span></a></cite><cite
              itemscope="" itemtype="http://schema.stenci.la/Cite"><a
                href="#bib67"><span>67</span><span>Schlüter et
                  al.</span><span>2016</span></a></cite></span>. Species such as corn (<em
            itemscope="" itemtype="http://schema.stenci.la/Emphasis">Zea mays</em>) <cite
            itemscope="" itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a
              href="#bib57"><span>57</span><span>Pick et al.</span><span>2011</span></a></cite> and
          great millet (<em itemscope="" itemtype="http://schema.stenci.la/Emphasis">Sorghum
            bicolor</em>) <cite itemscope="" itemtype="http://schema.stenci.la/Cite"
            data-citationmode="Narrative"><a href="#bib20"><span>20</span><span>Döring et
                al.</span><span>2016</span></a></cite> use NADP-ME, species like common millet (<em
            itemscope="" itemtype="http://schema.stenci.la/Emphasis">Panicum miliaceum</em>) <cite
            itemscope="" itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a
              href="#bib30"><span>30</span><span>Hatch</span><span>1987</span></a></cite> and
          African spinach (<em itemscope="" itemtype="http://schema.stenci.la/Emphasis">Gynandropsis
            gynandra</em>) <span itemscope="" itemtype="http://schema.stenci.la/CiteGroup"><cite
              itemscope="" itemtype="http://schema.stenci.la/Cite"><a
                href="#bib25"><span>25</span><span>Feodorova et
                  al.</span><span>2010</span></a></cite><cite itemscope=""
              itemtype="http://schema.stenci.la/Cite"><a
                href="#bib82"><span>82</span><span>Voznesenskaya et
                  al.</span><span>2007</span></a></cite></span> use NAD-ME and species such as
          guinea grass (<em itemscope="" itemtype="http://schema.stenci.la/Emphasis">Panicum
            maximum</em>) <cite itemscope="" itemtype="http://schema.stenci.la/Cite"
            data-citationmode="Narrative"><a href="#bib12"><span>12</span><span>Bräutigam et
                al.</span><span>2014</span></a></cite> use mainly PEP-CK with the evolutionary
          constraints leading to one or the other enzyme unknown. Mixed forms are only known to
          occur between a malic enzyme and PEP-CK but not between both malic enzymes <cite
            itemscope="" itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a
              href="#bib83"><span>83</span><span>Wang et al.</span><span>2014</span></a></cite>.
          After decarboxylation, the C3 acid diffuses back to the site of phosphoenolpyruvate
          carboxylase (PEPC) and is recycled for another C4 cycle by pyruvate phosphate dikinase
          (PPDK) <span itemscope="" itemtype="http://schema.stenci.la/CiteGroup"><cite itemscope=""
              itemtype="http://schema.stenci.la/Cite"><a
                href="#bib30"><span>30</span><span>Hatch</span><span>1987</span></a></cite><cite
              itemscope="" itemtype="http://schema.stenci.la/Cite"><a
                href="#bib67"><span>67</span><span>Schlüter et
                  al.</span><span>2016</span></a></cite></span>. All the enzymes involved in the C4
          cycle are also present in C3 plants <cite itemscope=""
            itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a
              href="#bib4"><span>4</span><span>Aubry et al.</span><span>2011</span></a></cite>. In
          its most typical form, this C4 cycle is distributed between different cell types in a leaf
          in an arrangement called Kranz anatomy <cite itemscope=""
            itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a
              href="#bib29"><span>29</span><span>Haberlandt and
                Engelmann</span><span>1904</span></a></cite>. Initial carbon fixation by PEPC occurs
          in the mesophyll cell, the outer layer of photosynthetic tissue. The secondary fixation by
          Rubisco after decarboxylation occurs in an inner layer of photosynthetic tissue, the
          bundle sheath which in turn surrounds the veins. Both cells are connected by plasmodesmata
          which are pores with limited transfer specificity between cells. A model which may test
          possible carbon fixation pathways at the molecular level thus requires two cell
          architectures connected by transport processes <cite itemscope=""
            itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a
              href="#bib15"><span>15</span><span>Bräutigam and
                Weber</span><span>2010</span></a></cite>.</p>
        <p itemscope="" itemtype="http://schema.stenci.la/Paragraph">CBM of genome-scale or close to
          it are well suited to study evolution (summarised in <cite itemscope=""
            itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a
              href="#bib55"><span>55</span><span>Papp et al.</span><span>2011</span></a></cite>).
          Evolution of different metabolic modes from a ground state, the metabolism of <em
            itemscope="" itemtype="http://schema.stenci.la/Emphasis">Escherichia coli</em>, such as
          glycerol usage <cite itemscope="" itemtype="http://schema.stenci.la/Cite"
            data-citationmode="Narrative"><a href="#bib39"><span>39</span><span>Lewis et
                al.</span><span>2010</span></a></cite> or endosymbiotic metabolism <cite
            itemscope="" itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a
              href="#bib54"><span>54</span><span>Pál et al.</span><span>2006</span></a></cite> have
          been successfully predicted. Metabolic maps of eukaryotic metabolism are of higher
          complexity compared to bacteria since they require information about intracellular
          compartmentation and intracellular transport <cite itemscope=""
            itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a
              href="#bib21"><span>21</span><span>Duarte</span><span>2004</span></a></cite> and may
          require multicellular approaches. In plants, aspects of complex metabolic pathways, such
          as the energetics of CAM photosynthesis <cite itemscope=""
            itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a
              href="#bib17"><span>17</span><span>Cheung et al.</span><span>2014</span></a></cite>,
          and fluxes in C3 and C4 metabolism <span itemscope=""
            itemtype="http://schema.stenci.la/CiteGroup"><cite itemscope=""
              itemtype="http://schema.stenci.la/Cite"><a href="#bib11"><span>11</span><span>Boyle
                  and Morgan</span><span>2009</span></a></cite><cite itemscope=""
              itemtype="http://schema.stenci.la/Cite"><a href="#bib28"><span>28</span><span>Gomes de
                  Oliveira Dal’Molin et al.</span><span>2011</span></a></cite><cite itemscope=""
              itemtype="http://schema.stenci.la/Cite"><a href="#bib19"><span>19</span><span>de
                  Oliveira Dal'Molin et al.</span><span>2010</span></a></cite><cite itemscope=""
              itemtype="http://schema.stenci.la/Cite"><a href="#bib2"><span>2</span><span>Arnold and
                  Nikoloski</span><span>2014</span></a></cite><cite itemscope=""
              itemtype="http://schema.stenci.la/Cite"><a href="#bib64"><span>64</span><span>Saha et
                  al.</span><span>2011</span></a></cite></span> have been elucidated with genome
          scale models. The C4 cycle is not predicted by these current C4 models unless the C4 cycle
          is forced by constraints <span itemscope=""
            itemtype="http://schema.stenci.la/CiteGroup"><cite itemscope=""
              itemtype="http://schema.stenci.la/Cite"><a href="#bib28"><span>28</span><span>Gomes de
                  Oliveira Dal’Molin et al.</span><span>2011</span></a></cite><cite itemscope=""
              itemtype="http://schema.stenci.la/Cite"><a href="#bib47"><span>47</span><span>Mallmann
                  et al.</span><span>2014</span></a></cite></span>. In the C4GEM model, the fluxes
          representing the C4 cycle are a priori constrained to the cell types <cite itemscope=""
            itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a
              href="#bib28"><span>28</span><span>Gomes de Oliveira Dal’Molin et
                al.</span><span>2011</span></a></cite>, and in the Mallmann model, the C4 fluxes are
          induced by activating flux through PEPC <cite itemscope=""
            itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a
              href="#bib47"><span>47</span><span>Mallmann et al.</span><span>2014</span></a></cite>.
          Models in which specific a priori constraints activated C4 were successfully used to study
          metabolism under conditions of photosynthesis, photorespiration, and respiration <cite
            itemscope="" itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a
              href="#bib64"><span>64</span><span>Saha et al.</span><span>2011</span></a></cite> and
          to study N-assimilation under varying conditions <cite itemscope=""
            itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a
              href="#bib71"><span>71</span><span>Simons et al.</span><span>2013</span></a></cite>.
          However, they are incapable of testing under which conditions the pathway may evolve.</p>
        <p itemscope="" itemtype="http://schema.stenci.la/Paragraph">Schematic models suggest that
          the C4 cycle evolves from its ancestral metabolic state C3 photosynthesis along a sequence
          of stages (summarised in <cite itemscope="" itemtype="http://schema.stenci.la/Cite"
            data-citationmode="Narrative"><a
              href="#bib62"><span>62</span><span>Sage</span><span>2004</span></a></cite>; <cite
            itemscope="" itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a
              href="#bib14"><span>14</span><span>Bräutigam and
                Gowik</span><span>2016</span></a></cite>). In the presence of tight vein spacing and
          of photosynthetically active bundle sheath cells (i.e. Kranz anatomy), a key intermediate
          in which the process of photorespiration is divided between cell types is thought to
          evolve <span itemscope="" itemtype="http://schema.stenci.la/CiteGroup"><cite itemscope=""
              itemtype="http://schema.stenci.la/Cite"><a
                href="#bib48"><span>48</span><span>Monson</span><span>1999</span></a></cite><cite
              itemscope="" itemtype="http://schema.stenci.la/Cite"><a
                href="#bib63"><span>63</span><span>Sage et
                  al.</span><span>2012</span></a></cite><cite itemscope=""
              itemtype="http://schema.stenci.la/Cite"><a href="#bib31"><span>31</span><span>Heckmann
                  et al.</span><span>2013</span></a></cite><cite itemscope=""
              itemtype="http://schema.stenci.la/Cite"><a
                href="#bib6"><span>6</span><span>Bauwe</span><span>2010</span></a></cite></span>.
          The metabolic fluxes in this intermediate suggest an immediate path towards C4
          photosynthesis <span itemscope="" itemtype="http://schema.stenci.la/CiteGroup"><cite
              itemscope="" itemtype="http://schema.stenci.la/Cite"><a
                href="#bib47"><span>47</span><span>Mallmann et
                  al.</span><span>2014</span></a></cite><cite itemscope=""
              itemtype="http://schema.stenci.la/Cite"><a
                href="#bib14"><span>14</span><span>Bräutigam and
                  Gowik</span><span>2016</span></a></cite></span>. <cite itemscope=""
            itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a
              href="#bib31"><span>31</span><span>Heckmann et al.</span><span>2013</span></a></cite>
          built a kinetic model in which the complex C4 cycle was represented by a single enzyme,
          PEPC. Assuming carbon assimilation as a proxy for fitness, the model showed that the
          evolution from a C3 progenitor species with Kranz-type anatomy towards C4 photosynthesis
          occurs in modular, individually adaptive steps on a Mount Fuji fitness landscape. It is
          frequently assumed that evolution of C4 photosynthesis requires water limitation <span
            itemscope="" itemtype="http://schema.stenci.la/CiteGroup"><cite itemscope=""
              itemtype="http://schema.stenci.la/Cite"><a
                href="#bib14"><span>14</span><span>Bräutigam and
                  Gowik</span><span>2016</span></a></cite><cite itemscope=""
              itemtype="http://schema.stenci.la/Cite"><a href="#bib31"><span>31</span><span>Heckmann
                  et al.</span><span>2013</span></a></cite><cite itemscope=""
              itemtype="http://schema.stenci.la/Cite"><a href="#bib47"><span>47</span><span>Mallmann
                  et al.</span><span>2014</span></a></cite></span>. However, ecophysiological
          research showed that C4 can likely evolve in wet habitats <span itemscope=""
            itemtype="http://schema.stenci.la/CiteGroup"><cite itemscope=""
              itemtype="http://schema.stenci.la/Cite"><a href="#bib53"><span>53</span><span>Osborne
                  and Freckleton</span><span>2009</span></a></cite><cite itemscope=""
              itemtype="http://schema.stenci.la/Cite"><a href="#bib44"><span>44</span><span>Lundgren
                  and Christin</span><span>2017</span></a></cite></span>. CBM presents a possible
          avenue to study the evolution of C4 photosynthesis including its metabolic complexity <em
            itemscope="" itemtype="http://schema.stenci.la/Emphasis">in silico</em>.</p>
        <p itemscope="" itemtype="http://schema.stenci.la/Paragraph">In this study, we establish a
          generic two-celled, constraint-based model starting from the <em itemscope=""
            itemtype="http://schema.stenci.la/Emphasis">Arabidopsis</em> core model <cite
            itemscope="" itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a
              href="#bib2"><span>2</span><span>Arnold and
                Nikoloski</span><span>2014</span></a></cite>. We test under which conditions and
          constraints C4 photosynthesis is predicted as the optimal solution. Finally, we test which
          constraints result in the prediction of the particular C4 modes with their different
          decarboxylation enzymes. In the process, we demonstrate that evolution is predictable at
          the molecular level in an eukaryotic system and define the selective pressures and
          limitations guiding the &#39;choice&#39; of metabolic flux.</p>
        <h2 itemscope="" itemtype="http://schema.stenci.la/Heading" id="materials-and-methods">
          Materials and methods</h2>
        <h3 itemscope="" itemtype="http://schema.stenci.la/Heading" id="flux-balance-analysis">Flux
          Balance Analysis</h3>
        <p itemscope="" itemtype="http://schema.stenci.la/Paragraph">Flux balance analysis (FBA) is
          a CBM approach <cite itemscope="" itemtype="http://schema.stenci.la/Cite"
            data-citationmode="Narrative"><a href="#bib52"><span>52</span><span>Orth et
                al.</span><span>2010</span></a></cite> to investigate the steady-state behaviour of
          a metabolic network defined by its stoichiometric matrix <span itemscope=""
            itemtype="http://schema.stenci.la/MathFragment"><span class="mjx-chtml"><span
                class="mjx-math" aria-label="S"><span class="mjx-mrow" aria-hidden="true"><span
                    class="mjx-mi"><span class="mjx-char MJXc-TeX-math-I"
                      style="padding-top: 0.519em; padding-bottom: 0.298em; padding-right: 0.032em;">S</span></span></span></span></span></span>.
          By employing linear programming, FBA allows computing an optimised flux distribution that
          minimises and/or maximises the synthesis and/or consumption rate of one specific
          metabolite or a combination of various metabolites. Next to the steady-state assumption
          and stoichiometric matrix <span itemscope=""
            itemtype="http://schema.stenci.la/MathFragment"><span class="mjx-chtml"><span
                class="mjx-math" aria-label="S"><span class="mjx-mrow" aria-hidden="true"><span
                    class="mjx-mi"><span class="mjx-char MJXc-TeX-math-I"
                      style="padding-top: 0.519em; padding-bottom: 0.298em; padding-right: 0.032em;">S</span></span></span></span></span></span>,
          FBA relies on the definition of the reaction directionality and reversibility, denoted by
          the lower bound <span itemscope="" itemtype="http://schema.stenci.la/MathFragment"><span
              class="mjx-chtml"><span class="mjx-math" aria-label="{v}_{min}"><span class="mjx-mrow"
                  aria-hidden="true"><span class="mjx-msubsup"><span class="mjx-base"><span
                        class="mjx-texatom"><span class="mjx-mrow"><span class="mjx-mi"><span
                              class="mjx-char MJXc-TeX-math-I"
                              style="padding-top: 0.225em; padding-bottom: 0.298em;">v</span></span></span></span></span><span
                      class="mjx-sub"
                      style="font-size: 70.7%; vertical-align: -0.212em; padding-right: 0.071em;"><span
                        class="mjx-texatom" style=""><span class="mjx-mrow"><span
                            class="mjx-mi"><span class="mjx-char MJXc-TeX-math-I"
                              style="padding-top: 0.225em; padding-bottom: 0.298em;">m</span></span><span
                            class="mjx-mi"><span class="mjx-char MJXc-TeX-math-I"
                              style="padding-top: 0.446em; padding-bottom: 0.298em;">i</span></span><span
                            class="mjx-mi"><span class="mjx-char MJXc-TeX-math-I"
                              style="padding-top: 0.225em; padding-bottom: 0.298em;">n</span></span></span></span></span></span></span></span></span></span>
          and upper bound <span itemscope="" itemtype="http://schema.stenci.la/MathFragment"><span
              class="mjx-chtml"><span class="mjx-math" aria-label="{v}_{max}"><span class="mjx-mrow"
                  aria-hidden="true"><span class="mjx-msubsup"><span class="mjx-base"><span
                        class="mjx-texatom"><span class="mjx-mrow"><span class="mjx-mi"><span
                              class="mjx-char MJXc-TeX-math-I"
                              style="padding-top: 0.225em; padding-bottom: 0.298em;">v</span></span></span></span></span><span
                      class="mjx-sub"
                      style="font-size: 70.7%; vertical-align: -0.212em; padding-right: 0.071em;"><span
                        class="mjx-texatom" style=""><span class="mjx-mrow"><span
                            class="mjx-mi"><span class="mjx-char MJXc-TeX-math-I"
                              style="padding-top: 0.225em; padding-bottom: 0.298em;">m</span></span><span
                            class="mjx-mi"><span class="mjx-char MJXc-TeX-math-I"
                              style="padding-top: 0.225em; padding-bottom: 0.298em;">a</span></span><span
                            class="mjx-mi"><span class="mjx-char MJXc-TeX-math-I"
                              style="padding-top: 0.225em; padding-bottom: 0.298em;">x</span></span></span></span></span></span></span></span></span></span>
          , as well as the definition of an objective function <span itemscope=""
            itemtype="http://schema.stenci.la/MathFragment"><span class="mjx-chtml"><span
                class="mjx-math" aria-label="z"><span class="mjx-mrow" aria-hidden="true"><span
                    class="mjx-mi"><span class="mjx-char MJXc-TeX-math-I"
                      style="padding-top: 0.225em; padding-bottom: 0.298em; padding-right: 0.003em;">z</span></span></span></span></span></span>.
          The objective function <span itemscope=""
            itemtype="http://schema.stenci.la/MathFragment"><span class="mjx-chtml"><span
                class="mjx-math" aria-label="z"><span class="mjx-mrow" aria-hidden="true"><span
                    class="mjx-mi"><span class="mjx-char MJXc-TeX-math-I"
                      style="padding-top: 0.225em; padding-bottom: 0.298em; padding-right: 0.003em;">z</span></span></span></span></span></span>
          defines a flux distribution <span itemscope=""
            itemtype="http://schema.stenci.la/MathFragment"><span class="mjx-chtml"><span
                class="mjx-math" aria-label="v"><span class="mjx-mrow" aria-hidden="true"><span
                    class="mjx-mi"><span class="mjx-char MJXc-TeX-math-I"
                      style="padding-top: 0.225em; padding-bottom: 0.298em;">v</span></span></span></span></span></span>,
          with respect to an objective <span itemscope=""
            itemtype="http://schema.stenci.la/MathFragment"><span class="mjx-chtml"><span
                class="mjx-math" aria-label="c"><span class="mjx-mrow" aria-hidden="true"><span
                    class="mjx-mi"><span class="mjx-char MJXc-TeX-math-I"
                      style="padding-top: 0.225em; padding-bottom: 0.298em;">c</span></span></span></span></span></span>.
        </p><span itemscope="" itemtype="http://schema.stenci.la/MathBlock"><span
            class="mjx-chtml MJXc-display" style="text-align: center;"><span class="mjx-math"
              aria-label="{\displaystyle \begin{array}{ll}\text{min/max}&amp; {z}_{{}_{FBA}}={c}^{T}v\\ \text{s.t.}\\ &amp; S\cdot v=0\\ &amp; {v}_{min}\le v\le {v}_{max}\end{array}}"><span
                class="mjx-mrow" aria-hidden="true"><span class="mjx-texatom"><span
                    class="mjx-mrow"><span class="mjx-mstyle"><span class="mjx-mrow"><span
                          class="mjx-mtable"
                          style="vertical-align: -2.455em; padding: 0px 0.167em;"><span
                            class="mjx-table"><span class="mjx-mtr" style="height: 1.411em;"><span
                                class="mjx-mtd"
                                style="padding: 0px 0.5em 0px 0px; text-align: left; width: 4.028em;"><span
                                  class="mjx-mrow" style="margin-top: -0.141em;"><span
                                    class="mjx-mtext"><span class="mjx-char MJXc-TeX-main-R"
                                      style="padding-top: 0.446em; padding-bottom: 0.593em;">min/max</span></span><span
                                    class="mjx-strut"></span></span></span><span class="mjx-mtd"
                                style="padding: 0px 0px 0px 0.5em; text-align: left; width: 6.881em;"><span
                                  class="mjx-mrow" style="margin-top: -0.141em;"><span
                                    class="mjx-msubsup"><span class="mjx-base"
                                      style="margin-right: -0.003em;"><span
                                        class="mjx-texatom"><span class="mjx-mrow"><span
                                            class="mjx-mi"><span class="mjx-char MJXc-TeX-math-I"
                                              style="padding-top: 0.225em; padding-bottom: 0.298em; padding-right: 0.003em;">z</span></span></span></span></span><span
                                      class="mjx-sub"
                                      style="font-size: 70.7%; vertical-align: -0.212em; padding-right: 0.071em;"><span
                                        class="mjx-texatom" style=""><span class="mjx-mrow"><span
                                            class="mjx-msubsup"><span class="mjx-base"><span
                                                class="mjx-texatom"><span
                                                  class="mjx-mrow"></span></span></span><span
                                              class="mjx-sub"
                                              style="font-size: 83.3%; vertical-align: -0.317em; padding-right: 0.06em;"><span
                                                class="mjx-texatom" style=""><span
                                                  class="mjx-mrow"><span class="mjx-mi"><span
                                                      class="mjx-char MJXc-TeX-math-I"
                                                      style="padding-top: 0.446em; padding-bottom: 0.298em; padding-right: 0.106em;">F</span></span><span
                                                    class="mjx-mi"><span
                                                      class="mjx-char MJXc-TeX-math-I"
                                                      style="padding-top: 0.446em; padding-bottom: 0.298em;">B</span></span><span
                                                    class="mjx-mi"><span
                                                      class="mjx-char MJXc-TeX-math-I"
                                                      style="padding-top: 0.519em; padding-bottom: 0.298em;">A</span></span></span></span></span></span></span></span></span></span><span
                                    class="mjx-mo MJXc-space3"><span
                                      class="mjx-char MJXc-TeX-main-R"
                                      style="padding-top: 0.077em; padding-bottom: 0.298em;">=</span></span><span
                                    class="mjx-msubsup MJXc-space3"><span class="mjx-base"><span
                                        class="mjx-texatom"><span class="mjx-mrow"><span
                                            class="mjx-mi"><span class="mjx-char MJXc-TeX-math-I"
                                              style="padding-top: 0.225em; padding-bottom: 0.298em;">c</span></span></span></span></span><span
                                      class="mjx-sup"
                                      style="font-size: 70.7%; vertical-align: 0.513em; padding-left: 0px; padding-right: 0.071em;"><span
                                        class="mjx-texatom" style=""><span class="mjx-mrow"><span
                                            class="mjx-mi"><span class="mjx-char MJXc-TeX-math-I"
                                              style="padding-top: 0.446em; padding-bottom: 0.298em; padding-right: 0.12em;">T</span></span></span></span></span></span><span
                                    class="mjx-mi"><span class="mjx-char MJXc-TeX-math-I"
                                      style="padding-top: 0.225em; padding-bottom: 0.298em;">v</span></span><span
                                    class="mjx-strut"></span></span></span></span><span
                              class="mjx-mtr" style="height: 1.4em;"><span class="mjx-mtd"
                                style="padding: 0.2em 0.5em 0px 0px; text-align: left;"><span
                                  class="mjx-mrow" style="margin-top: -0.2em;"><span
                                    class="mjx-mtext"><span class="mjx-char MJXc-TeX-main-R"
                                      style="padding-top: 0.298em; padding-bottom: 0.372em;">s.t.</span></span><span
                                    class="mjx-strut"></span></span></span><span class="mjx-mtd"
                                style="padding: 0.2em 0px 0px 0.5em; text-align: left;"><span
                                  style="margin-top: -0.2em;"></span></span></span><span
                              class="mjx-mtr" style="height: 1.4em;"><span class="mjx-mtd"
                                style="padding: 0.2em 0.5em 0px 0px; text-align: left;"><span
                                  class="mjx-mrow" style="margin-top: -0.2em;"><span
                                    class="mjx-strut"></span></span></span><span class="mjx-mtd"
                                style="padding: 0.2em 0px 0px 0.5em; text-align: left;"><span
                                  class="mjx-mrow" style="margin-top: -0.2em;"><span
                                    class="mjx-mi"><span class="mjx-char MJXc-TeX-math-I"
                                      style="padding-top: 0.519em; padding-bottom: 0.298em; padding-right: 0.032em;">S</span></span><span
                                    class="mjx-mo MJXc-space2"><span
                                      class="mjx-char MJXc-TeX-main-R"
                                      style="padding-top: 0.004em; padding-bottom: 0.298em;"></span></span><span
                                    class="mjx-mi MJXc-space2"><span
                                      class="mjx-char MJXc-TeX-math-I"
                                      style="padding-top: 0.225em; padding-bottom: 0.298em;">v</span></span><span
                                    class="mjx-mo MJXc-space3"><span
                                      class="mjx-char MJXc-TeX-main-R"
                                      style="padding-top: 0.077em; padding-bottom: 0.298em;">=</span></span><span
                                    class="mjx-mn MJXc-space3"><span
                                      class="mjx-char MJXc-TeX-main-R"
                                      style="padding-top: 0.372em; padding-bottom: 0.372em;">0</span></span><span
                                    class="mjx-strut"></span></span></span></span><span
                              class="mjx-mtr" style="height: 1.2em;"><span class="mjx-mtd"
                                style="padding: 0.2em 0.5em 0px 0px; text-align: left;"><span
                                  class="mjx-mrow" style="margin-top: -0.2em;"><span
                                    class="mjx-strut"></span></span></span><span class="mjx-mtd"
                                style="padding: 0.2em 0px 0px 0.5em; text-align: left;"><span
                                  class="mjx-mrow" style="margin-top: -0.2em;"><span
                                    class="mjx-msubsup"><span class="mjx-base"><span
                                        class="mjx-texatom"><span class="mjx-mrow"><span
                                            class="mjx-mi"><span class="mjx-char MJXc-TeX-math-I"
                                              style="padding-top: 0.225em; padding-bottom: 0.298em;">v</span></span></span></span></span><span
                                      class="mjx-sub"
                                      style="font-size: 70.7%; vertical-align: -0.212em; padding-right: 0.071em;"><span
                                        class="mjx-texatom" style=""><span class="mjx-mrow"><span
                                            class="mjx-mi"><span class="mjx-char MJXc-TeX-math-I"
                                              style="padding-top: 0.225em; padding-bottom: 0.298em;">m</span></span><span
                                            class="mjx-mi"><span class="mjx-char MJXc-TeX-math-I"
                                              style="padding-top: 0.446em; padding-bottom: 0.298em;">i</span></span><span
                                            class="mjx-mi"><span class="mjx-char MJXc-TeX-math-I"
                                              style="padding-top: 0.225em; padding-bottom: 0.298em;">n</span></span></span></span></span></span><span
                                    class="mjx-mo MJXc-space3"><span
                                      class="mjx-char MJXc-TeX-main-R"
                                      style="padding-top: 0.372em; padding-bottom: 0.446em;"></span></span><span
                                    class="mjx-mi MJXc-space3"><span
                                      class="mjx-char MJXc-TeX-math-I"
                                      style="padding-top: 0.225em; padding-bottom: 0.298em;">v</span></span><span
                                    class="mjx-mo MJXc-space3"><span
                                      class="mjx-char MJXc-TeX-main-R"
                                      style="padding-top: 0.372em; padding-bottom: 0.446em;"></span></span><span
                                    class="mjx-msubsup MJXc-space3"><span class="mjx-base"><span
                                        class="mjx-texatom"><span class="mjx-mrow"><span
                                            class="mjx-mi"><span class="mjx-char MJXc-TeX-math-I"
                                              style="padding-top: 0.225em; padding-bottom: 0.298em;">v</span></span></span></span></span><span
                                      class="mjx-sub"
                                      style="font-size: 70.7%; vertical-align: -0.212em; padding-right: 0.071em;"><span
                                        class="mjx-texatom" style=""><span class="mjx-mrow"><span
                                            class="mjx-mi"><span class="mjx-char MJXc-TeX-math-I"
                                              style="padding-top: 0.225em; padding-bottom: 0.298em;">m</span></span><span
                                            class="mjx-mi"><span class="mjx-char MJXc-TeX-math-I"
                                              style="padding-top: 0.225em; padding-bottom: 0.298em;">a</span></span><span
                                            class="mjx-mi"><span class="mjx-char MJXc-TeX-math-I"
                                              style="padding-top: 0.225em; padding-bottom: 0.298em;">x</span></span></span></span></span></span><span
                                    class="mjx-strut"></span></span></span></span></span></span></span></span></span></span></span></span></span></span>
        <p itemscope="" itemtype="http://schema.stenci.la/Paragraph">The degeneracy problem, the
          possible existence of alternate optimal solutions, is one of the major issues of
          constraint-based optimisation, such as FBA <cite itemscope=""
            itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a
              href="#bib46"><span>46</span><span>Mahadevan and
                Schilling</span><span>2003</span></a></cite>. To avoid this problem, we use the
          parsimonious version of FBA (pFBA) <cite itemscope=""
            itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a
              href="#bib39"><span>39</span><span>Lewis et al.</span><span>2010</span></a></cite>.
          This approach incorporates the flux parsimony as a constraint to find the solution with
          the minimum absolute flux value among the alternative optima, which is in agreement with
          the assumption that the cell is evolutionary optimised to allocate a minimum amount of
          resources to achieve its objective.</p><span itemscope=""
          itemtype="http://schema.stenci.la/MathBlock"><span class="mjx-chtml MJXc-display"
            style="text-align: center;"><span class="mjx-math"
              aria-label="\begin{array}{cc}\text{min/max}\hfill &amp; {z}_{{}_{pFBA}}=\sum \left|{v}_{i}\right|\hfill \\ \text{s.t.}\hfill &amp; \\ &amp; S\cdot v=0\hfill \\ &amp; {v}_{min}\le v\le {v}_{max}\hfill \\ &amp; {c}^{T}v={z}_{{}_{FBA}}\hfill \end{array}"><span
                class="mjx-mrow" aria-hidden="true"><span class="mjx-mtable"
                  style="vertical-align: -3.288em; padding: 0px 0.167em;"><span
                    class="mjx-table"><span class="mjx-mtr" style="height: 1.466em;"><span
                        class="mjx-mtd"
                        style="padding: 0px 0.5em 0px 0px; text-align: left; width: 4.028em;"><span
                          class="mjx-mrow" style="margin-top: -0.2em;"><span class="mjx-mtext"><span
                              class="mjx-char MJXc-TeX-main-R"
                              style="padding-top: 0.446em; padding-bottom: 0.593em;">min/max</span></span><span
                            class="mjx-strut"></span></span></span><span class="mjx-mtd"
                        style="padding: 0px 0px 0px 0.5em; text-align: left; width: 6.881em;"><span
                          class="mjx-mrow" style="margin-top: -0.2em;"><span
                            class="mjx-msubsup"><span class="mjx-base"
                              style="margin-right: -0.003em;"><span class="mjx-texatom"><span
                                  class="mjx-mrow"><span class="mjx-mi"><span
                                      class="mjx-char MJXc-TeX-math-I"
                                      style="padding-top: 0.225em; padding-bottom: 0.298em; padding-right: 0.003em;">z</span></span></span></span></span><span
                              class="mjx-sub"
                              style="font-size: 70.7%; vertical-align: -0.212em; padding-right: 0.071em;"><span
                                class="mjx-texatom" style=""><span class="mjx-mrow"><span
                                    class="mjx-msubsup"><span class="mjx-base"><span
                                        class="mjx-texatom"><span
                                          class="mjx-mrow"></span></span></span><span
                                      class="mjx-sub"
                                      style="font-size: 83.3%; vertical-align: -0.317em; padding-right: 0.06em;"><span
                                        class="mjx-texatom" style=""><span class="mjx-mrow"><span
                                            class="mjx-mi"><span class="mjx-char MJXc-TeX-math-I"
                                              style="padding-top: 0.225em; padding-bottom: 0.446em;">p</span></span><span
                                            class="mjx-mi"><span class="mjx-char MJXc-TeX-math-I"
                                              style="padding-top: 0.446em; padding-bottom: 0.298em; padding-right: 0.106em;">F</span></span><span
                                            class="mjx-mi"><span class="mjx-char MJXc-TeX-math-I"
                                              style="padding-top: 0.446em; padding-bottom: 0.298em;">B</span></span><span
                                            class="mjx-mi"><span class="mjx-char MJXc-TeX-math-I"
                                              style="padding-top: 0.519em; padding-bottom: 0.298em;">A</span></span></span></span></span></span></span></span></span></span><span
                            class="mjx-mo MJXc-space3"><span class="mjx-char MJXc-TeX-main-R"
                              style="padding-top: 0.077em; padding-bottom: 0.298em;">=</span></span><span
                            class="mjx-mo MJXc-space3"><span class="mjx-char MJXc-TeX-size1-R"
                              style="padding-top: 0.519em; padding-bottom: 0.519em;"></span></span><span
                            class="mjx-mrow MJXc-space1"><span class="mjx-mo"><span
                                class="mjx-char MJXc-TeX-main-R"
                                style="padding-top: 0.446em; padding-bottom: 0.593em;">|</span></span><span
                              class="mjx-msubsup"><span class="mjx-base"><span
                                  class="mjx-texatom"><span class="mjx-mrow"><span
                                      class="mjx-mi"><span class="mjx-char MJXc-TeX-math-I"
                                        style="padding-top: 0.225em; padding-bottom: 0.298em;">v</span></span></span></span></span><span
                                class="mjx-sub"
                                style="font-size: 70.7%; vertical-align: -0.212em; padding-right: 0.071em;"><span
                                  class="mjx-texatom" style=""><span class="mjx-mrow"><span
                                      class="mjx-mi"><span class="mjx-char MJXc-TeX-math-I"
                                        style="padding-top: 0.446em; padding-bottom: 0.298em;">i</span></span></span></span></span></span><span
                              class="mjx-mo"><span class="mjx-char MJXc-TeX-main-R"
                                style="padding-top: 0.446em; padding-bottom: 0.593em;">|</span></span></span><span
                            class="mjx-strut"></span></span></span></span><span class="mjx-mtr"
                      style="height: 1.4em;"><span class="mjx-mtd"
                        style="padding: 0.2em 0.5em 0px 0px; text-align: left;"><span
                          class="mjx-mrow" style="margin-top: -0.2em;"><span class="mjx-mtext"><span
                              class="mjx-char MJXc-TeX-main-R"
                              style="padding-top: 0.298em; padding-bottom: 0.372em;">s.t.</span></span><span
                            class="mjx-strut"></span></span></span><span class="mjx-mtd"
                        style="padding: 0.2em 0px 0px 0.5em;"><span class="mjx-mrow"
                          style="margin-top: -0.2em;"><span
                            class="mjx-strut"></span></span></span></span><span class="mjx-mtr"
                      style="height: 1.4em;"><span class="mjx-mtd"
                        style="padding: 0.2em 0.5em 0px 0px;"><span class="mjx-mrow"
                          style="margin-top: -0.2em;"><span
                            class="mjx-strut"></span></span></span><span class="mjx-mtd"
                        style="padding: 0.2em 0px 0px 0.5em; text-align: left;"><span
                          class="mjx-mrow" style="margin-top: -0.2em;"><span class="mjx-mi"><span
                              class="mjx-char MJXc-TeX-math-I"
                              style="padding-top: 0.519em; padding-bottom: 0.298em; padding-right: 0.032em;">S</span></span><span
                            class="mjx-mo MJXc-space2"><span class="mjx-char MJXc-TeX-main-R"
                              style="padding-top: 0.004em; padding-bottom: 0.298em;"></span></span><span
                            class="mjx-mi MJXc-space2"><span class="mjx-char MJXc-TeX-math-I"
                              style="padding-top: 0.225em; padding-bottom: 0.298em;">v</span></span><span
                            class="mjx-mo MJXc-space3"><span class="mjx-char MJXc-TeX-main-R"
                              style="padding-top: 0.077em; padding-bottom: 0.298em;">=</span></span><span
                            class="mjx-mn MJXc-space3"><span class="mjx-char MJXc-TeX-main-R"
                              style="padding-top: 0.372em; padding-bottom: 0.372em;">0</span></span><span
                            class="mjx-strut"></span></span></span></span><span class="mjx-mtr"
                      style="height: 1.4em;"><span class="mjx-mtd"
                        style="padding: 0.2em 0.5em 0px 0px;"><span class="mjx-mrow"
                          style="margin-top: -0.2em;"><span
                            class="mjx-strut"></span></span></span><span class="mjx-mtd"
                        style="padding: 0.2em 0px 0px 0.5em; text-align: left;"><span
                          class="mjx-mrow" style="margin-top: -0.2em;"><span
                            class="mjx-msubsup"><span class="mjx-base"><span
                                class="mjx-texatom"><span class="mjx-mrow"><span
                                    class="mjx-mi"><span class="mjx-char MJXc-TeX-math-I"
                                      style="padding-top: 0.225em; padding-bottom: 0.298em;">v</span></span></span></span></span><span
                              class="mjx-sub"
                              style="font-size: 70.7%; vertical-align: -0.212em; padding-right: 0.071em;"><span
                                class="mjx-texatom" style=""><span class="mjx-mrow"><span
                                    class="mjx-mi"><span class="mjx-char MJXc-TeX-math-I"
                                      style="padding-top: 0.225em; padding-bottom: 0.298em;">m</span></span><span
                                    class="mjx-mi"><span class="mjx-char MJXc-TeX-math-I"
                                      style="padding-top: 0.446em; padding-bottom: 0.298em;">i</span></span><span
                                    class="mjx-mi"><span class="mjx-char MJXc-TeX-math-I"
                                      style="padding-top: 0.225em; padding-bottom: 0.298em;">n</span></span></span></span></span></span><span
                            class="mjx-mo MJXc-space3"><span class="mjx-char MJXc-TeX-main-R"
                              style="padding-top: 0.372em; padding-bottom: 0.446em;"></span></span><span
                            class="mjx-mi MJXc-space3"><span class="mjx-char MJXc-TeX-math-I"
                              style="padding-top: 0.225em; padding-bottom: 0.298em;">v</span></span><span
                            class="mjx-mo MJXc-space3"><span class="mjx-char MJXc-TeX-main-R"
                              style="padding-top: 0.372em; padding-bottom: 0.446em;"></span></span><span
                            class="mjx-msubsup MJXc-space3"><span class="mjx-base"><span
                                class="mjx-texatom"><span class="mjx-mrow"><span
                                    class="mjx-mi"><span class="mjx-char MJXc-TeX-math-I"
                                      style="padding-top: 0.225em; padding-bottom: 0.298em;">v</span></span></span></span></span><span
                              class="mjx-sub"
                              style="font-size: 70.7%; vertical-align: -0.212em; padding-right: 0.071em;"><span
                                class="mjx-texatom" style=""><span class="mjx-mrow"><span
                                    class="mjx-mi"><span class="mjx-char MJXc-TeX-math-I"
                                      style="padding-top: 0.225em; padding-bottom: 0.298em;">m</span></span><span
                                    class="mjx-mi"><span class="mjx-char MJXc-TeX-math-I"
                                      style="padding-top: 0.225em; padding-bottom: 0.298em;">a</span></span><span
                                    class="mjx-mi"><span class="mjx-char MJXc-TeX-math-I"
                                      style="padding-top: 0.225em; padding-bottom: 0.298em;">x</span></span></span></span></span></span><span
                            class="mjx-strut"></span></span></span></span><span class="mjx-mtr"
                      style="height: 1.411em;"><span class="mjx-mtd"
                        style="padding: 0.2em 0.5em 0px 0px;"><span class="mjx-mrow"
                          style="margin-top: -0.141em;"><span
                            class="mjx-strut"></span></span></span><span class="mjx-mtd"
                        style="padding: 0.2em 0px 0px 0.5em; text-align: left;"><span
                          class="mjx-mrow" style="margin-top: -0.141em;"><span
                            class="mjx-msubsup"><span class="mjx-base"><span
                                class="mjx-texatom"><span class="mjx-mrow"><span
                                    class="mjx-mi"><span class="mjx-char MJXc-TeX-math-I"
                                      style="padding-top: 0.225em; padding-bottom: 0.298em;">c</span></span></span></span></span><span
                              class="mjx-sup"
                              style="font-size: 70.7%; vertical-align: 0.513em; padding-left: 0px; padding-right: 0.071em;"><span
                                class="mjx-texatom" style=""><span class="mjx-mrow"><span
                                    class="mjx-mi"><span class="mjx-char MJXc-TeX-math-I"
                                      style="padding-top: 0.446em; padding-bottom: 0.298em; padding-right: 0.12em;">T</span></span></span></span></span></span><span
                            class="mjx-mi"><span class="mjx-char MJXc-TeX-math-I"
                              style="padding-top: 0.225em; padding-bottom: 0.298em;">v</span></span><span
                            class="mjx-mo MJXc-space3"><span class="mjx-char MJXc-TeX-main-R"
                              style="padding-top: 0.077em; padding-bottom: 0.298em;">=</span></span><span
                            class="mjx-msubsup MJXc-space3"><span class="mjx-base"
                              style="margin-right: -0.003em;"><span class="mjx-texatom"><span
                                  class="mjx-mrow"><span class="mjx-mi"><span
                                      class="mjx-char MJXc-TeX-math-I"
                                      style="padding-top: 0.225em; padding-bottom: 0.298em; padding-right: 0.003em;">z</span></span></span></span></span><span
                              class="mjx-sub"
                              style="font-size: 70.7%; vertical-align: -0.212em; padding-right: 0.071em;"><span
                                class="mjx-texatom" style=""><span class="mjx-mrow"><span
                                    class="mjx-msubsup"><span class="mjx-base"><span
                                        class="mjx-texatom"><span
                                          class="mjx-mrow"></span></span></span><span
                                      class="mjx-sub"
                                      style="font-size: 83.3%; vertical-align: -0.317em; padding-right: 0.06em;"><span
                                        class="mjx-texatom" style=""><span class="mjx-mrow"><span
                                            class="mjx-mi"><span class="mjx-char MJXc-TeX-math-I"
                                              style="padding-top: 0.446em; padding-bottom: 0.298em; padding-right: 0.106em;">F</span></span><span
                                            class="mjx-mi"><span class="mjx-char MJXc-TeX-math-I"
                                              style="padding-top: 0.446em; padding-bottom: 0.298em;">B</span></span><span
                                            class="mjx-mi"><span class="mjx-char MJXc-TeX-math-I"
                                              style="padding-top: 0.519em; padding-bottom: 0.298em;">A</span></span></span></span></span></span></span></span></span></span><span
                            class="mjx-strut"></span></span></span></span></span></span></span></span></span></span>
        <p itemscope="" itemtype="http://schema.stenci.la/Paragraph">All FBA experiments in this
          study employ pFBA and are performed using the cobrapy module in a python 2.7 environment
          run on a personal computer (macOS Sierra, 4 GHz Intel Core i7, 32 GB 1867 MHz DDR3). All
          FBA experiments are available as jupyter notebooks in the supplementary material and can
          also be accessed and executed from the GitHub repository <a
            href="https://github.com/ma-blaetke/CBM_C3_C4_Metabolism" itemscope=""
            itemtype="http://schema.stenci.la/Link">https://github.com/ma-blaetke/CBM_C3_C4_Metabolism</a> (<cite
            itemscope="" itemtype="http://schema.stenci.la/Cite"><a
              href="#bib10"><span>10</span><span>Blätke</span><span>2019</span></a></cite>; copy
          archived at <a href="https://github.com/elifesciences-publications/CBM_C3_C4_Metabolism"
            itemscope=""
            itemtype="http://schema.stenci.la/Link">https://github.com/elifesciences-publications/CBM_C3_C4_Metabolism</a>).
        </p>
        <stencila-code-chunk itemscope="" itemtype="http://schema.stenci.la/CodeChunk"
          data-execution_count="1" data-programminglanguage="python">
          <pre class="language-python" itemscope="" itemtype="http://schema.stenci.la/CodeBlock"
            slot="text"><code>##### Import Modules #####

## numpy
import numpy as np

## pandas
import pandas as pd
pd.set_option(&#39;display.max_rows&#39;, None)
pd.set_option(&#39;display.max_columns&#39;, None)
pd.set_option(&#39;display.width&#39;, None)
pd.set_option(&#39;display.max_colwidth&#39;, None)

## plotly
import plotly
import plotly.graph_objects as go
from plotly.subplots import make_subplots

## cobra
import cobra

## escher
from escher import Builder

## ipython HTML display
from IPython.display import HTML

## goatools
from goatools import obo_parser

## tqdm
from tqdm.notebook import trange, tqdm

## string
import string

print(f&quot;Code Cell 1: Import Python Modules&quot;)</code></pre>
          <figure slot="outputs">
            <pre class="language-text" itemscope="" itemtype="http://schema.stenci.la/CodeBlock"><code>Code Cell 1: Import Python Modules
</code></pre>
          </figure>
        </stencila-code-chunk>
        <stencila-code-chunk itemscope="" itemtype="http://schema.stenci.la/CodeChunk"
          data-execution_count="2" data-programminglanguage="python">
          <pre class="language-python" itemscope="" itemtype="http://schema.stenci.la/CodeBlock"
            slot="text"><code>print(f&quot;Code Cell 2: Set Parameters&quot;)

##### Set Parameters #####

inf = float(1e6) </code></pre>
          <figure slot="outputs">
            <pre class="language-text" itemscope="" itemtype="http://schema.stenci.la/CodeBlock"><code>Code Cell 2: Set Parameters
</code></pre>
          </figure>
        </stencila-code-chunk>
        <stencila-code-chunk itemscope="" itemtype="http://schema.stenci.la/CodeChunk"
          data-execution_count="3" data-programminglanguage="python">
          <pre class="language-python" itemscope="" itemtype="http://schema.stenci.la/CodeBlock"
            slot="text"><code>print(f&quot;Code Cell 3: Define Functions&quot;)

##### Define Functions - SBML Model and FBA #####

## Load SBML model
def load_sbml_model():
    
    &#39;&#39;&#39;
    Return cobra model

            Parameters:

            Returns:
                    cobra_model (cobra.model): cobra model of specified sbm file
    &#39;&#39;&#39;
        
    sbml_file = &#39;elife-49305.ipython.src/2018-23-05-mb-genC3.sbml&#39;
    cobra_model = cobra.io.sbml.read_sbml_model(sbml_file)
    
    return cobra_model

## Add reactions to model
def add_rxn(name, D_mets, model, rev=True):
    
    &#39;&#39;&#39;
    Add new reaction to cobra model

            Parameters:
                    name (str): short name/id of reaction
                    D_mets (dict): dictionary of metabolites and their stoichimetric coeffcients attending at reaction
                    model (cobra.model): cobra model to add the reaction
                    rev (bool): reversiblity of reaction (default: True)

            Returns:
                    
    &#39;&#39;&#39;
    
    r_name = name
    r_obj = cobra.Reaction(rname)
    r_obj.name = r_name
    r_obj.id = r_name
    model.add_reaction(r_obj)
    r_obj.add_metabolites(D_mets)
    r_obj.objective_coefficient = 0
    r_obj.bounds = (-inf,inf) if rev else (0,inf)

## Set flux of a reaction to a fixed value
def set_fixed_flux(r_id, val, model):
    
    &#39;&#39;&#39;
    Set flux of reaction to a fixed value

            Parameters:
                    r_id (str): reaction id
                    val (float): flux value
                    model (cobra.model): cobra model to add the reaction

            Returns:
    &#39;&#39;&#39;
    
    r_obj = model.reactions.get_by_id(r_id)
    r_obj.bounds = (val,val)
    
## Set lower and upper flux bound of a reaction
def set_bounds(r_id, val_tuple, model):
    
    &#39;&#39;&#39;
    Set flux bounds of reaction

            Parameters:
                    r_id (str): reaction id
                    val_tuple (tuple): (lower_bound, upper_bound)
                    model (cobra.model): cobra model to add the reaction

            Returns:
    &#39;&#39;&#39;
    
    r_obj = model.reactions.get_by_id(r_id)
    r_obj.bounds = val_tuple

## Set flux ratio for two reactions
def set_fixed_flux_ratio(r_dict, name, model):
    
    &#39;&#39;&#39;
    Set flux ratio of two reactions

            Parameters:
                    r_dict (dict): rdictionary with two keys (reaction ids) and their proportion in the ratio {r_id1: prop1, r_ids2: prop2}
                    name (str): name of constraint
                    model (cobra.model): cobra model to add the reaction

            Returns:
    &#39;&#39;&#39;
        
    if len(r_dict) == 2:
        r_id1 = list(r_dict.keys())[0]
        r_obj1 = model.reactions.get_by_id(r_id1)
        r_v1 = list(r_dict.values())[0]
        r_id2 = list(r_dict.keys())[1]
        r_obj2 = model.reactions.get_by_id(r_id2)
        r_v2 = list(r_dict.values())[1]
        const = model.problem.Constraint(
            float(r_v1) * r_obj2.flux_expression - float(r_v2) * r_obj1.flux_expression, 
            lb = 0.0, 
            ub = 0.0, 
            name = name)
        model.add_cons_vars(const)
        return const</code></pre>
          <figure slot="outputs">
            <pre class="language-text" itemscope="" itemtype="http://schema.stenci.la/CodeBlock"><code>Code Cell 3: Define Functions
</code></pre>
          </figure>
        </stencila-code-chunk>
        <h3 itemscope="" itemtype="http://schema.stenci.la/Heading"
          id="generic-model-for-c3-metabolism">Generic model for C3 metabolism</h3>
        <h4 itemscope="" itemtype="http://schema.stenci.la/Heading" id="metabolic-model">Metabolic
          model</h4>
        <p itemscope="" itemtype="http://schema.stenci.la/Paragraph">The generic model representing
          the metabolism of a mesophyll cell of a mature photosynthetically active C3 leaf, further
          on called <em itemscope="" itemtype="http://schema.stenci.la/Emphasis">one-cell</em>
          model, is based on the <em itemscope=""
            itemtype="http://schema.stenci.la/Emphasis">Arabidopsis</em> core model <cite
            itemscope="" itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a
              href="#bib2"><span>2</span><span>Arnold and
                Nikoloski</span><span>2014</span></a></cite>. The model is compartmentalised into
          cytosol (c), chloroplast (h), mitochondria (m), and peroxisome (p). Each reaction in the
          <em itemscope="" itemtype="http://schema.stenci.la/Emphasis">Arabidopsis</em> core model
          <cite itemscope="" itemtype="http://schema.stenci.la/Cite"
            data-citationmode="Narrative"><a href="#bib2"><span>2</span><span>Arnold and
                Nikoloski</span><span>2014</span></a></cite> was compared with the corresponding
          entry in AraCyc <cite itemscope="" itemtype="http://schema.stenci.la/Cite"
            data-citationmode="Narrative"><a href="#bib49"><span>49</span><span>Mueller et
                al.</span><span>2003</span></a></cite>. Based on the given information, we corrected
          co-factors, gene associations, enzyme commission numbers and reversibility (information
          from BRENDA <cite itemscope="" itemtype="http://schema.stenci.la/Cite"><a
              href="#bib68"><span>68</span><span>Schomburg et al.</span><span>2002</span></a></cite>
          were included). The gene associations and their GO terms <cite itemscope=""
            itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a
              href="#bib3"><span>3</span><span>Ashburner et al.</span><span>2000</span></a></cite>
          of the cellular components were used to correct the location of reactions. Major additions
          to the model are the cyclic electron flow <cite itemscope=""
            itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a
              href="#bib70"><span>70</span><span>Shikanai</span><span>2016</span></a></cite>,
          alternative oxidases in mitochondria and chloroplast <cite itemscope=""
            itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a
              href="#bib81"><span>81</span><span>Vishwakarma et
                al.</span><span>2015</span></a></cite>, as well as several transport processes
          between the compartments and the cytosol <cite itemscope=""
            itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a
              href="#bib42"><span>42</span><span>Linka and Weber</span><span>2010</span></a></cite>.
          NAD-dependent dehydrogenase to oxidise malate is present in all compartments <span
            itemscope="" itemtype="http://schema.stenci.la/CiteGroup"><cite itemscope=""
              itemtype="http://schema.stenci.la/Cite"><a
                href="#bib27"><span>27</span><span>Gietl</span><span>1992</span></a></cite><cite
              itemscope="" itemtype="http://schema.stenci.la/Cite"><a
                href="#bib9"><span>9</span><span>Berkemeyer et
                  al.</span><span>1998</span></a></cite></span>, which excludes the interconversion
          of NAD and NADP by cycles through the nitrate reductase present in the <em itemscope=""
            itemtype="http://schema.stenci.la/Emphasis">Arabidopsis</em> core model. Correctly
          defining the protonation state of the metabolites in the various cellular compartments is
          a general drawback of metabolic models due to the lack of knowledge in that area. This
          issue mainly affects biochemical reactions and transport reactions involving protons. We
          added a sink/source reaction for protons in the form:</p><span itemscope=""
          itemtype="http://schema.stenci.la/MathBlock"><span class="mjx-chtml MJXc-display"
            style="text-align: center;"><span class="mjx-math"
              aria-label="\begin{array}{cc}\leftrightarrow H\mathrm{\_}\{x\}\hfill &amp; x=c,h,m,p\hfill \end{array}"><span
                class="mjx-mrow" aria-hidden="true"><span class="mjx-mtable"
                  style="vertical-align: -0.288em; padding: 0px 0.167em;"><span
                    class="mjx-table"><span class="mjx-mtr" style="height: 1.075em;"><span
                        class="mjx-mtd"
                        style="padding: 0px 0.5em 0px 0px; text-align: left; width: 4.238em;"><span
                          class="mjx-mrow" style="margin-top: -0.2em;"><span class="mjx-mo"><span
                              class="mjx-char MJXc-TeX-main-R"
                              style="padding-top: 0.225em; padding-bottom: 0.372em;"></span></span><span
                            class="mjx-mi MJXc-space3"><span class="mjx-char MJXc-TeX-math-I"
                              style="padding-top: 0.446em; padding-bottom: 0.298em; padding-right: 0.057em;">H</span></span><span
                            class="mjx-texatom"><span class="mjx-mrow"><span class="mjx-mi"><span
                                  class="mjx-char MJXc-TeX-main-R"
                                  style="margin-top: -0.291em; padding-bottom: 0.372em;">_</span></span></span></span><span
                            class="mjx-mo"><span class="mjx-char MJXc-TeX-main-R"
                              style="padding-top: 0.446em; padding-bottom: 0.593em;">{</span></span><span
                            class="mjx-mi"><span class="mjx-char MJXc-TeX-math-I"
                              style="padding-top: 0.225em; padding-bottom: 0.298em;">x</span></span><span
                            class="mjx-mo"><span class="mjx-char MJXc-TeX-main-R"
                              style="padding-top: 0.446em; padding-bottom: 0.593em;">}</span></span><span
                            class="mjx-strut"></span></span></span><span class="mjx-mtd"
                        style="padding: 0px 0px 0px 0.5em; text-align: left; width: 5.63em;"><span
                          class="mjx-mrow" style="margin-top: -0.2em;"><span class="mjx-mi"><span
                              class="mjx-char MJXc-TeX-math-I"
                              style="padding-top: 0.225em; padding-bottom: 0.298em;">x</span></span><span
                            class="mjx-mo MJXc-space3"><span class="mjx-char MJXc-TeX-main-R"
                              style="padding-top: 0.077em; padding-bottom: 0.298em;">=</span></span><span
                            class="mjx-mi MJXc-space3"><span class="mjx-char MJXc-TeX-math-I"
                              style="padding-top: 0.225em; padding-bottom: 0.298em;">c</span></span><span
                            class="mjx-mo"><span class="mjx-char MJXc-TeX-main-R"
                              style="margin-top: -0.144em; padding-bottom: 0.519em;">,</span></span><span
                            class="mjx-mi MJXc-space1"><span class="mjx-char MJXc-TeX-math-I"
                              style="padding-top: 0.446em; padding-bottom: 0.298em;">h</span></span><span
                            class="mjx-mo"><span class="mjx-char MJXc-TeX-main-R"
                              style="margin-top: -0.144em; padding-bottom: 0.519em;">,</span></span><span
                            class="mjx-mi MJXc-space1"><span class="mjx-char MJXc-TeX-math-I"
                              style="padding-top: 0.225em; padding-bottom: 0.298em;">m</span></span><span
                            class="mjx-mo"><span class="mjx-char MJXc-TeX-main-R"
                              style="margin-top: -0.144em; padding-bottom: 0.519em;">,</span></span><span
                            class="mjx-mi MJXc-space1"><span class="mjx-char MJXc-TeX-math-I"
                              style="padding-top: 0.225em; padding-bottom: 0.446em;">p</span></span><span
                            class="mjx-strut"></span></span></span></span></span></span></span></span></span></span>
        <p itemscope="" itemtype="http://schema.stenci.la/Paragraph">to all compartments to prevent
          futile fluxes of protons and other metabolites coupled through the proton transport. The
          curated <em itemscope="" itemtype="http://schema.stenci.la/Emphasis">one-cell</em> model
          is provided in <a href="#fig1sdata1" itemscope=""
            itemtype="http://schema.stenci.la/Link">Figure 1—source data 1</a>.</p>
        <stencila-code-chunk itemscope="" itemtype="http://schema.stenci.la/CodeChunk"
          data-execution_count="4" data-programminglanguage="python">
          <pre class="language-python" itemscope="" itemtype="http://schema.stenci.la/CodeBlock"
            slot="text"><code>print(f&quot;Code Cell 4: Load Metabolic Model of C3 Photosynthesis in Arabidposis thaliana&quot;)

##### Load model #####
c3_model = load_sbml_model()

# Model summary
c3_num_mets = len(c3_model.metabolites)
c3_num_rxn = len(c3_model.reactions)
c3_num_transport_rxn = len(c3_model.reactions.query(lambda rxn: (rxn.id.startswith(&#39;Tr_&#39;))))
c3_num_export_rxn = len(c3_model.reactions.query(lambda rxn: (rxn.id.startswith(&#39;Ex_&#39;))))
c3_num_import_rxn = len(c3_model.reactions.query(lambda rxn: (rxn.id.startswith(&#39;Im_&#39;))))

df_c3_model_summary = pd.DataFrame([c3_num_mets, c3_num_rxn, c3_num_transport_rxn, c3_num_export_rxn, c3_num_import_rxn],
                                   index=[&#39;total metabolites&#39;,&#39;total reactions&#39;,&#39;transport reactions&#39;, &#39;export reactions&#39; ,&#39;import reactions&#39;], columns=[&#39;Count&#39;])
df_c3_model_summary</code></pre>
          <figure slot="outputs">
            <pre class="language-text" itemscope="" itemtype="http://schema.stenci.la/CodeBlock"><code>Code Cell 4: Load Metabolic Model of C3 Photosynthesis in Arabidposis thaliana
</code></pre>
            <table itemscope="" itemtype="http://schema.org/Table">
              <thead>
                <tr itemscope="" itemtype="http://schema.stenci.la/TableRow">
                  <th itemscope="" itemtype="http://schema.stenci.la/TableCell"></th>
                  <th itemscope="" itemtype="http://schema.stenci.la/TableCell">Count</th>
                </tr>
              </thead>
              <tbody>
                <tr itemscope="" itemtype="http://schema.stenci.la/TableRow">
                  <td itemscope="" itemtype="http://schema.stenci.la/TableCell">total metabolites
                  </td>
                  <td itemscope="" itemtype="http://schema.stenci.la/TableCell"><span
                      data-itemtype="http://schema.org/Number">413</span></td>
                </tr>
                <tr itemscope="" itemtype="http://schema.stenci.la/TableRow">
                  <td itemscope="" itemtype="http://schema.stenci.la/TableCell">total reactions</td>
                  <td itemscope="" itemtype="http://schema.stenci.la/TableCell"><span
                      data-itemtype="http://schema.org/Number">572</span></td>
                </tr>
                <tr itemscope="" itemtype="http://schema.stenci.la/TableRow">
                  <td itemscope="" itemtype="http://schema.stenci.la/TableCell">transport reactions
                  </td>
                  <td itemscope="" itemtype="http://schema.stenci.la/TableCell"><span
                      data-itemtype="http://schema.org/Number">139</span></td>
                </tr>
                <tr itemscope="" itemtype="http://schema.stenci.la/TableRow">
                  <td itemscope="" itemtype="http://schema.stenci.la/TableCell">export reactions
                  </td>
                  <td itemscope="" itemtype="http://schema.stenci.la/TableCell"><span
                      data-itemtype="http://schema.org/Number">90</span></td>
                </tr>
                <tr itemscope="" itemtype="http://schema.stenci.la/TableRow">
                  <td itemscope="" itemtype="http://schema.stenci.la/TableCell">import reactions
                  </td>
                  <td itemscope="" itemtype="http://schema.stenci.la/TableCell"><span
                      data-itemtype="http://schema.org/Number">8</span></td>
                </tr>
              </tbody>
            </table>
          </figure>
        </stencila-code-chunk>
        <h4 itemscope="" itemtype="http://schema.stenci.la/Heading" id="import">Import</h4>
        <stencila-code-chunk itemscope="" itemtype="http://schema.stenci.la/CodeChunk"
          data-execution_count="5" data-programminglanguage="python">
          <pre class="language-python" itemscope="" itemtype="http://schema.stenci.la/CodeBlock"
            slot="text"><code>print(f&quot;Code Cell 5: Add Constraints on Metabolite Input&quot;)

##### Add constraints on metabolite uptake #####

## CONSTRAINT: CO2 uptake rate in C3 plants is about 20 μmol/(m2*s)
f_c3_CO2 = 20 #[μmol/(m2*s)] 
set_bounds(&#39;Im_CO2&#39;, (0, f_c3_CO2), c3_model)

## CONSTRAINT: max. photon consumption 1000 μE
f_c3_hnu = 1000 #[μE] 
set_bounds(&#39;Im_hnu&#39;, (0, f_c3_hnu), c3_model)

## CONSTRAINT:  Fluxes of other import reactions
set_bounds(&#39;Im_H2O&#39;, (-inf, inf), c3_model)
set_bounds(&#39;Im_H2S&#39;, (0.,0.), c3_model)
set_bounds(&#39;Im_NH4&#39;, (0., 0.), c3_model)
set_bounds(&#39;Im_NO3&#39;, (0., inf), c3_model)
set_bounds(&#39;Im_Pi&#39;, (0., inf), c3_model)
set_bounds(&#39;Im_SO4&#39;, (0., inf), c3_model)
set_bounds(&#39;Ex_O2&#39;, (-inf, inf), c3_model)
set_bounds(&#39;Ex_Suc&#39;, (0., inf), c3_model)
set_bounds(&#39;Ex_starch&#39;, (0., inf), c3_model)
set_bounds(&#39;Ex_AA&#39;, (0., inf), c3_model)

</code></pre>
          <figure slot="outputs">
            <pre class="language-text" itemscope="" itemtype="http://schema.stenci.la/CodeBlock"><code>Code Cell 5: Add Constraints on Metabolite Input
</code></pre>
          </figure>
        </stencila-code-chunk>
        <p itemscope="" itemtype="http://schema.stenci.la/Paragraph">As in <cite itemscope=""
            itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a
              href="#bib2"><span>2</span><span>Arnold and
                Nikoloski</span><span>2014</span></a></cite>, we assume photoautotrophic growth
          conditions. Only the import of light, water, CO<sub itemscope=""
            itemtype="http://schema.stenci.la/Subscript"><span
              data-itemtype="http://schema.org/Number">2</span></sub>, inorganic phosphate (<span
            itemscope="" itemtype="http://schema.stenci.la/MathFragment"><span
              class="mjx-chtml"><span class="mjx-math" aria-label="\mathrm{Pi}"><span
                  class="mjx-mrow" aria-hidden="true"><span class="mjx-texatom"><span
                      class="mjx-mrow"><span class="mjx-mi"><span class="mjx-char MJXc-TeX-main-R"
                          style="padding-top: 0.372em; padding-bottom: 0.372em;">P</span></span><span
                        class="mjx-mi"><span class="mjx-char MJXc-TeX-main-R"
                          style="padding-top: 0.372em; padding-bottom: 0.372em;">i</span></span></span></span></span></span></span></span>),
          nitrate/ammonium, and sulphates/hydrogen sulphide is allowed, compare <a href="#table3"
            itemscope="" itemtype="http://schema.stenci.la/Link">Table 3</a>. More specifically, we
          do only allow for nitrate uptake, since it is the main source (80%) of nitrogen in leaves
          <cite itemscope="" itemtype="http://schema.stenci.la/Cite"
            data-citationmode="Narrative"><a href="#bib45"><span>45</span><span>Macduff and
                Bakken</span><span>2003</span></a></cite>. The CO<sub itemscope=""
            itemtype="http://schema.stenci.la/Subscript"><span
              data-itemtype="http://schema.org/Number">2</span></sub> uptake is limited to
          <stencila-code-expression programming-language="py" itemscope=""
            itemtype="http://schema.stenci.la/CodeExpression"><code class="py"
              slot="text">f_c3_CO2</code><output slot="output"></output></stencila-code-expression>
          μmol/(m<sup itemscope="" itemtype="http://schema.stenci.la/Superscript"><span
              data-itemtype="http://schema.org/Number">2</span></sup>s) <cite itemscope=""
            itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a
              href="#bib37"><span>37</span><span>Lacher</span><span>2003</span></a></cite>.
          Therefore, the carbon input constrains the model.</p>
        <h4 itemscope="" itemtype="http://schema.stenci.la/Heading" id="export">Export</h4>
        <stencila-code-chunk itemscope="" itemtype="http://schema.stenci.la/CodeChunk"
          data-execution_count="6" data-programminglanguage="python">
          <pre class="language-python" itemscope="" itemtype="http://schema.stenci.la/CodeBlock"
            slot="text"><code>print(f&quot;Code Cell 6: Add Constraints on Metabolite Output&quot;)
##### Add constraints on metabolite secretion #####

## CONSTRAINT:  Fluxes of other import reactions
set_bounds(&#39;Ex_O2&#39;, (-inf, inf), c3_model)
set_bounds(&#39;Ex_Suc&#39;, (0., inf), c3_model)
set_bounds(&#39;Ex_starch&#39;, (0., inf), c3_model)
set_bounds(&#39;Ex_AA&#39;, (0., inf), c3_model)

## CONSTRAINT: Output of sucrose : total amino acid 
r_suc_aa = (2.2, 1.0)
const_c3_suc_aa = set_fixed_flux_ratio({&#39;Ex_Suc&#39;:r_suc_aa[0],&#39;Ex_AA&#39;:r_suc_aa[1]}, &#39;const_c3_suc_aa&#39;, c3_model)

## CONSTRAINT: Output of sucrose : starch
r_suc_starch = (1.0, 1.0)
const_c3_suc_starch = set_fixed_flux_ratio({&#39;Ex_Suc&#39;:r_suc_starch[0],&#39;Ex_starch&#39;:r_suc_starch[1]}, &#39;const_c3_suc_starch&#39;, c3_model)</code></pre>
          <figure slot="outputs">
            <pre class="language-text" itemscope="" itemtype="http://schema.stenci.la/CodeBlock"><code>Code Cell 6: Add Constraints on Metabolite Output
</code></pre>
          </figure>
        </stencila-code-chunk>
        <p itemscope="" itemtype="http://schema.stenci.la/Paragraph">In contrast to <cite
            itemscope="" itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a
              href="#bib2"><span>2</span><span>Arnold and
                Nikoloski</span><span>2014</span></a></cite>, we focus on mature, fully
          differentiated and photosynthetic active leaves supporting the growth of the plant through
          the export of nutrients in the phloem sap, mainly sucrose and amino acids. An output
          reaction for sucrose <em itemscope=""
            itemtype="http://schema.stenci.la/Emphasis">Ex_Suc</em> is already included in the
          model. An additional export reaction <em itemscope=""
            itemtype="http://schema.stenci.la/Emphasis">Ex_AA</em> represents the relative
          proportion of 18 amino acids in the phloem sap of <em itemscope=""
            itemtype="http://schema.stenci.la/Emphasis">Arabidopsis</em> as stoichiometric
          coefficients in accordance to experimentally measured data from <cite itemscope=""
            itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a
              href="#bib85"><span>85</span><span>Wilkinson and
                Douglas</span><span>2003</span></a></cite>. The ratio of exported sucrose : total
          amino acid is estimated to be <stencila-code-expression programming-language="py"
            itemscope="" itemtype="http://schema.stenci.la/CodeExpression"><code class="py"
              slot="text">r_suc_aa[0]</code><output slot="output"></output>
          </stencila-code-expression> : <stencila-code-expression programming-language="py"
            itemscope="" itemtype="http://schema.stenci.la/CodeExpression"><code class="py"
              slot="text">r_suc_aa[1]</code><output slot="output"></output>
          </stencila-code-expression><span data-itemtype="http://schema.org/Number">0</span><cite
            itemscope="" itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a
              href="#bib85"><span>85</span><span>Wilkinson and
                Douglas</span><span>2003</span></a></cite>. This ratio is included as a flux ratio
          constraint of the reactions <em itemscope=""
            itemtype="http://schema.stenci.la/Emphasis">Ex_Suc</em> and <em itemscope=""
            itemtype="http://schema.stenci.la/Emphasis">Ex_AA</em>. Furthermore, it is known that
          the export of sucrose and the formation of starch is approximately the same <cite
            itemscope="" itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a
              href="#bib74"><span>74</span><span>Stitt and
                Zeeman</span><span>2012</span></a></cite>, which is reflected by the flux ratio
          constraint <span itemscope="" itemtype="http://schema.stenci.la/MathFragment"><span
              class="mjx-chtml"><span class="mjx-math"
                aria-label="{v}_{Ex\mathrm{\_}Suc}:{v}_{Ex\mathrm{\_}starch}"><span class="mjx-mrow"
                  aria-hidden="true"><span class="mjx-msubsup"><span class="mjx-base"><span
                        class="mjx-texatom"><span class="mjx-mrow"><span class="mjx-mi"><span
                              class="mjx-char MJXc-TeX-math-I"
                              style="padding-top: 0.225em; padding-bottom: 0.298em;">v</span></span></span></span></span><span
                      class="mjx-sub"
                      style="font-size: 70.7%; vertical-align: -0.23em; padding-right: 0.071em;"><span
                        class="mjx-texatom" style=""><span class="mjx-mrow"><span
                            class="mjx-mi"><span class="mjx-char MJXc-TeX-math-I"
                              style="padding-top: 0.446em; padding-bottom: 0.298em; padding-right: 0.026em;">E</span></span><span
                            class="mjx-mi"><span class="mjx-char MJXc-TeX-math-I"
                              style="padding-top: 0.225em; padding-bottom: 0.298em;">x</span></span><span
                            class="mjx-texatom"><span class="mjx-mrow"><span class="mjx-mi"><span
                                  class="mjx-char MJXc-TeX-main-R"
                                  style="margin-top: -0.291em; padding-bottom: 0.372em;">_</span></span></span></span><span
                            class="mjx-mi"><span class="mjx-char MJXc-TeX-math-I"
                              style="padding-top: 0.519em; padding-bottom: 0.298em; padding-right: 0.032em;">S</span></span><span
                            class="mjx-mi"><span class="mjx-char MJXc-TeX-math-I"
                              style="padding-top: 0.225em; padding-bottom: 0.298em;">u</span></span><span
                            class="mjx-mi"><span class="mjx-char MJXc-TeX-math-I"
                              style="padding-top: 0.225em; padding-bottom: 0.298em;">c</span></span></span></span></span></span><span
                    class="mjx-mo MJXc-space3"><span class="mjx-char MJXc-TeX-main-R"
                      style="padding-top: 0.151em; padding-bottom: 0.372em;">:</span></span><span
                    class="mjx-msubsup MJXc-space3"><span class="mjx-base"><span
                        class="mjx-texatom"><span class="mjx-mrow"><span class="mjx-mi"><span
                              class="mjx-char MJXc-TeX-math-I"
                              style="padding-top: 0.225em; padding-bottom: 0.298em;">v</span></span></span></span></span><span
                      class="mjx-sub"
                      style="font-size: 70.7%; vertical-align: -0.219em; padding-right: 0.071em;"><span
                        class="mjx-texatom" style=""><span class="mjx-mrow"><span
                            class="mjx-mi"><span class="mjx-char MJXc-TeX-math-I"
                              style="padding-top: 0.446em; padding-bottom: 0.298em; padding-right: 0.026em;">E</span></span><span
                            class="mjx-mi"><span class="mjx-char MJXc-TeX-math-I"
                              style="padding-top: 0.225em; padding-bottom: 0.298em;">x</span></span><span
                            class="mjx-texatom"><span class="mjx-mrow"><span class="mjx-mi"><span
                                  class="mjx-char MJXc-TeX-main-R"
                                  style="margin-top: -0.291em; padding-bottom: 0.372em;">_</span></span></span></span><span
                            class="mjx-mi"><span class="mjx-char MJXc-TeX-math-I"
                              style="padding-top: 0.225em; padding-bottom: 0.298em;">s</span></span><span
                            class="mjx-mi"><span class="mjx-char MJXc-TeX-math-I"
                              style="padding-top: 0.372em; padding-bottom: 0.298em;">t</span></span><span
                            class="mjx-mi"><span class="mjx-char MJXc-TeX-math-I"
                              style="padding-top: 0.225em; padding-bottom: 0.298em;">a</span></span><span
                            class="mjx-mi"><span class="mjx-char MJXc-TeX-math-I"
                              style="padding-top: 0.225em; padding-bottom: 0.298em;">r</span></span><span
                            class="mjx-mi"><span class="mjx-char MJXc-TeX-math-I"
                              style="padding-top: 0.225em; padding-bottom: 0.298em;">c</span></span><span
                            class="mjx-mi"><span class="mjx-char MJXc-TeX-math-I"
                              style="padding-top: 0.446em; padding-bottom: 0.298em;">h</span></span></span></span></span></span></span></span></span></span>
          = <stencila-code-expression programming-language="py" itemscope=""
            itemtype="http://schema.stenci.la/CodeExpression"><code class="py"
              slot="text">r_suc_starch[0]</code><output slot="output"></output>
          </stencila-code-expression>:<stencila-code-expression programming-language="py"
            itemscope="" itemtype="http://schema.stenci.la/CodeExpression"><code class="py"
              slot="text">r_suc_starch[1]</code><output slot="output"></output>
          </stencila-code-expression>. The model allows for the export of water and oxygen. The flux
          of all other export reactions is set to 0, see <a href="#table3" itemscope=""
            itemtype="http://schema.stenci.la/Link">Table 3</a> for a summary.</p>
        <figure itemscope="" itemtype="http://schema.stenci.la/Figure" id="table3" title="Table 3.">
          <label data-itemprop="label">Table 3.</label>
          <stencila-code-chunk itemscope="" itemtype="http://schema.stenci.la/CodeChunk"
            data-execution_count="7" data-programminglanguage="python">
            <pre class="language-python" itemscope="" itemtype="http://schema.stenci.la/CodeBlock"
              slot="text"><code>print(f&quot;Code Cell 7: Create Table on Flux Boundary Constraints of Input and Output Reactions&quot;)
#### Table 3 ####

#{
#  &quot;caption&quot;: &quot;#### Flux boundary constraints of Im-/export reactions&quot;,
# &quot;id&quot;: &quot;table3&quot;,
#  &quot;label&quot;: &quot;Table 3.&quot;,
#  &quot;trusted&quot;: true
#}

index = c3_model.reactions.query(lambda rxn: (rxn.id.startswith(&#39;Ex_&#39;) or rxn.id.startswith(&#39;Im_&#39;))).list_attr(&#39;id&#39;)
lower_bounds = c3_model.reactions.query(lambda rxn: (rxn.id.startswith(&#39;Ex_&#39;) or rxn.id.startswith(&#39;Im_&#39;))).list_attr(&#39;lower_bound&#39;)
upper_bounds = c3_model.reactions.query(lambda rxn: (rxn.id.startswith(&#39;Ex_&#39;) or rxn.id.startswith(&#39;Im_&#39;))).list_attr(&#39;upper_bound&#39;)
cols = [&#39;Lower bound [μmol/(m^2^s)]&#39;, &#39;Upper bound [μmol/(m^2^s)]&#39; ]

df_ex_im_rxn_bounds = pd.DataFrame(np.array([lower_bounds, upper_bounds]).T,index=index, columns=cols)
df_ex_im_rxn_bounds.index.name = &#39;Reaction ID&#39;

df_ex_im_rxn_bounds.style.applymap(lambda val: &#39;color: red&#39; if val != 0 else &#39;color: black&#39;)</code></pre>
            <figure slot="outputs">
              <pre class="language-text" itemscope="" itemtype="http://schema.stenci.la/CodeBlock"><code>Code Cell 7: Create Table on Flux Boundary Constraints of Input and Output Reactions
</code></pre>
              <table id="T_b217f_" itemscope="" itemtype="http://schema.org/Table">
                <thead>
                  <tr itemscope="" itemtype="http://schema.stenci.la/TableRow">
                    <th itemscope="" itemtype="http://schema.stenci.la/TableCell"></th>
                    <th itemscope="" itemtype="http://schema.stenci.la/TableCell">Lower bound
                      [μmol/(m^2^s)]</th>
                    <th itemscope="" itemtype="http://schema.stenci.la/TableCell">Upper bound
                      [μmol/(m^2^s)]</th>
                  </tr>
                  <tr itemscope="" itemtype="http://schema.stenci.la/TableRow">
                    <th itemscope="" itemtype="http://schema.stenci.la/TableCell">Reaction ID</th>
                    <th itemscope="" itemtype="http://schema.stenci.la/TableCell"></th>
                    <th itemscope="" itemtype="http://schema.stenci.la/TableCell"></th>
                  </tr>
                </thead>
                <tbody>
                  <tr itemscope="" itemtype="http://schema.stenci.la/TableRow">
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell">Im_hnu</td>
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell"><span
                        data-itemtype="http://schema.org/Number">0</span></td>
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell"><span
                        data-itemtype="http://schema.org/Number">1000</span></td>
                  </tr>
                  <tr itemscope="" itemtype="http://schema.stenci.la/TableRow">
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell">Im_CO2</td>
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell"><span
                        data-itemtype="http://schema.org/Number">0</span></td>
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell"><span
                        data-itemtype="http://schema.org/Number">20</span></td>
                  </tr>
                  <tr itemscope="" itemtype="http://schema.stenci.la/TableRow">
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell">Im_H2O</td>
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell"><span
                        data-itemtype="http://schema.org/Number">-1000000</span></td>
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell"><span
                        data-itemtype="http://schema.org/Number">1000000</span></td>
                  </tr>
                  <tr itemscope="" itemtype="http://schema.stenci.la/TableRow">
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell">Im_Pi</td>
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell"><span
                        data-itemtype="http://schema.org/Number">0</span></td>
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell"><span
                        data-itemtype="http://schema.org/Number">1000000</span></td>
                  </tr>
                  <tr itemscope="" itemtype="http://schema.stenci.la/TableRow">
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell">Im_NO3</td>
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell"><span
                        data-itemtype="http://schema.org/Number">0</span></td>
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell"><span
                        data-itemtype="http://schema.org/Number">1000000</span></td>
                  </tr>
                  <tr itemscope="" itemtype="http://schema.stenci.la/TableRow">
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell">Im_NH4</td>
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell"><span
                        data-itemtype="http://schema.org/Number">0</span></td>
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell"><span
                        data-itemtype="http://schema.org/Number">0</span></td>
                  </tr>
                  <tr itemscope="" itemtype="http://schema.stenci.la/TableRow">
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell">Im_SO4</td>
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell"><span
                        data-itemtype="http://schema.org/Number">0</span></td>
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell"><span
                        data-itemtype="http://schema.org/Number">1000000</span></td>
                  </tr>
                  <tr itemscope="" itemtype="http://schema.stenci.la/TableRow">
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell">Im_H2S</td>
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell"><span
                        data-itemtype="http://schema.org/Number">0</span></td>
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell"><span
                        data-itemtype="http://schema.org/Number">0</span></td>
                  </tr>
                  <tr itemscope="" itemtype="http://schema.stenci.la/TableRow">
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell">Ex_O2</td>
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell"><span
                        data-itemtype="http://schema.org/Number">-1000000</span></td>
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell"><span
                        data-itemtype="http://schema.org/Number">1000000</span></td>
                  </tr>
                  <tr itemscope="" itemtype="http://schema.stenci.la/TableRow">
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell">Ex_Ala_c</td>
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell"><span
                        data-itemtype="http://schema.org/Number">0</span></td>
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell"><span
                        data-itemtype="http://schema.org/Number">0</span></td>
                  </tr>
                  <tr itemscope="" itemtype="http://schema.stenci.la/TableRow">
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell">Ex_Ala_h</td>
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell"><span
                        data-itemtype="http://schema.org/Number">0</span></td>
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell"><span
                        data-itemtype="http://schema.org/Number">0</span></td>
                  </tr>
                  <tr itemscope="" itemtype="http://schema.stenci.la/TableRow">
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell">Ex_Ala_m</td>
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell"><span
                        data-itemtype="http://schema.org/Number">0</span></td>
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell"><span
                        data-itemtype="http://schema.org/Number">0</span></td>
                  </tr>
                  <tr itemscope="" itemtype="http://schema.stenci.la/TableRow">
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell">Ex_Ala_p</td>
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell"><span
                        data-itemtype="http://schema.org/Number">0</span></td>
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell"><span
                        data-itemtype="http://schema.org/Number">0</span></td>
                  </tr>
                  <tr itemscope="" itemtype="http://schema.stenci.la/TableRow">
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell">Ex_Arg_c</td>
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell"><span
                        data-itemtype="http://schema.org/Number">0</span></td>
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell"><span
                        data-itemtype="http://schema.org/Number">0</span></td>
                  </tr>
                  <tr itemscope="" itemtype="http://schema.stenci.la/TableRow">
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell">Ex_Arg_h</td>
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell"><span
                        data-itemtype="http://schema.org/Number">0</span></td>
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell"><span
                        data-itemtype="http://schema.org/Number">0</span></td>
                  </tr>
                  <tr itemscope="" itemtype="http://schema.stenci.la/TableRow">
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell">Ex_Arg_m</td>
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell"><span
                        data-itemtype="http://schema.org/Number">0</span></td>
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell"><span
                        data-itemtype="http://schema.org/Number">0</span></td>
                  </tr>
                  <tr itemscope="" itemtype="http://schema.stenci.la/TableRow">
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell">Ex_Arg_p</td>
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell"><span
                        data-itemtype="http://schema.org/Number">0</span></td>
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell"><span
                        data-itemtype="http://schema.org/Number">0</span></td>
                  </tr>
                  <tr itemscope="" itemtype="http://schema.stenci.la/TableRow">
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell">Ex_Asn_c</td>
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell"><span
                        data-itemtype="http://schema.org/Number">0</span></td>
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell"><span
                        data-itemtype="http://schema.org/Number">0</span></td>
                  </tr>
                  <tr itemscope="" itemtype="http://schema.stenci.la/TableRow">
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell">Ex_Asn_h</td>
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell"><span
                        data-itemtype="http://schema.org/Number">0</span></td>
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell"><span
                        data-itemtype="http://schema.org/Number">0</span></td>
                  </tr>
                  <tr itemscope="" itemtype="http://schema.stenci.la/TableRow">
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell">Ex_Asn_m</td>
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell"><span
                        data-itemtype="http://schema.org/Number">0</span></td>
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell"><span
                        data-itemtype="http://schema.org/Number">0</span></td>
                  </tr>
                  <tr itemscope="" itemtype="http://schema.stenci.la/TableRow">
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell">Ex_Asn_p</td>
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell"><span
                        data-itemtype="http://schema.org/Number">0</span></td>
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell"><span
                        data-itemtype="http://schema.org/Number">0</span></td>
                  </tr>
                  <tr itemscope="" itemtype="http://schema.stenci.la/TableRow">
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell">Ex_Asp_c</td>
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell"><span
                        data-itemtype="http://schema.org/Number">0</span></td>
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell"><span
                        data-itemtype="http://schema.org/Number">0</span></td>
                  </tr>
                  <tr itemscope="" itemtype="http://schema.stenci.la/TableRow">
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell">Ex_Asp_h</td>
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell"><span
                        data-itemtype="http://schema.org/Number">0</span></td>
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell"><span
                        data-itemtype="http://schema.org/Number">0</span></td>
                  </tr>
                  <tr itemscope="" itemtype="http://schema.stenci.la/TableRow">
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell">Ex_Asp_m</td>
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell"><span
                        data-itemtype="http://schema.org/Number">0</span></td>
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell"><span
                        data-itemtype="http://schema.org/Number">0</span></td>
                  </tr>
                  <tr itemscope="" itemtype="http://schema.stenci.la/TableRow">
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell">Ex_Asp_p</td>
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell"><span
                        data-itemtype="http://schema.org/Number">0</span></td>
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell"><span
                        data-itemtype="http://schema.org/Number">0</span></td>
                  </tr>
                  <tr itemscope="" itemtype="http://schema.stenci.la/TableRow">
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell">Ex_Cys_c</td>
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell"><span
                        data-itemtype="http://schema.org/Number">0</span></td>
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell"><span
                        data-itemtype="http://schema.org/Number">0</span></td>
                  </tr>
                  <tr itemscope="" itemtype="http://schema.stenci.la/TableRow">
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell">Ex_Cys_h</td>
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell"><span
                        data-itemtype="http://schema.org/Number">0</span></td>
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell"><span
                        data-itemtype="http://schema.org/Number">0</span></td>
                  </tr>
                  <tr itemscope="" itemtype="http://schema.stenci.la/TableRow">
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell">Ex_Cys_m</td>
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell"><span
                        data-itemtype="http://schema.org/Number">0</span></td>
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell"><span
                        data-itemtype="http://schema.org/Number">0</span></td>
                  </tr>
                  <tr itemscope="" itemtype="http://schema.stenci.la/TableRow">
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell">Ex_Cys_p</td>
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell"><span
                        data-itemtype="http://schema.org/Number">0</span></td>
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell"><span
                        data-itemtype="http://schema.org/Number">0</span></td>
                  </tr>
                  <tr itemscope="" itemtype="http://schema.stenci.la/TableRow">
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell">Ex_Gln_c</td>
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell"><span
                        data-itemtype="http://schema.org/Number">0</span></td>
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell"><span
                        data-itemtype="http://schema.org/Number">0</span></td>
                  </tr>
                  <tr itemscope="" itemtype="http://schema.stenci.la/TableRow">
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell">Ex_Gln_h</td>
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell"><span
                        data-itemtype="http://schema.org/Number">0</span></td>
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell"><span
                        data-itemtype="http://schema.org/Number">0</span></td>
                  </tr>
                  <tr itemscope="" itemtype="http://schema.stenci.la/TableRow">
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell">Ex_Gln_m</td>
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell"><span
                        data-itemtype="http://schema.org/Number">0</span></td>
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell"><span
                        data-itemtype="http://schema.org/Number">0</span></td>
                  </tr>
                  <tr itemscope="" itemtype="http://schema.stenci.la/TableRow">
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell">Ex_Gln_p</td>
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell"><span
                        data-itemtype="http://schema.org/Number">0</span></td>
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell"><span
                        data-itemtype="http://schema.org/Number">0</span></td>
                  </tr>
                  <tr itemscope="" itemtype="http://schema.stenci.la/TableRow">
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell">Ex_Glu_c</td>
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell"><span
                        data-itemtype="http://schema.org/Number">0</span></td>
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell"><span
                        data-itemtype="http://schema.org/Number">0</span></td>
                  </tr>
                  <tr itemscope="" itemtype="http://schema.stenci.la/TableRow">
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell">Ex_Glu_h</td>
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell"><span
                        data-itemtype="http://schema.org/Number">0</span></td>
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell"><span
                        data-itemtype="http://schema.org/Number">0</span></td>
                  </tr>
                  <tr itemscope="" itemtype="http://schema.stenci.la/TableRow">
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell">Ex_Glu_m</td>
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell"><span
                        data-itemtype="http://schema.org/Number">0</span></td>
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell"><span
                        data-itemtype="http://schema.org/Number">0</span></td>
                  </tr>
                  <tr itemscope="" itemtype="http://schema.stenci.la/TableRow">
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell">Ex_Glu_p</td>
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell"><span
                        data-itemtype="http://schema.org/Number">0</span></td>
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell"><span
                        data-itemtype="http://schema.org/Number">0</span></td>
                  </tr>
                  <tr itemscope="" itemtype="http://schema.stenci.la/TableRow">
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell">Ex_Gly_c</td>
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell"><span
                        data-itemtype="http://schema.org/Number">0</span></td>
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell"><span
                        data-itemtype="http://schema.org/Number">0</span></td>
                  </tr>
                  <tr itemscope="" itemtype="http://schema.stenci.la/TableRow">
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell">Ex_Gly_h</td>
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell"><span
                        data-itemtype="http://schema.org/Number">0</span></td>
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell"><span
                        data-itemtype="http://schema.org/Number">0</span></td>
                  </tr>
                  <tr itemscope="" itemtype="http://schema.stenci.la/TableRow">
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell">Ex_Gly_m</td>
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell"><span
                        data-itemtype="http://schema.org/Number">0</span></td>
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell"><span
                        data-itemtype="http://schema.org/Number">0</span></td>
                  </tr>
                  <tr itemscope="" itemtype="http://schema.stenci.la/TableRow">
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell">Ex_Gly_p</td>
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell"><span
                        data-itemtype="http://schema.org/Number">0</span></td>
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell"><span
                        data-itemtype="http://schema.org/Number">0</span></td>
                  </tr>
                  <tr itemscope="" itemtype="http://schema.stenci.la/TableRow">
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell">Ex_His_c</td>
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell"><span
                        data-itemtype="http://schema.org/Number">0</span></td>
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell"><span
                        data-itemtype="http://schema.org/Number">0</span></td>
                  </tr>
                  <tr itemscope="" itemtype="http://schema.stenci.la/TableRow">
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell">Ex_His_h</td>
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell"><span
                        data-itemtype="http://schema.org/Number">0</span></td>
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell"><span
                        data-itemtype="http://schema.org/Number">0</span></td>
                  </tr>
                  <tr itemscope="" itemtype="http://schema.stenci.la/TableRow">
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell">Ex_His_m</td>
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell"><span
                        data-itemtype="http://schema.org/Number">0</span></td>
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell"><span
                        data-itemtype="http://schema.org/Number">0</span></td>
                  </tr>
                  <tr itemscope="" itemtype="http://schema.stenci.la/TableRow">
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell">Ex_His_p</td>
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell"><span
                        data-itemtype="http://schema.org/Number">0</span></td>
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell"><span
                        data-itemtype="http://schema.org/Number">0</span></td>
                  </tr>
                  <tr itemscope="" itemtype="http://schema.stenci.la/TableRow">
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell">Ex_Ile_c</td>
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell"><span
                        data-itemtype="http://schema.org/Number">0</span></td>
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell"><span
                        data-itemtype="http://schema.org/Number">0</span></td>
                  </tr>
                  <tr itemscope="" itemtype="http://schema.stenci.la/TableRow">
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell">Ex_Ile_h</td>
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell"><span
                        data-itemtype="http://schema.org/Number">0</span></td>
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell"><span
                        data-itemtype="http://schema.org/Number">0</span></td>
                  </tr>
                  <tr itemscope="" itemtype="http://schema.stenci.la/TableRow">
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell">Ex_Ile_m</td>
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell"><span
                        data-itemtype="http://schema.org/Number">0</span></td>
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell"><span
                        data-itemtype="http://schema.org/Number">0</span></td>
                  </tr>
                  <tr itemscope="" itemtype="http://schema.stenci.la/TableRow">
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell">Ex_Ile_p</td>
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell"><span
                        data-itemtype="http://schema.org/Number">0</span></td>
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell"><span
                        data-itemtype="http://schema.org/Number">0</span></td>
                  </tr>
                  <tr itemscope="" itemtype="http://schema.stenci.la/TableRow">
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell">Ex_Leu_c</td>
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell"><span
                        data-itemtype="http://schema.org/Number">0</span></td>
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell"><span
                        data-itemtype="http://schema.org/Number">0</span></td>
                  </tr>
                  <tr itemscope="" itemtype="http://schema.stenci.la/TableRow">
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell">Ex_Leu_h</td>
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell"><span
                        data-itemtype="http://schema.org/Number">0</span></td>
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell"><span
                        data-itemtype="http://schema.org/Number">0</span></td>
                  </tr>
                  <tr itemscope="" itemtype="http://schema.stenci.la/TableRow">
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell">Ex_Leu_m</td>
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell"><span
                        data-itemtype="http://schema.org/Number">0</span></td>
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell"><span
                        data-itemtype="http://schema.org/Number">0</span></td>
                  </tr>
                  <tr itemscope="" itemtype="http://schema.stenci.la/TableRow">
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell">Ex_Leu_p</td>
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell"><span
                        data-itemtype="http://schema.org/Number">0</span></td>
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell"><span
                        data-itemtype="http://schema.org/Number">0</span></td>
                  </tr>
                  <tr itemscope="" itemtype="http://schema.stenci.la/TableRow">
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell">Ex_Lys_c</td>
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell"><span
                        data-itemtype="http://schema.org/Number">0</span></td>
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell"><span
                        data-itemtype="http://schema.org/Number">0</span></td>
                  </tr>
                  <tr itemscope="" itemtype="http://schema.stenci.la/TableRow">
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell">Ex_Lys_h</td>
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell"><span
                        data-itemtype="http://schema.org/Number">0</span></td>
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell"><span
                        data-itemtype="http://schema.org/Number">0</span></td>
                  </tr>
                  <tr itemscope="" itemtype="http://schema.stenci.la/TableRow">
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell">Ex_Lys_m</td>
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell"><span
                        data-itemtype="http://schema.org/Number">0</span></td>
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell"><span
                        data-itemtype="http://schema.org/Number">0</span></td>
                  </tr>
                  <tr itemscope="" itemtype="http://schema.stenci.la/TableRow">
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell">Ex_Lys_p</td>
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell"><span
                        data-itemtype="http://schema.org/Number">0</span></td>
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell"><span
                        data-itemtype="http://schema.org/Number">0</span></td>
                  </tr>
                  <tr itemscope="" itemtype="http://schema.stenci.la/TableRow">
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell">Ex_Met_c</td>
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell"><span
                        data-itemtype="http://schema.org/Number">0</span></td>
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell"><span
                        data-itemtype="http://schema.org/Number">0</span></td>
                  </tr>
                  <tr itemscope="" itemtype="http://schema.stenci.la/TableRow">
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell">Ex_Met_h</td>
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell"><span
                        data-itemtype="http://schema.org/Number">0</span></td>
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell"><span
                        data-itemtype="http://schema.org/Number">0</span></td>
                  </tr>
                  <tr itemscope="" itemtype="http://schema.stenci.la/TableRow">
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell">Ex_Met_m</td>
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell"><span
                        data-itemtype="http://schema.org/Number">0</span></td>
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell"><span
                        data-itemtype="http://schema.org/Number">0</span></td>
                  </tr>
                  <tr itemscope="" itemtype="http://schema.stenci.la/TableRow">
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell">Ex_Met_p</td>
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell"><span
                        data-itemtype="http://schema.org/Number">0</span></td>
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell"><span
                        data-itemtype="http://schema.org/Number">0</span></td>
                  </tr>
                  <tr itemscope="" itemtype="http://schema.stenci.la/TableRow">
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell">Ex_Phe_c</td>
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell"><span
                        data-itemtype="http://schema.org/Number">0</span></td>
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell"><span
                        data-itemtype="http://schema.org/Number">0</span></td>
                  </tr>
                  <tr itemscope="" itemtype="http://schema.stenci.la/TableRow">
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell">Ex_Phe_h</td>
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell"><span
                        data-itemtype="http://schema.org/Number">0</span></td>
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell"><span
                        data-itemtype="http://schema.org/Number">0</span></td>
                  </tr>
                  <tr itemscope="" itemtype="http://schema.stenci.la/TableRow">
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell">Ex_Phe_m</td>
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell"><span
                        data-itemtype="http://schema.org/Number">0</span></td>
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell"><span
                        data-itemtype="http://schema.org/Number">0</span></td>
                  </tr>
                  <tr itemscope="" itemtype="http://schema.stenci.la/TableRow">
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell">Ex_Phe_p</td>
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell"><span
                        data-itemtype="http://schema.org/Number">0</span></td>
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell"><span
                        data-itemtype="http://schema.org/Number">0</span></td>
                  </tr>
                  <tr itemscope="" itemtype="http://schema.stenci.la/TableRow">
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell">Ex_Pro_c</td>
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell"><span
                        data-itemtype="http://schema.org/Number">0</span></td>
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell"><span
                        data-itemtype="http://schema.org/Number">0</span></td>
                  </tr>
                  <tr itemscope="" itemtype="http://schema.stenci.la/TableRow">
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell">Ex_Pro_h</td>
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell"><span
                        data-itemtype="http://schema.org/Number">0</span></td>
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell"><span
                        data-itemtype="http://schema.org/Number">0</span></td>
                  </tr>
                  <tr itemscope="" itemtype="http://schema.stenci.la/TableRow">
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell">Ex_Pro_m</td>
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell"><span
                        data-itemtype="http://schema.org/Number">0</span></td>
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell"><span
                        data-itemtype="http://schema.org/Number">0</span></td>
                  </tr>
                  <tr itemscope="" itemtype="http://schema.stenci.la/TableRow">
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell">Ex_Pro_p</td>
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell"><span
                        data-itemtype="http://schema.org/Number">0</span></td>
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell"><span
                        data-itemtype="http://schema.org/Number">0</span></td>
                  </tr>
                  <tr itemscope="" itemtype="http://schema.stenci.la/TableRow">
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell">Ex_Ser_c</td>
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell"><span
                        data-itemtype="http://schema.org/Number">0</span></td>
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell"><span
                        data-itemtype="http://schema.org/Number">0</span></td>
                  </tr>
                  <tr itemscope="" itemtype="http://schema.stenci.la/TableRow">
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell">Ex_Ser_h</td>
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell"><span
                        data-itemtype="http://schema.org/Number">0</span></td>
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell"><span
                        data-itemtype="http://schema.org/Number">0</span></td>
                  </tr>
                  <tr itemscope="" itemtype="http://schema.stenci.la/TableRow">
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell">Ex_Ser_m</td>
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell"><span
                        data-itemtype="http://schema.org/Number">0</span></td>
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell"><span
                        data-itemtype="http://schema.org/Number">0</span></td>
                  </tr>
                  <tr itemscope="" itemtype="http://schema.stenci.la/TableRow">
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell">Ex_Ser_p</td>
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell"><span
                        data-itemtype="http://schema.org/Number">0</span></td>
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell"><span
                        data-itemtype="http://schema.org/Number">0</span></td>
                  </tr>
                  <tr itemscope="" itemtype="http://schema.stenci.la/TableRow">
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell">Ex_Thr_c</td>
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell"><span
                        data-itemtype="http://schema.org/Number">0</span></td>
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell"><span
                        data-itemtype="http://schema.org/Number">0</span></td>
                  </tr>
                  <tr itemscope="" itemtype="http://schema.stenci.la/TableRow">
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell">Ex_Thr_h</td>
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell"><span
                        data-itemtype="http://schema.org/Number">0</span></td>
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell"><span
                        data-itemtype="http://schema.org/Number">0</span></td>
                  </tr>
                  <tr itemscope="" itemtype="http://schema.stenci.la/TableRow">
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell">Ex_Thr_m</td>
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell"><span
                        data-itemtype="http://schema.org/Number">0</span></td>
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell"><span
                        data-itemtype="http://schema.org/Number">0</span></td>
                  </tr>
                  <tr itemscope="" itemtype="http://schema.stenci.la/TableRow">
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell">Ex_Thr_p</td>
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell"><span
                        data-itemtype="http://schema.org/Number">0</span></td>
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell"><span
                        data-itemtype="http://schema.org/Number">0</span></td>
                  </tr>
                  <tr itemscope="" itemtype="http://schema.stenci.la/TableRow">
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell">Ex_Trp_c</td>
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell"><span
                        data-itemtype="http://schema.org/Number">0</span></td>
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell"><span
                        data-itemtype="http://schema.org/Number">0</span></td>
                  </tr>
                  <tr itemscope="" itemtype="http://schema.stenci.la/TableRow">
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell">Ex_Trp_h</td>
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell"><span
                        data-itemtype="http://schema.org/Number">0</span></td>
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell"><span
                        data-itemtype="http://schema.org/Number">0</span></td>
                  </tr>
                  <tr itemscope="" itemtype="http://schema.stenci.la/TableRow">
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell">Ex_Trp_m</td>
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell"><span
                        data-itemtype="http://schema.org/Number">0</span></td>
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell"><span
                        data-itemtype="http://schema.org/Number">0</span></td>
                  </tr>
                  <tr itemscope="" itemtype="http://schema.stenci.la/TableRow">
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell">Ex_Trp_p</td>
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell"><span
                        data-itemtype="http://schema.org/Number">0</span></td>
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell"><span
                        data-itemtype="http://schema.org/Number">0</span></td>
                  </tr>
                  <tr itemscope="" itemtype="http://schema.stenci.la/TableRow">
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell">Ex_Tyr_c</td>
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell"><span
                        data-itemtype="http://schema.org/Number">0</span></td>
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell"><span
                        data-itemtype="http://schema.org/Number">0</span></td>
                  </tr>
                  <tr itemscope="" itemtype="http://schema.stenci.la/TableRow">
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell">Ex_Tyr_h</td>
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell"><span
                        data-itemtype="http://schema.org/Number">0</span></td>
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell"><span
                        data-itemtype="http://schema.org/Number">0</span></td>
                  </tr>
                  <tr itemscope="" itemtype="http://schema.stenci.la/TableRow">
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell">Ex_Tyr_m</td>
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell"><span
                        data-itemtype="http://schema.org/Number">0</span></td>
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell"><span
                        data-itemtype="http://schema.org/Number">0</span></td>
                  </tr>
                  <tr itemscope="" itemtype="http://schema.stenci.la/TableRow">
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell">Ex_Tyr_p</td>
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell"><span
                        data-itemtype="http://schema.org/Number">0</span></td>
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell"><span
                        data-itemtype="http://schema.org/Number">0</span></td>
                  </tr>
                  <tr itemscope="" itemtype="http://schema.stenci.la/TableRow">
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell">Ex_Val_c</td>
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell"><span
                        data-itemtype="http://schema.org/Number">0</span></td>
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell"><span
                        data-itemtype="http://schema.org/Number">0</span></td>
                  </tr>
                  <tr itemscope="" itemtype="http://schema.stenci.la/TableRow">
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell">Ex_Val_h</td>
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell"><span
                        data-itemtype="http://schema.org/Number">0</span></td>
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell"><span
                        data-itemtype="http://schema.org/Number">0</span></td>
                  </tr>
                  <tr itemscope="" itemtype="http://schema.stenci.la/TableRow">
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell">Ex_Val_m</td>
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell"><span
                        data-itemtype="http://schema.org/Number">0</span></td>
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell"><span
                        data-itemtype="http://schema.org/Number">0</span></td>
                  </tr>
                  <tr itemscope="" itemtype="http://schema.stenci.la/TableRow">
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell">Ex_Val_p</td>
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell"><span
                        data-itemtype="http://schema.org/Number">0</span></td>
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell"><span
                        data-itemtype="http://schema.org/Number">0</span></td>
                  </tr>
                  <tr itemscope="" itemtype="http://schema.stenci.la/TableRow">
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell">Ex_starch</td>
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell"><span
                        data-itemtype="http://schema.org/Number">0</span></td>
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell"><span
                        data-itemtype="http://schema.org/Number">1000000</span></td>
                  </tr>
                  <tr itemscope="" itemtype="http://schema.stenci.la/TableRow">
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell">Ex_Glc</td>
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell"><span
                        data-itemtype="http://schema.org/Number">0</span></td>
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell"><span
                        data-itemtype="http://schema.org/Number">0</span></td>
                  </tr>
                  <tr itemscope="" itemtype="http://schema.stenci.la/TableRow">
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell">Ex_Frc</td>
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell"><span
                        data-itemtype="http://schema.org/Number">0</span></td>
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell"><span
                        data-itemtype="http://schema.org/Number">0</span></td>
                  </tr>
                  <tr itemscope="" itemtype="http://schema.stenci.la/TableRow">
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell">Ex_Suc</td>
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell"><span
                        data-itemtype="http://schema.org/Number">0</span></td>
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell"><span
                        data-itemtype="http://schema.org/Number">1000000</span></td>
                  </tr>
                  <tr itemscope="" itemtype="http://schema.stenci.la/TableRow">
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell">Ex_cellulose</td>
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell"><span
                        data-itemtype="http://schema.org/Number">0</span></td>
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell"><span
                        data-itemtype="http://schema.org/Number">0</span></td>
                  </tr>
                  <tr itemscope="" itemtype="http://schema.stenci.la/TableRow">
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell">Ex_Mas</td>
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell"><span
                        data-itemtype="http://schema.org/Number">0</span></td>
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell"><span
                        data-itemtype="http://schema.org/Number">0</span></td>
                  </tr>
                  <tr itemscope="" itemtype="http://schema.stenci.la/TableRow">
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell">Ex_MACP</td>
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell"><span
                        data-itemtype="http://schema.org/Number">0</span></td>
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell"><span
                        data-itemtype="http://schema.org/Number">0</span></td>
                  </tr>
                  <tr itemscope="" itemtype="http://schema.stenci.la/TableRow">
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell">Ex_Tre</td>
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell"><span
                        data-itemtype="http://schema.org/Number">0</span></td>
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell"><span
                        data-itemtype="http://schema.org/Number">0</span></td>
                  </tr>
                  <tr itemscope="" itemtype="http://schema.stenci.la/TableRow">
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell">Ex_AA</td>
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell"><span
                        data-itemtype="http://schema.org/Number">0</span></td>
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell"><span
                        data-itemtype="http://schema.org/Number">1000000</span></td>
                  </tr>
                </tbody>
              </table>
            </figure>
          </stencila-code-chunk>
          <figcaption>
            <h5 itemscope="" itemtype="http://schema.stenci.la/Heading"
              id="flux-boundary-constraints-of-im-export-reactions">Flux boundary constraints of
              Im-/export reactions</h5>
          </figcaption>
        </figure>
        <h4 itemscope="" itemtype="http://schema.stenci.la/Heading" id="additional-constraints">
          Additional Constraints</h4>
        <stencila-code-chunk itemscope="" itemtype="http://schema.stenci.la/CodeChunk"
          data-execution_count="8" data-programminglanguage="python">
          <pre class="language-python" itemscope="" itemtype="http://schema.stenci.la/CodeBlock"
            slot="text"><code>print(f&quot;Code Cell 8: Add Constraints on ATP maintenance costs&quot;)

## CONSTRAINT: Maintenance cost
atp_cost_L3_m = 0.009111187245501572 #Mitochondria-L3-ATP Cost [µmol*s-1*m-2]
atp_cost_L3_h = 0.15270708327974447 #Chloroplast-L3-ATP Cost [µmol*s-1*m-2]
atp_cost_L3_p = 0.0076669066992201855 #Peroxisome-L3-ATP Cost [µmol*s-1*m-2]
atp_cost_L3_c = 0.042683072918274702 #Cytosl/Other-L3-ATP Cost [µmol*s-1*m-2]

set_fixed_flux(&#39;NGAM_c&#39;,atp_cost_L3_c + atp_cost_L3_p, c3_model)
set_fixed_flux(&#39;NGAM_m&#39;,atp_cost_L3_m, c3_model)
set_fixed_flux(&#39;NGAM_h&#39;,atp_cost_L3_h, c3_model)</code></pre>
          <figure slot="outputs">
            <pre class="language-text" itemscope="" itemtype="http://schema.stenci.la/CodeBlock"><code>Code Cell 8: Add Constraints on ATP maintenance costs
</code></pre>
          </figure>
        </stencila-code-chunk>
        <figure itemscope="" itemtype="http://schema.stenci.la/Figure" id="table4" title="Table 4.">
          <label data-itemprop="label">Table 4.</label>
          <stencila-code-chunk itemscope="" itemtype="http://schema.stenci.la/CodeChunk"
            data-execution_count="9" data-programminglanguage="python">
            <pre class="language-python" itemscope="" itemtype="http://schema.stenci.la/CodeBlock"
              slot="text"><code>#### Table 4 ####

#{
#  &quot;caption&quot;: &quot;#### Maintenance costs by compartment&quot;,
#  &quot;id&quot;: &quot;table4&quot;,
#  &quot;label&quot;: &quot;Table 4.&quot;,
#  &quot;trusted&quot;: true
#}

df_maintenance = pd.DataFrame([atp_cost_L3_c,atp_cost_L3_h, atp_cost_L3_m, atp_cost_L3_p], index=[&#39;cytosol&#39;,&#39;chloroplast&#39;,&#39;mitochondria&#39;,&#39;peroxisome&#39;], columns=[&#39;Flux [μmol/(m^2^s)]&#39;])
df_maintenance.index.name = &#39;Compartment&#39;
df_maintenance</code></pre>
            <figure slot="outputs">
              <table itemscope="" itemtype="http://schema.org/Table">
                <thead>
                  <tr itemscope="" itemtype="http://schema.stenci.la/TableRow">
                    <th itemscope="" itemtype="http://schema.stenci.la/TableCell"></th>
                    <th itemscope="" itemtype="http://schema.stenci.la/TableCell">Flux
                      [μmol/(m^2^s)]</th>
                  </tr>
                  <tr itemscope="" itemtype="http://schema.stenci.la/TableRow">
                    <th itemscope="" itemtype="http://schema.stenci.la/TableCell">Compartment</th>
                    <th itemscope="" itemtype="http://schema.stenci.la/TableCell"></th>
                  </tr>
                </thead>
                <tbody>
                  <tr itemscope="" itemtype="http://schema.stenci.la/TableRow">
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell">cytosol</td>
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell"><span
                        data-itemtype="http://schema.org/Number">0.042683</span></td>
                  </tr>
                  <tr itemscope="" itemtype="http://schema.stenci.la/TableRow">
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell">chloroplast</td>
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell"><span
                        data-itemtype="http://schema.org/Number">0.152707</span></td>
                  </tr>
                  <tr itemscope="" itemtype="http://schema.stenci.la/TableRow">
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell">mitochondria</td>
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell"><span
                        data-itemtype="http://schema.org/Number">0.009111</span></td>
                  </tr>
                  <tr itemscope="" itemtype="http://schema.stenci.la/TableRow">
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell">peroxisome</td>
                    <td itemscope="" itemtype="http://schema.stenci.la/TableCell"><span
                        data-itemtype="http://schema.org/Number">0.007667</span></td>
                  </tr>
                </tbody>
              </table>
            </figure>
          </stencila-code-chunk>
          <figcaption>
            <h5 itemscope="" itemtype="http://schema.stenci.la/Heading"
              id="maintenance-costs-by-compartment">Maintenance costs by compartment</h5>
          </figcaption>
        </figure>
        <p itemscope="" itemtype="http://schema.stenci.la/Paragraph">We explicitly include the
          maintenance costs in our model to cover the amounts of ATP that is used to degradation and
          re-synthesis proteins for each compartment. <cite itemscope=""
            itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a
              href="#bib40"><span>40</span><span>Li et al.</span><span>2017</span></a></cite>
          specifies the ATP costs for protein degradation and synthesis of each compartment of a
          mature <em itemscope="" itemtype="http://schema.stenci.la/Emphasis">Arabidopsis</em> leaf.
          Based on the given data, we were able to calculate the flux rates to constrain the
          maintenance reactions in each compartment (<a href="#table4" itemscope=""
            itemtype="http://schema.stenci.la/Link">Table 4</a>).</p>
        <p itemscope="" itemtype="http://schema.stenci.la/Paragraph">The <em itemscope=""
            itemtype="http://schema.stenci.la/Emphasis">one-cell</em> model contains maintenance
          reactions only for the cytsol (<em itemscope=""
            itemtype="http://schema.stenci.la/Emphasis">NGAM_c</em>), chloroplast (<em itemscope=""
            itemtype="http://schema.stenci.la/Emphasis">NGAM_h</em>) and mitochondria (<em
            itemscope="" itemtype="http://schema.stenci.la/Emphasis">NGAM_m</em>) in the form:</p>
        <span itemscope="" itemtype="http://schema.stenci.la/MathBlock"><span
            class="mjx-chtml MJXc-display" style="text-align: center;"><span class="mjx-math"
              aria-label="\begin{array}{cc}ATP\mathrm{\_}\{x\}+H2O\mathrm{\_}\{x\}\to ADP\mathrm{\_}\{x\}+H\mathrm{\_}\{x\}+Pi\mathrm{\_}\{x\}\hfill &amp; x=c,h,m\hfill \end{array}"><span
                class="mjx-mrow" aria-hidden="true"><span class="mjx-mtable"
                  style="vertical-align: -0.288em; padding: 0px 0.167em;"><span
                    class="mjx-table"><span class="mjx-mtr" style="height: 1.075em;"><span
                        class="mjx-mtd"
                        style="padding: 0px 0.5em 0px 0px; text-align: left; width: 24.252em;"><span
                          class="mjx-mrow" style="margin-top: -0.2em;"><span class="mjx-mi"><span
                              class="mjx-char MJXc-TeX-math-I"
                              style="padding-top: 0.519em; padding-bottom: 0.298em;">A</span></span><span
                            class="mjx-mi"><span class="mjx-char MJXc-TeX-math-I"
                              style="padding-top: 0.446em; padding-bottom: 0.298em; padding-right: 0.12em;">T</span></span><span
                            class="mjx-mi"><span class="mjx-char MJXc-TeX-math-I"
                              style="padding-top: 0.446em; padding-bottom: 0.298em; padding-right: 0.109em;">P</span></span><span
                            class="mjx-texatom"><span class="mjx-mrow"><span class="mjx-mi"><span
                                  class="mjx-char MJXc-TeX-main-R"
                                  style="margin-top: -0.291em; padding-bottom: 0.372em;">_</span></span></span></span><span
                            class="mjx-mo"><span class="mjx-char MJXc-TeX-main-R"
                              style="padding-top: 0.446em; padding-bottom: 0.593em;">{</span></span><span
                            class="mjx-mi"><span class="mjx-char MJXc-TeX-math-I"
                              style="padding-top: 0.225em; padding-bottom: 0.298em;">x</span></span><span
                            class="mjx-mo"><span class="mjx-char MJXc-TeX-main-R"
                              style="padding-top: 0.446em; padding-bottom: 0.593em;">}</span></span><span
                            class="mjx-mo MJXc-space2"><span class="mjx-char MJXc-TeX-main-R"
                              style="padding-top: 0.298em; padding-bottom: 0.446em;">+</span></span><span
                            class="mjx-mi MJXc-space2"><span class="mjx-char MJXc-TeX-math-I"
                              style="padding-top: 0.446em; padding-bottom: 0.298em; padding-right: 0.057em;">H</span></span><span
                            class="mjx-mn"><span class="mjx-char MJXc-TeX-main-R"
                              style="padding-top: 0.372em; padding-bottom: 0.372em;">2</span></span><span
                            class="mjx-mi"><span class="mjx-char MJXc-TeX-math-I"
                              style="padding-top: 0.519em; padding-bottom: 0.298em;">O</span></span><span
                            class="mjx-texatom"><span class="mjx-mrow"><span class="mjx-mi"><span
                                  class="mjx-char MJXc-TeX-main-R"
                                  style="margin-top: -0.291em; padding-bottom: 0.372em;">_</span></span></span></span><span
                            class="mjx-mo"><span class="mjx-char MJXc-TeX-main-R"
                              style="padding-top: 0.446em; padding-bottom: 0.593em;">{</span></span><span
                            class="mjx-mi"><span class="mjx-char MJXc-TeX-math-I"
                              style="padding-top: 0.225em; padding-bottom: 0.298em;">x</span></span><span
                            class="mjx-mo"><span class="mjx-char MJXc-TeX-main-R"
                              style="padding-top: 0.446em; padding-bottom: 0.593em;">}</span></span><span
                            class="mjx-mo MJXc-space3"><span class="mjx-char MJXc-TeX-main-R"
                              style="padding-top: 0.225em; padding-bottom: 0.372em;"></span></span><span
                            class="mjx-mi MJXc-space3"><span class="mjx-char MJXc-TeX-math-I"
                              style="padding-top: 0.519em; padding-bottom: 0.298em;">A</span></span><span
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                              style="padding-top: 0.446em; padding-bottom: 0.298em;">D</span></span><span
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                              style="padding-top: 0.446em; padding-bottom: 0.298em; padding-right: 0.109em;">P</span></span><span
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                                  class="mjx-char MJXc-TeX-main-R"
                                  style="margin-top: -0.291em; padding-bottom: 0.372em;">_</span></span></span></span><span
                            class="mjx-mo"><span class="mjx-char MJXc-TeX-main-R"
                              style="padding-top: 0.446em; padding-bottom: 0.593em;">{</span></span><span
                            class="mjx-mi"><span class="mjx-char MJXc-TeX-math-I"
                              style="padding-top: 0.225em; padding-bottom: 0.298em;">x</span></span><span
                            class="mjx-mo"><span class="mjx-char MJXc-TeX-main-R"
                              style="padding-top: 0.446em; padding-bottom: 0.593em;">}</span></span><span
                            class="mjx-mo MJXc-space2"><span class="mjx-char MJXc-TeX-main-R"
                              style="padding-top: 0.298em; padding-bottom: 0.446em;">+</span></span><span
                            class="mjx-mi MJXc-space2"><span class="mjx-char MJXc-TeX-math-I"
                              style="padding-top: 0.446em; padding-bottom: 0.298em; padding-right: 0.057em;">H</span></span><span
                            class="mjx-texatom"><span class="mjx-mrow"><span class="mjx-mi"><span
                                  class="mjx-char MJXc-TeX-main-R"
                                  style="margin-top: -0.291em; padding-bottom: 0.372em;">_</span></span></span></span><span
                            class="mjx-mo"><span class="mjx-char MJXc-TeX-main-R"
                              style="padding-top: 0.446em; padding-bottom: 0.593em;">{</span></span><span
                            class="mjx-mi"><span class="mjx-char MJXc-TeX-math-I"
                              style="padding-top: 0.225em; padding-bottom: 0.298em;">x</span></span><span
                            class="mjx-mo"><span class="mjx-char MJXc-TeX-main-R"
                              style="padding-top: 0.446em; padding-bottom: 0.593em;">}</span></span><span
                            class="mjx-mo MJXc-space2"><span class="mjx-char MJXc-TeX-main-R"
                              style="padding-top: 0.298em; padding-bottom: 0.446em;">+</span></span><span
                            class="mjx-mi MJXc-space2"><span class="mjx-char MJXc-TeX-math-I"
                              style="padding-top: 0.446em; padding-bottom: 0.298em; padding-right: 0.109em;">P</span></span><span
                            class="mjx-mi"><span class="mjx-char MJXc-TeX-math-I"
                              style="padding-top: 0.446em; padding-bottom: 0.298em;">i</span></span><span
                            class="mjx-texatom"><span class="mjx-mrow"><span class="mjx-mi"><span
                                  class="mjx-char MJXc-TeX-main-R"
                                  style="margin-top: -0.291em; padding-bottom: 0.372em;">_</span></span></span></span><span
                            class="mjx-mo"><span class="mjx-char MJXc-TeX-main-R"
                              style="padding-top: 0.446em; padding-bottom: 0.593em;">{</span></span><span
                            class="mjx-mi"><span class="mjx-char MJXc-TeX-math-I"
                              style="padding-top: 0.225em; padding-bottom: 0.298em;">x</span></span><span
                            class="mjx-mo"><span class="mjx-char MJXc-TeX-main-R"
                              style="padding-top: 0.446em; padding-bottom: 0.593em;">}</span></span><span
                            class="mjx-strut"></span></span></span><span class="mjx-mtd"
                        style="padding: 0px 0px 0px 0.5em; text-align: left; width: 4.682em;"><span
                          class="mjx-mrow" style="margin-top: -0.2em;"><span class="mjx-mi"><span
                              class="mjx-char MJXc-TeX-math-I"
                              style="padding-top: 0.225em; padding-bottom: 0.298em;">x</span></span><span
                            class="mjx-mo MJXc-space3"><span class="mjx-char MJXc-TeX-main-R"
                              style="padding-top: 0.077em; padding-bottom: 0.298em;">=</span></span><span
                            class="mjx-mi MJXc-space3"><span class="mjx-char MJXc-TeX-math-I"
                              style="padding-top: 0.225em; padding-bottom: 0.298em;">c</span></span><span
                            class="mjx-mo"><span class="mjx-char MJXc-TeX-main-R"
                              style="margin-top: -0.144em; padding-bottom: 0.519em;">,</span></span><span
                            class="mjx-mi MJXc-space1"><span class="mjx-char MJXc-TeX-math-I"
                              style="padding-top: 0.446em; padding-bottom: 0.298em;">h</span></span><span
                            class="mjx-mo"><span class="mjx-char MJXc-TeX-main-R"
                              style="margin-top: -0.144em; padding-bottom: 0.519em;">,</span></span><span
                            class="mjx-mi MJXc-space1"><span class="mjx-char MJXc-TeX-math-I"
                              style="padding-top: 0.225em; padding-bottom: 0.298em;">m</span></span><span
                            class="mjx-strut"></span></span></span></span></span></span></span></span></span></span>
        <p itemscope="" itemtype="http://schema.stenci.la/Paragraph">An equivalent maintenance
          reaction cannot be formulated for the peroxisome since in the <em itemscope=""
            itemtype="http://schema.stenci.la/Emphasis">one-cell</em> model ATP/ADP are not included
          as peroxisomal metabolites. The flux through the maintenance reactions is fixed to the
          determined maintenance costs given in <a href="#table4" itemscope=""
            itemtype="http://schema.stenci.la/Link">Table 4</a>. The peroxisomal maintenance costs
          are added to the cytosolic maintenance costs.</p>
        <stencila-code-chunk itemscope="" itemtype="http://schema.stenci.la/CodeChunk"
          data-execution_count="10" data-programminglanguage="python">
          <pre class="language-python" itemscope="" itemtype="http://schema.stenci.la/CodeBlock"
            slot="text"><code>print(f&quot;Code Cell 10: Add Constraint on Rubisco Oxygenation : Decarboxylation Ratio&quot;)

## CONSTRAINT: oxygenation : decarboxylation = 1 : 10
r_c3_rbc_rbo = (10.0, 1.0)
const_c3_rbc_rbo = set_fixed_flux_ratio({&#39;RBC_h&#39;:r_c3_rbc_rbo[0],&#39;RBO_h&#39;:r_c3_rbc_rbo[1]}, &#39;const_c3_rbc_rbo&#39;, c3_model)</code></pre>
          <figure slot="outputs">
            <pre class="language-text" itemscope="" itemtype="http://schema.stenci.la/CodeBlock"><code>Code Cell 10: Add Constraint on Rubisco Oxygenation : Decarboxylation Ratio
</code></pre>
          </figure>
        </stencila-code-chunk>
        <p itemscope="" itemtype="http://schema.stenci.la/Paragraph">The CO<sub itemscope=""
            itemtype="http://schema.stenci.la/Subscript"><span
              data-itemtype="http://schema.org/Number">2</span></sub> and O<sub itemscope=""
            itemtype="http://schema.stenci.la/Subscript"><span
              data-itemtype="http://schema.org/Number">2</span></sub> partial pressures determine
          the ratio of the oxygenation : carboxylation rate of Rubisco (given by reactions <em
            itemscope="" itemtype="http://schema.stenci.la/Emphasis">RBO_h</em> and <em itemscope=""
            itemtype="http://schema.stenci.la/Emphasis">RBC_h</em>) and can be described by the
          mathematical expression:</p><span itemscope=""
          itemtype="http://schema.stenci.la/MathBlock"><span class="mjx-chtml MJXc-display"
            style="text-align: center;"><span class="mjx-math"
              aria-label="{\displaystyle \frac{{v}_{RBO\mathrm{\_}h}}{{v}_{RBC\mathrm{\_}h}}=\frac{1}{{S}_{R}}\cdot \frac{{p}_{{O}_{2}}}{{p}_{C{O}_{2}}},}"><span
                class="mjx-mrow" aria-hidden="true"><span class="mjx-texatom"><span
                    class="mjx-mrow"><span class="mjx-mstyle"><span class="mjx-mrow"><span
                          class="mjx-mfrac"><span class="mjx-box MJXc-stacked"
                            style="width: 3.094em; padding: 0px 0.12em;"><span class="mjx-numerator"
                              style="width: 3.094em; top: -1.232em;"><span class="mjx-msubsup"><span
                                  class="mjx-base"><span class="mjx-texatom"><span
                                      class="mjx-mrow"><span class="mjx-mi"><span
                                          class="mjx-char MJXc-TeX-math-I"
                                          style="padding-top: 0.225em; padding-bottom: 0.298em;">v</span></span></span></span></span><span
                                  class="mjx-sub"
                                  style="font-size: 70.7%; vertical-align: -0.229em; padding-right: 0.071em;"><span
                                    class="mjx-texatom" style=""><span class="mjx-mrow"><span
                                        class="mjx-mi"><span class="mjx-char MJXc-TeX-math-I"
                                          style="padding-top: 0.446em; padding-bottom: 0.298em;">R</span></span><span
                                        class="mjx-mi"><span class="mjx-char MJXc-TeX-math-I"
                                          style="padding-top: 0.446em; padding-bottom: 0.298em;">B</span></span><span
                                        class="mjx-mi"><span class="mjx-char MJXc-TeX-math-I"
                                          style="padding-top: 0.519em; padding-bottom: 0.298em;">O</span></span><span
                                        class="mjx-texatom"><span class="mjx-mrow"><span
                                            class="mjx-mi"><span class="mjx-char MJXc-TeX-main-R"
                                              style="margin-top: -0.291em; padding-bottom: 0.372em;">_</span></span></span></span><span
                                        class="mjx-mi"><span class="mjx-char MJXc-TeX-math-I"
                                          style="padding-top: 0.446em; padding-bottom: 0.298em;">h</span></span></span></span></span></span></span><span
                              class="mjx-denominator" style="width: 3.094em; bottom: -0.91em;"><span
                                class="mjx-msubsup"><span class="mjx-base"><span
                                    class="mjx-texatom"><span class="mjx-mrow"><span
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                                          style="padding-top: 0.225em; padding-bottom: 0.298em;">v</span></span></span></span></span><span
                                  class="mjx-sub"
                                  style="font-size: 70.7%; vertical-align: -0.23em; padding-right: 0.071em;"><span
                                    class="mjx-texatom" style=""><span class="mjx-mrow"><span
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                                          style="padding-top: 0.446em; padding-bottom: 0.298em;">R</span></span><span
                                        class="mjx-mi"><span class="mjx-char MJXc-TeX-math-I"
                                          style="padding-top: 0.446em; padding-bottom: 0.298em;">B</span></span><span
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                                          style="padding-top: 0.519em; padding-bottom: 0.298em; padding-right: 0.045em;">C</span></span><span
                                        class="mjx-texatom"><span class="mjx-mrow"><span
                                            class="mjx-mi"><span class="mjx-char MJXc-TeX-main-R"
                                              style="margin-top: -0.291em; padding-bottom: 0.372em;">_</span></span></span></span><span
                                        class="mjx-mi"><span class="mjx-char MJXc-TeX-math-I"
                                          style="padding-top: 0.446em; padding-bottom: 0.298em;">h</span></span></span></span></span></span></span><span
                              style="border-bottom: 1.3px solid; top: -0.296em; width: 3.094em;"
                              class="mjx-line"></span></span><span
                            style="height: 2.142em; vertical-align: -0.91em;"
                            class="mjx-vsize"></span></span><span class="mjx-mo MJXc-space3"><span
                            class="mjx-char MJXc-TeX-main-R"
                            style="padding-top: 0.077em; padding-bottom: 0.298em;">=</span></span><span
                          class="mjx-mfrac MJXc-space3"><span class="mjx-box MJXc-stacked"
                            style="width: 1.385em; padding: 0px 0.12em;"><span class="mjx-numerator"
                              style="width: 1.385em; top: -1.368em;"><span class="mjx-mn"><span
                                  class="mjx-char MJXc-TeX-main-R"
                                  style="padding-top: 0.372em; padding-bottom: 0.372em;">1</span></span></span><span
                              class="mjx-denominator"
                              style="width: 1.385em; bottom: -0.953em;"><span
                                class="mjx-msubsup"><span class="mjx-base"
                                  style="margin-right: -0.032em;"><span class="mjx-texatom"><span
                                      class="mjx-mrow"><span class="mjx-mi"><span
                                          class="mjx-char MJXc-TeX-math-I"
                                          style="padding-top: 0.519em; padding-bottom: 0.298em; padding-right: 0.032em;">S</span></span></span></span></span><span
                                  class="mjx-sub"
                                  style="font-size: 70.7%; vertical-align: -0.212em; padding-right: 0.071em;"><span
                                    class="mjx-texatom" style=""><span class="mjx-mrow"><span
                                        class="mjx-mi"><span class="mjx-char MJXc-TeX-math-I"
                                          style="padding-top: 0.446em; padding-bottom: 0.298em;">R</span></span></span></span></span></span></span><span
                              style="border-bottom: 1.3px solid; top: -0.296em; width: 1.385em;"
                              class="mjx-line"></span></span><span
                            style="height: 2.32em; vertical-align: -0.953em;"
                            class="mjx-vsize"></span></span><span class="mjx-mo MJXc-space2"><span
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                            style="padding-top: 0.004em; padding-bottom: 0.298em;"></span></span><span
                          class="mjx-mfrac MJXc-space2"><span class="mjx-box MJXc-stacked"
                            style="width: 2.139em; padding: 0px 0.12em;"><span class="mjx-numerator"
                              style="width: 2.139em; top: -1.433em;"><span class="mjx-msubsup"><span
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                                          style="padding-top: 0.225em; padding-bottom: 0.446em;">p</span></span></span></span></span><span
                                  class="mjx-sub"
                                  style="font-size: 70.7%; vertical-align: -0.36em; padding-right: 0.071em;"><span
                                    class="mjx-texatom" style=""><span class="mjx-mrow"><span
                                        class="mjx-msubsup"><span class="mjx-base"><span
                                            class="mjx-texatom"><span class="mjx-mrow"><span
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                                                  class="mjx-char MJXc-TeX-math-I"
                                                  style="padding-top: 0.519em; padding-bottom: 0.298em;">O</span></span></span></span></span><span
                                          class="mjx-sub"
                                          style="font-size: 83.3%; vertical-align: -0.267em; padding-right: 0.06em;"><span
                                            class="mjx-texatom" style=""><span
                                              class="mjx-mrow"><span class="mjx-mn"><span
                                                  class="mjx-char MJXc-TeX-main-R"
                                                  style="padding-top: 0.372em; padding-bottom: 0.372em;">2</span></span></span></span></span></span></span></span></span></span></span><span
                              class="mjx-denominator"
                              style="width: 2.139em; bottom: -1.112em;"><span
                                class="mjx-msubsup"><span class="mjx-base"><span
                                    class="mjx-texatom"><span class="mjx-mrow"><span
                                        class="mjx-mi"><span class="mjx-char MJXc-TeX-math-I"
                                          style="padding-top: 0.225em; padding-bottom: 0.446em;">p</span></span></span></span></span><span
                                  class="mjx-sub"
                                  style="font-size: 70.7%; vertical-align: -0.36em; padding-right: 0.071em;"><span
                                    class="mjx-texatom" style=""><span class="mjx-mrow"><span
                                        class="mjx-mi"><span class="mjx-char MJXc-TeX-math-I"
                                          style="padding-top: 0.519em; padding-bottom: 0.298em; padding-right: 0.045em;">C</span></span><span
                                        class="mjx-msubsup"><span class="mjx-base"><span
                                            class="mjx-texatom"><span class="mjx-mrow"><span
                                                class="mjx-mi"><span
                                                  class="mjx-char MJXc-TeX-math-I"
                                                  style="padding-top: 0.519em; padding-bottom: 0.298em;">O</span></span></span></span></span><span
                                          class="mjx-sub"
                                          style="font-size: 83.3%; vertical-align: -0.267em; padding-right: 0.06em;"><span
                                            class="mjx-texatom" style=""><span
                                              class="mjx-mrow"><span class="mjx-mn"><span
                                                  class="mjx-char MJXc-TeX-main-R"
                                                  style="padding-top: 0.372em; padding-bottom: 0.372em;">2</span></span></span></span></span></span></span></span></span></span></span><span
                              style="border-bottom: 1.3px solid; top: -0.296em; width: 2.139em;"
                              class="mjx-line"></span></span><span
                            style="height: 2.546em; vertical-align: -1.112em;"
                            class="mjx-vsize"></span></span><span class="mjx-mo"><span
                            class="mjx-char MJXc-TeX-main-R"
                            style="margin-top: -0.144em; padding-bottom: 0.519em;">,</span></span></span></span></span></span></span></span></span></span>
        <p itemscope="" itemtype="http://schema.stenci.la/Paragraph">where <span itemscope=""
            itemtype="http://schema.stenci.la/MathFragment"><span class="mjx-chtml"><span
                class="mjx-math" aria-label="{S}_{R}"><span class="mjx-mrow"
                  aria-hidden="true"><span class="mjx-msubsup"><span class="mjx-base"
                      style="margin-right: -0.032em;"><span class="mjx-texatom"><span
                          class="mjx-mrow"><span class="mjx-mi"><span
                              class="mjx-char MJXc-TeX-math-I"
                              style="padding-top: 0.519em; padding-bottom: 0.298em; padding-right: 0.032em;">S</span></span></span></span></span><span
                      class="mjx-sub"
                      style="font-size: 70.7%; vertical-align: -0.212em; padding-right: 0.071em;"><span
                        class="mjx-texatom" style=""><span class="mjx-mrow"><span
                            class="mjx-mi"><span class="mjx-char MJXc-TeX-math-I"
                              style="padding-top: 0.446em; padding-bottom: 0.298em;">R</span></span></span></span></span></span></span></span></span></span>
          specifies the ability of Rubisco to bind CO<sub itemscope=""
            itemtype="http://schema.stenci.la/Subscript"><span
              data-itemtype="http://schema.org/Number">2</span></sub> over O<sub itemscope=""
            itemtype="http://schema.stenci.la/Subscript"><span
              data-itemtype="http://schema.org/Number">2</span></sub>. In the case of a mature leave
          and ambient CO<sub itemscope="" itemtype="http://schema.stenci.la/Subscript"><span
              data-itemtype="http://schema.org/Number">2</span></sub> and O<sub itemscope=""
            itemtype="http://schema.stenci.la/Subscript"><span
              data-itemtype="http://schema.org/Number">2</span></sub> partial pressures in temperate
          regions with adequate water supply, the ratio <span itemscope=""
            itemtype="http://schema.stenci.la/MathFragment"><span class="mjx-chtml"><span
                class="mjx-math" aria-label="{v}_{RB{O}_{h}}/{v}_{RB{C}_{h}}"><span class="mjx-mrow"
                  aria-hidden="true"><span class="mjx-msubsup"><span class="mjx-base"><span
                        class="mjx-texatom"><span class="mjx-mrow"><span class="mjx-mi"><span
                              class="mjx-char MJXc-TeX-math-I"
                              style="padding-top: 0.225em; padding-bottom: 0.298em;">v</span></span></span></span></span><span
                      class="mjx-sub"
                      style="font-size: 70.7%; vertical-align: -0.229em; padding-right: 0.071em;"><span
                        class="mjx-texatom" style=""><span class="mjx-mrow"><span
                            class="mjx-mi"><span class="mjx-char MJXc-TeX-math-I"
                              style="padding-top: 0.446em; padding-bottom: 0.298em;">R</span></span><span
                            class="mjx-mi"><span class="mjx-char MJXc-TeX-math-I"
                              style="padding-top: 0.446em; padding-bottom: 0.298em;">B</span></span><span
                            class="mjx-msubsup"><span class="mjx-base"><span
                                class="mjx-texatom"><span class="mjx-mrow"><span
                                    class="mjx-mi"><span class="mjx-char MJXc-TeX-math-I"
                                      style="padding-top: 0.519em; padding-bottom: 0.298em;">O</span></span></span></span></span><span
                              class="mjx-sub"
                              style="font-size: 83.3%; vertical-align: -0.295em; padding-right: 0.06em;"><span
                                class="mjx-texatom" style=""><span class="mjx-mrow"><span
                                    class="mjx-mi"><span class="mjx-char MJXc-TeX-math-I"
                                      style="padding-top: 0.446em; padding-bottom: 0.298em;">h</span></span></span></span></span></span></span></span></span></span><span
                    class="mjx-texatom"><span class="mjx-mrow"><span class="mjx-mo"><span
                          class="mjx-char MJXc-TeX-main-R"
                          style="padding-top: 0.446em; padding-bottom: 0.593em;">/</span></span></span></span><span
                    class="mjx-msubsup"><span class="mjx-base"><span class="mjx-texatom"><span
                          class="mjx-mrow"><span class="mjx-mi"><span
                              class="mjx-char MJXc-TeX-math-I"
                              style="padding-top: 0.225em; padding-bottom: 0.298em;">v</span></span></span></span></span><span
                      class="mjx-sub"
                      style="font-size: 70.7%; vertical-align: -0.23em; padding-right: 0.071em;"><span
                        class="mjx-texatom" style=""><span class="mjx-mrow"><span
                            class="mjx-mi"><span class="mjx-char MJXc-TeX-math-I"
                              style="padding-top: 0.446em; padding-bottom: 0.298em;">R</span></span><span
                            class="mjx-mi"><span class="mjx-char MJXc-TeX-math-I"
                              style="padding-top: 0.446em; padding-bottom: 0.298em;">B</span></span><span
                            class="mjx-msubsup"><span class="mjx-base"
                              style="margin-right: -0.045em;"><span class="mjx-texatom"><span
                                  class="mjx-mrow"><span class="mjx-mi"><span
                                      class="mjx-char MJXc-TeX-math-I"
                                      style="padding-top: 0.519em; padding-bottom: 0.298em; padding-right: 0.045em;">C</span></span></span></span></span><span
                              class="mjx-sub"
                              style="font-size: 83.3%; vertical-align: -0.295em; padding-right: 0.06em;"><span
                                class="mjx-texatom" style=""><span class="mjx-mrow"><span
                                    class="mjx-mi"><span class="mjx-char MJXc-TeX-math-I"
                                      style="padding-top: 0.446em; padding-bottom: 0.298em;">h</span></span></span></span></span></span></span></span></span></span></span></span></span></span>
          is fixed and is predicted to be <stencila-code-expression programming-language="py"
            itemscope="" itemtype="http://schema.stenci.la/CodeExpression"><code class="py"
              slot="text">int(r_c3_rbc_rbo[0])</code><output slot="output"></output>
          </stencila-code-expression>:<stencila-code-expression programming-language="py"
            itemscope="" itemtype="http://schema.stenci.la/CodeExpression"><code class="py"
              slot="text">int(r_c3_rbc_rbo[1])</code><output slot="output"></output>
          </stencila-code-expression>, which is encoded by an additional flux ratio constraint.</p>
        <stencila-code-chunk itemscope="" itemtype="http://schema.stenci.la/CodeChunk"
          data-execution_count="11" data-programminglanguage="python">
          <pre class="language-python" itemscope="" itemtype="http://schema.stenci.la/CodeBlock"
            slot="text"><code>print(f&quot;Code Cell 11: Add Constraint on NADPH dehydrogenase and plastoquinol oxidase&quot;)

## CONSTRAINT: fluxes through the chloroplastic NADPH dehydrogenase and plastoquinol oxidase were set to zero 
#because the contributions of NADPH dehydrogenase (Yamamoto et al., 2011) and plastoquinol oxidase 
#(Josse et al., 2000) to photosynthesis are thought to be minor.
set_bounds(&#39;AOX4_h&#39;,(0,0), c3_model)
set_bounds(&#39;iCitDHNADP_h&#39;,(0,0), c3_model)</code></pre>
          <figure slot="outputs">
            <pre class="language-text" itemscope="" itemtype="http://schema.stenci.la/CodeBlock"><code>Code Cell 11: Add Constraint on NADPH dehydrogenase and plastoquinol oxidase
</code></pre>
          </figure>
        </stencila-code-chunk>
        <p itemscope="" itemtype="http://schema.stenci.la/Paragraph">We assume no flux for the
          chloroplastic NADPH dehydrogenase (<em itemscope=""
            itemtype="http://schema.stenci.la/Emphasis">iCitDHNADP_h</em>) and plastoquinol oxidase
          (<em itemscope="" itemtype="http://schema.stenci.la/Emphasis">AOX4_h</em>) because <cite
            itemscope="" itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a
              href="#bib33"><span>33</span><span>Josse et al.</span><span>2000</span></a></cite> and
          <cite itemscope="" itemtype="http://schema.stenci.la/Cite"
            data-citationmode="Narrative"><a href="#bib88"><span>88</span><span>Yamamoto et
                al.</span><span>2011</span></a></cite> have shown that their effect on the
          photosynthesis is minor.</p>
        <stencila-code-chunk itemscope="" itemtype="http://schema.stenci.la/CodeChunk"
          data-execution_count="12" data-programminglanguage="python">
          <pre class="language-python" itemscope="" itemtype="http://schema.stenci.la/CodeBlock"
            slot="text"><code>print(f&quot;Code Cell 12: Add additional Transport Constraints&quot;)

## CONSTRAINT: NTT is only active at night
set_fixed_flux(&#39;Tr_NTT&#39;,0, c3_model)

## CONSTRAINT: No uncoupled pyruvate transport
set_bounds(&#39;Tr_Pyr1&#39;,(0,0), c3_model)
set_bounds(&#39;Tr_Pyr2&#39;,(0,0), c3_model)

## CONSTRAINT: 
set_bounds(&#39;G6PDH_h&#39;, (0.,0.), c3_model)
set_bounds(&#39;PPIF6PK_c&#39;, (0,0.), c3_model)</code></pre>
          <figure slot="outputs">
            <pre class="language-text" itemscope="" itemtype="http://schema.stenci.la/CodeBlock"><code>Code Cell 12: Add additional Transport Constraints
</code></pre>
          </figure>
        </stencila-code-chunk>
        <h4 itemscope="" itemtype="http://schema.stenci.la/Heading" id="objective">Objective</h4>
        <stencila-code-chunk itemscope="" itemtype="http://schema.stenci.la/CodeChunk"
          data-execution_count="13" data-programminglanguage="python">
          <pre class="language-python" itemscope="" itemtype="http://schema.stenci.la/CodeBlock"
            slot="text"><code>print(f&quot;Code Cell 13: Add Objective - Optimize Sucrose Output&quot;)

## Optimize/Maximize sucrose output
r_c3_opt_id = &quot;Ex_Suc&quot;
r_c3_opt_obj = c3_model.reactions.get_by_id(r_c3_opt_id)
r_c3_opt_obj.objective_coefficient = 1.</code></pre>
          <figure slot="outputs">
            <pre class="language-text" itemscope="" itemtype="http://schema.stenci.la/CodeBlock"><code>Code Cell 13: Add Objective - Optimize Sucrose Output
</code></pre>
          </figure>
        </stencila-code-chunk>
        <p itemscope="" itemtype="http://schema.stenci.la/Paragraph">In accordance with the
          assumption of mature, fully differentiated and photosynthetic active leaf, the model’s
          objective is to maximise the phloem sap output defined by reactions
          <stencila-code-expression programming-language="py" itemscope=""
            itemtype="http://schema.stenci.la/CodeExpression"><code class="py"
              slot="text">r_c3_opt_id</code><output slot="output"></output>
          </stencila-code-expression>. Additionally, we assume that the involved plant cells put
          only a minimal metabolic effort, in the form of energy and resources, into the production
          of phloem sap as possible. This assumption is in correspondence with minimising the
          nitrogen investment by reducing the number of enzymes that are active in a metabolic
          network. Therefore, we perform a parsimonious FBA to minimise the total flux.</p>
        <p itemscope="" itemtype="http://schema.stenci.la/Paragraph">For enhanced compliance with
          the recent standards of the systems biology community, the <em itemscope=""
            itemtype="http://schema.stenci.la/Emphasis">one-cell</em> model is encoded in SBML level
          3. Meta-information on subsystems, publications, cross-references are provided as evidence
          code in the form of MIRIAM URI’s. FBA related information, gene association rules, charge
          and formula of a species element are encoded using the Flux Balance Constraints package
          developed for SBML level 3. All fluxes in the model are consistently defined
          as μmol/(m<sup itemscope="" itemtype="http://schema.stenci.la/Superscript"><span
              data-itemtype="http://schema.org/Number">2</span></sup>s).</p>
        <h3 itemscope="" itemtype="http://schema.stenci.la/Heading"
          id="generic-model-for-c4-metabolism">Generic model for C4 metabolism</h3>
        <h4 itemscope="" itemtype="http://schema.stenci.la/Heading" id="metabolic-model-1">Metabolic
          model</h4>
        <stencila-code-chunk itemscope="" itemtype="http://schema.stenci.la/CodeChunk"
          data-execution_count="14" data-programminglanguage="python">
          <pre class="language-python" itemscope="" itemtype="http://schema.stenci.la/CodeBlock"
            slot="text"><code>print(f&quot;Code Cell 14: Initiate  Metabolic Model of C4 Photosynthesis&quot;)

## Intitialize C4 model
c4_model = cobra.Model(&#39;c4_model&#39;)

## Define cell types
cell_types = [&#39;M&#39;, &#39;B&#39;]

## Duplicate metabolites
for m in c3_model.metabolites:
    for cell in cell_types:
        m_dt = cobra.Metabolite(&#39;[&#39;+cell+&#39;]_&#39;+m.id, name = m.formula, compartment = m.compartment)
        c4_model.add_metabolites([m_dt])

## Duplicate reactions
for r_c3_obj in c3_model.reactions:
    for cell in cell_types:
        r_c4_obj = cobra.Reaction(&#39;[&#39;+cell+&#39;]_&#39;+r_c3_obj.id)
        r_c4_obj.name = r_c3_obj.name
        r_c4_obj.subsystem = r_c3_obj.subsystem
        r_c4_obj.bounds = r_c3_obj.bounds
        c4_model.add_reaction(r_c4_obj)
        r_c4_obj.add_metabolites({&#39;[&#39;+cell+&#39;]_&#39;+m_c3_obj.id: r_c3_obj.get_coefficient(m_c3_obj) for m_c3_obj in r_c3_obj.metabolites})
        
## Model Summary
c4_num_mets = len(c4_model.metabolites)
c4_num_rxn = len(c4_model.reactions)

df_c4_model_summary = pd.DataFrame([c4_num_mets, c4_num_rxn], index=[&#39;Number of metabolites&#39;,&#39;Number of reactions&#39;], columns=[&#39;Count&#39;])
df_c4_model_summary</code></pre>
          <figure slot="outputs">
            <pre class="language-text" itemscope="" itemtype="http://schema.stenci.la/CodeBlock"><code>Code Cell 14: Initiate  Metabolic Model of C4 Photosynthesis
</code></pre>
            <table itemscope="" itemtype="http://schema.org/Table">
              <thead>
                <tr itemscope="" itemtype="http://schema.stenci.la/TableRow">
                  <th itemscope="" itemtype="http://schema.stenci.la/TableCell"></th>
                  <th itemscope="" itemtype="http://schema.stenci.la/TableCell">Count</th>
                </tr>
              </thead>
              <tbody>
                <tr itemscope="" itemtype="http://schema.stenci.la/TableRow">
                  <td itemscope="" itemtype="http://schema.stenci.la/TableCell">Number of
                    metabolites</td>
                  <td itemscope="" itemtype="http://schema.stenci.la/TableCell"><span
                      data-itemtype="http://schema.org/Number">826</span></td>
                </tr>
                <tr itemscope="" itemtype="http://schema.stenci.la/TableRow">
                  <td itemscope="" itemtype="http://schema.stenci.la/TableCell">Number of reactions
                  </td>
                  <td itemscope="" itemtype="http://schema.stenci.la/TableCell"><span
                      data-itemtype="http://schema.org/Number">1144</span></td>
                </tr>
              </tbody>
            </table>
          </figure>
        </stencila-code-chunk>
        <p itemscope="" itemtype="http://schema.stenci.la/Paragraph">The generic model of C4
          metabolism, short <em itemscope=""
            itemtype="http://schema.stenci.la/Emphasis">two-cell</em> model, comprises two copies of
          the <em itemscope="" itemtype="http://schema.stenci.la/Emphasis">one-cell</em> model to
          represent one mesophyll and one bundle sheath cell. Reactions and metabolites belonging to
          the metabolic network of the mesophyll are indicated with the prefix <em itemscope=""
            itemtype="http://schema.stenci.la/Emphasis">[M]</em>, whereas the prefix for the bundle
          sheath is <em itemscope="" itemtype="http://schema.stenci.la/Emphasis">[B]</em>. The
          separate mesophyll and bundle sheath networks are connected via reversible transport
          reactions of the cytosolic metabolites indicated with the prefix <em itemscope=""
            itemtype="http://schema.stenci.la/Emphasis">[MB]</em>, <a href="#fig2" itemscope=""
            itemtype="http://schema.stenci.la/Link">Figure 2</a>. The C4 evolution not only confined
          Rubisco to the bundle sheath cells, the CO<sub itemscope=""
            itemtype="http://schema.stenci.la/Subscript"><span
              data-itemtype="http://schema.org/Number">2</span></sub> concentrating mechanism
          steadily supplies Rubisco with CO<sub itemscope=""
            itemtype="http://schema.stenci.la/Subscript"><span
              data-itemtype="http://schema.org/Number">2</span></sub> in such a way that the
          oxygenation rate is negligible. Therefore, the bundle sheath network is equipped with two
          Rubisco populations. The native Rubisco population binds external CO<sub itemscope=""
            itemtype="http://schema.stenci.la/Subscript"><span
              data-itemtype="http://schema.org/Number">2</span></sub> and adheres to forced
          oxygenation : carboxylation ratios, where the optimised evolutionary population binds only
          internal CO<sub itemscope="" itemtype="http://schema.stenci.la/Subscript"><span
              data-itemtype="http://schema.org/Number">2</span></sub> and the carboxylation occurs
          independently of the oxygenation. External CO<sub itemscope=""
            itemtype="http://schema.stenci.la/Subscript"><span
              data-itemtype="http://schema.org/Number">2</span></sub> is defined as _[B]_CO2_ex_<em
            itemscope="" itemtype="http://schema.stenci.la/Emphasis">{_c,h</em>} supplied by the
          mesophyll network. Internal CO<sub itemscope=""
            itemtype="http://schema.stenci.la/Subscript"><span
              data-itemtype="http://schema.org/Number">2</span></sub> given by _[B]_CO2_<em
            itemscope="" itemtype="http://schema.stenci.la/Emphasis">{_c,h,m</em>} originates from
          reactions in the bundle sheath network producing CO<sub itemscope=""
            itemtype="http://schema.stenci.la/Subscript"><span
              data-itemtype="http://schema.org/Number">2</span></sub>. External CO<sub itemscope=""
            itemtype="http://schema.stenci.la/Subscript"><span
              data-itemtype="http://schema.org/Number">2</span></sub> in the bundle sheath network
          is only allowed to move to the chloroplast <em itemscope=""
            itemtype="http://schema.stenci.la/Emphasis">[B]_Tr_CO2h_Ex</em> and to react with
          Rubisco <em itemscope="" itemtype="http://schema.stenci.la/Emphasis">[B]_RBC_h_Ex</em>.
          The differentiation of two Rubisco populations binding either external or internal CO<sub
            itemscope="" itemtype="http://schema.stenci.la/Subscript"><span
              data-itemtype="http://schema.org/Number">2</span></sub> approximates the
          concentration-dependent shift of the oxygenation : carboxylation ratio.</p>
        <h4 itemscope="" itemtype="http://schema.stenci.la/Heading" id="imports">Imports</h4>
        <stencila-code-chunk itemscope="" itemtype="http://schema.stenci.la/CodeChunk"
          data-execution_count="15" data-programminglanguage="python">
          <pre class="language-python" itemscope="" itemtype="http://schema.stenci.la/CodeBlock"
            slot="text"><code>print(f&quot;Code Cell 15: Adapt CO2 Input Constraint&quot;)


## CONSTRAINT: CO2 uptake rate uin C4 plants is higher, about 40 μmol/(m2*s)
f_C4_CO2_M = 40 #[μmol/(m2*s)] 
set_bounds(&#39;[M]_Im_CO2&#39;, (0, f_C4_CO2_M), c4_model)

## CONSTRAINT: No CO2 uptake in bundle sheat cells due to suberin layer in cell membranes
f_C4_CO2_B = 0 #[μmol/(m2*s)] 
set_fixed_flux(&#39;[B]_Im_CO2&#39;, f_C4_CO2_B, c4_model)

## Other constraints on inputs are directly transfered from the c3 model</code></pre>
          <figure slot="outputs">
            <pre class="language-text" itemscope="" itemtype="http://schema.stenci.la/CodeBlock"><code>Code Cell 15: Adapt CO2 Input Constraint
</code></pre>
          </figure>
        </stencila-code-chunk>
        <p itemscope="" itemtype="http://schema.stenci.la/Paragraph">As for the <em itemscope=""
            itemtype="http://schema.stenci.la/Emphasis">one-cell</em> model, we assume
          photoautotrophic growth conditions, see <a href="#table3" itemscope=""
            itemtype="http://schema.stenci.la/Link">Table 3</a>. During C4 evolution the CO<sub
            itemscope="" itemtype="http://schema.stenci.la/Subscript"><span
              data-itemtype="http://schema.org/Number">2</span></sub> assimilation became more
          efficient allowing higher CO<sub itemscope=""
            itemtype="http://schema.stenci.la/Subscript"><span
              data-itemtype="http://schema.org/Number">2</span></sub> assimilation rates. <em
            itemscope="" itemtype="http://schema.stenci.la/Emphasis">Zea mays</em> achieves up to
          <stencila-code-expression programming-language="py" itemscope=""
            itemtype="http://schema.stenci.la/CodeExpression"><code class="py"
              slot="text">f_C4_CO2_M</code><output slot="output"></output>
          </stencila-code-expression> μmol/(m<sup itemscope=""
            itemtype="http://schema.stenci.la/Superscript"><span
              data-itemtype="http://schema.org/Number">2</span></sup>s) ([M]_Im_CO<sub itemscope=""
            itemtype="http://schema.stenci.la/Subscript"><span
              data-itemtype="http://schema.org/Number">2</span></sub>) <cite itemscope=""
            itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a
              href="#bib60"><span>60</span><span>Rozema</span><span>1993</span></a></cite>. We
          assume that the CO<sub itemscope="" itemtype="http://schema.stenci.la/Subscript"><span
              data-itemtype="http://schema.org/Number">2</span></sub> uptake from the environment by
          the bundle sheath has to be bridged by the mesophyll. Therefore, the input flux of
          [B]_Im_CO<sub itemscope="" itemtype="http://schema.stenci.la/Subscript"><span
              data-itemtype="http://schema.org/Number">2</span></sub> is set to
          <stencila-code-expression programming-language="py" itemscope=""
            itemtype="http://schema.stenci.la/CodeExpression"><code class="py"
              slot="text">f_C4_CO2_B</code><output slot="output"></output>
          </stencila-code-expression>.</p>
        <h4 itemscope="" itemtype="http://schema.stenci.la/Heading" id="exports">Exports</h4>
        <stencila-code-chunk itemscope="" itemtype="http://schema.stenci.la/CodeChunk"
          data-execution_count="16" data-programminglanguage="python">
          <pre class="language-python" itemscope="" itemtype="http://schema.stenci.la/CodeBlock"
            slot="text"><code>print(f&quot;Code Cell 16: Adapt Output Constraints&quot;)

## CONSTRAINT: Output of sucrose : total amino acid and sucrose : starch
r_suc_aa = (2.2, 1.0)
const_c4_suc_aa_b = set_fixed_flux_ratio({&#39;[B]_Ex_Suc&#39;:r_suc_aa[0],&#39;[B]_Ex_AA&#39;:r_suc_aa[1]}, &#39;const_c4_suc_aa_b&#39;, c4_model)
const_c4_suc_aa_m = set_fixed_flux_ratio({&#39;[M]_Ex_Suc&#39;:r_suc_aa[0],&#39;[M]_Ex_AA&#39;:r_suc_aa[1]}, &#39;const_c4_suc_aa_m&#39;, c4_model)

r_suc_starch = (1.0, 1.0)
const_c4_suc_starch_b = set_fixed_flux_ratio({&#39;[B]_Ex_Suc&#39;:r_suc_starch[0],&#39;[B]_Ex_starch&#39;:r_suc_starch[1]}, &#39;const_c4_suc_starch_b&#39;, c4_model)
const_c4_suc_starch_m = set_fixed_flux_ratio({&#39;[M]_Ex_Suc&#39;:r_suc_starch[0],&#39;[M]_Ex_starch&#39;:r_suc_starch[1]}, &#39;const_c4_suc_starch_m&#39;, c4_model)


## Other constraints on outputs are directly transfered from the c4 model</code></pre>
          <figure slot="outputs">
            <pre class="language-text" itemscope="" itemtype="http://schema.stenci.la/CodeBlock"><code>Code Cell 16: Adapt Output Constraints
</code></pre>
          </figure>
        </stencila-code-chunk>
        <p itemscope="" itemtype="http://schema.stenci.la/Paragraph">The outputs of the <em
            itemscope="" itemtype="http://schema.stenci.la/Emphasis">one-cell</em> model are
          transferred to the mesophyll and bundle sheath network, as well as the corresponding flux
          ratios, see <a href="#table3" itemscope="" itemtype="http://schema.stenci.la/Link">Table
            3</a>.</p>
        <h4 itemscope="" itemtype="http://schema.stenci.la/Heading" id="additional-constraints-1">
          Additional Constraints</h4>
        <stencila-code-chunk itemscope="" itemtype="http://schema.stenci.la/CodeChunk"
          data-execution_count="17" data-programminglanguage="python">
          <pre class="language-python" itemscope="" itemtype="http://schema.stenci.la/CodeBlock"
            slot="text"><code>print(f&quot;Code Cell 17: Add Metabolite Exchange Reactions&quot;)


## Metabolites excluded from M/BS exchange
no_transport = [&#39;NO3&#39;,&#39;NO2&#39;, &#39;O2&#39;,&#39;Na&#39;, &#39;H2S&#39;, &#39;SO4&#39;,
                &#39;H2O&#39;,&#39;FBP&#39;,&#39;F26BP&#39;,&#39;DPGA&#39;,&#39;H&#39;,&#39;ACD&#39;,&#39;AC&#39;,&#39;M_DASH_THF&#39;, &#39;5M_DASH_THF&#39;, &#39;H_DASH_Cys&#39;, &#39;aH_DASH_Cys&#39;, &#39;ORO&#39;, &#39;DHO&#39;,
                &#39;GABA&#39;,&#39;A_DASH_Ser&#39;,&#39;PRPP&#39;,&#39;AD&#39;,&#39;THF&#39;,&#39;DHF&#39;,&#39;ADN&#39;,&#39;Mas&#39;,&#39;CoA&#39;,&#39;GluP&#39;,
                &#39;A_DASH_CoA&#39;,&#39;cellulose1&#39;,&#39;cellulose2&#39;,&#39;cellulose3&#39;,&#39;starch1&#39;,
                &#39;starch2&#39;,&#39;starch3&#39;,&#39;TRXox&#39;,&#39;TRXrd&#39;,&#39;Glu_DASH_SeA&#39;,&#39;T6P&#39;,&#39;aMet&#39;,
                &#39;PPi&#39;, &#39;P5C&#39;, &#39;NH4&#39;, &#39;Pi&#39;, &#39;CO2&#39;, &#39;OAA&#39;,&#39;HCO3&#39;, 
                &#39;UTP&#39;, &#39;UDP&#39;, &#39;UDPG&#39;, &#39;ATP&#39;, &#39;ADP&#39;, &#39;AMP&#39;, &#39;IMP&#39;, &#39;XMP&#39;, 
                &#39;GTP&#39;, &#39;GDP&#39;, &#39;GMP&#39;, &#39;OMP&#39;, &#39;UMP&#39;, &#39;CTP&#39;, &#39;GDP&#39;, &#39;CDP&#39;, &#39;dADP&#39;, 
                &#39;dCDP&#39;, &#39;dGDP&#39;, &#39;dUDP&#39;, &#39;dUTP&#39;, &#39;dUMP&#39;, &#39;dTMP&#39;, &#39;dTDP&#39;, &#39;GTP&#39;, 
                &#39;dATP&#39;, &#39;dCTP&#39;, &#39;dGTP&#39;, &#39;dTTP&#39;, &#39;NAD&#39;, &#39;NADH&#39;, &#39;NADP&#39;, &#39;NADPH&#39;]

## dd M/BS exchange reactions
L_r_transport = []
for m_c3_obj in c3_model.metabolites:
    if m_c3_obj.id[-1:] == &#39;c&#39; and m_c3_obj.id[:-2] not in no_transport:
        r_c4_obj = cobra.Reaction(&#39;[MB]_&#39;+m_c3_obj.id)
        r_c4_obj.name = &#39;[MB]_&#39;+m_c3_obj.id
        r_c4_obj.subsystem = &#39;Exchange&#39;
        r_c4_obj.bounds = (-inf, inf)
        c4_model.add_reaction(r_c4_obj)
        r_c4_obj.add_metabolites({&#39;[M]_&#39;+m_c3_obj.id: -1,&#39;[B]_&#39;+m_c3_obj.id: 1 })
        L_r_transport.append(&#39;[MB]_&#39;+m_c3_obj.id)
        
## Model Summary
c4_num_mets = len(c4_model.metabolites)
c4_num_rxn = len(c4_model.reactions)

df_c4_model_summary = pd.DataFrame([c4_num_mets, c4_num_rxn], index=[&#39;Number of metabolites&#39;,&#39;Number of reactions&#39;], columns=[&#39;Count&#39;])
df_c4_model_summary</code></pre>
          <figure slot="outputs">
            <pre class="language-text" itemscope="" itemtype="http://schema.stenci.la/CodeBlock"><code>Code Cell 17: Add Metabolite Exchange Reactions
</code></pre>
            <table itemscope="" itemtype="http://schema.org/Table">
              <thead>
                <tr itemscope="" itemtype="http://schema.stenci.la/TableRow">
                  <th itemscope="" itemtype="http://schema.stenci.la/TableCell"></th>
                  <th itemscope="" itemtype="http://schema.stenci.la/TableCell">Count</th>
                </tr>
              </thead>
              <tbody>
                <tr itemscope="" itemtype="http://schema.stenci.la/TableRow">
                  <td itemscope="" itemtype="http://schema.stenci.la/TableCell">Number of
                    metabolites</td>
                  <td itemscope="" itemtype="http://schema.stenci.la/TableCell"><span
                      data-itemtype="http://schema.org/Number">826</span></td>
                </tr>
                <tr itemscope="" itemtype="http://schema.stenci.la/TableRow">
                  <td itemscope="" itemtype="http://schema.stenci.la/TableCell">Number of reactions
                  </td>
                  <td itemscope="" itemtype="http://schema.stenci.la/TableCell"><span
                      data-itemtype="http://schema.org/Number">1188</span></td>
                </tr>
              </tbody>
            </table>
          </figure>
        </stencila-code-chunk>
        <p itemscope="" itemtype="http://schema.stenci.la/Paragraph">The mesophyll and bundle sheath
          networks are connected by a range of cytosolic transport metabolites including amino
          acids, sugars (glucose, fructose, sucrose, trehalose, ribose), single phosphorylated sugar
          (glucose-6-phosphate, glucose-1-phosphate, fructose-6-phosphate, sucrose-6-phosphate),
          mono-/di-/tri-carboxylic acids (phosphoenolpyruvate, pyruvate, citrate, cis-aconitate,
          isocitrate, <span itemscope="" itemtype="http://schema.stenci.la/MathFragment"><span
              class="mjx-chtml"><span class="mjx-math" aria-label="\alpha"><span class="mjx-mrow"
                  aria-hidden="true"><span class="mjx-mi"><span class="mjx-char MJXc-TeX-math-I"
                      style="padding-top: 0.225em; padding-bottom: 0.298em;">α</span></span></span></span></span></span>-ketoglutarate,
          succinate, fumarate, malate), glyceric acids (2-Phosphoglycerate, 3-Phosphoglycerate),
          glycolate, glycerate, glyceraldehyde-3-phosphate, di-hydroxyacetone-phosphate and CO<sub
            itemscope="" itemtype="http://schema.stenci.la/Subscript"><span
              data-itemtype="http://schema.org/Number">2</span></sub>. Nucleotides, NAD/NADH,
          NADP/NADPH, pyrophosphate, inorganic phosphate are not considered as transport
          metabolites. Oxaloacetate has been excluded as transport metabolite since concentrations
          of oxaloacetate are very low <em itemscope=""
            itemtype="http://schema.stenci.la/Emphasis">in vivo</em> and it is reasonably unstable
          in aqueous solutions. Other small molecules that can be imported by the bundle sheath from
          the environment, as well as protons and HCO<sub itemscope=""
            itemtype="http://schema.stenci.la/Subscript"><span
              data-itemtype="http://schema.org/Number">3</span></sub><sup itemscope=""
            itemtype="http://schema.stenci.la/Superscript">-</sup>, are not exchanged between the
          two cell types.</p>
        <stencila-code-chunk itemscope="" itemtype="http://schema.stenci.la/CodeChunk"
          data-execution_count="18" data-programminglanguage="python">
          <pre class="language-python" itemscope="" itemtype="http://schema.stenci.la/CodeBlock"
            slot="text"><code>print(f&quot;Code Cell 18: Add Constraints for CO2 Uptake in BS Cells and Rubisco Carboxylation : Oxygenation Ratio&quot;)


## CONSTRAINT: Add external CO2 species to bundle sheath
#(the original CO2 species is treated as internal CO2)
m_list_CO_Ex= [&#39;[B]_CO2_ex_c&#39;,&#39;[B]_CO2_ex_h&#39;]

for m_id in m_list_CO_Ex:
    m_obj = cobra.Metabolite(m_id)
    c4_model.add_metabolites(m_obj)

## CONSTRAINT: Copy reactions &#39;Tr_CO2h&#39;, &#39;RBC_h&#39; and replace internal CO2 with external CO2 in the copied reactions 
r_list_CO_Ex = [&#39;Tr_CO2h&#39;, &#39;RBC_h&#39;]

for r_id in r_list_CO_Ex:
    r_obj = c4_model.reactions.get_by_id(&#39;[B]_&#39;+r_id)
    r_obj_Ex = cobra.Reaction(r_obj.id+&#39;_Ex&#39;)
    r_obj_Ex.name = r_obj.id+&#39;_Ex&#39;
    r_obj_Ex.subsystem = r_obj.subsystem
    r_obj_Ex.bounds = r_obj.bounds
    c4_model.add_reaction(r_obj_Ex)
    r_obj_Ex.add_metabolites({m_obj.id if not m_obj.id[:-2] == &#39;[B]_CO2&#39; else &#39;[B]_CO2_ex&#39;+m_obj.id[-2:]: r_obj.get_coefficient(m_obj) 
                                  for m_obj in r_obj.metabolites})

## CONSTRAINT: CO2 exchange between mesophyll and bundle sheat
r_c4_obj = cobra.Reaction(&#39;[MB]_CO2_c&#39;)
r_c4_obj.name = &#39;[MB]_CO2_c&#39;
r_c4_obj.subsystem = &#39;Exchange&#39;
r_c4_obj.bounds = (-inf, inf)
c4_model.add_reaction(r_c4_obj)
r_c4_obj.add_metabolites({&#39;[M]_CO2_c&#39;: -1,&#39;[B]_CO2_ex_c&#39;: 1 })
L_r_transport.append(&#39;[MB]_CO2_c&#39;)

## CONSTRAINT: oxygenation : carboxylation = 1 : 3
r_c4_rbc_rbo = (3.0, 1.0)
const_c4_rbc_rbo_b = set_fixed_flux_ratio({&#39;[B]_RBC_h_Ex&#39;:r_c4_rbc_rbo[0],&#39;[B]_RBO_h&#39;:r_c4_rbc_rbo[1]}, &#39;const_c4_rbc_rbo_b&#39;, c4_model)
const_c4_rbc_rbo_m = set_fixed_flux_ratio({&#39;[M]_RBC_h&#39;:r_c4_rbc_rbo[0],&#39;[M]_RBO_h&#39;:r_c4_rbc_rbo[1]}, &#39;const_c4_rbc_rbo_m&#39;, c4_model)

#Model Summary
c4_num_mets = len(c4_model.metabolites)
c4_num_rxn = len(c4_model.reactions)

df_c4_model_summary = pd.DataFrame([c4_num_mets, c4_num_rxn], index=[&#39;Number of metabolites&#39;,&#39;Number of reactions&#39;], columns=[&#39;Count&#39;])
df_c4_model_summary</code></pre>
          <figure slot="outputs">
            <pre class="language-text" itemscope="" itemtype="http://schema.stenci.la/CodeBlock"><code>Code Cell 18: Add Constraints for CO2 Uptake in BS Cells and Rubisco Carboxylation : Oxygenation Ratio
</code></pre>
            <table itemscope="" itemtype="http://schema.org/Table">
              <thead>
                <tr itemscope="" itemtype="http://schema.stenci.la/TableRow">
                  <th itemscope="" itemtype="http://schema.stenci.la/TableCell"></th>
                  <th itemscope="" itemtype="http://schema.stenci.la/TableCell">Count</th>
                </tr>
              </thead>
              <tbody>
                <tr itemscope="" itemtype="http://schema.stenci.la/TableRow">
                  <td itemscope="" itemtype="http://schema.stenci.la/TableCell">Number of
                    metabolites</td>
                  <td itemscope="" itemtype="http://schema.stenci.la/TableCell"><span
                      data-itemtype="http://schema.org/Number">828</span></td>
                </tr>
                <tr itemscope="" itemtype="http://schema.stenci.la/TableRow">
                  <td itemscope="" itemtype="http://schema.stenci.la/TableCell">Number of reactions
                  </td>
                  <td itemscope="" itemtype="http://schema.stenci.la/TableCell"><span
                      data-itemtype="http://schema.org/Number">1191</span></td>
                </tr>
              </tbody>
            </table>
          </figure>
        </stencila-code-chunk>
        <p itemscope="" itemtype="http://schema.stenci.la/Paragraph">The ATP costs for cell
          maintenance in the <em itemscope="" itemtype="http://schema.stenci.la/Emphasis">genC3</em>
          model are assigned to both cell types in the <em itemscope=""
            itemtype="http://schema.stenci.la/Emphasis">two-cell</em> model. Due to declining CO<sub
            itemscope="" itemtype="http://schema.stenci.la/Subscript"><span
              data-itemtype="http://schema.org/Number">2</span></sub> concentrations over
          evolutionary time and/or adverse conditions which close the stromata, the oxygenation :
          carboxylation ratio of the native Rubisco population in the bundle sheath and the
          mesophyll is increased and can be predicted as <stencila-code-expression
            programming-language="py" itemscope=""
            itemtype="http://schema.stenci.la/CodeExpression"><code class="py"
              slot="text">r_c4_rbc_rbo[0]</code><output slot="output"></output>
          </stencila-code-expression> : <stencila-code-expression programming-language="py"
            itemscope="" itemtype="http://schema.stenci.la/CodeExpression"><code class="py"
              slot="text">r_c4_rbc_rbo[1]</code><output slot="output"></output>
          </stencila-code-expression>, the corresponding flux ratios are adapted accordingly.</p>
        <stencila-code-chunk itemscope="" itemtype="http://schema.stenci.la/CodeChunk"
          data-execution_count="19" data-programminglanguage="python">
          <pre class="language-python" itemscope="" itemtype="http://schema.stenci.la/CodeBlock"
            slot="text"><code>print(f&quot;Code Cell 19: Add Constraint on Photon Uptake&quot;)

## Reaction variables for light uptake
B_Im_hnu = c4_model.reactions.get_by_id(&quot;[B]_Im_hnu&quot;)
M_Im_hnu = c4_model.reactions.get_by_id(&quot;[M]_Im_hnu&quot;)

## CONSTRAINT: Total Photon uptake limited to 1000 µE
f_c4_hnu_ub = 1000 #[μE] 
f_c4_hnu_lb = 0 #[μE] 

const_hnu_sum = c4_model.problem.Constraint(
    B_Im_hnu.flux_expression + M_Im_hnu.flux_expression,
    lb = f_c4_hnu_lb,
    ub = f_c4_hnu_ub,
    name = &#39;const_hnu_sum&#39;,
)

c4_model.add_cons_vars(const_hnu_sum)

## CONSTRAINT: Total Photon uptake by bundle sheath must be less equal than in mesophyll
const_hnu_ratio = c4_model.problem.Constraint( 
    M_Im_hnu.flux_expression - B_Im_hnu.flux_expression, 
    lb = f_c4_hnu_lb, 
    ub = f_c4_hnu_ub, 
    name = &#39;const_hnu_ratio&#39;)

c4_model.add_cons_vars(const_hnu_ratio)</code></pre>
          <figure slot="outputs">
            <pre class="language-text" itemscope="" itemtype="http://schema.stenci.la/CodeBlock"><code>Code Cell 19: Add Constraint on Photon Uptake
</code></pre>
          </figure>
        </stencila-code-chunk>
        <p itemscope="" itemtype="http://schema.stenci.la/Paragraph">Furthermore, we assume that the
          total photon uptake in the mesophyll and bundle sheath is in the range of 
          <stencila-code-expression programming-language="py" itemscope=""
            itemtype="http://schema.stenci.la/CodeExpression"><code class="py"
              slot="text">f_c4_hnu_lb</code><output slot="output"></output>
          </stencila-code-expression> μmol/(m<sup itemscope=""
            itemtype="http://schema.stenci.la/Superscript"><span
              data-itemtype="http://schema.org/Number">2</span></sup>s) to <stencila-code-expression
            programming-language="py" itemscope=""
            itemtype="http://schema.stenci.la/CodeExpression"><code class="py"
              slot="text">f_c4_hnu_lb</code><output slot="output"></output>
          </stencila-code-expression> μmol/(m<sup itemscope=""
            itemtype="http://schema.stenci.la/Superscript"><span
              data-itemtype="http://schema.org/Number">2</span></sup>s). Since they are more central
          in the leaf, the photon uptake by the bundle sheath must be equal or less compared to the
          mesophyll.</p>
        <h4 itemscope="" itemtype="http://schema.stenci.la/Heading" id="objective-1">Objective</h4>
        <stencila-code-chunk itemscope="" itemtype="http://schema.stenci.la/CodeChunk"
          data-execution_count="20" data-programminglanguage="python">
          <pre class="language-python" itemscope="" itemtype="http://schema.stenci.la/CodeBlock"
            slot="text"><code>print(f&quot;Code Cell 20: Add Objective to Optimize Sucrose Output&quot;)

## Optimize/Maximize sucrose output
r_opt_id = &quot;[B]_Ex_Suc&quot;
r_opt_obj = c4_model.reactions.get_by_id(r_opt_id)
r_opt_obj.objective_coefficient = 1.</code></pre>
          <figure slot="outputs">
            <pre class="language-text" itemscope="" itemtype="http://schema.stenci.la/CodeBlock"><code>Code Cell 20: Add Objective to Optimize Sucrose Output
</code></pre>
          </figure>
        </stencila-code-chunk>
        <p itemscope="" itemtype="http://schema.stenci.la/Paragraph">The maximisation of the phloem
          sap output through the bundle sheath and the minimisation of the metabolic effort are kept
          as objectives in the <em itemscope=""
            itemtype="http://schema.stenci.la/Emphasis">two-cell</em> model.</p>
        <h2 itemscope="" itemtype="http://schema.stenci.la/Heading" id="results">Results</h2>
        <h3 itemscope="" itemtype="http://schema.stenci.la/Heading"
          id="the-curated-arabidopsis-core-model-predicts-physiological-results">The curated <em
            itemscope="" itemtype="http://schema.stenci.la/Emphasis">Arabidopsis</em> core model
          predicts physiological results</h3>
        <p itemscope="" itemtype="http://schema.stenci.la/Paragraph">Flux balance analysis requires
          five types of information, the metabolic map of the organism, the input, the output, a set
          of constraints (i.e. limitations on input, directionality of reactions, forced flux
          through reactions), and optimisation criteria for the algorithm which approximate the
          selective pressures the metabolism evolved under. In this context, inputs define the
          resources that need to be taken up by the metabolic network to fulfil a particular
          metabolic function, which is related to the outputs, for example the synthesis of
          metabolites part of the biomass or other specific products. In CBM, the objective is most
          likely related to the in- and/or outputs.</p>
        <p itemscope="" itemtype="http://schema.stenci.la/Paragraph">For reconstruction of the C3
          metabolic map we curated the <em itemscope=""
            itemtype="http://schema.stenci.la/Emphasis">Arabidopsis</em> core model <cite
            itemscope="" itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a
              href="#bib2"><span>2</span><span>Arnold and
                Nikoloski</span><span>2014</span></a></cite> manually (<a href="#table1"
            itemscope="" itemtype="http://schema.stenci.la/Link">Table 1</a>) to represent the
          metabolism of a mesophyll cell in a mature photosynthetically active leaf of a C3 plant ,
          further on called <em itemscope=""
            itemtype="http://schema.stenci.la/Emphasis">one-cell</em> model (provided in <a
            href="#fig1sdata1" itemscope="" itemtype="http://schema.stenci.la/Link">Figure 1—source
            data 1</a>). The <em itemscope=""
            itemtype="http://schema.stenci.la/Emphasis">Arabidopsis</em> core model is a
          bottom-up-assembled, large-scale model relying solely on <em itemscope=""
            itemtype="http://schema.stenci.la/Emphasis">Arabidopsis</em>-specific annotations and
          the inclusion of only manually curated reactions of the primary metabolism. The <em
            itemscope="" itemtype="http://schema.stenci.la/Emphasis">Arabidopsis</em> core model is
          accurate with respect to mass and energy conservation, allowing optimal nutrient
          utilisation and biochemically sound predictions <cite itemscope=""
            itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a
              href="#bib2"><span>2</span><span>Arnold and
                Nikoloski</span><span>2014</span></a></cite>.</p>
        <table id="table1" itemscope="" itemtype="http://schema.org/Table">
          <caption><label data-itemprop="label">Table 1.</label>
            <div itemprop="caption">
              <h4 itemscope="" itemtype="http://schema.stenci.la/Heading"
                id="curation-of-the-arabidopsis-core-model-from-narrative-bib2">Curation of the <em
                  itemscope="" itemtype="http://schema.stenci.la/Emphasis">Arabidopsis</em> core
                model from <cite itemscope="" itemtype="http://schema.stenci.la/Cite"
                  data-citationmode="Narrative"><a href="#bib2"><span>2</span><span>Arnold and
                      Nikoloski</span><span>2014</span></a></cite>.</h4>
            </div>
          </caption>
          <thead>
            <tr itemscope="" itemtype="http://schema.stenci.la/TableRow">
              <th itemscope="" itemtype="http://schema.stenci.la/TableCell"><em itemscope=""
                  itemtype="http://schema.stenci.la/Emphasis">Arabidopsis core model</em></th>
              <th itemscope="" itemtype="http://schema.stenci.la/TableCell">Observation</th>
              <th itemscope="" itemtype="http://schema.stenci.la/TableCell"><em itemscope=""
                  itemtype="http://schema.stenci.la/Emphasis">one-cell model</em></th>
              <th itemscope="" itemtype="http://schema.stenci.la/TableCell">Reference</th>
            </tr>
          </thead>
          <tbody>
            <tr itemscope="" itemtype="http://schema.stenci.la/TableRow">
              <td itemscope="" itemtype="http://schema.stenci.la/TableCell">NADP-dependent malate
                dehydrogenases in all compartments</td>
              <td itemscope="" itemtype="http://schema.stenci.la/TableCell">cycles through nitrate
                reductase to interconvert NAD and NADP</td>
              <td itemscope="" itemtype="http://schema.stenci.la/TableCell">NAD-dependent malate
                dehydrogenases in all compartments, NADP-dependent malate dehydrogenase only in
                chloroplast</td>
              <td itemscope="" itemtype="http://schema.stenci.la/TableCell">(<cite itemscope=""
                  itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a
                    href="#bib76"><span>76</span><span>Swarbreck et
                      al.</span><span>2008</span></a></cite>)</td>
            </tr>
            <tr itemscope="" itemtype="http://schema.stenci.la/TableRow">
              <td itemscope="" itemtype="http://schema.stenci.la/TableCell">Cyclic electron flow
              </td>
              <td itemscope="" itemtype="http://schema.stenci.la/TableCell">absence of cyclic
                electron flow</td>
              <td itemscope="" itemtype="http://schema.stenci.la/TableCell">added</td>
              <td itemscope="" itemtype="http://schema.stenci.la/TableCell">(<cite itemscope=""
                  itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a
                    href="#bib70"><span>70</span><span>Shikanai</span><span>2016</span></a></cite>)
              </td>
            </tr>
            <tr itemscope="" itemtype="http://schema.stenci.la/TableRow">
              <td itemscope="" itemtype="http://schema.stenci.la/TableCell">Alternative oxidase</td>
              <td itemscope="" itemtype="http://schema.stenci.la/TableCell">missing alternative
                routes for electrons to pass the electron transport chain to reduce oxygen</td>
              <td itemscope="" itemtype="http://schema.stenci.la/TableCell">added alternative
                oxidase reactions to the chloroplast and mitochondria</td>
              <td itemscope="" itemtype="http://schema.stenci.la/TableCell">(<cite itemscope=""
                  itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a
                    href="#bib81"><span>81</span><span>Vishwakarma et
                      al.</span><span>2015</span></a></cite>)</td>
            </tr>
            <tr itemscope="" itemtype="http://schema.stenci.la/TableRow">
              <td itemscope="" itemtype="http://schema.stenci.la/TableCell">Alanine transferase</td>
              <td itemscope="" itemtype="http://schema.stenci.la/TableCell">No alanine transferase
                in cytosol Alanine transferase</td>
              <td itemscope="" itemtype="http://schema.stenci.la/TableCell">added</td>
              <td itemscope="" itemtype="http://schema.stenci.la/TableCell">(<cite itemscope=""
                  itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a
                    href="#bib41"><span>41</span><span>Liepman and
                      Olsen</span><span>2003</span></a></cite>)</td>
            </tr>
            <tr itemscope="" itemtype="http://schema.stenci.la/TableRow">
              <td itemscope="" itemtype="http://schema.stenci.la/TableCell">Transport chloroplast
              </td>
              <td itemscope="" itemtype="http://schema.stenci.la/TableCell">no maltose transporter
                by MEX1</td>
              <td itemscope="" itemtype="http://schema.stenci.la/TableCell">added</td>
              <td itemscope="" itemtype="http://schema.stenci.la/TableCell">(<cite itemscope=""
                  itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a
                    href="#bib42"><span>42</span><span>Linka and
                      Weber</span><span>2010</span></a></cite>)</td>
            </tr>
            <tr itemscope="" itemtype="http://schema.stenci.la/TableRow">
              <td itemscope="" itemtype="http://schema.stenci.la/TableCell"></td>
              <td itemscope="" itemtype="http://schema.stenci.la/TableCell">no glucose transporter
                by MEX1 and pGlcT MEX1</td>
              <td itemscope="" itemtype="http://schema.stenci.la/TableCell">added</td>
              <td itemscope="" itemtype="http://schema.stenci.la/TableCell"></td>
            </tr>
            <tr itemscope="" itemtype="http://schema.stenci.la/TableRow">
              <td itemscope="" itemtype="http://schema.stenci.la/TableCell"></td>
              <td itemscope="" itemtype="http://schema.stenci.la/TableCell">no unidirectional
                transport of ATP, ADP, AMP by BT-like</td>
              <td itemscope="" itemtype="http://schema.stenci.la/TableCell">added</td>
              <td itemscope="" itemtype="http://schema.stenci.la/TableCell"></td>
            </tr>
            <tr itemscope="" itemtype="http://schema.stenci.la/TableRow">
              <td itemscope="" itemtype="http://schema.stenci.la/TableCell"></td>
              <td itemscope="" itemtype="http://schema.stenci.la/TableCell">no Mal/OAA, Mal/Pyr, and
                Mal/Glu exchange by DiTs</td>
              <td itemscope="" itemtype="http://schema.stenci.la/TableCell">added</td>
              <td itemscope="" itemtype="http://schema.stenci.la/TableCell"></td>
            </tr>
            <tr itemscope="" itemtype="http://schema.stenci.la/TableRow">
              <td itemscope="" itemtype="http://schema.stenci.la/TableCell"></td>
              <td itemscope="" itemtype="http://schema.stenci.la/TableCell">no folate transporter by
                FBT and FOLT1</td>
              <td itemscope="" itemtype="http://schema.stenci.la/TableCell">added</td>
              <td itemscope="" itemtype="http://schema.stenci.la/TableCell"></td>
            </tr>
            <tr itemscope="" itemtype="http://schema.stenci.la/TableRow">
              <td itemscope="" itemtype="http://schema.stenci.la/TableCell">Transport Mitochondria
              </td>
              <td itemscope="" itemtype="http://schema.stenci.la/TableCell">no Mal/OAA, Cit/iCit,
                Mal/KG exchange by DTC</td>
              <td itemscope="" itemtype="http://schema.stenci.la/TableCell">added</td>
              <td itemscope="" itemtype="http://schema.stenci.la/TableCell">(<cite itemscope=""
                  itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a
                    href="#bib42"><span>42</span><span>Linka and
                      Weber</span><span>2010</span></a></cite>)</td>
            </tr>
            <tr itemscope="" itemtype="http://schema.stenci.la/TableRow">
              <td itemscope="" itemtype="http://schema.stenci.la/TableCell"></td>
              <td itemscope="" itemtype="http://schema.stenci.la/TableCell">no H+ importer by UCPs
                import</td>
              <td itemscope="" itemtype="http://schema.stenci.la/TableCell">added</td>
              <td itemscope="" itemtype="http://schema.stenci.la/TableCell"></td>
            </tr>
            <tr itemscope="" itemtype="http://schema.stenci.la/TableRow">
              <td itemscope="" itemtype="http://schema.stenci.la/TableCell"></td>
              <td itemscope="" itemtype="http://schema.stenci.la/TableCell">no OAA/Pi exchange by
                DIC1-3</td>
              <td itemscope="" itemtype="http://schema.stenci.la/TableCell">added</td>
              <td itemscope="" itemtype="http://schema.stenci.la/TableCell"></td>
            </tr>
            <tr itemscope="" itemtype="http://schema.stenci.la/TableRow">
              <td itemscope="" itemtype="http://schema.stenci.la/TableCell"></td>
              <td itemscope="" itemtype="http://schema.stenci.la/TableCell">no ATP/Pi exchange by
                APCs</td>
              <td itemscope="" itemtype="http://schema.stenci.la/TableCell">added</td>
              <td itemscope="" itemtype="http://schema.stenci.la/TableCell"></td>
            </tr>
            <tr itemscope="" itemtype="http://schema.stenci.la/TableRow">
              <td itemscope="" itemtype="http://schema.stenci.la/TableCell"></td>
              <td itemscope="" itemtype="http://schema.stenci.la/TableCell">no NAD/ADP and NAD/AMP
                exchange by NDT2</td>
              <td itemscope="" itemtype="http://schema.stenci.la/TableCell">added</td>
              <td itemscope="" itemtype="http://schema.stenci.la/TableCell"></td>
            </tr>
            <tr itemscope="" itemtype="http://schema.stenci.la/TableRow">
              <td itemscope="" itemtype="http://schema.stenci.la/TableCell"></td>
              <td itemscope="" itemtype="http://schema.stenci.la/TableCell">no ThPP/ATP exchange by
                TPCs</td>
              <td itemscope="" itemtype="http://schema.stenci.la/TableCell">added</td>
              <td itemscope="" itemtype="http://schema.stenci.la/TableCell"></td>
            </tr>
            <tr itemscope="" itemtype="http://schema.stenci.la/TableRow">
              <td itemscope="" itemtype="http://schema.stenci.la/TableCell"></td>
              <td itemscope="" itemtype="http://schema.stenci.la/TableCell">no Asp/Glu by AGCs</td>
              <td itemscope="" itemtype="http://schema.stenci.la/TableCell">added</td>
              <td itemscope="" itemtype="http://schema.stenci.la/TableCell"></td>
            </tr>
            <tr itemscope="" itemtype="http://schema.stenci.la/TableRow">
              <td itemscope="" itemtype="http://schema.stenci.la/TableCell"></td>
              <td itemscope="" itemtype="http://schema.stenci.la/TableCell">no uncoupled Ala
                exchange</td>
              <td itemscope="" itemtype="http://schema.stenci.la/TableCell">added</td>
              <td itemscope="" itemtype="http://schema.stenci.la/TableCell"></td>
            </tr>
            <tr itemscope="" itemtype="http://schema.stenci.la/TableRow">
              <td itemscope="" itemtype="http://schema.stenci.la/TableCell">Transport peroxisome
              </td>
              <td itemscope="" itemtype="http://schema.stenci.la/TableCell">missing NAD/NADH,
                NAD/ADP, NAD/AMP exchange by PXN</td>
              <td itemscope="" itemtype="http://schema.stenci.la/TableCell">added</td>
              <td itemscope="" itemtype="http://schema.stenci.la/TableCell">(<cite itemscope=""
                  itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a
                    href="#bib42"><span>42</span><span>Linka and
                      Weber</span><span>2010</span></a></cite>)</td>
            </tr>
            <tr itemscope="" itemtype="http://schema.stenci.la/TableRow">
              <td itemscope="" itemtype="http://schema.stenci.la/TableCell"></td>
              <td itemscope="" itemtype="http://schema.stenci.la/TableCell">no ATP/ADP and ATP/AMP
                exchange by PNCs</td>
              <td itemscope="" itemtype="http://schema.stenci.la/TableCell">added</td>
              <td itemscope="" itemtype="http://schema.stenci.la/TableCell"></td>
            </tr>
            <tr itemscope="" itemtype="http://schema.stenci.la/TableRow">
              <td itemscope="" itemtype="http://schema.stenci.la/TableCell">H<sup itemscope=""
                  itemtype="http://schema.stenci.la/Superscript">+</sup> sinks/sources</td>
              <td itemscope="" itemtype="http://schema.stenci.la/TableCell">H<sup itemscope=""
                  itemtype="http://schema.stenci.la/Superscript">+</sup> sinks/source reaction for
                the cytosol and futile transport cycles introduced by H<sup itemscope=""
                  itemtype="http://schema.stenci.la/Superscript">+</sup> -coupled transport
                reactions</td>
              <td itemscope="" itemtype="http://schema.stenci.la/TableCell">H<sup itemscope=""
                  itemtype="http://schema.stenci.la/Superscript">+</sup> sinks/source reaction added
                for each compartment</td>
              <td itemscope="" itemtype="http://schema.stenci.la/TableCell"></td>
            </tr>
            <tr itemscope="" itemtype="http://schema.stenci.la/TableRow">
              <td itemscope="" itemtype="http://schema.stenci.la/TableCell">ATPase stoichiometry
              </td>
              <td itemscope="" itemtype="http://schema.stenci.la/TableCell">False H<sup itemscope=""
                  itemtype="http://schema.stenci.la/Superscript">+</sup>/ATP ratios for the
                plastidal and mitochondrial ATP synthase</td>
              <td itemscope="" itemtype="http://schema.stenci.la/TableCell">H<sup itemscope=""
                  itemtype="http://schema.stenci.la/Superscript">+</sup>/ATP ratio set to 3 : 1
                (chloroplast) and 4:1 (mitochondria)</td>
              <td itemscope="" itemtype="http://schema.stenci.la/TableCell">(<cite itemscope=""
                  itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a
                    href="#bib56"><span>56</span><span>Petersen et
                      al.</span><span>2012</span></a></cite>; <cite itemscope=""
                  itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a
                    href="#bib79"><span>79</span><span>Turina et
                      al.</span><span>2016</span></a></cite>)</td>
            </tr>
            <tr itemscope="" itemtype="http://schema.stenci.la/TableRow">
              <td itemscope="" itemtype="http://schema.stenci.la/TableCell">Alanine/aspartate
                transferase</td>
              <td itemscope="" itemtype="http://schema.stenci.la/TableCell">no direct conversion of
                alanine and aspartate</td>
              <td itemscope="" itemtype="http://schema.stenci.la/TableCell">added to cytosol,
                chloroplast and mitochondria</td>
              <td itemscope="" itemtype="http://schema.stenci.la/TableCell">(<cite itemscope=""
                  itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a
                    href="#bib69"><span>69</span><span>Schultz and
                      Coruzzi</span><span>1995</span></a></cite>; <cite itemscope=""
                  itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a
                    href="#bib22"><span>22</span><span>Duff et
                      al.</span><span>2012</span></a></cite>)</td>
            </tr>
          </tbody>
        </table>
        <p itemscope="" itemtype="http://schema.stenci.la/Paragraph">For the inputs, we considered a
          photoautotrophic growth scenario with a fixed CO<sub itemscope=""
            itemtype="http://schema.stenci.la/Subscript"><span
              data-itemtype="http://schema.org/Number">2</span></sub> uptake of about
          <stencila-code-expression programming-language="py" itemscope=""
            itemtype="http://schema.stenci.la/CodeExpression"><code class="py"
              slot="text">f_c3_CO2</code><output slot="output"></output></stencila-code-expression>
           μmol/(m<sup itemscope="" itemtype="http://schema.stenci.la/Superscript"><span
              data-itemtype="http://schema.org/Number">2</span></sup>s) <cite itemscope=""
            itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a
              href="#bib37"><span>37</span><span>Lacher</span><span>2003</span></a></cite>. Light,
          sulphates, and phosphate are freely available. Due to the observation that nitrate is the
          main source (80%) of nitrogen in leaves in many species <cite itemscope=""
            itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a
              href="#bib45"><span>45</span><span>Macduff and
                Bakken</span><span>2003</span></a></cite>, we set nitrate as the sole nitrogen
          source. If both ammonia and nitrate are allowed, the model will inevitably predict the
          physiologically incorrect sole use of ammonia since fewer reactions and less energy are
          required to convert it into glutamate, the universal amino group currency in plants. Water
          and oxygen can be freely exchanged with the environment in both directions.</p>
        <p itemscope="" itemtype="http://schema.stenci.la/Paragraph">To compute the output, we
          assume a mature fully differentiated and photosynthetically active leaf, which is
          optimised for the synthesis and export of sucrose and amino acids to the phloem under
          minimal metabolic effort. Following the examples of models in bacteria, many plant models
          use a biomass function which assumes that the leaf is required to build itself <span
            itemscope="" itemtype="http://schema.stenci.la/CiteGroup"><cite itemscope=""
              itemtype="http://schema.stenci.la/Cite"><a href="#bib19"><span>19</span><span>de
                  Oliveira Dal'Molin et al.</span><span>2010</span></a></cite><cite itemscope=""
              itemtype="http://schema.stenci.la/Cite"><a href="#bib2"><span>2</span><span>Arnold and
                  Nikoloski</span><span>2014</span></a></cite><cite itemscope=""
              itemtype="http://schema.stenci.la/Cite"><a href="#bib64"><span>64</span><span>Saha et
                  al.</span><span>2011</span></a></cite></span> using photoautotrophic that is <cite
            itemscope="" itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a
              href="#bib2"><span>2</span><span>Arnold and
                Nikoloski</span><span>2014</span></a></cite> or heterotrophic that is <cite
            itemscope="" itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a
              href="#bib17"><span>17</span><span>Cheung et al.</span><span>2014</span></a></cite>
          energy and molecule supply. In plants, however, leaves transition from a sink phase in
          which they build themselves from metabolites delivered by the phloem to a source phase in
          which they produce metabolites for other organs including sink leaves <cite itemscope=""
            itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a
              href="#bib78"><span>78</span><span>Turgeon</span><span>1989</span></a></cite>. The
          composition of <em itemscope=""
            itemtype="http://schema.stenci.la/Emphasis">Arabidopsis</em> phloem exudate <cite
            itemscope="" itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a
              href="#bib85"><span>85</span><span>Wilkinson and
                Douglas</span><span>2003</span></a></cite> was used to constrain the relative
          proportions of the 18 amino acids and the ratio of sucrose : total amino acids (r<em
            itemscope="" itemtype="http://schema.stenci.la/Emphasis">suc_aa<a href="" itemscope=""
              itemtype="http://schema.stenci.la/Link"><span
                data-itemtype="http://schema.org/Number">0</span></a> : r_suc_aa<a href=""
              itemscope="" itemtype="http://schema.stenci.la/Link"><span
                data-itemtype="http://schema.org/Number">1</span></a>). To account for daily carbon
            storage as starch for export during the night, we assume that half of the assimilated
            carbon is stored in the _one-cell</em> model. We explicitly account for maintenance
          costs by the use of a generic ATPase and use the measured ATP costs for protein
          degradation and synthesis of a mature <em itemscope=""
            itemtype="http://schema.stenci.la/Emphasis">Arabidopsis</em> leaf <cite itemscope=""
            itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a
              href="#bib40"><span>40</span><span>Li et al.</span><span>2017</span></a></cite> as a
          constraint. We initially assume a low photorespiratory flux according to the ambient
          CO<sub itemscope="" itemtype="http://schema.stenci.la/Subscript"><span
              data-itemtype="http://schema.org/Number">2</span></sub> and O<sub itemscope=""
            itemtype="http://schema.stenci.la/Subscript"><span
              data-itemtype="http://schema.org/Number">2</span></sub> partial pressures considering
          no heat, drought, salt or osmotic stress which may alter the ratio towards higher flux
          towards the oxygenation reaction.</p>
        <p itemscope="" itemtype="http://schema.stenci.la/Paragraph">To develop a largely
          unconstrained model and detect possible errors in the metabolic map, we initially kept the
          model unconstrained with regard to fixed fluxes, flux ratios, and reaction directions.
          Different model iterations were run in (re-)design, simulate, validate cycles against
          known physiology with errors sequentially eliminated and a minimal set of constraints
          required for a C3 model recapitulating extant plant metabolism determined. After each
          change, the CBM predicted all fluxes which were output as a table and manually examined
          (for example see <a href="#fig1sdata2" itemscope=""
            itemtype="http://schema.stenci.la/Link">Figure 1—source data 2</a>).</p>
        <p itemscope="" itemtype="http://schema.stenci.la/Paragraph">The initial FBA resulted in
          carbon fixation by enzymes such as the malic enzymes which, in reality, are constrained by
          the kinetics of the enzymes towards decarboxylation. All decarboxylation reactions were
          made unidirectional towards decarboxylation to prevent erroneous carbon fixation in the
          flux distribution. The next iteration of FBA predicted loops through nitrate reductases
          which ultimately converted NADH to NADPH. We traced this loop to an error in the initial
          model, in which malate dehydrogenases in the cytosol and mitochondrion were NADP-dependent
          instead of NAD-dependent. After correction of the co-factor in the <em itemscope=""
            itemtype="http://schema.stenci.la/Emphasis">one-cell</em> model, the loops through
          nitrate reductases were no longer observed. Another iteration predicted excessive flux
          through the mitochondrial membrane where multiple metabolites were exchanged and
          identified missing transport processes as the likely reason. Based on <cite itemscope=""
            itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a
              href="#bib42"><span>42</span><span>Linka and Weber</span><span>2010</span></a></cite>,
          we added known fluxes across the mitochondrial and plastidic envelope membranes which
          remedied the excessive fluxes in the solution. The chloroplastic ADP/ATP carrier protein
          is constrained to zero flux since its mutant is only affected during the night but not if
          light is available <cite itemscope="" itemtype="http://schema.stenci.la/Cite"
            data-citationmode="Narrative"><a href="#bib59"><span>59</span><span>Reiser et
                al.</span><span>2004</span></a></cite>.</p>
        <p itemscope="" itemtype="http://schema.stenci.la/Paragraph">The obtained flux distribution
          still contained excessive fluxes through multiple transport proteins across internal
          membranes which ultimately transferred protons between the organelles and the cytosol.
          Since for most if not all transport proteins the precise protonation state of metabolites
          during transport is unknown and hence cannot be correctly integrated into the model, we
          allowed protons to appear and disappear as needed in all compartments. This provision
          precludes conclusions about the energetics of membrane transport. ATP generation occurred
          in a distorted way distributed across different organelles which were traced to the H<sup
            itemscope="" itemtype="http://schema.stenci.la/Superscript">+</sup> consumption of the
          ATPases in mitochondria and chloroplasts. The stoichiometry was altered to to 3:1
          (chloroplast) and 4:1 (mitochondria) <span itemscope=""
            itemtype="http://schema.stenci.la/CiteGroup"><cite itemscope=""
              itemtype="http://schema.stenci.la/Cite"><a href="#bib56"><span>56</span><span>Petersen
                  et al.</span><span>2012</span></a></cite><cite itemscope=""
              itemtype="http://schema.stenci.la/Cite"><a href="#bib79"><span>79</span><span>Turina
                  et al.</span><span>2016</span></a></cite></span>. We assume no flux for the
          chloroplastic NADPH dehydrogenase and plastoquinol oxidase because <span itemscope=""
            itemtype="http://schema.stenci.la/CiteGroup"><cite itemscope=""
              itemtype="http://schema.stenci.la/Cite"><a href="#bib33"><span>33</span><span>Josse et
                  al.</span><span>2000</span></a></cite><cite itemscope=""
              itemtype="http://schema.stenci.la/Cite"><a href="#bib88"><span>88</span><span>Yamamoto
                  et al.</span><span>2011</span></a></cite></span> have shown that their effect on
          photosynthesis is minor.</p>
        <p itemscope="" itemtype="http://schema.stenci.la/Paragraph">In preparation for modelling
          the C4 cycle, we ensured that all reactions known to occur in C4 (i.e. malate/pyruvate
          exchange, likely via DiT2 in maize <cite itemscope=""
            itemtype="http://schema.stenci.la/Cite"><a href="#bib84"><span>84</span><span>Weissmann
                et al.</span><span>2016</span></a></cite>, possibly promiscuous amino transferases
          <cite itemscope="" itemtype="http://schema.stenci.la/Cite"><a
              href="#bib22"><span>22</span><span>Duff et al.</span><span>2012</span></a></cite>) are
          present in the <em itemscope="" itemtype="http://schema.stenci.la/Emphasis">one-cell</em>
          model, since <cite itemscope="" itemtype="http://schema.stenci.la/Cite"
            data-citationmode="Narrative"><a href="#bib4"><span>4</span><span>Aubry et
                al.</span><span>2011</span></a></cite> showed that all genes encoding enzymes and
          transporters underlying the C4 metabolism are already present in the genome of C3 plants.
          We integrated cyclic electron flow <cite itemscope=""
            itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a
              href="#bib70"><span>70</span><span>Shikanai</span><span>2016</span></a></cite> and
          alternative oxidases in the mitochondria <cite itemscope=""
            itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a
              href="#bib81"><span>81</span><span>Vishwakarma et
                al.</span><span>2015</span></a></cite>, since both have been hypothesised to be
          important during the evolution and/or execution of the C4 cycle. Models and analysis
          workflows provided as jupyter notebooks <cite itemscope=""
            itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a
              href="#bib77"><span>77</span><span>Thomas et al.</span><span>2016</span></a></cite>
          are available as supplementary material or can be accessed on GitHub <a
            href="https://github.com/ma-blaetke/CBM_C3_C4_Metabolism" itemscope=""
            itemtype="http://schema.stenci.la/Link">https://github.com/ma-blaetke/CBM_C3_C4_Metabolism</a> (<cite
            itemscope="" itemtype="http://schema.stenci.la/Cite"><a
              href="#bib10"><span>10</span><span>Blätke</span><span>2019</span></a></cite>; copy
          archived at <a href="https://github.com/elifesciences-publications/CBM_C3_C4_Metabolism"
            itemscope=""
            itemtype="http://schema.stenci.la/Link">https://github.com/elifesciences-publications/CBM_C3_C4_Metabolism</a>).
        </p>
        <p itemscope="" itemtype="http://schema.stenci.la/Paragraph">The <em itemscope=""
            itemtype="http://schema.stenci.la/Emphasis">one-cell</em> model comprises in total
          <stencila-code-expression programming-language="py" itemscope=""
            itemtype="http://schema.stenci.la/CodeExpression"><code class="py"
              slot="text">c3_num_mets</code><output slot="output"></output>
          </stencila-code-expression> metabolites and <stencila-code-expression
            programming-language="py" itemscope=""
            itemtype="http://schema.stenci.la/CodeExpression"><code class="py"
              slot="text">c3_num_rxn</code><output slot="output"></output>
          </stencila-code-expression> reactions, whereof <stencila-code-expression
            programming-language="py" itemscope=""
            itemtype="http://schema.stenci.la/CodeExpression"><code class="py"
              slot="text">c3_num_transport_rxn</code><output slot="output"></output>
          </stencila-code-expression> are internal transporters, <stencila-code-expression
            programming-language="py" itemscope=""
            itemtype="http://schema.stenci.la/CodeExpression"><code class="py"
              slot="text">c3_num_export_rxn</code><output slot="output"></output>
          </stencila-code-expression> are export and <stencila-code-expression
            programming-language="py" itemscope=""
            itemtype="http://schema.stenci.la/CodeExpression"><code class="py"
              slot="text">c3_num_import_rxn</code><output slot="output"></output>
          </stencila-code-expression> import reactions (see also below), which are involved in 59
          subsystems. <a href="#fig1" itemscope="" itemtype="http://schema.stenci.la/Link">Figure
            1</a> provides an overview of the primary subsystems according to <cite itemscope=""
            itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a
              href="#bib2"><span>2</span><span>Arnold and
                Nikoloski</span><span>2014</span></a></cite>.</p>
        <figure itemscope="" itemtype="http://schema.stenci.la/Figure" id="fig1" title="Figure 1.">
          <label data-itemprop="label">Figure 1.</label><img src="index.html.media/fig1.jpg" alt=""
            itemscope="" itemtype="http://schema.org/ImageObject">
          <figcaption>
            <h4 itemscope="" itemtype="http://schema.stenci.la/Heading"
              id="schematic-representation-of-the-primary-subsystems-in-the-one-cell-model-and-the-used-inputoutput-constraints-adapted-from-narrative-bib2">
              Schematic representation of the primary subsystems in the <em itemscope=""
                itemtype="http://schema.stenci.la/Emphasis">one-cell</em> model and the used
              input/output constraints; adapted from <cite itemscope=""
                itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a
                  href="#bib2"><span>2</span><span>Arnold and
                    Nikoloski</span><span>2014</span></a></cite>.</h4>
          </figcaption>
        </figure>
        <stencila-code-chunk itemscope="" itemtype="http://schema.stenci.la/CodeChunk"
          data-execution_count="21" data-programminglanguage="python">
          <pre class="language-python" itemscope="" itemtype="http://schema.stenci.la/CodeBlock"
            slot="text"><code>######################################################################################################################################
######################################################################################################################################
##############                                        EXPERIMENT 1: Effect of CO2 Uptake                                ##############
######################################################################################################################################
######################################################################################################################################
print(f&quot;Code Cell 21: Experiment 1 -- Effect of CO2 Uptake&quot;)

#Create copy of c3 model
c3_model_exp1 = c3_model.copy()

#Optimize/Maximize sucrose output
result_exp1_1_fba = c3_model_exp1.optimize(&#39;maximize&#39;) #perform FBA

#Optimize/Minimize total flux
if result_exp1_1_fba.status == &#39;optimal&#39;: 
    result_exp1_1_pfba = cobra.flux_analysis.parsimonious.pfba(c3_model_exp1)
    
#Fetch flux for CO2 uptake
v_co2_exp1 = result_exp1_1_pfba.fluxes[&#39;Im_CO2&#39;]

#Array defining proprtion of CO2 uptake 
co2_ratios_exp1 = np.linspace(0,1,21)

df_result_exp1 = pd.DataFrame()
#Iterate over proportions of CO2 uptake
for co2_ratio in tqdm(co2_ratios_exp1):
    
    #Fix upper flux bound for photon uptake
    set_bounds(&#39;Im_CO2&#39;, (0, v_co2_exp1 * co2_ratio), c3_model_exp1)
    
    #Optimize/Maximize sucrose output
    result_exp1_2_fba = c3_model_exp1.optimize(&#39;maximize&#39;) #perform FBA
    
    #Optimize/Minimize total flux
    if result_exp1_2_fba.status == &#39;optimal&#39;: # check if feasible
        result_exp1_2_pfba  = cobra.flux_analysis.parsimonious.pfba(c3_model_exp1) #perform pFBA
        if result_exp1_2_pfba.status == &#39;optimal&#39;:
            df_result_exp1[v_co2_exp1 * co2_ratio] = result_exp1_2_pfba.fluxes</code></pre>
          <figure slot="outputs">
            <pre class="language-text" itemscope="" itemtype="http://schema.stenci.la/CodeBlock"><code>Code Cell 21: Experiment 1 -- Effect of CO2 Uptake
</code></pre>
            <pre class="language-text" itemscope=""
              itemtype="http://schema.stenci.la/CodeBlock"><code>  0%|          | 0/21 [00:00&lt;?, ?it/s]</code></pre>
          </figure>
        </stencila-code-chunk>
        <figure itemscope="" itemtype="http://schema.stenci.la/Figure" id="fig1s1"
          title="Figure 1—figure supplement 1"><label data-itemprop="label">Figure 1—figure
            supplement 1</label>
          <stencila-code-chunk itemscope="" itemtype="http://schema.stenci.la/CodeChunk"
            data-execution_count="22" data-programminglanguage="python">
            <pre class="language-python" itemscope="" itemtype="http://schema.stenci.la/CodeBlock"
              slot="text"><code>######################################################################
##############        Figure 1—figure supplement 1      ##############
######################################################################

#{
#  &quot;caption&quot;: &quot;#### Effect of CO~2. Dependence of the phloem output on CO~2~ input flux in the range 0 μmol/(m^2^s)–20 μmol/(m^2^s). Sucrose and starch are produced in the same amounts, each of them consists of 12 C-atoms.&quot;,
#  &quot;id&quot;: &quot;fig1s1&quot;,
#  &quot;label&quot;: &quot;Figure 1—figure supplement 1&quot;,
#  &quot;trusted&quot;: true
#}

#Define reactions of interest by id
r_ids_exp1 = [&#39;Ex_Suc&#39;,&#39;Ex_AA&#39;]

#Create figure
fig_exp1 = go.Figure()

#Add traces for reactions of interest
for r_id in r_ids_exp1:
    
    #Create trace
    trace = go.Scatter(
        y = df_result_exp1.loc[r_id,:],
        x = df_result_exp1.columns,
        name = r_id,
        mode = &#39;lines+markers&#39;,
        )
    
    #Add trace
    fig_exp1.add_trace(trace)

#Update xaxes
fig_exp1.update_xaxes(
    title = dict(
        text = &#39;CO\u2082 Uptake [µmol/(m\u00B2s)]&#39;,
        font = dict(size=18)
    ),
    tickfont = dict(size=16)
)

#Update yaxes
fig_exp1.update_yaxes(
    title = dict(
        text = &#39;Flux [µmol/(m\u00B2s)]&#39;,
        font = dict(size=18)
    ),
    tickfont = dict(size=16)
)

#Update layout
fig_exp1.update_layout(
    width=1000, 
    height=500,
    title = dict(
        text=&#39;&lt;b&gt;Phloem Export&lt;/b&gt;&#39;,
        x=0.5,
        font=dict(size=20)
    ),
    legend=dict(
        font=dict(size=18),
    )
)

#Show figure
fig_exp1.show()</code></pre>
            <figure slot="outputs"><span data-itemtype="http://schema.stenci.la/Null">null</span>
              <stencila-image-plotly>
                <picture>
                  <script type="application/vnd.plotly.v1+json">
                    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          <figcaption>
            <h5 itemscope="" itemtype="http://schema.stenci.la/Heading"
              id="effect-of-co2-dependence-of-the-phloem-output-on-co2-input-flux-in-the-range-0-μmolm2s20-μmolm2s-sucrose-and-starch-are-produced-in-the-same-amounts-each-of-them-consists-of-12-c-atoms">
              Effect of CO<sub itemscope="" itemtype="http://schema.stenci.la/Subscript">2.
                Dependence of the phloem output on CO</sub>2~ input flux in the range 0 μmol/(m<sup
                itemscope="" itemtype="http://schema.stenci.la/Superscript"><span
                  data-itemtype="http://schema.org/Number">2</span></sup>s)–20 μmol/(m<sup
                itemscope="" itemtype="http://schema.stenci.la/Superscript"><span
                  data-itemtype="http://schema.org/Number">2</span></sup>s). Sucrose and starch are
              produced in the same amounts, each of them consists of 12 C-atoms.</h5>
          </figcaption>
        </figure>
        <stencila-code-chunk itemscope="" itemtype="http://schema.stenci.la/CodeChunk"
          data-execution_count="23" data-programminglanguage="python">
          <pre class="language-python" itemscope="" itemtype="http://schema.stenci.la/CodeBlock"
            slot="text"><code>######################################################################################################################################
######################################################################################################################################
##############                                           EXPERIMENT 2: Effect of PPFD                                   ##############
######################################################################################################################################
######################################################################################################################################
print(f&quot;Code Cell 23: Experiment 2 -- Effect of PPFD&quot;)

#Create copy of c3 model
c3_model_exp2 = c3_model.copy()

#Optimize/Maximize sucrose output
result_exp2_1_fba = c3_model_exp2.optimize(&#39;maximize&#39;) #perform FBA

#Optimize/Minimize total flux
if result_exp2_1_fba.status == &#39;optimal&#39;: 
    result_exp2_1_pfba = cobra.flux_analysis.parsimonious.pfba(c3_model_exp2)
    
#Fetch flux for photon uptake
v_hnu_exp2 = result_exp2_1_pfba.fluxes[&#39;Im_hnu&#39;]

#Array defining proprtion of photon uptake 
hnu_ratios_exp2 = np.linspace(0,2,21)

df_result_exp2 = pd.DataFrame()

#Iterate over proportions of photon uptake
for hnu_ratio in tqdm(hnu_ratios_exp2):
    
    #Fix upper flux bound for photon uptake
    set_bounds(&#39;Im_hnu&#39;, (v_hnu_exp2 * hnu_ratio, v_hnu_exp2 * hnu_ratio), c3_model_exp2)
    
    #Optimize/Maximize sucrose output
    result_exp2_2_fba = c3_model_exp2.optimize(&#39;maximize&#39;) #perform FBA
    
    #Optimize/Minimize total flux
    if result_exp2_2_fba.status == &#39;optimal&#39;: # check if feasible
        result_exp2_2_pfba  = cobra.flux_analysis.parsimonious.pfba(c3_model_exp2) #perform pFBA
        if result_exp2_2_pfba.status == &#39;optimal&#39;:
            df_result_exp2[v_hnu_exp2 * hnu_ratio] = result_exp2_2_pfba.fluxes</code></pre>
          <figure slot="outputs">
            <pre class="language-text" itemscope="" itemtype="http://schema.stenci.la/CodeBlock"><code>Code Cell 23: Experiment 2 -- Effect of PPFD
</code></pre>
            <pre class="language-text" itemscope=""
              itemtype="http://schema.stenci.la/CodeBlock"><code>  0%|          | 0/21 [00:00&lt;?, ?it/s]</code></pre>
            <pre class="language-text" itemscope="" itemtype="http://schema.stenci.la/CodeBlock"><code>/Users/blaetke/opt/anaconda3/envs/elife-49305-era/lib/python3.9/site-packages/cobra/util/solver.py:508: UserWarning:

Solver status is &#39;infeasible&#39;.

</code></pre>
          </figure>
        </stencila-code-chunk>
        <figure itemscope="" itemtype="http://schema.stenci.la/Figure" id="fig1s2"
          title="Figure 1—figure supplement 2"><label data-itemprop="label">Figure 1—figure
            supplement 2</label>
          <stencila-code-chunk itemscope="" itemtype="http://schema.stenci.la/CodeChunk"
            data-execution_count="24" data-programminglanguage="python">
            <pre class="language-python" itemscope="" itemtype="http://schema.stenci.la/CodeBlock"
              slot="text"><code>######################################################################
##############         Figure 1—figure supplement 2     ##############
######################################################################

#{
#  &quot;caption&quot;: &quot;#### PPFD variation. Dependence of phloem output on the PPFD in the range 0 μmol/(m^2^s)–400 μmol/(m^2^s). Sucrose and starch are produced in the same amounts, each of them consists of 12 C-atoms.&quot;,
#  &quot;id&quot;: &quot;fig1s2,
#  &quot;label&quot;: &quot;Figure 1—figure supplement 2&quot;,
#  &quot;trusted&quot;: true
#}

#Define reactions of interest by id
r_ids_exp2 = [&#39;Ex_Suc&#39;,&#39;Ex_AA&#39;]

#Create figure
fig_exp2 = go.Figure()

#Add traces for reactions of interest
for r_id in r_ids_exp2:
    
    #Create trace
    trace = go.Scatter(
        y = df_result_exp2.loc[r_id,:],
        x = df_result_exp2.columns,
        name = r_id,
        mode = &#39;lines+markers&#39;,
        )
    
    #Add trace
    fig_exp2.add_trace(trace)

#Update xaxes
fig_exp2.update_xaxes(
    title = dict(
        text = &#39;&lt;b&gt;PPFD [µE]&lt;/b&gt;&#39;,
        font = dict(size=18)
    ),
    tickfont = dict(size=16)
)

#Update yaxes
fig_exp2.update_yaxes(
    title = dict(
        text = &#39;Flux [µmol/(m\u00B2s)]&#39;,
        font = dict(size=18)
    ),
    tickfont = dict(size=16)
)

#Update layout
fig_exp2.update_layout(
    width=1000, 
    height=500,
    title = dict(
        text=&#39;Phloem Export&#39;,
        x=0.5,
        font=dict(size=20)
    ),
    legend=dict(
        font=dict(size=18),
    )
)

#Show figure
fig_exp2.show()</code></pre>
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                  </script><img src="index.html.media/1" alt="" itemscope=""
                    itemtype="http://schema.org/ImageObject">
                </picture>
              </stencila-image-plotly>
            </figure>
          </stencila-code-chunk>
          <figcaption>
            <h5 itemscope="" itemtype="http://schema.stenci.la/Heading"
              id="ppfd-variation-dependence-of-phloem-output-on-the-ppfd-in-the-range-0-μmolm2s400-μmolm2s-sucrose-and-starch-are-produced-in-the-same-amounts-each-of-them-consists-of-12-c-atoms">
              PPFD variation. Dependence of phloem output on the PPFD in the range 0 μmol/(m<sup
                itemscope="" itemtype="http://schema.stenci.la/Superscript"><span
                  data-itemtype="http://schema.org/Number">2</span></sup>s)–400 μmol/(m<sup
                itemscope="" itemtype="http://schema.stenci.la/Superscript"><span
                  data-itemtype="http://schema.org/Number">2</span></sup>s). Sucrose and starch are
              produced in the same amounts, each of them consists of 12 C-atoms.</h5>
          </figcaption>
        </figure>
        <stencila-code-chunk itemscope="" itemtype="http://schema.stenci.la/CodeChunk"
          data-execution_count="25" data-programminglanguage="python">
          <pre class="language-python" itemscope="" itemtype="http://schema.stenci.la/CodeBlock"
            slot="text"><code>######################################################################################################################################
######################################################################################################################################
##############                                      EXPERIMENT 3: Simulate C3 Fluxes                                    ##############
######################################################################################################################################
######################################################################################################################################

print(f&quot;Code Cell 25: Experiment 3 -- Simulate C3 Fluxes&quot;)

#Create copy of c3 model
c3_model_exp3 = c3_model.copy()

#Optimize/Maximize sucrose output
result_exp3_fba = c3_model_exp3.optimize(&#39;maximize&#39;)

#Optimize/Minimize total flux
if result_exp3_fba.status == &#39;optimal&#39;: 
    result_exp3_pfba = cobra.flux_analysis.parsimonious.pfba(c3_model_exp3)
    
#Load GO Term Database
goDB = obo_parser.GODag(&#39;elife-49305.ipython.src/go_basic.obo&#39;)

def get_go_term(go_ids):
    if isinstance(go_ids, list):
        go_terms = [goDB[go_id].name for go_id in go_ids]
    else:
        go_terms= [goDB[go_ids].name] 
    return go_terms


#Filter all biochemical reactions
c3_biochem_rxn = c3_model_exp3.reactions.query(lambda x: ~x.id.startswith(&#39;Tr&#39;) and ~x.id.startswith(&#39;Ex&#39;) and ~x.id.startswith(&#39;Im&#39;))

#Grab annotation provided for the biochemical reactions, keep only GO IDs
df_anno = pd.DataFrame(
    c3_biochem_rxn.list_attr(&#39;annotation&#39;), 
    index=c3_biochem_rxn.list_attr(&#39;id&#39;)
).drop([&#39;doi&#39;,&#39;ec-code&#39;,&#39;kegg.reaction&#39;,&#39;pubmed&#39;,&#39;isbn&#39;], axis=1)


#Get GO Terms of GO IDs
df_anno[&#39;go term&#39;] = df_anno[&#39;go&#39;].apply(lambda go_ids: get_go_term(go_ids))

#Create Dataframe mapping GO IDs to biochemical reactions
df_go_term = pd.DataFrame(False,index=df_anno.index, columns=set(df_anno[&#39;go term&#39;].sum()))

for r_id in df_anno.index:
    df_go_term.loc[r_id,df_anno.loc[r_id,&#39;go term&#39;][0]] = True
    
#Define metabolites of interest (here energy equivalents)
met_classes = [&#39;ATP&#39;,&#39;NADH&#39;, &#39;NADPH&#39;]

#Set up list to store Go terms
go_terms = []

#Search for all GO terms related to the reactions the metabolites are involved in
for met_class in met_classes:
    
    #Find the specific metabolite ids in all compartments
    met_ids = c3_model_exp3.metabolites.query(lambda x: x.id.startswith(met_class)).list_attr(&#39;id&#39;)
    
    #Find reactions reactions and assigned GO Terms for all the metabolites
    for met_id in met_ids:
        #Get reaction ids
        rxn_ids = [r_obj.id for r_obj in c3_model_exp3.metabolites.get_by_id(met_id).reactions]
        #Get &amp; collect GO Terms
        go_terms += df_anno.loc[df_anno.index.intersection(rxn_ids),&#39;go term&#39;].sum()

#Create final list of all unique GO Terms
go_terms = list(set(go_terms))
go_terms = sorted(go_terms) + [&#39;Others&#39;]</code></pre>
          <figure slot="outputs">
            <pre class="language-text" itemscope="" itemtype="http://schema.stenci.la/CodeBlock"><code>Code Cell 25: Experiment 3 -- Simulate C3 Fluxes
elife-49305.ipython.src/go_basic.obo: fmt(1.2) rel(2017-10-20) 47,002 GO Terms
</code></pre>
          </figure>
        </stencila-code-chunk>
        <figure itemscope="" itemtype="http://schema.stenci.la/Figure" id="fig1s3"
          title="Figure 1—figure supplement 3"><label data-itemprop="label">Figure 1—figure
            supplement 3</label>
          <stencila-code-chunk itemscope="" itemtype="http://schema.stenci.la/CodeChunk"
            data-scrolled="false" data-execution_count="26" data-programminglanguage="python">
            <pre class="language-python" itemscope="" itemtype="http://schema.stenci.la/CodeBlock"
              slot="text"><code>##############################################################################
##############             Figure 1—figure supplement 3         ##############
##############################################################################

#{
#  &quot;caption&quot;: &quot;### Energy Flux Distribution in the _one-cell_ Model. (**A**) ATP production and consumption, (**B**) NADPH production and consumption, (**C**) NADH production and consumption.&quot;,
#  &quot;id&quot;: &quot;fig1s3&quot;,
#  &quot;label&quot;: &quot;Figure 1—figure supplement 3&quot;,
#  &quot;trusted&quot;: true
#}

def get_flux_by_go_term(df_rxn_go_term, rxn_fluxes):
    
    &#39;&#39;&#39;
    Return flux sum for go terms

            Parameters:
                    rxn_fluxes (dict): dictionary of reactions  and their flux values
                    df_rxn_go_term (dataframe): dataframe relating reactions and go terms

            Returns:
                    df_rxn_go_term_flux (dataframe): go term related to prodcution of a specified energy equivalent and flux sum
    &#39;&#39;&#39;
    
    #Convert dictionary of reaction fluxes into series 
    s_rxn_fluxes = pd.Series(rxn_fluxes)
    
    #Get flux sum
    flux_sum = s_rxn_fluxes.sum()
    
    #Determine flux sum for go terms
    s_rxn_fluxes = s_rxn_fluxes[s_rxn_fluxes / flux_sum &gt; 0.01]
    df_rxn_go_term = df_rxn_go_term.loc[s_rxn_fluxes.index, df_rxn_go_term.loc[s_rxn_fluxes.index,:].sum() &gt; 0]
    df_rxn_go_term_flux = df_rxn_go_term.mul(s_rxn_fluxes[s_rxn_fluxes.index.intersection(df_rxn_go_term.index)],axis=0).sum()
    df_rxn_go_term_flux[&#39;Others&#39;] = flux_sum - df_rxn_go_term_flux.sum()
    
    return df_rxn_go_term_flux

def prod_cons_charts(met_id, rxn_fluxes_prod, rxn_fluxes_cons, go_terms, df_rxn_go_term):
    
    &#39;&#39;&#39;
    Creates figure with two pie charts for production and consumption of a specified energy equivalent,
    and returns two lists of go terms related to the production and consumption

            Parameters:
                    met_id (int): metabolite id of the energy equivalent
                    rxn_fluxes_prod (dict): dictionary of reactions producing the energy equivalents and their flux values
                    rxn_fluxes_cons (dict): dictionary of reactions consuming the energy equivalents and their flux values
                    go_terms (list): list of all go terms
                    df_rxn_go_term (dataframe): dataframe relating reactions and go terms

            Returns:
                    trace_prod (plotly trace): pie chart of go term related to prodcution
                    go_term_fluxes_prod (dataframe): go term related to prodcution of a specified energy equivalent and flux sum
                    trace_cons (plotly trace): pie chart of go term related to consumption
                    go_term_fluxes_cons (dataframe):  go term related to consumption of a specified energy equivalent and flux sum
    &#39;&#39;&#39;
    
    #Get GO terms for production and consuption of energy equivalent
    go_term_fluxes_prod = get_flux_by_go_term(df_rxn_go_term, rxn_fluxes_prod)
    go_term_fluxes_cons = get_flux_by_go_term(df_rxn_go_term, rxn_fluxes_cons)
    
    #Create index of GO terms for production and consuption of energy equivalent 
    go_term_index = go_term_fluxes_prod.index.union(go_term_fluxes_cons.index)
    
    #Create trace for GO terms of energy equivalent production
    trace_prod = go.Pie(
        labels = go_term_fluxes_prod.index,
        values = go_term_fluxes_prod,
        marker=dict(line=dict(color=&#39;#FFF&#39;, width=1)),
    )
    

    #Create trace for GO terms of energy equivalent consumption
    trace_cons = go.Pie(
        labels = go_term_fluxes_cons.index,
        values = go_term_fluxes_cons,
        marker=dict(line=dict(color=&#39;#FFF&#39;, width=1)),
    )

    return trace_prod, go_term_fluxes_prod, trace_cons, go_term_fluxes_cons

#Set up Dataframe to store total production and consumption flux per metabolite class
df_prod_cons = pd.DataFrame(index=met_classes, columns=[&#39;Production&#39;, &#39;Consumption&#39;])
df_prod_cons_percentage = pd.DataFrame(index=go_terms)


#Create figure with subplots
fig_exp31 = make_subplots(
    rows=3, 
    cols=2, 
    subplot_titles = [&#39;&lt;b&gt;Production&lt;/b&gt;&#39;,&#39;&lt;b&gt;Consumption&lt;/b&gt;&#39;] * 3,
    row_titles = [f&#39;&lt;b&gt;({list(string.ascii_uppercase)[i]}) {met_class}&lt;/b&gt;&#39; 
                      for i, met_class in enumerate(met_classes)],
    specs=[[{&quot;type&quot;: &quot;pie&quot;}, {&quot;type&quot;: &quot;pie&quot;}],[{&quot;type&quot;: &quot;pie&quot;}, {&quot;type&quot;: &quot;pie&quot;}],[{&quot;type&quot;: &quot;pie&quot;}, {&quot;type&quot;: &quot;pie&quot;}]],
    vertical_spacing = 0.0
)
        
#Create pie charts reflecting the proportions of a metabolite class produced or consumped by Go Terms
for i, met_class in enumerate(met_classes):
    
    #Find the specific metabolite ids in all compartments
    met_ids = c3_model_exp3.metabolites.query(lambda x: x.id.startswith(met_class)).list_attr(&#39;id&#39;)
    
    #Set up dictionaries to hold fluxes producing or consuming a certain metabolite
    fluxes_prod_met_class = {}
    fluxes_cons_met_class = {}
        
    for met_id in met_ids:
        
        #Get reactions and fluxes producing or consuming a certain metabolite (excluding transport, import and export reactions)
        fluxes = {r_obj.id: result_exp3_pfba.fluxes[r_obj.id] * r_obj.get_coefficient(met_id)
                   for r_obj in c3_model_exp3.metabolites.get_by_id(met_id).reactions 
                   if not r_obj.id[:3] in [&#39;Tr_&#39;, &#39;Im_&#39;, &#39;Ex_&#39;]}
        
        #Extract reactions and fluxes producing a certain metabolite
        fluxes_prod = {r_id: abs(flux) for r_id, flux in fluxes.items() if flux &gt; 0}
        fluxes_prod_met_class = {**fluxes_prod_met_class, **fluxes_prod}
        
        #Extract reactions and fluxes consuming a certain metabolite
        fluxes_cons = {r_id: abs(flux) for r_id, flux in fluxes.items() if flux &lt; 0}
        fluxes_cons_met_class = {**fluxes_cons_met_class, **fluxes_cons}
    
    #Store total fluxes
    df_prod_cons.loc[met_class] = [sum(fluxes_prod_met_class.values()), sum(fluxes_cons_met_class.values())]
    
    
    #Plot porportion of fluxes by Go Terms
    trace_prod, go_term_fluxes_prod, trace_cons, go_term_fluxes_cons = prod_cons_charts(met_class, fluxes_prod_met_class, fluxes_cons_met_class, go_terms, df_go_term)
    
    #Add trace to figure
    #fig_exp31.append_trace(trace_go,i+1,1)
    
    #Add trace to figure
    fig_exp31.append_trace(trace_prod,i+1,1)
    
    #Add trace to figure
    fig_exp31.append_trace(trace_cons,i+1,2)
    
    
    #save flux sum for GO Terms
    df_prod_cons_percentage[f&#39;{met_class}_prod&#39;] = np.nan
    df_prod_cons_percentage[f&#39;{met_class}_prod&#39;].loc[go_term_fluxes_prod.index] = go_term_fluxes_prod
    
    df_prod_cons_percentage[f&#39;{met_class}_cons&#39;] = np.nan
    df_prod_cons_percentage[f&#39;{met_class}_cons&#39;].loc[go_term_fluxes_cons.index] = go_term_fluxes_cons
    
df_prod_cons_percentage.dropna(how=&#39;all&#39;, inplace=True)
df_prod_cons_percentage = df_prod_cons_percentage / df_prod_cons_percentage.sum() * 100
     
df_prod_percentage = df_prod_cons.loc[:,&#39;Production&#39;] / df_prod_cons[&#39;Production&#39;].sum() * 100

#Update traces
fig_exp31.update_traces(
    textposition=&#39;inside&#39;, 
    textinfo=&#39;percent&#39;
)

#Get all GO terms in figure
go_term_labels = list(set([label  for trace in fig_exp31[&#39;data&#39;]  for label in trace[&#39;labels&#39;]]))

#Create list colors according to number of GO terms in figure
colors = [&#39;hsl(&#39;+str(h)+&#39;,50%&#39;+&#39;,50%)&#39; for h in np.linspace(0, 360, len(go_term_labels))]

#Assign color to GO term
go_term_color = pd.Series(index=go_term_labels, data=colors)

#Re-color trace in figure 
for trace in fig_exp31[&#39;data&#39;]: 
    trace[&#39;marker&#39;][&#39;colors&#39;] = go_term_color[trace[&#39;labels&#39;]].values
    
#Update annotations
fig_exp31.update_annotations(
    font=dict(size=18)
)

#Re-position subfigure enumeration
for anno in fig_exp31[&#39;layout&#39;][&#39;annotations&#39;]:
    if anno[&#39;xanchor&#39;] == &#39;left&#39;:
        anno[&#39;x&#39;] = 0.1 
        anno[&#39;y&#39;] = anno[&#39;y&#39;] + 0.15 
        anno[&#39;textangle&#39;] = 0
        anno[&#39;xanchor&#39;] = &#39;right&#39;
    if &#39;Production&#39; in anno[&#39;text&#39;] or &#39;Consumption&#39; in anno[&#39;text&#39;]:
        anno[&#39;y&#39;] = anno[&#39;y&#39;] - 0.025 
    
#Update layout
fig_exp31.update_layout(
    uniformtext_minsize=12, 
    uniformtext_mode=&#39;hide&#39;, 
    width=1000, 
    height=2000,
    legend=dict(
        font=dict(size=18),
        orientation = &#39;h&#39;,
        xanchor = &#39;center&#39;,
        x = 0.5,
        y = 0.05
        
    )
)
    
#Show figure
fig_exp31.show()</code></pre>
            <figure slot="outputs">
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                  </script><img src="index.html.media/2" alt="" itemscope=""
                    itemtype="http://schema.org/ImageObject">
                </picture>
              </stencila-image-plotly>
            </figure>
          </stencila-code-chunk>
          <figcaption>
            <h4 itemscope="" itemtype="http://schema.stenci.la/Heading"
              id="energy-flux-distribution-in-the-one-cell-model-a-atp-production-and-consumption-b-nadph-production-and-consumption-c-nadh-production-and-consumption">
              Energy Flux Distribution in the <em itemscope=""
                itemtype="http://schema.stenci.la/Emphasis">one-cell</em> Model. (<strong
                itemscope="" itemtype="http://schema.stenci.la/Strong">A</strong>) ATP production
              and consumption, (<strong itemscope=""
                itemtype="http://schema.stenci.la/Strong">B</strong>) NADPH production and
              consumption, (<strong itemscope=""
                itemtype="http://schema.stenci.la/Strong">C</strong>) NADH production and
              consumption.</h4>
          </figcaption>
        </figure>
        <figure itemscope="" itemtype="http://schema.stenci.la/Figure" id="fig1s4"
          title="Figure 1—figure supplement 4"><label data-itemprop="label">Figure 1—figure
            supplement 4</label>
          <stencila-code-chunk itemscope="" itemtype="http://schema.stenci.la/CodeChunk"
            data-execution_count="27" data-programminglanguage="python">
            <pre class="language-python" itemscope="" itemtype="http://schema.stenci.la/CodeBlock"
              slot="text"><code>#########################################################################
##############          Figure 1—figure supplement 4       ##############
#########################################################################

#{
#  &quot;caption&quot;: &quot;### Energy Flux Distribution in the _one-cell_ Model. (**A**) proportion of ATP, NADPH, NADH used as energy equivalent, (**B**) proportion of respiratory ATP used for maintenance.&quot;,
#  &quot;id&quot;: &quot;fig1s4&quot;,
#  &quot;label&quot;: &quot;Figure 1—figure supplement 4&quot;,
#  &quot;trusted&quot;: true
#}

#Create figure with subplots
fig_exp32 = make_subplots(
    rows=1, 
    cols=2, 
    subplot_titles = [&#39;&lt;b&gt;(A) Proportion of Energy Equivalents&lt;/b&gt;&#39;, &#39;&lt;b&gt;(B) Proportion of Respiratory ATP&lt;/b&gt;&#39;],
    specs=[[{&quot;type&quot;: &quot;pie&quot;}, {&quot;type&quot;: &quot;pie&quot;}]])

#Create trace for energy equivalents
trace_energy = go.Pie(
    labels = df_prod_cons.index,
    values = df_prod_cons[&#39;Production&#39;],
    marker=dict(line=dict(color=&#39;#FFF&#39;, width=1)),
    showlegend = False,
)

#Add trace to figure
fig_exp32.append_trace(trace_energy,1,1)

#Create trace for energy maintenace in respect to respiratory ATP
rxn_maintenance = [&#39;NGAM_h&#39;,&#39;NGAM_c&#39;,&#39;NGAM_m&#39;]
rxn_maintenance_flux = [result_exp3_pfba.fluxes[r_id] for r_id in rxn_maintenance]
atp_flux = result_exp3_pfba.fluxes[&#39;cplx5_m&#39;] * c3_model_exp3.reactions.get_by_id(&#39;cplx5_m&#39;).get_coefficient(&#39;ATP_m&#39;)
atp_maintenance_percentage = sum(rxn_maintenance_flux) / (atp_flux) * 100

trace_maintenance = go.Pie(
    labels = rxn_maintenance + [&#39;Others&#39;],
    values = rxn_maintenance_flux + [atp_flux - sum(rxn_maintenance_flux)],
    textfont=dict(size=18,family=&#39;Arial&#39;,),
    marker=dict(line=dict(color=&#39;#FFF&#39;, width=1)),
    showlegend = False,
)

#Add trace to figure
fig_exp32.append_trace(trace_maintenance,1,2)

#Update traces
fig_exp32.update_traces(
    textposition=&#39;outside&#39;, 
    textinfo=&#39;percent+label&#39;
)

#Update annotations
fig_exp32.update_annotations(
        font=dict(size=18)
    )

#Update layout
fig_exp32.update_layout(
    uniformtext_minsize=16, 
    uniformtext_mode=&#39;hide&#39;, 
    width=1000, 
    height=500)

#Show figure    
fig_exp32.show()</code></pre>
            <figure slot="outputs">
              <stencila-image-plotly>
                <picture>
                  <script type="application/vnd.plotly.v1+json">
                    {"config":{"plotlyServerURL":"https://plot.ly"},"data":[{"domain":{"x":[0,0.45],"y":[0,1]},"labels":["ATP","NADH","NADPH"],"marker":{"line":{"color":"#FFF","width":1}},"showlegend":false,"textinfo":"percent+label","textposition":"outside","type":"pie","values":[74.74335847413222,3.0325701440631625,46.72929396794722]},{"domain":{"x":[0.55,1],"y":[0,1]},"labels":["NGAM_h","NGAM_c","NGAM_m","Others"],"marker":{"line":{"color":"#FFF","width":1}},"showlegend":false,"textfont":{"family":"Arial","size":18},"textinfo":"percent+label","textposition":"outside","type":"pie","values":[0.152707083279744,0.0503499796174949,0.00911118724550157,0.5430984651403115]}],"layout":{"annotations":[{"font":{"size":18},"showarrow":false,"text":"<b>(A) Proportion of Energy Equivalents</b>","x":0.225,"xanchor":"center","xref":"paper","y":1,"yanchor":"bottom","yref":"paper"},{"font":{"size":18},"showarrow":false,"text":"<b>(B) Proportion of Respiratory 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                  </script><img src="index.html.media/3" alt="" itemscope=""
                    itemtype="http://schema.org/ImageObject">
                </picture>
              </stencila-image-plotly>
            </figure>
          </stencila-code-chunk>
          <figcaption>
            <h4 itemscope="" itemtype="http://schema.stenci.la/Heading"
              id="energy-flux-distribution-in-the-one-cell-model-a-proportion-of-atp-nadph-nadh-used-as-energy-equivalent-b-proportion-of-respiratory-atp-used-for-maintenance">
              Energy Flux Distribution in the <em itemscope=""
                itemtype="http://schema.stenci.la/Emphasis">one-cell</em> Model. (<strong
                itemscope="" itemtype="http://schema.stenci.la/Strong">A</strong>) proportion of
              ATP, NADPH, NADH used as energy equivalent, (<strong itemscope=""
                itemtype="http://schema.stenci.la/Strong">B</strong>) proportion of respiratory ATP
              used for maintenance.</h4>
          </figcaption>
        </figure>
        <p itemscope="" itemtype="http://schema.stenci.la/Paragraph">The <em itemscope=""
            itemtype="http://schema.stenci.la/Emphasis">one-cell</em> model requires a
          photosynthetic photon flux density (PPFD) of <stencila-code-expression
            programming-language="py" itemscope=""
            itemtype="http://schema.stenci.la/CodeExpression"><code class="py"
              slot="text">round(result_exp3_pfba['Im_hnu'],2)</code><output slot="output"></output>
          </stencila-code-expression> μmol/(m<sup itemscope=""
            itemtype="http://schema.stenci.la/Superscript"><span
              data-itemtype="http://schema.org/Number">2</span></sup>s) (<a href="#table2"
            itemscope="" itemtype="http://schema.stenci.la/Link">Table 2</a>). The <em itemscope=""
            itemtype="http://schema.stenci.la/Emphasis">one-cell</em> model takes up the maximal
          amount of CO<sub itemscope="" itemtype="http://schema.stenci.la/Subscript"><span
              data-itemtype="http://schema.org/Number">2</span></sub> to produce the maximum amount
          of phloem sap, as well as <stencila-code-expression programming-language="py" itemscope=""
            itemtype="http://schema.stenci.la/CodeExpression"><code class="py"
              slot="text">round(result_exp3_pfba['Im_NO3'],2)</code><output slot="output"></output>
          </stencila-code-expression> μmol/(m<sup itemscope=""
            itemtype="http://schema.stenci.la/Superscript"><span
              data-itemtype="http://schema.org/Number">2</span></sup>s) of NO<sub itemscope=""
            itemtype="http://schema.stenci.la/Subscript"><span
              data-itemtype="http://schema.org/Number">3</span></sub><sup itemscope=""
            itemtype="http://schema.stenci.la/Superscript">-</sup> and <stencila-code-expression
            programming-language="py" itemscope=""
            itemtype="http://schema.stenci.la/CodeExpression"><code class="py"
              slot="text">round(result_exp3_pfba['Im_H2O'],2)</code><output slot="output"></output>
          </stencila-code-expression> μmol/(m<sup itemscope=""
            itemtype="http://schema.stenci.la/Superscript"><span
              data-itemtype="http://schema.org/Number">2</span></sup>s) of H<sub itemscope=""
            itemtype="http://schema.stenci.la/Subscript"><span
              data-itemtype="http://schema.org/Number">2</span></sub>O. According to the assumed
          ratio of sucrose and amino acids in the phloem sap, the flux of sucrose predicted by the
          model is <stencila-code-expression programming-language="py" itemscope=""
            itemtype="http://schema.stenci.la/CodeExpression"><code class="py"
              slot="text">round(result_exp3_pfba['Ex_Suc'],2)</code><output slot="output"></output>
          </stencila-code-expression> μmol/(m<sup itemscope=""
            itemtype="http://schema.stenci.la/Superscript"><span
              data-itemtype="http://schema.org/Number">2</span></sup>s) and of amino acids
          <stencila-code-expression programming-language="py" itemscope=""
            itemtype="http://schema.stenci.la/CodeExpression"><code class="py"
              slot="text">round(result_exp3_pfba['Ex_AA'],2)</code><output slot="output"></output>
          </stencila-code-expression> μmol/(m<sup itemscope=""
            itemtype="http://schema.stenci.la/Superscript"><span
              data-itemtype="http://schema.org/Number">2</span></sup>s). The rate of oxygen supply
          by the network is <stencila-code-expression programming-language="py" itemscope=""
            itemtype="http://schema.stenci.la/CodeExpression"><code class="py"
              slot="text">round(result_exp3_pfba['Ex_O2'],2)</code><output slot="output"></output>
          </stencila-code-expression> μmol/(m<sup itemscope=""
            itemtype="http://schema.stenci.la/Superscript"><span
              data-itemtype="http://schema.org/Number">2</span></sup>s). Part of the complete flux
          table is displayed in <a href="#table2" itemscope=""
            itemtype="http://schema.stenci.la/Link">Table 2</a>; the full table is available, see <a
            href="#fig1sdata2" itemscope="" itemtype="http://schema.stenci.la/Link">Figure 1—source
            data 2</a>. The flux table of all reactions did not display circular fluxes, and the
          reactions were within expected physiological ranges (<a href="#fig1sdata2" itemscope=""
            itemtype="http://schema.stenci.la/Link">Figure 1—source data 2</a>).</p>
        <table id="table2" itemscope="" itemtype="http://schema.org/Table">
          <caption><label data-itemprop="label">Table 2.</label>
            <div itemprop="caption">
              <h4 itemscope="" itemtype="http://schema.stenci.la/Heading"
                id="inputoutput-fluxes-of-one-cell-model-in-comparison-to-physiological-observations">
                Input/output fluxes of <em itemscope=""
                  itemtype="http://schema.stenci.la/Emphasis">one-cell</em> model in comparison to
                physiological observations.</h4>
            </div>
          </caption>
          <thead>
            <tr itemscope="" itemtype="http://schema.stenci.la/TableRow">
              <th itemscope="" itemtype="http://schema.stenci.la/TableCell">Molecular Species</th>
              <th itemscope="" itemtype="http://schema.stenci.la/TableCell">Flux [µmol/(m<sup
                  itemscope="" itemtype="http://schema.stenci.la/Superscript">2</sup>/s)]</th>
              <th itemscope="" itemtype="http://schema.stenci.la/TableCell">
                Physiological Range [µmol/(m<sup itemscope=""
                  itemtype="http://schema.stenci.la/Superscript">2</sup>/s)]</th>
              <th itemscope="" itemtype="http://schema.stenci.la/TableCell">Reference</th>
            </tr>
          </thead>
          <tbody>
            <tr itemscope="" itemtype="http://schema.stenci.la/TableRow">
              <td itemscope="" itemtype="http://schema.stenci.la/TableCell">(i) Inputs</td>
              <td itemscope="" itemtype="http://schema.stenci.la/TableCell"></td>
              <td itemscope="" itemtype="http://schema.stenci.la/TableCell"></td>
              <td itemscope="" itemtype="http://schema.stenci.la/TableCell"></td>
            </tr>
            <tr itemscope="" itemtype="http://schema.stenci.la/TableRow">
              <td itemscope="" itemtype="http://schema.stenci.la/TableCell">Photons</td>
              <td itemscope="" itemtype="http://schema.stenci.la/TableCell">
                <stencila-code-expression programming-language="py" itemscope=""
                  itemtype="http://schema.stenci.la/CodeExpression"><code class="py"
                    slot="text">round(result_exp3_pfba['Im_hnu'],2)</code><output
                    slot="output"></output></stencila-code-expression>
              </td>
              <td itemscope="" itemtype="http://schema.stenci.la/TableCell">100 - 400</td>
              <td itemscope="" itemtype="http://schema.stenci.la/TableCell"><cite itemscope=""
                  itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a
                    href="#bib5"><span>5</span><span>Bailey et
                      al.</span><span>2001</span></a></cite></td>
            </tr>
            <tr itemscope="" itemtype="http://schema.stenci.la/TableRow">
              <td itemscope="" itemtype="http://schema.stenci.la/TableCell">CO2</td>
              <td itemscope="" itemtype="http://schema.stenci.la/TableCell">
                <stencila-code-expression programming-language="py" itemscope=""
                  itemtype="http://schema.stenci.la/CodeExpression"><code class="py"
                    slot="text">round(result_exp3_pfba['Im_CO2'],2)</code><output
                    slot="output"></output></stencila-code-expression>
              </td>
              <td itemscope="" itemtype="http://schema.stenci.la/TableCell"><span
                  data-itemtype="http://schema.org/Number">20</span></td>
              <td itemscope="" itemtype="http://schema.stenci.la/TableCell"><cite itemscope=""
                  itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a
                    href="#bib37"><span>37</span><span>Lacher</span><span>2003</span></a></cite>
              </td>
            </tr>
            <tr itemscope="" itemtype="http://schema.stenci.la/TableRow">
              <td itemscope="" itemtype="http://schema.stenci.la/TableCell">NO<sub itemscope=""
                  itemtype="http://schema.stenci.la/Subscript">3</sub><sup itemscope=""
                  itemtype="http://schema.stenci.la/Superscript">-</sup></td>
              <td itemscope="" itemtype="http://schema.stenci.la/TableCell">
                <stencila-code-expression programming-language="py" itemscope=""
                  itemtype="http://schema.stenci.la/CodeExpression"><code class="py"
                    slot="text">round(result_exp3_pfba['Im_NO3'],2)</code><output
                    slot="output"></output></stencila-code-expression>
              </td>
              <td itemscope="" itemtype="http://schema.stenci.la/TableCell">0.11 - 0.18</td>
              <td itemscope="" itemtype="http://schema.stenci.la/TableCell"><cite itemscope=""
                  itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a
                    href="#bib35"><span>35</span><span>Kiba et
                      al.</span><span>2012</span></a></cite></td>
            </tr>
            <tr itemscope="" itemtype="http://schema.stenci.la/TableRow">
              <td itemscope="" itemtype="http://schema.stenci.la/TableCell">H<sub itemscope=""
                  itemtype="http://schema.stenci.la/Subscript">2</sub>O</td>
              <td itemscope="" itemtype="http://schema.stenci.la/TableCell">
                <stencila-code-expression programming-language="py" itemscope=""
                  itemtype="http://schema.stenci.la/CodeExpression"><code class="py"
                    slot="text">round(result_exp3_pfba['Im_H2O'],2)</code><output
                    slot="output"></output></stencila-code-expression>
              </td>
              <td itemscope="" itemtype="http://schema.stenci.la/TableCell">-</td>
              <td itemscope="" itemtype="http://schema.stenci.la/TableCell"></td>
            </tr>
            <tr itemscope="" itemtype="http://schema.stenci.la/TableRow">
              <td itemscope="" itemtype="http://schema.stenci.la/TableCell">(ii) Outputs</td>
              <td itemscope="" itemtype="http://schema.stenci.la/TableCell"></td>
              <td itemscope="" itemtype="http://schema.stenci.la/TableCell"></td>
              <td itemscope="" itemtype="http://schema.stenci.la/TableCell"></td>
            </tr>
            <tr itemscope="" itemtype="http://schema.stenci.la/TableRow">
              <td itemscope="" itemtype="http://schema.stenci.la/TableCell">O<sub itemscope=""
                  itemtype="http://schema.stenci.la/Subscript">2</sub></td>
              <td itemscope="" itemtype="http://schema.stenci.la/TableCell">
                <stencila-code-expression programming-language="py" itemscope=""
                  itemtype="http://schema.stenci.la/CodeExpression"><code class="py"
                    slot="text">round(result_exp3_pfba['Ex_O2'],2)</code><output
                    slot="output"></output></stencila-code-expression>
              </td>
              <td itemscope="" itemtype="http://schema.stenci.la/TableCell"><span
                  data-itemtype="http://schema.org/Number">16.5</span></td>
              <td itemscope="" itemtype="http://schema.stenci.la/TableCell"><cite itemscope=""
                  itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a
                    href="#bib75"><span>75</span><span>Sun et al.</span><span>1999</span></a></cite>
              </td>
            </tr>
            <tr itemscope="" itemtype="http://schema.stenci.la/TableRow">
              <td itemscope="" itemtype="http://schema.stenci.la/TableCell">Amino Acids</td>
              <td itemscope="" itemtype="http://schema.stenci.la/TableCell">
                <stencila-code-expression programming-language="py" itemscope=""
                  itemtype="http://schema.stenci.la/CodeExpression"><code class="py"
                    slot="text">round(result_exp3_pfba['Ex_AA'],2)</code><output
                    slot="output"></output></stencila-code-expression>
              </td>
              <td itemscope="" itemtype="http://schema.stenci.la/TableCell">-</td>
              <td itemscope="" itemtype="http://schema.stenci.la/TableCell"></td>
            </tr>
            <tr itemscope="" itemtype="http://schema.stenci.la/TableRow">
              <td itemscope="" itemtype="http://schema.stenci.la/TableCell">Sucrose/Starch</td>
              <td itemscope="" itemtype="http://schema.stenci.la/TableCell">
                <stencila-code-expression programming-language="py" itemscope=""
                  itemtype="http://schema.stenci.la/CodeExpression"><code class="py"
                    slot="text">round(result_exp3_pfba['Ex_Suc'],2)</code><output
                    slot="output"></output></stencila-code-expression>
              </td>
              <td itemscope="" itemtype="http://schema.stenci.la/TableCell">-</td>
              <td itemscope="" itemtype="http://schema.stenci.la/TableCell"></td>
            </tr>
          </tbody>
        </table>
        <p itemscope="" itemtype="http://schema.stenci.la/Paragraph">The CO<sub itemscope=""
            itemtype="http://schema.stenci.la/Subscript"><span
              data-itemtype="http://schema.org/Number">2</span></sub> uptake rate and the phloem sap
          output have a positive linear relationship, see <a href="#fig1s1" itemscope=""
            itemtype="http://schema.stenci.la/Link">Figure 1—figure supplement 1</a>. The same is
          true for the correlation of the PPFD and phloem sap output in the range of 100 μmol/(m<sup
            itemscope="" itemtype="http://schema.stenci.la/Superscript"><span
              data-itemtype="http://schema.org/Number">2</span></sup>s)–200 μmol/(m<sup itemscope=""
            itemtype="http://schema.stenci.la/Superscript"><span
              data-itemtype="http://schema.org/Number">2</span></sup>s), see <a href="#fig1s2"
            itemscope="" itemtype="http://schema.stenci.la/Link">Figure 1—figure supplement 2)</a>.
          Above 200 μmol/(m<sup itemscope="" itemtype="http://schema.stenci.la/Superscript"><span
              data-itemtype="http://schema.org/Number">2</span></sup>s), the CO<sub itemscope=""
            itemtype="http://schema.stenci.la/Subscript"><span
              data-itemtype="http://schema.org/Number">2</span></sub> uptake rate acts as a limiting
          factor restricting the increase of phloem sap production. If either the PPFD or the CO<sub
            itemscope="" itemtype="http://schema.stenci.la/Subscript"><span
              data-itemtype="http://schema.org/Number">2</span></sub> uptake rate is zero, the
          phloem sap cannot be produced, compare <a href="#fig1s1" itemscope=""
            itemtype="http://schema.stenci.la/Link">Figure 1—figure supplement 1</a> and <a
            href="#fig1s2" itemscope="" itemtype="http://schema.stenci.la/Link">Figure 1—figure
            supplement 2</a>. Most of the metabolic processes use ATP/ADP as main energy equivalent
          (<stencila-code-expression programming-language="py" itemscope=""
            itemtype="http://schema.stenci.la/CodeExpression"><code class="py"
              slot="text">round(df_prod_percentage['ATP'],2)</code><output slot="output"></output>
          </stencila-code-expression>%), followed by NADP/NADPH (<stencila-code-expression
            programming-language="py" itemscope=""
            itemtype="http://schema.stenci.la/CodeExpression"><code class="py"
              slot="text">round(df_prod_percentage['NADPH'],2)</code><output slot="output"></output>
          </stencila-code-expression>%) and NAD/NADH (<stencila-code-expression
            programming-language="py" itemscope=""
            itemtype="http://schema.stenci.la/CodeExpression"><code class="py"
              slot="text">round(df_prod_percentage['NADH'],2)</code><output slot="output"></output>
          </stencila-code-expression>%), see <a href="#fig1s4" itemscope=""
            itemtype="http://schema.stenci.la/Link">Figure 1—figure supplement 4(A)</a>. Nearly all
          ATP is produced by the light reactions (<stencila-code-expression
            programming-language="py" itemscope=""
            itemtype="http://schema.stenci.la/CodeExpression"><code class="py"
              slot="text">round(df_prod_cons_percentage.loc['photosynthesis, light
              reaction','ATP_prod'],2)</code><output slot="output"></output>
          </stencila-code-expression>%) and consumed by the reductive pentose phosphate cycle (
          <stencila-code-expression programming-language="py" itemscope=""
            itemtype="http://schema.stenci.la/CodeExpression"><code class="py"
              slot="text">round(df_prod_cons_percentage.loc['reductive pentose-phosphate
              cycle','ATP_cons'],2)</code><output slot="output"></output></stencila-code-expression>
          %), see <a href="#fig1s3" itemscope="" itemtype="http://schema.stenci.la/Link">Figure
            1—figure supplement 3(A)</a>. The oxidative phosphorylation produces only (
          <stencila-code-expression programming-language="py" itemscope=""
            itemtype="http://schema.stenci.la/CodeExpression"><code class="py"
              slot="text">round(df_prod_cons_percentage.loc['oxidative
              phosphorylation','ATP_prod'],2)</code><output slot="output"></output>
          </stencila-code-expression>%) of ATP. In proportion, the maintenance cost for protein
          synthesis and degradation makeup <stencila-code-expression programming-language="py"
            itemscope="" itemtype="http://schema.stenci.la/CodeExpression"><code class="py"
              slot="text">round(atp_maintenance_percentage,2)</code><output slot="output"></output>
          </stencila-code-expression>% of the respiratory ATP produced by the oxidative
          phosphorylation (<a href="#fig1s4" itemscope=""
            itemtype="http://schema.stenci.la/Link">Figure 1—figure supplement 4(B)</a>). Similarly,
          nearly all NADPH is produced by the light reaction (<stencila-code-expression
            programming-language="py" itemscope=""
            itemtype="http://schema.stenci.la/CodeExpression"><code class="py"
              slot="text">round(df_prod_cons_percentage.loc['photosynthesis, light
              reaction','NADPH_prod'],2)</code><output slot="output"></output>
          </stencila-code-expression>%), which is consumed by the reductive pentose-phosphate cycle
          (<stencila-code-expression programming-language="py" itemscope=""
            itemtype="http://schema.stenci.la/CodeExpression"><code class="py"
              slot="text">round(df_prod_cons_percentage.loc['reductive pentose-phosphate
              cycle','NADPH_cons'],2)</code><output slot="output"></output>
          </stencila-code-expression>%) as well (<a href="#fig1s3" itemscope=""
            itemtype="http://schema.stenci.la/Link">Figure 1—figure supplement 3(C)</a>). The
          canonical glycolysis and photorespiration produce nearly equal amounts of NADH,
          <stencila-code-expression programming-language="py" itemscope=""
            itemtype="http://schema.stenci.la/CodeExpression"><code class="py"
              slot="text">round(df_prod_cons_percentage.loc['canonical
              glycolysis','NADH_prod'],2)</code><output slot="output"></output>
          </stencila-code-expression>% and <stencila-code-expression programming-language="py"
            itemscope="" itemtype="http://schema.stenci.la/CodeExpression"><code class="py"
              slot="text">round(df_prod_cons_percentage.loc['photorespiration','NADH_prod'],2)</code><output
              slot="output"></output></stencila-code-expression>%), significantly less NADH is
          produced through gluconeogenesis (<stencila-code-expression programming-language="py"
            itemscope="" itemtype="http://schema.stenci.la/CodeExpression"><code class="py"
              slot="text">round(df_prod_cons_percentage.loc['gluconeogenesis','NADH_prod'],2)</code><output
              slot="output"></output></stencila-code-expression>%) and pyruvate dehydrogenase
          activity (<stencila-code-expression programming-language="py" itemscope=""
            itemtype="http://schema.stenci.la/CodeExpression"><code class="py"
              slot="text">round(df_prod_cons_percentage.loc['pyruvate dehydrogenase
              activity','NADH_prod'],2)</code><output slot="output"></output>
          </stencila-code-expression>)%. Photorespiration (<stencila-code-expression
            programming-language="py" itemscope=""
            itemtype="http://schema.stenci.la/CodeExpression"><code class="py"
              slot="text">round(df_prod_cons_percentage.loc['photorespiration','NADH_cons'],2)</code><output
              slot="output"></output></stencila-code-expression>%), Nitrate assimilation (
          <stencila-code-expression programming-language="py" itemscope=""
            itemtype="http://schema.stenci.la/CodeExpression"><code class="py"
              slot="text">round(df_prod_cons_percentage.loc['nitrate
              assimilation','NADH_cons'],2)</code><output slot="output"></output>
          </stencila-code-expression>%), glutamate biosynthesis (<stencila-code-expression
            programming-language="py" itemscope=""
            itemtype="http://schema.stenci.la/CodeExpression"><code class="py"
              slot="text">round(df_prod_cons_percentage.loc['glutamate biosynthetic
              process','NADH_cons'],2)</code><output slot="output"></output>
          </stencila-code-expression>%), glyoxylate cycle (<stencila-code-expression
            programming-language="py" itemscope=""
            itemtype="http://schema.stenci.la/CodeExpression"><code class="py"
              slot="text">round(df_prod_cons_percentage.loc['glyoxylate
              cycle','NADH_cons'],2)</code><output slot="output"></output>
          </stencila-code-expression>%) and alternative respiration (<stencila-code-expression
            programming-language="py" itemscope=""
            itemtype="http://schema.stenci.la/CodeExpression"><code class="py"
              slot="text">round(df_prod_cons_percentage.loc['alternative
              respiration','NADH_cons'],2)</code><output slot="output"></output>
          </stencila-code-expression>%) consume the produced NADH (<a href="#fig1s3" itemscope=""
            itemtype="http://schema.stenci.la/Link">Figure 1—figure supplement 3(B)</a>).</p>
        <h3 itemscope="" itemtype="http://schema.stenci.la/Heading"
          id="a-c4-cycle-is-predicted-under-resource-limitation">A C4 cycle is predicted under
          resource limitation</h3>
        <p itemscope="" itemtype="http://schema.stenci.la/Paragraph">To rebuild the characteristic
          physiology of C4 leaves, we duplicated the <em itemscope=""
            itemtype="http://schema.stenci.la/Emphasis">one-cell</em> model and connected the two
          network copies by bi-directional transport of cytosolic metabolites including amino acids,
          sugars, single phosphorylated sugars, mono-/di-/tri-carboxylic acids, glyceric acids,
          glycolate, glycerate, glyceraldehyde-3-phosphate, di-hydroxyacetone-phosphate and CO<sub
            itemscope="" itemtype="http://schema.stenci.la/Subscript"><span
              data-itemtype="http://schema.org/Number">2</span></sub>, see Materials and methods for
          details. Since CBM is limited to static model analysis, we introduced two Rubisco
          populations in the bundle sheath network to approximate CO<sub itemscope=""
            itemtype="http://schema.stenci.la/Subscript"><span
              data-itemtype="http://schema.org/Number">2</span></sub> concentration-dependent
          changes in the oxygenation : carboxylation ratio of Rubisco (<span itemscope=""
            itemtype="http://schema.stenci.la/MathFragment"><span class="mjx-chtml"><span
                class="mjx-math" aria-label="{v}_{RBO}/{v}_{RBC}"><span class="mjx-mrow"
                  aria-hidden="true"><span class="mjx-msubsup"><span class="mjx-base"><span
                        class="mjx-texatom"><span class="mjx-mrow"><span class="mjx-mi"><span
                              class="mjx-char MJXc-TeX-math-I"
                              style="padding-top: 0.225em; padding-bottom: 0.298em;">v</span></span></span></span></span><span
                      class="mjx-sub"
                      style="font-size: 70.7%; vertical-align: -0.229em; padding-right: 0.071em;"><span
                        class="mjx-texatom" style=""><span class="mjx-mrow"><span
                            class="mjx-mi"><span class="mjx-char MJXc-TeX-math-I"
                              style="padding-top: 0.446em; padding-bottom: 0.298em;">R</span></span><span
                            class="mjx-mi"><span class="mjx-char MJXc-TeX-math-I"
                              style="padding-top: 0.446em; padding-bottom: 0.298em;">B</span></span><span
                            class="mjx-mi"><span class="mjx-char MJXc-TeX-math-I"
                              style="padding-top: 0.519em; padding-bottom: 0.298em;">O</span></span></span></span></span></span><span
                    class="mjx-texatom"><span class="mjx-mrow"><span class="mjx-mo"><span
                          class="mjx-char MJXc-TeX-main-R"
                          style="padding-top: 0.446em; padding-bottom: 0.593em;">/</span></span></span></span><span
                    class="mjx-msubsup"><span class="mjx-base"><span class="mjx-texatom"><span
                          class="mjx-mrow"><span class="mjx-mi"><span
                              class="mjx-char MJXc-TeX-math-I"
                              style="padding-top: 0.225em; padding-bottom: 0.298em;">v</span></span></span></span></span><span
                      class="mjx-sub"
                      style="font-size: 70.7%; vertical-align: -0.23em; padding-right: 0.071em;"><span
                        class="mjx-texatom" style=""><span class="mjx-mrow"><span
                            class="mjx-mi"><span class="mjx-char MJXc-TeX-math-I"
                              style="padding-top: 0.446em; padding-bottom: 0.298em;">R</span></span><span
                            class="mjx-mi"><span class="mjx-char MJXc-TeX-math-I"
                              style="padding-top: 0.446em; padding-bottom: 0.298em;">B</span></span><span
                            class="mjx-mi"><span class="mjx-char MJXc-TeX-math-I"
                              style="padding-top: 0.519em; padding-bottom: 0.298em; padding-right: 0.045em;">C</span></span></span></span></span></span></span></span></span></span>)
          itself. We kept the native constrained Rubisco population that is forced to undertake
          oxygenation reactions and added a CCM-dependent Rubisco population which can only
          carboxylate ribulose 1,5-bisphosphate. The CCM-dependent Rubisco population is only able
          to use CO<sub itemscope="" itemtype="http://schema.stenci.la/Subscript"><span
              data-itemtype="http://schema.org/Number">2</span></sub> produced by the bundle sheath
          network but not environmental CO<sub itemscope=""
            itemtype="http://schema.stenci.la/Subscript"><span
              data-itemtype="http://schema.org/Number">2</span></sub> released by the mesophyll. C4
          plants have a higher CO<sub itemscope=""
            itemtype="http://schema.stenci.la/Subscript"><span
              data-itemtype="http://schema.org/Number">2</span></sub> consumption and thus, an
          increased CO<sub itemscope="" itemtype="http://schema.stenci.la/Subscript"><span
              data-itemtype="http://schema.org/Number">2</span></sub> uptake of
          <stencila-code-expression programming-language="py" itemscope=""
            itemtype="http://schema.stenci.la/CodeExpression"><code class="py"
              slot="text">f_C4_CO2_M</code><output slot="output"></output>
          </stencila-code-expression> μmol/(m<sup itemscope=""
            itemtype="http://schema.stenci.la/Superscript"><span
              data-itemtype="http://schema.org/Number">2</span></sup>s) was allowed <cite
            itemscope="" itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a
              href="#bib38"><span>38</span><span>Leakey et al.</span><span>2006</span></a></cite>.
          All other constraints and the objective of the <em itemscope=""
            itemtype="http://schema.stenci.la/Emphasis">one-cell</em> model are maintained in the
          <em itemscope="" itemtype="http://schema.stenci.la/Emphasis">two-cell</em> model, see <a
            href="#fig2" itemscope="" itemtype="http://schema.stenci.la/Link">Figure 2</a>.</p>
        <figure itemscope="" itemtype="http://schema.stenci.la/Figure" id="fig2" title="Figure 2.">
          <label data-itemprop="label">Figure 2.</label><img src="index.html.media/fig2.jpg" alt=""
            itemscope="" itemtype="http://schema.org/ImageObject">
          <figcaption>
            <h4 itemscope="" itemtype="http://schema.stenci.la/Heading"
              id="schematic-representation-of-the-primary-subsystems-in-the-two-cell-model-and-the-used-inputoutput-constraints-adapted-from-narrative-bib2">
              Schematic representation of the primary subsystems in the <em itemscope=""
                itemtype="http://schema.stenci.la/Emphasis">two-cell</em> model and the used
              input/output constraints; adapted from <cite itemscope=""
                itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a
                  href="#bib2"><span>2</span><span>Arnold and
                    Nikoloski</span><span>2014</span></a></cite>.</h4>
          </figcaption>
        </figure>
        <stencila-code-chunk itemscope="" itemtype="http://schema.stenci.la/CodeChunk"
          data-execution_count="28" data-programminglanguage="python">
          <pre class="language-python" itemscope="" itemtype="http://schema.stenci.la/CodeBlock"
            slot="text"><code>######################################################################################################################################
######################################################################################################################################
##############             EXPERIMENT 4: Effect of Photorespiratory Flux (Decarboxylation-Oxygenation Ratio)            ##############
######################################################################################################################################
######################################################################################################################################

print(f&quot;Code Cell 28: Experiment 4-- Effect of Photorespiratory Flux (Decarboxylation-Oxygenation Ratio)&quot;)

#Remove original c4_rbc_rbo constraints from c4 model
c4_model.remove_cons_vars(const_c4_rbc_rbo_b)
c4_model.remove_cons_vars(const_c4_rbc_rbo_m)

#Create copy of c4 model
c4_model_exp4 = c4_model.copy() 

#Reaction Variables
B_Ex_Suc = c4_model_exp4.reactions.get_by_id(&quot;[B]_Ex_Suc&quot;)

#Proportions of Decarboxylation in the RBC : RBO ratio
rbc_proportions = np.arange(1,10.25,0.25)

#Initiate dataframe to save results
df_result_exp4 = pd.DataFrame(index=c4_model.reactions.list_attr(&#39;id&#39;), columns=rbc_proportions)

#Iterate over 
for rbc_value in tqdm(rbc_proportions):
        
    #Set c4_rbc_rbo constraints
    r_c4_rbc_rbo = (float(rbc_value), 1.0)
    const_c4_rbc_rbo_b_exp4 = set_fixed_flux_ratio({&#39;[B]_RBC_h_Ex&#39;:r_c4_rbc_rbo[0],&#39;[B]_RBO_h&#39;:r_c4_rbc_rbo[1]}, &#39;const_c4_rbc_rbo_b_exp4&#39;, c4_model_exp4)
    const_c4_rbc_rbo_m_exp4 = set_fixed_flux_ratio({&#39;[M]_RBC_h&#39;:r_c4_rbc_rbo[0],&#39;[M]_RBO_h&#39;:r_c4_rbc_rbo[1]}, &#39;const_c4_rbc_rbo_m_exp4&#39;, c4_model_exp4)

    #Optimize/Maximize sucrose output
    B_Ex_Suc.objective_coefficient = 1.
    result_exp4_fba = c4_model_exp4.optimize(&#39;maximize&#39;)
        
    #Optimize/Minimize total flux
    if result_exp4_fba.status == &#39;optimal&#39;: 
        result_exp4_pfba = cobra.flux_analysis.parsimonious.pfba(c4_model_exp4)
        df_result_exp4[rbc_value] = result_exp4_pfba.fluxes       
    
    #Remove c4_rbc_rbo constraint 
    c4_model_exp4.remove_cons_vars(const_c4_rbc_rbo_b_exp4)
    c4_model_exp4.remove_cons_vars(const_c4_rbc_rbo_m_exp4)

#Reset original c4_rbc_rbo constraints in c4 model
c4_model.add_cons_vars(const_c4_rbc_rbo_b)
c4_model.add_cons_vars(const_c4_rbc_rbo_m)</code></pre>
          <figure slot="outputs">
            <pre class="language-text" itemscope="" itemtype="http://schema.stenci.la/CodeBlock"><code>Code Cell 28: Experiment 4-- Effect of Photorespiratory Flux (Decarboxylation-Oxygenation Ratio)
</code></pre>
            <pre class="language-text" itemscope=""
              itemtype="http://schema.stenci.la/CodeBlock"><code>  0%|          | 0/37 [00:00&lt;?, ?it/s]</code></pre>
          </figure>
        </stencila-code-chunk>
        <p itemscope="" itemtype="http://schema.stenci.la/Paragraph">Initially, we optimised for the
          classical objective function of minimal total flux through the metabolic network at
          different levels of photorespiration. These different levels of photorespiration integrate
          changes to external CO<sub itemscope="" itemtype="http://schema.stenci.la/Subscript"><span
              data-itemtype="http://schema.org/Number">2</span></sub> concentration and stomatal
          opening status which is governed by plant water status and biotic interactions. From the
          complete flux distribution, we extracted fluxes of PEPC and PPDK, the decarboxylation
          enzymes, Rubisco and metabolite transporter between the two cells to ascertain the
          presence of a C4 cycle, see <a href="#fig3" itemscope=""
            itemtype="http://schema.stenci.la/Link">Figure 3</a>, <a href="#fig4" itemscope=""
            itemtype="http://schema.stenci.la/Link">Figure 3</a>, and <a href="#fig3s1" itemscope=""
            itemtype="http://schema.stenci.la/Link">Figure 3—figure supplement 1</a>. At low
          photorespiratory levels, flux through PEPC is barely detectable (<a href="#fig3"
            itemscope="" itemtype="http://schema.stenci.la/Link">Figure 3(A)</a>). If
          photorespiration increases to moderate levels, flux through PEPC can be predicted and
          increases to <stencila-code-expression programming-language="py" itemscope=""
            itemtype="http://schema.stenci.la/CodeExpression"><code class="py"
              slot="text">f_C4_CO2_M</code><output slot="output"></output>
          </stencila-code-expression> μmol/(m<sup itemscope=""
            itemtype="http://schema.stenci.la/Superscript"><span
              data-itemtype="http://schema.org/Number">2</span></sup>s), that is all CO<sub
            itemscope="" itemtype="http://schema.stenci.la/Subscript"><span
              data-itemtype="http://schema.org/Number">2</span></sub> is funnelled through PEPC, for
          high photorespiratory fluxes. Concomitant with flux through PEPC, the activity of the
          decarboxylation enzymes changes (<a href="#fig3" itemscope=""
            itemtype="http://schema.stenci.la/Link">Figure 3(B)</a>). At low to intermediate levels
          of photorespiratory flux, glycine decarboxylase complex activity is predicted to shuttle
          CO<sub itemscope="" itemtype="http://schema.stenci.la/Subscript"><span
              data-itemtype="http://schema.org/Number">2</span></sub> to the bundle sheath at up to
          <stencila-code-expression programming-language="py" itemscope=""
            itemtype="http://schema.stenci.la/CodeExpression"><code class="py"
              slot="text">round(df_result_exp4.loc['[B]_GlyDH_m'].max(),2)</code><output
              slot="output"></output></stencila-code-expression> μmol/(m<sup itemscope=""
            itemtype="http://schema.stenci.la/Superscript"><span
              data-itemtype="http://schema.org/Number">2</span></sup>s). Decarboxylation of C4 acids
          is initially mostly mediated by PEP-CK and is largely taken over by NADP-ME at high fluxes
          through photorespiration. Flux through NAD-ME is very low under all photorespiration
          levels. The decarboxylation enzymes dictate flux through the different Rubiscos in the
          model (<a href="#fig3" itemscope="" itemtype="http://schema.stenci.la/Link">Figure
            3(C)</a>). At low photorespiratory flux, both the Rubiscos in mesophyll and bundle
          sheath are active. Only very little flux occurs through the CCM-dependent Rubisco, which
          is a result of the glycine decarboxylase (<a href="#fig3" itemscope=""
            itemtype="http://schema.stenci.la/Link">Figure 3(B)</a>). With increasing
          photorespiratory flux, this flux through glycine decarboxylase increases (<a href="#fig3"
            itemscope="" itemtype="http://schema.stenci.la/Link">Figure 3(B)</a>) and therefore,
          total Rubisco activity exceeds the carbon intake flux (<a href="#fig3" itemscope=""
            itemtype="http://schema.stenci.la/Link">Figure 3(C)</a>). Carbon fixation switches to
          the CCM-dependent Rubisco with increasing flux through PEPC (<a href="#fig3" itemscope=""
            itemtype="http://schema.stenci.la/Link">Figure 3(A)</a>) and the classic C4 cycle
          decarboxylation enzymes (<a href="#fig3" itemscope=""
            itemtype="http://schema.stenci.la/Link">Figure 3(B)</a>). Flux through PPDK mostly
          reflects flux through PEPC (<a href="#fig3" itemscope=""
            itemtype="http://schema.stenci.la/Link">Figure 3(D)</a>). The transport fluxes between
          the cells change with changing photosynthetic mode (<a href="#fig4" itemscope=""
            itemtype="http://schema.stenci.la/Link">Figure 4</a>).</p>
        <figure itemscope="" itemtype="http://schema.stenci.la/Figure" id="fig3" title="Figure 3.">
          <label data-itemprop="label">Figure 3.</label>
          <stencila-code-chunk itemscope="" itemtype="http://schema.stenci.la/CodeChunk"
            data-execution_count="29" data-programminglanguage="python">
            <pre class="language-python" itemscope="" itemtype="http://schema.stenci.la/CodeBlock"
              slot="text"><code>########################################################
##############           Figure 3.        ##############
########################################################

#{
#  &quot;caption&quot;: &quot;### Effect of oxygenation : carboxylation ratio on the major steps in C4 cycle, including (**A**) activity of phosphoenolpyruvate carboxylase (PEPC), (**B**) activity of Rubisco, (**C**) activity of the decarboxylation enzymes, (**D**) activity of pyruvate phosphate dikinase (PPDK)&quot;,
#  &quot;id&quot;: &quot;fig3&quot;,
#  &quot;label&quot;: &quot;Figure 3.&quot;,
#  &quot;trusted&quot;: true
#}

#Dictionary of c4 cycle reactions and their subplots
D_rxn ={
    (1,1): {&#39;rxn&#39;:[&#39;[M]_PEPC2_c&#39;, &#39;[B]_PEPC2_c&#39;], &#39;title&#39;:&#39;PEPC&#39;},
    (1,2): {&#39;rxn&#39;:[&#39;[B]_MalDH4_h&#39;, &#39;[B]_MalDH2_m&#39;, &#39;[B]_PEPC1_c&#39;, &#39;[B]_GlyDH_m&#39; ], &#39;title&#39;: &#39;Decarboxylation Enzymes&#39;},
    (2,1): {&#39;rxn&#39;:[&#39;[M]_RBC_h&#39;, &#39;[B]_RBC_h_Ex&#39;, &#39;[B]_RBC_h&#39;], &#39;title&#39;: &#39;Decarboxylation by Rubisco&#39;},
    (2,2): {&#39;rxn&#39;:[&#39;[M]_PyrPiDK_h&#39;,&#39;[B]_PyrPiDK_h&#39; ], &#39;title&#39;:&#39;PPDK&#39;}
}

#Dictionary of legend names for c4 cycle reactions
D_rxn_alias = {
    &#39;[M]_PEPC2_c&#39;: (&#39;Mesophyll&#39;,0),
    &#39;[B]_PEPC2_c&#39;: (&#39;Bundle Sheath&#39;,1),
    &#39;[B]_MalDH4_h&#39;: (&#39;NADP-ME&#39;,6),
    &#39;[B]_MalDH2_m&#39;: (&#39;NAD-ME&#39;,7),
    &#39;[B]_PEPC1_c&#39;: (&#39;PEPCK&#39;,8),
    &#39;[B]_GlyDH_m&#39;: (&#39;Gly DH&#39;,9),
    &#39;[M]_RBC_h&#39;: (&#39;Mesophyll, constrained&#39;,0), 
    &#39;[B]_RBC_h_Ex&#39;: (&#39;Bundle sheath, constrained&#39;,1), 
    &#39;[B]_RBC_h&#39;: (&#39;Bundle sheath, unconstrained&#39;,2),
    &#39;[M]_PyrPiDK_h&#39;: (&#39;Mesophyll&#39;,0),
    &#39;[B]_PyrPiDK_h&#39;:(&#39;Bundle Sheath&#39;,1)
    
}

#Create figure with subplots
fig_exp41 = make_subplots(rows=2, 
                    cols=2, 
                    subplot_titles=[f&quot;&lt;b&gt;({list(string.ascii_uppercase)[i]}) {info[&#39;title&#39;]}&lt;/b&gt;&quot; for i, info in enumerate(D_rxn.values())],
                    y_title= &#39;Flux [µmol/(m\u00B2s)]&#39;,
                    x_title= &#39;Rubisco Carboxylation : Oxygenation Ratio&#39;,
                    #horizontal_spacing=0,
                    vertical_spacing=0.1,
                    specs=[[{},{}],[{},{}]]
                    
                   )

#Add traces to the figure
for i, item in enumerate(D_rxn.items()):
    
    pos_x_y = item[0]
    info = item[1]
    
    #Add empty trace to group legend by title
    trace = go.Scatter(
        x = [None],
        y = [None],
        name = f&quot;&lt;b&gt;({list(string.ascii_uppercase)[i]}) {info[&#39;title&#39;]}&lt;/b&gt;&quot;,
        legendgroup = info[&#39;title&#39;],
        mode = &#39;markers&#39;,
        marker = dict(color=&#39;black&#39;, symbol=&#39;triangle-right&#39;)
    )
    fig_exp41.add_trace(trace,pos_x_y[0],pos_x_y[1])
    
    for r_id in info[&#39;rxn&#39;]:
        
        #Add trace with an a reaction group
        trace = go.Bar(
            x = df_result_exp4.columns,
            y = abs(df_result_exp4.loc[r_id]),
            name = D_rxn_alias[r_id][0],
            legendgroup=info[&#39;title&#39;],
            marker=dict(color = plotly.colors.DEFAULT_PLOTLY_COLORS[D_rxn_alias[r_id][1]])
        )
        fig_exp41.add_trace(trace,pos_x_y[0],pos_x_y[1])

#Update xaxes
fig_exp41.update_xaxes(
    tickfont=dict(size=16),
    ticksuffix=&#39; : 1&#39;,
    tickangle=25,
    title=dict(font=dict(size=18))
)

#Update yaxes
fig_exp41.update_yaxes(
    tickfont=dict(size=16),
    title=dict(font=dict(size=18))
)

#Update annotations
fig_exp41.update_annotations(font=dict(size=18))

#Update layout
fig_exp41.update_layout(
    barmode=&#39;stack&#39;,
    width= 1000,
    height=800,
    legend=dict(
        orientation = &#39;v&#39;,
        tracegroupgap = 10,
        traceorder=&#39;grouped&#39;,
        font=dict(size=18),
    )
)


#Show figure
fig_exp41.show()</code></pre>
            <figure slot="outputs">
              <stencila-image-plotly>
                <picture>
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                  </script><img src="index.html.media/4" alt="" itemscope=""
                    itemtype="http://schema.org/ImageObject">
                </picture>
              </stencila-image-plotly>
            </figure>
          </stencila-code-chunk>
          <figcaption>
            <h4 itemscope="" itemtype="http://schema.stenci.la/Heading"
              id="effect-of-oxygenation--carboxylation-ratio-on-the-major-steps-in-c4-cycle-including-a-activity-of-phosphoenolpyruvate-carboxylase-pepc-b-activity-of-rubisco-c-activity-of-the-decarboxylation-enzymes-d-activity-of-pyruvate-phosphate-dikinase-ppdk">
              Effect of oxygenation : carboxylation ratio on the major steps in C4 cycle, including
              (<strong itemscope="" itemtype="http://schema.stenci.la/Strong">A</strong>) activity
              of phosphoenolpyruvate carboxylase (PEPC), (<strong itemscope=""
                itemtype="http://schema.stenci.la/Strong">B</strong>) activity of Rubisco, (<strong
                itemscope="" itemtype="http://schema.stenci.la/Strong">C</strong>) activity of the
              decarboxylation enzymes, (<strong itemscope=""
                itemtype="http://schema.stenci.la/Strong">D</strong>) activity of pyruvate phosphate
              dikinase (PPDK)</h4>
          </figcaption>
        </figure>
        <figure itemscope="" itemtype="http://schema.stenci.la/Figure" id="fig4" title="Figure 4.">
          <label data-itemprop="label">Figure 4.</label>
          <stencila-code-chunk itemscope="" itemtype="http://schema.stenci.la/CodeChunk"
            data-execution_count="30" data-programminglanguage="python">
            <pre class="language-python" itemscope="" itemtype="http://schema.stenci.la/CodeBlock"
              slot="text"><code>########################################################
##############         Figure 4.          ##############
########################################################

#(E) and (F) have been integrated into one graph

#{
#  &quot;caption&quot;: &quot;### Effect of oxygenation : carboxylation ratio on the metabolite exchange between mesophyll and bundle sheath cells in the in C4 cycle. Positive fluxes represent the transport of metabolites from the mesophyll to the bundle sheath, negative fluxes indicate the transport of metabolites from the bundle sheath to the meophyll.&quot;,
#  &quot;id&quot;: &quot;fig4&quot;,
#  &quot;label&quot;: &quot;Figure 4.&quot;,
#  &quot;trusted&quot;: true
#}

#Get exchange reaction between mesophyll and bundle sheath
rxn_mb_transport = c4_model.reactions.query(lambda rxn: (rxn.id.startswith(&#39;[MB]&#39;))).list_attr(&#39;id&#39;)

#Extract fluxes for exchange reactions
df_result_exp4_transport = df_result_exp4.loc[rxn_mb_transport]
df_result_exp4_transport = df_result_exp4_transport[(abs(df_result_exp4_transport) &gt; 0.01).sum(axis=1) &gt; 0]
df_result_exp4_transport = df_result_exp4_transport.reindex(df_result_exp4_transport.mean(axis=1).sort_values().index)

#Create Figure
fig_exp42 = go.Figure()

#Add trace
trace = go.Heatmap(
    x=df_result_exp4_transport.columns,
    y=df_result_exp4_transport.index.str.split(&#39;_&#39;,expand=True).get_level_values(1),
    z=df_result_exp4_transport,
    colorscale = &#39;RdBu_R&#39;,
    colorbar = dict(
        tickfont=dict(size=16),
        title = dict(
            text=&#39;&lt;b&gt;Flux [µmol/(m\u00B2s)]&lt;/b&gt;&#39;, 
            side=&#39;right&#39;, 
            font=dict(size=18)
        )
    )
)

fig_exp42.add_trace(trace)

#Update axes
fig_exp42.update_xaxes(
    tickfont=dict(size=16),
    ticksuffix=&#39; : 1&#39;,
    tickangle=25,
    title= dict(text=&#39;Rubisco Carboxylation : Oxygenation Ratio&#39;,font=dict(size=18))
)

#Update yaxes
fig_exp42.update_yaxes(
    tickfont=dict(size=16)
)

#Update layout
fig_exp42.update_layout(
    barmode=&#39;stack&#39;,
    width= 1000,
    height=800,
    title = dict(
        text = &#39;&lt;b&gt;(E) + (F) Transport between Mesophyll and Bundle Sheath&lt;/b&gt;&#39;,
        font = dict(size=18),
        x = 0.5
    )
    )

#Show figure
fig_exp42.show()</code></pre>
            <figure slot="outputs">
              <stencila-image-plotly>
                <picture>
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                  </script><img src="index.html.media/5" alt="" itemscope=""
                    itemtype="http://schema.org/ImageObject">
                </picture>
              </stencila-image-plotly>
            </figure>
          </stencila-code-chunk>
          <figcaption>
            <h4 itemscope="" itemtype="http://schema.stenci.la/Heading"
              id="effect-of-oxygenation--carboxylation-ratio-on-the-metabolite-exchange-between-mesophyll-and-bundle-sheath-cells-in-the-in-c4-cycle-positive-fluxes-represent-the-transport-of-metabolites-from-the-mesophyll-to-the-bundle-sheath-negative-fluxes-indicate-the-transport-of-metabolites-from-the-bundle-sheath-to-the-meophyll">
              Effect of oxygenation : carboxylation ratio on the metabolite exchange between
              mesophyll and bundle sheath cells in the in C4 cycle. Positive fluxes represent the
              transport of metabolites from the mesophyll to the bundle sheath, negative fluxes
              indicate the transport of metabolites from the bundle sheath to the meophyll.</h4>
          </figcaption>
        </figure>
        <figure itemscope="" itemtype="http://schema.stenci.la/Figure" id="fig3s1"
          title="Figure 3—figure supplement 1"><label data-itemprop="label">Figure 3—figure
            supplement 1</label>
          <stencila-code-chunk itemscope="" itemtype="http://schema.stenci.la/CodeChunk"
            data-execution_count="31" data-programminglanguage="python">
            <pre class="language-python" itemscope="" itemtype="http://schema.stenci.la/CodeBlock"
              slot="text"><code>##############################################################
##############      FIGURE 3 - Supplement 1     ##############
##############################################################

#{
#  &quot;caption&quot;: &quot;### Flux maps illustrating the effect of the oxygenation : carboxylation ratio of Rubisco on the C3-C4 trajectory. Flux maps illustrating the effect of the proportion of photorespiratory flux through Rubisco. (**A**) Low photorespiratory flux; (**B**) Moderate photorespiratory flux; and (**C**) High photorespiratory flux. (Arc width and colour are set relative to flux values in μmol/(m^2^s), grey arcs - no flux).&quot;,
#  &quot;id&quot;: &quot;fig3s1&quot;,
#  &quot;label&quot;: &quot;Figure 3—figure supplement 1&quot;,
#  &quot;trusted&quot;: true
#}

# Rubisco carboxylation proportion to display on metabolic map
show_co2_proportion = {1: &#39;High Photorespiratory Flux&#39;,3: &#39;Moderate Photorespiratory Flux&#39;, 10: &#39;Low Photorespiratory Flux&#39;}

# Build metabolic map and add flux solution for each ratio
for i, co2_proportion in enumerate(sorted(show_co2_proportion.keys(), reverse=True)):
    
    #Build map
    b = Builder(
    map_json = &#39;elife-49305.ipython.src/2018-06-29-mb-C4-RBO-RBC-Ratio.json&#39;,
    reaction_styles = [&#39;color&#39;,&#39;size&#39;, &#39;text&#39;],
    reaction_data = df_result_exp4[co2_proportion],
    reaction_scale = [
        {&#39;type&#39;: &#39;value&#39;, &#39;value&#39;: 40, &#39;color&#39;: &#39;#ff0000&#39;, &#39;size&#39;: 25},
        {&#39;type&#39;: &#39;value&#39;, &#39;value&#39;: -40, &#39;color&#39;: &#39;#ff0000&#39;, &#39;size&#39;: 25},
        {&#39;type&#39;: &#39;value&#39;, &#39;value&#39;: 20, &#39;color&#39;: &#39;#209123&#39;, &#39;size&#39;: 20},
        {&#39;type&#39;: &#39;value&#39;, &#39;value&#39;: -20, &#39;color&#39;: &#39;#209123&#39;, &#39;size&#39;: 20},
        {&#39;type&#39;: &#39;value&#39;, &#39;value&#39;: 0.01, &#39;color&#39;: &#39;#9696ff&#39;, &#39;size&#39;: 5},
        {&#39;type&#39;: &#39;value&#39;, &#39;value&#39;: -0.01, &#39;color&#39;: &#39;#9696ff&#39;, &#39;size&#39;: 5},
        {&#39;type&#39;: &#39;value&#39;, &#39;value&#39;: 0, &#39;color&#39;: &#39;#ccc&#39;, &#39;size&#39;: 3}],
    menu = False,
    height = 1000
    )
    
    #Add title
    print(f&quot;({list(string.ascii_uppercase)[i]}) {show_co2_proportion[co2_proportion]}&quot;)
    
    #Display
    display(b)</code></pre>
            <figure slot="outputs">
              <pre class="language-text" itemscope="" itemtype="http://schema.stenci.la/CodeBlock"><code>(A) Low Photorespiratory Flux
</code></pre>
              <pre><output>Builder(height=1000, menu=False, reaction_data={'[M]_PSII_h': 29.0802147928429, '[B]_PSII_h': 18.8652312755852…</output></pre>
              <pre class="language-text" itemscope="" itemtype="http://schema.stenci.la/CodeBlock"><code>(B) Moderate Photorespiratory Flux
</code></pre>
              <pre><output>Builder(height=1000, menu=False, reaction_data={'[M]_PSII_h': 39.33216931971396, '[B]_PSII_h': 18.001451340808…</output></pre>
              <pre class="language-text" itemscope="" itemtype="http://schema.stenci.la/CodeBlock"><code>(C) High Photorespiratory Flux
</code></pre>
              <pre><output>Builder(height=1000, menu=False, reaction_data={'[M]_PSII_h': 24.226697390955252, '[B]_PSII_h': 18.17818261340…</output></pre>
            </figure>
          </stencila-code-chunk>
          <figcaption>
            <h4 itemscope="" itemtype="http://schema.stenci.la/Heading"
              id="flux-maps-illustrating-the-effect-of-the-oxygenation--carboxylation-ratio-of-rubisco-on-the-c3-c4-trajectory-flux-maps-illustrating-the-effect-of-the-proportion-of-photorespiratory-flux-through-rubisco-a-low-photorespiratory-flux-b-moderate-photorespiratory-flux-and-c-high-photorespiratory-flux-arc-width-and-colour-are-set-relative-to-flux-values-in-μmolm2s-grey-arcs---no-flux">
              Flux maps illustrating the effect of the oxygenation : carboxylation ratio of Rubisco
              on the C3-C4 trajectory. Flux maps illustrating the effect of the proportion of
              photorespiratory flux through Rubisco. (<strong itemscope=""
                itemtype="http://schema.stenci.la/Strong">A</strong>) Low photorespiratory flux;
              (<strong itemscope="" itemtype="http://schema.stenci.la/Strong">B</strong>) Moderate
              photorespiratory flux; and (<strong itemscope=""
                itemtype="http://schema.stenci.la/Strong">C</strong>) High photorespiratory flux.
              (Arc width and colour are set relative to flux values in μmol/(m<sup itemscope=""
                itemtype="http://schema.stenci.la/Superscript"><span
                  data-itemtype="http://schema.org/Number">2</span></sup>s), grey arcs - no flux).
            </h4>
          </figcaption>
        </figure>
        <p itemscope="" itemtype="http://schema.stenci.la/Paragraph">At low rates of
          photorespiration when PEPC is barely active, the only flux towards the bundle sheath is
          CO<sub itemscope="" itemtype="http://schema.stenci.la/Subscript"><span
              data-itemtype="http://schema.org/Number">2</span></sub> diffusion with no fluxes
          towards the mesophyll (<a href="#fig4" itemscope=""
            itemtype="http://schema.stenci.la/Link">Figure 4</a>). In the intermediate phase
          glycolate and glycerate are predicted to be transported and a low-level C4 cycle dependent
          on the transport of aspartate, malate, PEP and alanine operates (<a href="#fig4"
            itemscope="" itemtype="http://schema.stenci.la/Link">Figure 4</a>). In case of high
          photorespiratory rates, the exchange between mesophyll and bundle sheath is mainly carried
          by malate and pyruvate (<a href="#fig4" itemscope=""
            itemtype="http://schema.stenci.la/Link">Figure 4</a>). Flux through PPDK (<a
            href="#fig3" itemscope="" itemtype="http://schema.stenci.la/Link">Figure 3(D)</a>) is
          lower than flux through PEPC (<a href="#fig3" itemscope=""
            itemtype="http://schema.stenci.la/Link">Figure 3(A)</a>) at the intermediate stage.
          Evolution of C4 photosynthesis with NADP-ME as the major decarboxylation enzyme is
          predicted if the photorespiratory flux is high and model optimised for minimal total flux,
          in other words, resource limitation.</p>
        <h3 itemscope="" itemtype="http://schema.stenci.la/Heading"
          id="c4-modes-with-different-decarboxylation-enzymes-result-from-different-set-of-constraints">
          C4 modes with different decarboxylation enzymes result from different set of constraints
        </h3>
        <stencila-code-chunk itemscope="" itemtype="http://schema.stenci.la/CodeChunk"
          data-execution_count="32" data-programminglanguage="python">
          <pre class="language-python" itemscope="" itemtype="http://schema.stenci.la/CodeBlock"
            slot="text"><code>######################################################################################################################################
######################################################################################################################################
##############                        EXPERIMENT 5: Effect of Decarboxylation Enzymes on the c4 mode                    ##############
######################################################################################################################################
######################################################################################################################################

print(f&quot;Code Cell 32: Experiment 5 -- Effect of Decarboxylation Enzymes on the c4 mode&quot;)
    
#Create copy of c4 model
c4_model_exp5 = c4_model.copy() 

#Reaction variables
B_Ex_Suc = c4_model_exp5.reactions.get_by_id(&quot;[B]_Ex_Suc&quot;)
B_RBO = c4_model_exp5.reactions.get_by_id(&quot;[B]_RBO_h&quot;)
M_RBO = c4_model_exp5.reactions.get_by_id(&quot;[M]_RBO_h&quot;)

#Decarboxylation reactions ids and names of enzymes
c4_mode_r_id_enzyme = {&#39;[B]_MalDH4_h&#39;: &#39;NADP-ME&#39;,  &#39;[B]_PEPC1_c&#39;: &#39;PEP-CK&#39;, &#39;[B]_MalDH2_m&#39;: &#39;NAD-ME&#39;,}

#Set flux through decarboxylation enzymes two zero 
for c4_mode_r_id in c4_mode_r_id_enzyme.keys():
    set_fixed_flux(c4_mode_r_id, 0, c4_model_exp5)

#Initialise data frames to hold results
df_result_exp5_pfba = pd.DataFrame(index=c4_model.reactions.list_attr(&#39;id&#39;), columns=c4_mode_r_id_enzyme.keys(), dtype=&#39;float64&#39;)
df_result_exp5_pfva = pd.DataFrame(index=c4_model.reactions.list_attr(&#39;id&#39;), columns=c4_mode_r_id_enzyme.keys(), dtype=&#39;float64&#39;)

#Get exchange reaction between mesophyll and bundle sheath
rxn_mb_transport = c4_model.reactions.query(lambda rxn: (rxn.id.startswith(&#39;[MB]&#39;))).list_attr(&#39;id&#39;)

#Perform FBA experiment for each decarboxylation enzyme
for c4_mode_r_id in tqdm(c4_mode_r_id_enzyme.keys()):
    
    #Allow non-zero flux for current decarboxylation enzyme
    set_bounds(c4_mode_r_id, (0,inf), c4_model_exp5)   
    
    #Optimization - Maximize sucrose output &amp; Minimize Oxygenation by Rubisco
    B_Ex_Suc.objective_coefficient = 1.
    B_RBO.objective_coefficient = -1.
    M_RBO.objective_coefficient = -1.
    result_exp5_fba = c4_model_exp5.optimize(&#39;maximize&#39;)
        
    #Optimize/Minimize total flux
    if result_exp5_fba.status == &#39;optimal&#39;: 
        
        result_exp5_pfba = cobra.flux_analysis.parsimonious.pfba(c4_model_exp5)
        df_result_exp5_pfba[c4_mode_r_id] = result_exp5_pfba.fluxes
        
        pfba_factor = 0.015
        
        result_exp5_pfva = cobra.flux_analysis.flux_variability_analysis(c4_model_exp5,reaction_list=rxn_mb_transport, pfba_factor= 1 + pfba_factor) 
        df_result_exp5_pfva[c4_mode_r_id] = result_exp5_pfva.apply(lambda x: (x[0], x[1]), axis=1)

    #Reset flux of current decarboxylation enzyme to zero
    set_fixed_flux(c4_mode_r_id, 0, c4_model_exp5)</code></pre>
          <figure slot="outputs">
            <pre class="language-text" itemscope="" itemtype="http://schema.stenci.la/CodeBlock"><code>Code Cell 32: Experiment 5 -- Effect of Decarboxylation Enzymes on the c4 mode
</code></pre>
            <pre class="language-text" itemscope=""
              itemtype="http://schema.stenci.la/CodeBlock"><code>  0%|          | 0/3 [00:00&lt;?, ?it/s]</code></pre>
          </figure>
        </stencila-code-chunk>
        <p itemscope="" itemtype="http://schema.stenci.la/Paragraph">Among the known independent
          evolutionary events leading to C4 photosynthesis, 20 are towards NAD-ME while 21 occurred
          towards NADP-ME <cite itemscope="" itemtype="http://schema.stenci.la/Cite"
            data-citationmode="Narrative"><a
              href="#bib62"><span>62</span><span>Sage</span><span>2004</span></a></cite>. PEP-CK is
          dominant or at least co-dominant only in <em itemscope=""
            itemtype="http://schema.stenci.la/Emphasis">Panicum maximum</em><span
            data-itemtype="http://schema.org/Number">0</span><cite itemscope=""
            itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a
              href="#bib12"><span>12</span><span>Bräutigam et
                al.</span><span>2014</span></a></cite>, <em itemscope=""
            itemtype="http://schema.stenci.la/Emphasis">Alloteropsis semialata semialata</em><span
            data-itemtype="http://schema.org/Number">0</span><cite itemscope=""
            itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a
              href="#bib18"><span>18</span><span>Christin et al.</span><span>2012</span></a></cite>,
          and in the <em itemscope=""
            itemtype="http://schema.stenci.la/Emphasis">Chloridoideae</em><span
            data-itemtype="http://schema.org/Number">0</span><cite itemscope=""
            itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a
              href="#bib62"><span>62</span><span>Sage</span><span>2004</span></a></cite>. To analyse
          whether the predicted evolution of the C4 cycle is independent of a particular
          decarboxylation enzyme, we performed three separate experiments, where only one
          decarboxylation enzyme can be active at a time. The other decarboxylation enzymes were
          de-activated by constraining the reaction flux to zero resulting in three different
          predictions, one for each decarboxylation enzyme. The flux distributions obtained under
          the assumption of oxygenation : carboxylation ratio of 1 : 3 and minimisation of
          photorespiration as an additional objective predicts the emergence of a C4 cycle for each
          known decarboxylation enzyme. To visualise the possible C4 fluxes, the flux distribution
          for candidate C4 cycle enzymes was extracted from each of the three predictions and
          visualised as arc width and color (<a href="#fig4" itemscope=""
            itemtype="http://schema.stenci.la/Link">Figure 4</a>). While the flux distribution in
          the mesophyll is identical for three predicted C4 cycles of the decarboxylation enzymes,
          it is diverse in the bundle sheath due to the different localisation of the
          decarboxylation and related transport processes, see <a href="#fig4" itemscope=""
            itemtype="http://schema.stenci.la/Link">Figure 4</a>. The flux distribution does not
          completely mimic the variation in transfer acids known from laboratory experiments <cite
            itemscope="" itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a
              href="#bib30"><span>30</span><span>Hatch</span><span>1987</span></a></cite> since all
          of the decarboxylation enzymes use the malate/pyruvate shuttle. In the case of NAD-ME and
          PEP-CK, the <em itemscope="" itemtype="http://schema.stenci.la/Emphasis">two-cell</em>
          model also predicts a supplementary flux through the aspartate/alanine shuttle. We tested
          whether transfer acids other than malate and pyruvate are feasible and explored the
          near-optimal space. To this end, the model predictions are repeated, allowing deviation
          from the optimal solution and the changes recorded. Deviations from the optimal solution
          are visualised as error bars (<a href="#fig5" itemscope=""
            itemtype="http://schema.stenci.la/Link">Figure 5</a>). Performing a flux variability
          analysis (FVA) and allowing the minimal total flux to differ by <stencila-code-expression
            programming-language="py" itemscope=""
            itemtype="http://schema.stenci.la/CodeExpression"><code class="py"
              slot="text">pfba_factor * 100</code><output slot="output"></output>
          </stencila-code-expression>%, predicts that for most metabolites which are transferred
          between mesophyll and bundle sheath, the variability is similar for all three
          decarboxylation types. For the NAD-ME and PEP-CK types, changes in the near-optimal space
          were observed for the transfer acids malate, aspartate, pyruvate and alanine. Minor
          differences were present for triose phosphates and phosphoglycerates as well as for PEP.
          For the NADP-ME type, FVA identifies only minor variation (<a href="#fig5" itemscope=""
            itemtype="http://schema.stenci.la/Link">Figure 5</a>). In the case of NAD-ME but not in
          the case of NADP-ME the activity of the malate/pyruvate shuttle can be taken over by the
          aspartate/alanine shuttle and partly taken over in case of PEP-CK, see <a href="#fig5"
            itemscope="" itemtype="http://schema.stenci.la/Link">Figure 5</a>. The aspartate/alanine
          shuttle is thus only a near-optimal solution when the model and by proxy evolutionary
          constraints are resource efficiency and minimal photorespiration.</p>
        <figure itemscope="" itemtype="http://schema.stenci.la/Figure" id="fig5" title="Figure 5.">
          <label data-itemprop="label">Figure 5.</label>
          <stencila-code-chunk itemscope="" itemtype="http://schema.stenci.la/CodeChunk"
            data-execution_count="33" data-programminglanguage="python">
            <pre class="language-python" itemscope="" itemtype="http://schema.stenci.la/CodeBlock"
              slot="text"><code>########################################################
##############          Figure 5.         ##############
########################################################

#{
#  &quot;caption&quot;: &quot;### Flux maps illustrating the effect of the C4 mode. (**A**) NADP-ME, (**B**) PEP-CK, (**C**) NAD-ME.  (Arc width and colour are set relative to flux values in μmol/(m^2^s), grey arcs - no flux).&quot;,
#  &quot;id&quot;: &quot;fig5&quot;,
#  &quot;label&quot;: &quot;Figure 5.&quot;,
#  &quot;trusted&quot;: true
#}

# Build metabolic map and add flux solution for decarboxylation enzyme
for i, c4_mode_r_id in enumerate(c4_mode_r_id_enzyme.keys()):
    
    #Build map
    b = Builder(
    map_json = &#39;elife-49305.ipython.src/2018-06-29-mb-C4-Map-Decarb-Enzymes.json&#39;,
    reaction_styles = [&#39;color&#39;,&#39;size&#39;, &#39;text&#39;],
    reaction_data = df_result_exp5_pfba[c4_mode_r_id],
    reaction_scale = [
        {&#39;type&#39;: &#39;value&#39;, &#39;value&#39;: 40, &#39;color&#39;: &#39;#ff0000&#39;, &#39;size&#39;: 25},
        {&#39;type&#39;: &#39;value&#39;, &#39;value&#39;: -40, &#39;color&#39;: &#39;#ff0000&#39;, &#39;size&#39;: 25},
        {&#39;type&#39;: &#39;value&#39;, &#39;value&#39;: 20, &#39;color&#39;: &#39;#209123&#39;, &#39;size&#39;: 20},
        {&#39;type&#39;: &#39;value&#39;, &#39;value&#39;: -20, &#39;color&#39;: &#39;#209123&#39;, &#39;size&#39;: 20},
        {&#39;type&#39;: &#39;value&#39;, &#39;value&#39;: 0.01, &#39;color&#39;: &#39;#9696ff&#39;, &#39;size&#39;: 5},
        {&#39;type&#39;: &#39;value&#39;, &#39;value&#39;: -0.01, &#39;color&#39;: &#39;#9696ff&#39;, &#39;size&#39;: 5},
        {&#39;type&#39;: &#39;value&#39;, &#39;value&#39;: 0, &#39;color&#39;: &#39;#ccc&#39;, &#39;size&#39;: 3}],
    menu = False,
    height = 1000
    )
    
    #Add title
    print(f&quot;({list(string.ascii_uppercase)[i]}) {c4_mode_r_id_enzyme[c4_mode_r_id]}&quot;)
    
    #Display
    display(b)</code></pre>
            <figure slot="outputs">
              <pre class="language-text" itemscope="" itemtype="http://schema.stenci.la/CodeBlock"><code>(A) NADP-ME
</code></pre>
              <pre><output>Builder(height=1000, menu=False, reaction_data={'[M]_PSII_h': 24.504287664634546, '[B]_PSII_h': 17.85320161911…</output></pre>
              <pre class="language-text" itemscope="" itemtype="http://schema.stenci.la/CodeBlock"><code>(B) PEP-CK
</code></pre>
              <pre><output>Builder(height=1000, menu=False, reaction_data={'[M]_PSII_h': 24.049836369752107, '[B]_PSII_h': 19.40600140368…</output></pre>
              <pre class="language-text" itemscope="" itemtype="http://schema.stenci.la/CodeBlock"><code>(C) NAD-ME
</code></pre>
              <pre><output>Builder(height=1000, menu=False, reaction_data={'[M]_PSII_h': 23.350924803251964, '[B]_PSII_h': 19.36547262051…</output></pre>
            </figure>
          </stencila-code-chunk>
          <figcaption>
            <h4 itemscope="" itemtype="http://schema.stenci.la/Heading"
              id="flux-maps-illustrating-the-effect-of-the-c4-mode-a-nadp-me-b-pep-ck-c-nad-me--arc-width-and-colour-are-set-relative-to-flux-values-in-μmolm2s-grey-arcs---no-flux">
              Flux maps illustrating the effect of the C4 mode. (<strong itemscope=""
                itemtype="http://schema.stenci.la/Strong">A</strong>) NADP-ME, (<strong itemscope=""
                itemtype="http://schema.stenci.la/Strong">B</strong>) PEP-CK, (<strong itemscope=""
                itemtype="http://schema.stenci.la/Strong">C</strong>) NAD-ME. (Arc width and colour
              are set relative to flux values in μmol/(m<sup itemscope=""
                itemtype="http://schema.stenci.la/Superscript"><span
                  data-itemtype="http://schema.org/Number">2</span></sup>s), grey arcs - no flux).
            </h4>
          </figcaption>
        </figure>
        <figure itemscope="" itemtype="http://schema.stenci.la/Figure" id="fig5" title="Figure 5.">
          <label data-itemprop="label">Figure 5.</label>
          <stencila-code-chunk itemscope="" itemtype="http://schema.stenci.la/CodeChunk"
            data-execution_count="34" data-programminglanguage="python">
            <pre class="language-python" itemscope="" itemtype="http://schema.stenci.la/CodeBlock"
              slot="text"><code>########################################################
##############          Figure 6.         ##############
########################################################

#{
#  &quot;caption&quot;: &quot;### Flux variability analysis of metabolite exchange with 1.5% deviation of the total flux minimum. The upper bar defines the maximum exchange flux, while the lower bar defines the minimum exchange flux, points indicate the value of the original flux solution under minimal metabolic effort constraint. Positive flux values correspond to the transport direction from mesophyll to bundle sheath, negative values to the transport direction from bundle sheath to mesophyll.&quot;,
#  &quot;id&quot;: &quot;fig6&quot;,
#  &quot;label&quot;: &quot;Figure 6.&quot;,
#  &quot;trusted&quot;: true
#}

#Create figure with subplots
fig_exp5 = make_subplots(
    rows = 3,
    cols = 1,
    subplot_titles = [f&quot;&lt;b&gt;FVA with {enzyme} (pFBA Factor = 1.5 %)&lt;/b&gt;&quot; for enzyme in c4_mode_r_id_enzyme.values()],
    y_title = &#39;Flux [µmol/(m\u00B2s)]&#39;,
    x_title = &#39;Exchange Metabolites&#39;,
    vertical_spacing=0.1
)

#Get exchange reaction between mesophyll and bundle sheath
rxn_mb_transport = c4_model.reactions.query(lambda rxn: (rxn.id.startswith(&#39;[MB]&#39;))).list_attr(&#39;id&#39;)

#Add subplot for each decarboxylation enzyme
for i, c4_mode_r_id in enumerate(c4_mode_r_id_enzyme.keys()):
    
    #Create trace
    trace = go.Scatter(
        x = df_result_exp5_pfba.loc[rxn_mb_transport,:].index.str.split(&#39;_&#39;,expand=True).get_level_values(1), 
        y = df_result_exp5_pfba.loc[rxn_mb_transport,c4_mode_r_id],
        mode = &#39;markers&#39;,
        name = c4_mode_r_id,
        error_y = dict(
            type = &#39;data&#39;, 
            symmetric = False,
            array = df_result_exp5_pfva.loc[rxn_mb_transport,c4_mode_r_id].apply(lambda x: x[1]) - df_result_exp5_pfba.loc[rxn_mb_transport,c4_mode_r_id],
            arrayminus = df_result_exp5_pfba.loc[rxn_mb_transport,c4_mode_r_id] - df_result_exp5_pfva.loc[rxn_mb_transport,c4_mode_r_id].apply(lambda x: x[0]),
        )
    )
    
    #Add trace
    fig_exp5.add_trace(trace,i+1,1)
    
    #Add annotation - postive flux: transport from mesophyll to bundle sheath
    anno_ms2bs = dict(
        x=-1,
        y=25,
        xref=f&quot;x{i+1}&quot;,
        yref=f&quot;y{i+1}&quot;,
        text=  u&quot;[M] \u2192 [BS]&quot;,
        textangle= -90,
        showarrow=False,
        font=dict(
            size=6,
            color=&#39;#000000&#39;
        ),
        align=&#39;center&#39;,         
    )
    
    fig_exp5.add_annotation(anno_ms2bs)
    
    #Add annotation - negative flux: transport from  bundle sheath to mesophyll
    anno_ms2bs = dict(
        x=-1,
        y=-25,
        xref=f&quot;x{i+1}&quot;,
        yref=f&quot;y{i+1}&quot;,
        text= &#39;[BS] \u2192 [M]&#39;,
        textangle= -90,
        showarrow=False,
        font=dict(
            size=6,
            color=&#39;#000000&#39;
        ),
        align=&#39;center&#39;,         
    )
    
    fig_exp5.add_annotation(anno_ms2bs)

#Update xaxes
fig_exp5.update_xaxes(
    tickangle = 35,
    tickfont=dict(size=16)
)

#Update yaxes
fig_exp5.update_yaxes(
    range = [-50, 50],
    tickfont=dict(size=16)
)

#Update layout
fig_exp5.update_layout(
    width = 1000,
    height = 1000,
    showlegend = False
)

#Update annotations
fig_exp5.update_annotations(font=dict(size=18))

#Show figure  
fig_exp5.show()</code></pre>
            <figure slot="outputs">
              <stencila-image-plotly>
                <picture>
                  <script type="application/vnd.plotly.v1+json">
                    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                  </script><img src="index.html.media/6" alt="" itemscope=""
                    itemtype="http://schema.org/ImageObject">
                </picture>
              </stencila-image-plotly>
            </figure>
          </stencila-code-chunk>
          <figcaption>
            <p itemscope="" itemtype="http://schema.stenci.la/Paragraph">Flux variability analysis
              of metabolite exchange with 1.5% deviation of the total flux minimum. The upper bar
              defines the maximum exchange flux, while the lower bar defines the minimum exchange
              flux, points indicate the value of the original flux solution under minimal metabolic
              effort constraint. Positive flux values correspond to the transport direction from
              mesophyll to bundle sheath, negative values to the transport direction from bundle
              sheath to mesophyll.</p>
          </figcaption>
        </figure>
        <stencila-code-chunk itemscope="" itemtype="http://schema.stenci.la/CodeChunk" data-tags=""
          data-execution_count="35" data-programminglanguage="python">
          <pre class="language-python" itemscope="" itemtype="http://schema.stenci.la/CodeBlock"
            slot="text"><code>######################################################################################################################################
######################################################################################################################################
##############         EXPERIMENT 6: Effect of PPFD and Photon Distribution betwenn Mesophyll and Bundle Sheath         ##############
######################################################################################################################################
######################################################################################################################################

print(f&quot;Code Cell 35: Experiment 6 -- Effect of PPFD and Photon Distribution betwenn Mesophyll and Bundle Sheath&quot;)

#Remove original constraints on PPFD from c4 model
c4_model.remove_cons_vars(const_hnu_sum) 
c4_model.remove_cons_vars(const_hnu_ratio) 

#Create copy of c4 model
c4_model_exp6 = c4_model.copy() 

#Reaction variables
B_Ex_Suc = c4_model_exp6.reactions.get_by_id(&quot;[B]_Ex_Suc&quot;)
M_Im_hnu = c4_model_exp6.reactions.get_by_id(&quot;[M]_Im_hnu&quot;)
B_Im_hnu = c4_model_exp6.reactions.get_by_id(&quot;[B]_Im_hnu&quot;)
B_RBO = c4_model_exp6.reactions.get_by_id(&quot;[B]_RBO_h&quot;)
M_RBO = c4_model_exp6.reactions.get_by_id(&quot;[M]_RBO_h&quot;)

#Decarboxylation reactions ids and names of enzymes
c4_mode_r_id_enzyme = {&#39;[B]_MalDH4_h&#39;: &#39;NADP-ME&#39;, &#39;[B]_PEPC1_c&#39;: &#39;PEP-CK&#39;, &#39;[B]_MalDH2_m&#39;: &#39;NAD-ME&#39;, }

#Porportions of light hitting the mesophyll
#ds_prop_light_m = pd.Series(np.arange(0.5,10.1,0.1))
ds_prop_light_m = pd.Series(np.concatenate((np.arange(0.5,1.,0.1, dtype=&#39;float64&#39;),np.arange(1,11,1)),axis = 0))
min_ppfd_bs_m = 1/ds_prop_light_m.max()
max_ppfd_bs_m = 1/ds_prop_light_m.min()


#Maximum light uptake
hnu_max = 1000

#Total PPFD
#ds_ppfd = pd.Series(np.arange(0,hnu_max+50,50))
ds_ppfd = pd.Series(sorted(np.concatenate((np.array([0,50,150]),np.arange(100,hnu_max+100,100)), axis=None)))


#Initialise dataframes to hold results
light_index = pd.MultiIndex.from_product([ds_ppfd.values,ds_prop_light_m.values, ], names=[&quot;PPFD&quot;, &quot;prop_light_M&quot;])
df_result_exp6 = pd.DataFrame(index=c4_model.reactions.list_attr(&#39;id&#39;), columns=light_index, dtype=&#39;float64&#39;)

#Test all combinations of PPFD and photon distribution
for light in tqdm(light_index):

    #Add constraint on PPFD
    ppfd = light[0]
    const_hnu1_exp6 = c4_model_exp6.problem.Constraint( 
        1.0 * M_Im_hnu.flux_expression +  1.0 * B_Im_hnu.flux_expression, 
        lb = float(ppfd), 
        ub = float(ppfd),
        name = &#39;const_hnu1_exp6&#39;,    
    )
    c4_model_exp6.add_cons_vars(const_hnu1_exp6)
    
    #Add contraint on light distribution
    prop = light[1]
    const_hnu2_exp6 = set_fixed_flux_ratio({&#39;[M]_Im_hnu&#39;:prop,&#39;[B]_Im_hnu&#39;:1}, &#39;const_hnu2_exp6&#39;, c4_model_exp6)
        
    #Optimization - Maximize sucrose output &amp; Minimize Oxygenation by Rubisco
    B_Ex_Suc.objective_coefficient = 1.
    B_RBO.objective_coefficient = -1.
    M_RBO.objective_coefficient = -1.
    result_exp6_fba = c4_model_exp6.optimize(&#39;maximize&#39;)
    
        
    #Optimize/Minimize total flux
    if result_exp6_fba.status == &#39;optimal&#39;: 
        
        result_exp6_pfba = cobra.flux_analysis.parsimonious.pfba(c4_model_exp6)
        df_result_exp6[light] = result_exp6_pfba.fluxes
        
   #Remove light constraints
    c4_model_exp6.remove_cons_vars(const_hnu1_exp6) 
    c4_model_exp6.remove_cons_vars(const_hnu2_exp6) 

#Add original constraints on PPFD back to c4 model 
c4_model.add_cons_vars(const_hnu_sum) 
c4_model.add_cons_vars(const_hnu_ratio)

#Transpose results
df_result_exp6 = df_result_exp6.T</code></pre>
          <figure slot="outputs">
            <pre class="language-text" itemscope="" itemtype="http://schema.stenci.la/CodeBlock"><code>Code Cell 35: Experiment 6 -- Effect of PPFD and Photon Distribution betwenn Mesophyll and Bundle Sheath
</code></pre>
            <pre class="language-text" itemscope=""
              itemtype="http://schema.stenci.la/CodeBlock"><code>  0%|          | 0/195 [00:00&lt;?, ?it/s]</code></pre>
            <pre class="language-text" itemscope="" itemtype="http://schema.stenci.la/CodeBlock"><code>/Users/blaetke/opt/anaconda3/envs/elife-49305-era/lib/python3.9/site-packages/cobra/util/solver.py:508: UserWarning:

Solver status is &#39;infeasible&#39;.

</code></pre>
          </figure>
        </stencila-code-chunk>
        <p itemscope="" itemtype="http://schema.stenci.la/Paragraph">To analyse the effect of other
          conditions on the particular C4 state, we apply the minimisation of photorespiration as an
          additional objective to minimal total flux. Since NAD-ME and PEP-CK type plants use amino
          acids as transfer acids in nature, nitrogen availability has been tagged as a possible
          evolutionary constraint that selects for decarboxylation by NAD-ME or PEP-CK. When nitrate
          uptake was limiting, the optimal solution to the model predicted overall reduced flux
          towards the phloem output (<a href="#fig8s1" itemscope=""
            itemtype="http://schema.stenci.la/Link">Figure 8—figure supplement 1</a>) but reactions
          were predicted to occur in the same proportions as predicted for unlimited nitrate uptake.
          Flux through NADP-ME and supplementary flux through PEP-CK dropped proportionally, since
          restricting nitrogen limits the export of all metabolites from the system and reduced
          CO<sub itemscope="" itemtype="http://schema.stenci.la/Subscript"><span
              data-itemtype="http://schema.org/Number">2</span></sub> uptake is observed (<a
            href="#fig8s1" itemscope="" itemtype="http://schema.stenci.la/Link">Figure 8—figure
            supplement 1</a>). Similarly, limiting water or CO<sub itemscope=""
            itemtype="http://schema.stenci.la/Subscript"><span
              data-itemtype="http://schema.org/Number">2</span></sub> uptake into the model resulted
          in overall reduced flux towards the phloem output, see <a href="#fig8s2" itemscope=""
            itemtype="http://schema.stenci.la/Link">Figure 8—figure supplement 2</a> and <a
            href="#fig8s3" itemscope="" itemtype="http://schema.stenci.la/Link">Figure 8—figure
            supplement 3</a>, but reactions were predicted to occur in the same proportions as
          predicted for unlimited uptake.</p>
        <figure itemscope="" itemtype="http://schema.stenci.la/Figure" id="fig7" title="Figure 7.">
          <label data-itemprop="label">Figure 7.</label>
          <stencila-code-chunk itemscope="" itemtype="http://schema.stenci.la/CodeChunk"
            data-execution_count="36" data-programminglanguage="python">
            <pre class="language-python" itemscope="" itemtype="http://schema.stenci.la/CodeBlock"
              slot="text"><code>########################################################
##############            FIGURE 7        ##############
########################################################

#{
#  &quot;caption&quot;: &quot;### Effect of total PPFD on CO~2~ uptake rate in C4 mode.&quot;,
#  &quot;id&quot;: &quot;fig7&quot;,
#  &quot;label&quot;: &quot;Figure 7.&quot;,
#  &quot;trusted&quot;: true
#}

#Create figure
fig_exp61 = go.Figure()

#Create trace for co2 flux
trace = go.Scatter(
    y = df_result_exp6.mean(axis=0, level=&#39;PPFD&#39;)[&#39;[M]_Im_CO2&#39;],
    x = df_result_exp6.index.get_level_values(0).unique(),
    error_y=dict(
        array = df_result_exp6.var(axis=0, level=&#39;PPFD&#39;)[&#39;[M]_Im_CO2&#39;]
    )

)

#Add trace to figure
fig_exp61.add_trace(trace)

#Update yaxes
fig_exp61.update_yaxes(
    title = dict(text=&#39;Flux [µmol/(m\u00B2s)]&#39;,font=dict(size=18)),
    tickfont=dict(size=16)
)

#Update xaxes
fig_exp61.update_xaxes(
    title = dict(text=&#39;PPFD [µE]&#39;,font=dict(size=18)),
    tickangle = 35,
    tickfont=dict(size=16),
    #type=&#39;category&#39;
)

#Update layout
fig_exp61.update_layout(
    width = 1000,
    height = 500,
    title = dict(text=&#39;&lt;b&gt;CO\u2082 Uptake&lt;/b&gt;&#39;, font=dict(size=20), x=0.5)
)

#Show figure
fig_exp61.show()</code></pre>
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            </figure>
          </stencila-code-chunk>
          <figcaption>
            <h4 itemscope="" itemtype="http://schema.stenci.la/Heading"
              id="effect-of-total-ppfd-on-co2-uptake-rate-in-c4-mode">Effect of total PPFD on CO<sub
                itemscope="" itemtype="http://schema.stenci.la/Subscript"><span
                  data-itemtype="http://schema.org/Number">2</span></sub> uptake rate in C4 mode.
            </h4>
          </figcaption>
        </figure>
        <figure itemscope="" itemtype="http://schema.stenci.la/Figure" id="fig8" title="Figure 8.">
          <label data-itemprop="label">Figure 8.</label>
          <stencila-code-chunk itemscope="" itemtype="http://schema.stenci.la/CodeChunk"
            data-execution_count="37" data-programminglanguage="python">
            <pre class="language-python" itemscope="" itemtype="http://schema.stenci.la/CodeBlock"
              slot="text"><code>########################################################
##############           FIGURE 8         ##############
########################################################

#{
#  &quot;caption&quot;: &quot;### Effect of light on the C4 mode. Heat-maps illustrating the activity of the decarboxylation enzymes PEP-CK, NADP-ME, and NAD-ME relative to the CO~2~ uptake rate in dependence of the total PPFD and the photon distribution among mesophyll and bundle sheath.&quot;,
#  &quot;id&quot;: &quot;fig8&quot;,
#  &quot;label&quot;: &quot;Figure 8.&quot;,
#  &quot;trusted&quot;: true
#}

#Create figure with subplot
fig_exp62 = make_subplots(
    rows=3, 
    cols=1, 
    subplot_titles = [f&quot;&lt;b&gt;{enzyme}&lt;/b&gt;&quot; for enzyme in c4_mode_r_id_enzyme.values()],
    y_title= &#39;PPFD [BS] : PPFD [M]&#39;,
    x_title = &#39;Total PPFD [µE]&#39;,
    vertical_spacing = 0.1

)

#Get co2 flux for all combinations
df_im_co2 = df_result_exp6[&#39;[M]_Im_CO2&#39;].reset_index().pivot_table(index=&#39;PPFD&#39;, columns=&#39;prop_light_M&#39;, aggfunc=&#39;mean&#39;)

#Get low light threshold
low_light_thres = df_im_co2[((df_im_co2.round(1) &lt; df_im_co2.round(1).max().max()).sum(axis=1) != 0)].index.max()

#Save heatmap dataframes
dfs_r_id_co2 = {}

#Add subplot for each decarboxylation enzyme
for i, r_id in enumerate(c4_mode_r_id_enzyme.keys()):
    
    #Get decarboxylation flux of current enzyme for all combinations
    df_r_id = df_result_exp6[r_id].reset_index().pivot_table(index=&#39;PPFD&#39;, columns=&#39;prop_light_M&#39;, aggfunc=&#39;mean&#39;) 
    
    dfs_r_id_co2[r_id] = pd.DataFrame(df_r_id.values/df_im_co2.values, index = df_r_id.index, columns = df_r_id.columns)
    
    #Create trace
    trace = go.Heatmap(
        z = (df_r_id.values/df_im_co2.values).T,
        x = df_im_co2.index,
        y = [round(value,1) for value in df_im_co2.columns.get_level_values(1)],
        colorbar = dict(
            title = dict(
                text = &#39;Decarboxylation Rate : CO\u2082 Uptake Rate&#39;,
                font = dict(size=18)
            ),
            xpad = 20,
            titleside = &#39;right&#39;
        ),
        zmin = 0,
        zmax = 1,
        name= c4_mode_r_id_enzyme[r_id],
        showscale = True if i == 0 else False,
    )
    
    #Add trace to figure
    fig_exp62.append_trace(trace,i+1,1)

#Max percentage of decarboylation by NADPME under low light
nadpme_max_low_light = dfs_r_id_co2[&#39;[B]_MalDH4_h&#39;].loc[:low_light_thres].max().max().round(2) * 100

#Update yaxes
fig_exp62.update_yaxes(
    tickprefix= &#39;1:&#39;,
    tickfont=dict(size=16),
    type=&#39;category&#39;
)

#Update xaxes
fig_exp62.update_xaxes(
    tickangle = 35,
    tickvals=df_im_co2.index,
    tickfont=dict(size=16),
    type=&#39;category&#39;
)

#Update annotations
fig_exp62.update_annotations(
    font=dict(size=20)
)

#Update layout
fig_exp62.update_layout(
    width = 600,
    height = 1000
)

#Show figure
fig_exp62.show()</code></pre>
            <figure slot="outputs">
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                <picture>
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                  </script><img src="index.html.media/8" alt="" itemscope=""
                    itemtype="http://schema.org/ImageObject">
                </picture>
              </stencila-image-plotly>
            </figure>
          </stencila-code-chunk>
          <figcaption>
            <h4 itemscope="" itemtype="http://schema.stenci.la/Heading"
              id="effect-of-light-on-the-c4-mode-heat-maps-illustrating-the-activity-of-the-decarboxylation-enzymes-pep-ck-nadp-me-and-nad-me-relative-to-the-co2-uptake-rate-in-dependence-of-the-total-ppfd-and-the-photon-distribution-among-mesophyll-and-bundle-sheath">
              Effect of light on the C4 mode. Heat-maps illustrating the activity of the
              decarboxylation enzymes PEP-CK, NADP-ME, and NAD-ME relative to the CO<sub
                itemscope="" itemtype="http://schema.stenci.la/Subscript"><span
                  data-itemtype="http://schema.org/Number">2</span></sub> uptake rate in dependence
              of the total PPFD and the photon distribution among mesophyll and bundle sheath.</h4>
          </figcaption>
        </figure>
        <p itemscope="" itemtype="http://schema.stenci.la/Paragraph">Given that C4 plants sometimes
          optimise light availability to the bundle sheath <cite itemscope=""
            itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a
              href="#bib8"><span>8</span><span>Bellasio and
                Lundgren</span><span>2016</span></a></cite> we next explored light availability and
          light distribution. The model prediction is re-run with changes in the constraints, and
          the resulting tables of fluxes are queried for CO<sub itemscope=""
            itemtype="http://schema.stenci.la/Subscript"><span
              data-itemtype="http://schema.org/Number">2</span></sub> uptake and fluxes through the
          decarboxylation enzymes. In the experiment, we varied the total PPFD between
          <stencila-code-expression programming-language="py" itemscope=""
            itemtype="http://schema.stenci.la/CodeExpression"><code class="py"
              slot="text">ds_ppfd.min()</code><output slot="output"></output>
          </stencila-code-expression> μmol/(m<sup itemscope=""
            itemtype="http://schema.stenci.la/Superscript"><span
              data-itemtype="http://schema.org/Number">2</span></sup>s) to <stencila-code-expression
            programming-language="py" itemscope=""
            itemtype="http://schema.stenci.la/CodeExpression"><code class="py"
              slot="text">ds_ppfd.max()</code><output slot="output"></output>
          </stencila-code-expression> μmol/(m<sup itemscope=""
            itemtype="http://schema.stenci.la/Superscript"><span
              data-itemtype="http://schema.org/Number">2</span></sup>s) and photon distribution
          <span itemscope="" itemtype="http://schema.stenci.la/MathFragment"><span
              class="mjx-chtml"><span class="mjx-math" aria-label="PPF{D}_{B}"><span
                  class="mjx-mrow" aria-hidden="true"><span class="mjx-mi"><span
                      class="mjx-char MJXc-TeX-math-I"
                      style="padding-top: 0.446em; padding-bottom: 0.298em; padding-right: 0.109em;">P</span></span><span
                    class="mjx-mi"><span class="mjx-char MJXc-TeX-math-I"
                      style="padding-top: 0.446em; padding-bottom: 0.298em; padding-right: 0.109em;">P</span></span><span
                    class="mjx-mi"><span class="mjx-char MJXc-TeX-math-I"
                      style="padding-top: 0.446em; padding-bottom: 0.298em; padding-right: 0.106em;">F</span></span><span
                    class="mjx-msubsup"><span class="mjx-base"><span class="mjx-texatom"><span
                          class="mjx-mrow"><span class="mjx-mi"><span
                              class="mjx-char MJXc-TeX-math-I"
                              style="padding-top: 0.446em; padding-bottom: 0.298em;">D</span></span></span></span></span><span
                      class="mjx-sub"
                      style="font-size: 70.7%; vertical-align: -0.212em; padding-right: 0.071em;"><span
                        class="mjx-texatom" style=""><span class="mjx-mrow"><span
                            class="mjx-mi"><span class="mjx-char MJXc-TeX-math-I"
                              style="padding-top: 0.446em; padding-bottom: 0.298em;">B</span></span></span></span></span></span></span></span></span></span>
          / <span itemscope="" itemtype="http://schema.stenci.la/MathFragment"><span
              class="mjx-chtml"><span class="mjx-math" aria-label="PPF{D}_{M}"><span
                  class="mjx-mrow" aria-hidden="true"><span class="mjx-mi"><span
                      class="mjx-char MJXc-TeX-math-I"
                      style="padding-top: 0.446em; padding-bottom: 0.298em; padding-right: 0.109em;">P</span></span><span
                    class="mjx-mi"><span class="mjx-char MJXc-TeX-math-I"
                      style="padding-top: 0.446em; padding-bottom: 0.298em; padding-right: 0.109em;">P</span></span><span
                    class="mjx-mi"><span class="mjx-char MJXc-TeX-math-I"
                      style="padding-top: 0.446em; padding-bottom: 0.298em; padding-right: 0.106em;">F</span></span><span
                    class="mjx-msubsup"><span class="mjx-base"><span class="mjx-texatom"><span
                          class="mjx-mrow"><span class="mjx-mi"><span
                              class="mjx-char MJXc-TeX-math-I"
                              style="padding-top: 0.446em; padding-bottom: 0.298em;">D</span></span></span></span></span><span
                      class="mjx-sub"
                      style="font-size: 70.7%; vertical-align: -0.212em; padding-right: 0.071em;"><span
                        class="mjx-texatom" style=""><span class="mjx-mrow"><span
                            class="mjx-mi"><span class="mjx-char MJXc-TeX-math-I"
                              style="padding-top: 0.446em; padding-bottom: 0.298em; padding-right: 0.081em;">M</span></span></span></span></span></span></span></span></span></span>
          in the range between <stencila-code-expression programming-language="py" itemscope=""
            itemtype="http://schema.stenci.la/CodeExpression"><code class="py"
              slot="text">min_ppfd_bs_m</code><output slot="output"></output>
          </stencila-code-expression> to <stencila-code-expression programming-language="py"
            itemscope="" itemtype="http://schema.stenci.la/CodeExpression"><code class="py"
              slot="text">max_ppfd_bs_m</code><output slot="output"></output>
          </stencila-code-expression>, see <a href="#fig7" itemscope=""
            itemtype="http://schema.stenci.la/Link">Figure 7</a> and <a href="#fig8" itemscope=""
            itemtype="http://schema.stenci.la/Link">Figure 8</a>. Under light limitation, if the
          total PPFD is lower than <stencila-code-expression programming-language="py" itemscope=""
            itemtype="http://schema.stenci.la/CodeExpression"><code class="py"
              slot="text">low_light_thres</code><output slot="output"></output>
          </stencila-code-expression> μmol/(m<sup itemscope=""
            itemtype="http://schema.stenci.la/Superscript"><span
              data-itemtype="http://schema.org/Number">2</span></sup>s) , the CO<sub itemscope=""
            itemtype="http://schema.stenci.la/Subscript"><span
              data-itemtype="http://schema.org/Number">2</span></sub> uptake rate is reduced,
          leading to a decreased activity of the decarboxylation enzymes (<a href="#fig7"
            itemscope="" itemtype="http://schema.stenci.la/Link">Figure 7</a>). PEP-CK is used in
          the optimal solutions active under light-limiting conditions (<a href="#fig8" itemscope=""
            itemtype="http://schema.stenci.la/Link">Figure 8</a>). Under limiting light conditions,
          photon distribution with a higher proportion in the bundle sheath shifts decarboxylation
          towards NADP-ME but only to up to <stencila-code-expression programming-language="py"
            itemscope="" itemtype="http://schema.stenci.la/CodeExpression"><code class="py"
              slot="text">nadpme_max_low_light</code><output slot="output"></output>
          </stencila-code-expression>%. Under non-limiting conditions, the distribution of light
          availability determines the optimal decarboxylation enzyme. NADP-ME is the preferred
          decarboxylation enzyme with supplemental contributions by PEP-CK if light availability is
          near the threshold of <stencila-code-expression programming-language="py" itemscope=""
            itemtype="http://schema.stenci.la/CodeExpression"><code class="py"
              slot="text">low_light_thres</code><output slot="output"></output>
          </stencila-code-expression> μmol/(m<sup itemscope=""
            itemtype="http://schema.stenci.la/Superscript"><span
              data-itemtype="http://schema.org/Number">2</span></sup>s) or if at least twice as many
          photons are absorbed by the mesophyll. Excess light availability and a higher proportion
          of photons reaching the bundle sheath leads to optimal solutions which favour PEP-CK as
          the decarboxylation enzyme. In the case of very high light availability and an abrupt
          shift towards the bundle sheath, NAD-ME becomes the optimal solution (<a href="#fig8"
            itemscope="" itemtype="http://schema.stenci.la/Link">Figure 8</a>). NAD-ME is the least
          favourable enzyme overall, only low activity is predicted under extreme light conditions,
          where the bundle sheath absorbs equal or more photons than the mesophyll (<a href="#fig8"
            itemscope="" itemtype="http://schema.stenci.la/Link">Figure 8</a>). PEP-CK complements
          the activity of NADP-ME and NAD-ME to 100% in many conditions, meaning the <em
            itemscope="" itemtype="http://schema.stenci.la/Emphasis">two-cell</em> model also
          predicts the co-existence of PEP-CK/NADP-ME and PEP-CK/NAD-ME mode, while the flux
          distribution indicates no parallel use of NAD-ME and NADP-ME, compare <a href="#fig8"
            itemscope="" itemtype="http://schema.stenci.la/Link">Figure 8</a>.</p>
        <stencila-code-chunk itemscope="" itemtype="http://schema.stenci.la/CodeChunk"
          data-execution_count="38" data-programminglanguage="python">
          <pre class="language-python" itemscope="" itemtype="http://schema.stenci.la/CodeBlock"
            slot="text"><code>######################################################################################################################################
######################################################################################################################################
##############                              EXPERIMENT 7: Effect of N-Limtation on C4 Mode                              ##############
######################################################################################################################################
######################################################################################################################################

print(f&quot;Code Cell 38: Experiment 7 -- Effect of N-Limtation on C4 Mode&quot;)

#Create copy of c4 model
c4_model_exp7 = c4_model.copy() 

#Reaction variables
B_Ex_Suc = c4_model_exp7.reactions.get_by_id(&quot;[B]_Ex_Suc&quot;)
M_Im_NO3 = c4_model_exp7.reactions.get_by_id(&quot;[M]_Im_NO3&quot;)
B_Im_NO3 = c4_model_exp7.reactions.get_by_id(&quot;[B]_Im_NO3&quot;)
B_RBO = c4_model_exp7.reactions.get_by_id(&quot;[B]_RBO_h&quot;)
M_RBO = c4_model_exp7.reactions.get_by_id(&quot;[M]_RBO_h&quot;)

#Decarboxylation reactions ids and names of enzymes
c4_mode_r_id_enzyme = {&#39;[B]_MalDH2_m&#39;: &#39;NAD-ME&#39;,  &#39;[B]_PEPC1_c&#39;: &#39;PEP-CK&#39;, &#39;[B]_MalDH4_h&#39;: &#39;NADP-ME&#39;}

#NO3 flux
no3_flux = np.arange(0,2.1,0.2)

#Initialise dataframes to hold results
df_result_exp7 = pd.DataFrame(index=c4_model.reactions.list_attr(&#39;id&#39;), columns=no3_flux, dtype=&#39;float64&#39;)

#Test all no3 values
for no3_value in tqdm(df_result_exp7.columns):

    #Add N-limitation constraint 
    const_NO3_exp7 = c4_model_exp7.problem.Constraint( M_Im_NO3.flux_expression + B_Im_NO3.flux_expression,
                                        lb = no3_value, ub = no3_value, name=&#39;const_NO3_exp7&#39;)
    c4_model_exp7.add_cons_vars(const_NO3_exp7)
        
    #Optimization - Maximize sucrose output &amp; Minimize Oxygenation by Rubisco
    B_Ex_Suc.objective_coefficient = 1.
    B_RBO.objective_coefficient = -1.
    M_RBO.objective_coefficient = -1.
    result_exp7_fba = c4_model_exp7.optimize(&#39;maximize&#39;)
    
        
    #Optimize/Minimize total flux
    if result_exp7_fba.status == &#39;optimal&#39;: 
        
        result_exp7_pfba = cobra.flux_analysis.parsimonious.pfba(c4_model_exp7)
        df_result_exp7[no3_value] = result_exp7_pfba.fluxes
        
   #Remove N-limitation constraint
    c4_model_exp7.remove_cons_vars(const_NO3_exp7) </code></pre>
          <figure slot="outputs">
            <pre class="language-text" itemscope="" itemtype="http://schema.stenci.la/CodeBlock"><code>Code Cell 38: Experiment 7 -- Effect of N-Limtation on C4 Mode
</code></pre>
            <pre class="language-text" itemscope=""
              itemtype="http://schema.stenci.la/CodeBlock"><code>  0%|          | 0/11 [00:00&lt;?, ?it/s]</code></pre>
            <pre class="language-text" itemscope="" itemtype="http://schema.stenci.la/CodeBlock"><code>/Users/blaetke/opt/anaconda3/envs/elife-49305-era/lib/python3.9/site-packages/cobra/util/solver.py:508: UserWarning:

Solver status is &#39;infeasible&#39;.

</code></pre>
          </figure>
        </stencila-code-chunk>
        <figure itemscope="" itemtype="http://schema.stenci.la/Figure" id="fig8s1"
          title="Figure 8—figure supplement 1"><label data-itemprop="label">Figure 8—figure
            supplement 1</label>
          <stencila-code-chunk itemscope="" itemtype="http://schema.stenci.la/CodeChunk"
            data-execution_count="39" data-programminglanguage="python">
            <pre class="language-python" itemscope="" itemtype="http://schema.stenci.la/CodeBlock"
              slot="text"><code>##################################################################################
##############              Figure 8—figure supplement 1            ##############
##################################################################################

#{
#  &quot;caption&quot;: &quot;### Effect of NO~3~^-^ limitation on the flux through the different decarboxylation enzymes.&quot;,
#  &quot;id&quot;: &quot;fig8s1,
#  &quot;label&quot;: &quot;Figure 8—figure supplement 1&quot;,
#  &quot;trusted&quot;: true
#}

#Create figure
fig_exp7 = go.Figure()

#Add trace for each decarboxylation enzyme
for i, r_id in enumerate(c4_mode_r_id_enzyme.keys()):
    
    #Create bar plot trace
    trace = go.Bar(
        y = df_result_exp7.loc[r_id],
        x = df_result_exp7.columns,
        name = c4_mode_r_id_enzyme[r_id]
    )
    
    #Add trace to figure
    fig_exp7.add_trace(trace)

#Update xaxes
fig_exp7.update_xaxes(
    title = dict(
        text = &#39;NO\u2083 Uptake Rate [µmol/(m\u00B2s)]&#39;,
        font = dict(size=18)
    ),
    tickfont = dict(size=16)  
)

#Update yaxes
fig_exp7.update_yaxes(
    range = [0, 40],
    title = dict(
        text = &#39;Flux [µmol/(m\u00B2s)]&#39;,
        font = dict(size=18)
    ),
    tickfont = dict(size=16)  
)

#Update layout
fig_exp7.update_layout(
    barmode=&#39;stack&#39;,
    width= 1000,
    height=500,
    legend=dict(
        font=dict(size=18),
    )
)

#Show figure
fig_exp7.show()</code></pre>
            <figure slot="outputs">
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                  </script><img src="index.html.media/9" alt="" itemscope=""
                    itemtype="http://schema.org/ImageObject">
                </picture>
              </stencila-image-plotly>
            </figure>
          </stencila-code-chunk>
          <figcaption>
            <h4 itemscope="" itemtype="http://schema.stenci.la/Heading"
              id="effect-of-no3--limitation-on-the-flux-through-the-different-decarboxylation-enzymes">
              Effect of NO<sub itemscope="" itemtype="http://schema.stenci.la/Subscript"><span
                  data-itemtype="http://schema.org/Number">3</span></sub><sup itemscope=""
                itemtype="http://schema.stenci.la/Superscript">-</sup> limitation on the flux
              through the different decarboxylation enzymes.</h4>
          </figcaption>
        </figure>
        <stencila-code-chunk itemscope="" itemtype="http://schema.stenci.la/CodeChunk"
          data-execution_count="40" data-programminglanguage="python">
          <pre class="language-python" itemscope="" itemtype="http://schema.stenci.la/CodeBlock"
            slot="text"><code>######################################################################################################################################
######################################################################################################################################
##############                             EXPERIMENT 8: Effect of H2O-Limtation on C4 Mode                             ##############
######################################################################################################################################
######################################################################################################################################

print(f&quot;Code Cell 40: Experiment 8 -- Effect of H2O-Limtation on C4 Mode&quot;)

#Create copy of c4 model
c4_model_exp8 = c4_model.copy() 

#Reaction variables
B_Ex_Suc = c4_model_exp8.reactions.get_by_id(&quot;[B]_Ex_Suc&quot;)
M_Im_H2O = c4_model_exp8.reactions.get_by_id(&quot;[M]_Im_H2O&quot;)
B_Im_H2O = c4_model_exp8.reactions.get_by_id(&quot;[B]_Im_H2O&quot;)
B_RBO = c4_model_exp8.reactions.get_by_id(&quot;[B]_RBO_h&quot;)
M_RBO = c4_model_exp8.reactions.get_by_id(&quot;[M]_RBO_h&quot;)

#Decarboxylation reactions ids and names of enzymes
c4_mode_r_id_enzyme = {&#39;[B]_MalDH2_m&#39;: &#39;NAD-ME&#39;,  &#39;[B]_PEPC1_c&#39;: &#39;PEP-CK&#39;, &#39;[B]_MalDH4_h&#39;: &#39;NADP-ME&#39;}

#H2O flux
h2O_flux = np.arange(0,45,5)

#Initialise dataframes to hold results
df_result_exp8 = pd.DataFrame(index=c4_model.reactions.list_attr(&#39;id&#39;), columns=h2O_flux, dtype=&#39;float64&#39;)

#Test all H2O values
for h2o_value in tqdm(df_result_exp8.columns):

    #Add max H2O-uptake constraint 
    const_h2o_exp8 = c4_model_exp8.problem.Constraint( M_Im_H2O.flux_expression + B_Im_H2O.flux_expression,
                                        lb = 0, ub = h2o_value, name=&#39;const_h2o_exp8&#39;)
    c4_model_exp8.add_cons_vars(const_h2o_exp8)
        
    #Optimize/Maximize sucrose output
    B_Ex_Suc.objective_coefficient = 1.
    B_RBO.objective_coefficient = -1.
    M_RBO.objective_coefficient = -1.
    result_exp8_fba = c4_model_exp8.optimize(&#39;maximize&#39;)
    
    #Optimize/Minimize total flux
    if result_exp8_fba.status == &#39;optimal&#39;: 
        
        result_exp8_pfba = cobra.flux_analysis.parsimonious.pfba(c4_model_exp8)
        df_result_exp8[h2o_value] = result_exp8_pfba.fluxes
        
   #Remove max H2O-uptake constraint 
    c4_model_exp8.remove_cons_vars(const_h2o_exp8) </code></pre>
          <figure slot="outputs">
            <pre class="language-text" itemscope="" itemtype="http://schema.stenci.la/CodeBlock"><code>Code Cell 40: Experiment 8 -- Effect of H2O-Limtation on C4 Mode
</code></pre>
            <pre class="language-text" itemscope=""
              itemtype="http://schema.stenci.la/CodeBlock"><code>  0%|          | 0/9 [00:00&lt;?, ?it/s]</code></pre>
          </figure>
        </stencila-code-chunk>
        <figure itemscope="" itemtype="http://schema.stenci.la/Figure" id="fig8s2"
          title="Figure 8—figure supplement 2"><label data-itemprop="label">Figure 8—figure
            supplement 2</label>
          <stencila-code-chunk itemscope="" itemtype="http://schema.stenci.la/CodeChunk"
            data-execution_count="41" data-programminglanguage="python">
            <pre class="language-python" itemscope="" itemtype="http://schema.stenci.la/CodeBlock"
              slot="text"><code>##################################################################################
##############              Figure 8—figure supplement 2            ##############
##################################################################################

#{
#  &quot;caption&quot;: &quot;### Effect of H~2~O imitation on the flux through the different decarboxylation enzymes.&quot;,
#  &quot;id&quot;: &quot;fig8s2&quot;,
#  &quot;label&quot;: &quot;Figure 8—figure supplement 2&quot;,
#  &quot;trusted&quot;: true
#}
    
#Create figure
fig_exp8 = go.Figure()

#Add trace for each decarboxylation enzyme
for i, r_id in enumerate(c4_mode_r_id_enzyme.keys()):
    
    #Create bar plot trace
    trace = go.Bar(
        y = df_result_exp8.loc[r_id],
        x = df_result_exp8.columns,
        name = c4_mode_r_id_enzyme[r_id]
    )
    
    #Add trace to figure
    fig_exp8.add_trace(trace)

#Update xaxes
fig_exp8.update_xaxes(
    title = dict(
        text = &#39;Max H\u2082O Uptake Rate [µmol/(m\u00B2s)]&#39;,
        font = dict(size=18)
    ),
    tickfont = dict(size=16)  
)

#Update yaxes
fig_exp8.update_yaxes(
    range = [0, 40],
    title = dict(
        text = &#39;Flux [µmol/(m\u00B2s)]&#39;,
        font = dict(size=18)
    ),
    tickfont = dict(size=16)  
)

#Update layout
fig_exp8.update_layout(
    barmode=&#39;stack&#39;,
    width= 1000,
    height=500,
    legend=dict(
        font=dict(size=18),
    )
)

#Show figure
fig_exp8.show()</code></pre>
            <figure slot="outputs">
              <stencila-image-plotly>
                <picture>
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          <figcaption>
            <h4 itemscope="" itemtype="http://schema.stenci.la/Heading"
              id="effect-of-h2o-imitation-on-the-flux-through-the-different-decarboxylation-enzymes">
              Effect of H<sub itemscope="" itemtype="http://schema.stenci.la/Subscript"><span
                  data-itemtype="http://schema.org/Number">2</span></sub>O imitation on the flux
              through the different decarboxylation enzymes.</h4>
          </figcaption>
        </figure>
        <stencila-code-chunk itemscope="" itemtype="http://schema.stenci.la/CodeChunk"
          data-execution_count="42" data-programminglanguage="python">
          <pre class="language-python" itemscope="" itemtype="http://schema.stenci.la/CodeBlock"
            slot="text"><code>######################################################################################################################################
######################################################################################################################################
##############                             EXPERIMENT 9: Effect of CO2-Limtation on C4 Mode                             ##############
######################################################################################################################################
######################################################################################################################################

print(f&quot;Code Cell 42: Experiment 9 -- Effect of CO2-Limtation on C4 Mode&quot;)

#Create copy of c4 model
c4_model_exp9 = c4_model.copy() 

#Reaction variables
B_Ex_Suc = c4_model_exp9.reactions.get_by_id(&quot;[B]_Ex_Suc&quot;)
M_Im_CO2 = c4_model_exp9.reactions.get_by_id(&quot;[M]_Im_CO2&quot;)
B_Im_CO2 = c4_model_exp9.reactions.get_by_id(&quot;[B]_Im_CO2&quot;)
B_RBO = c4_model_exp9.reactions.get_by_id(&quot;[B]_RBO_h&quot;)
M_RBO = c4_model_exp9.reactions.get_by_id(&quot;[M]_RBO_h&quot;)

#Decarboxylation reactions ids and names of enzymes
c4_mode_r_id_enzyme = {&#39;[B]_MalDH2_m&#39;: &#39;NAD-ME&#39;,  &#39;[B]_PEPC1_c&#39;: &#39;PEP-CK&#39;, &#39;[B]_MalDH4_h&#39;: &#39;NADP-ME&#39;}

#H2O flux
co2_flux = np.arange(0,45,5)

#Initialise dataframes to hold results
df_result_exp9 = pd.DataFrame(index=c4_model.reactions.list_attr(&#39;id&#39;), columns=co2_flux, dtype=&#39;float64&#39;)

#Test all CO2 values
for co2_value in tqdm(df_result_exp9.columns):

    #Add max CO2-uptake constraint 
    const_co2_exp9 = c4_model_exp9.problem.Constraint( M_Im_CO2.flux_expression + B_Im_CO2.flux_expression,
                                        lb = 0, ub = co2_value, name=&#39;const_co2_exp9&#39;)
    c4_model_exp9.add_cons_vars(const_co2_exp9)
        
    #Optimize/Maximize sucrose output
    B_Ex_Suc.objective_coefficient = 1.
    B_RBO.objective_coefficient = -1.
    M_RBO.objective_coefficient = -1.
    result_exp9_fba = c4_model_exp9.optimize(&#39;maximize&#39;)
    
    #Optimize/Minimize total flux
    if result_exp9_fba.status == &#39;optimal&#39;: 
        
        result_exp9_pfba = cobra.flux_analysis.parsimonious.pfba(c4_model_exp9)
        df_result_exp9[co2_value] = result_exp9_pfba.fluxes
        
   #Remove max CO2-uptake constraint
    c4_model_exp9.remove_cons_vars(const_co2_exp9) </code></pre>
          <figure slot="outputs">
            <pre class="language-text" itemscope="" itemtype="http://schema.stenci.la/CodeBlock"><code>Code Cell 42: Experiment 9 -- Effect of CO2-Limtation on C4 Mode
</code></pre>
            <pre class="language-text" itemscope=""
              itemtype="http://schema.stenci.la/CodeBlock"><code>  0%|          | 0/9 [00:00&lt;?, ?it/s]</code></pre>
          </figure>
        </stencila-code-chunk>
        <figure itemscope="" itemtype="http://schema.stenci.la/Figure" id="fig8s3"
          title="Figure 8—figure supplement 3"><label data-itemprop="label">Figure 8—figure
            supplement 3</label>
          <stencila-code-chunk itemscope="" itemtype="http://schema.stenci.la/CodeChunk"
            data-execution_count="43" data-programminglanguage="python">
            <pre class="language-python" itemscope="" itemtype="http://schema.stenci.la/CodeBlock"
              slot="text"><code>##################################################################################
##############              Figure 8—figure supplement 3            ##############
##################################################################################

#{
#  &quot;caption&quot;: &quot;### Effect of CO~2~ limitation on the flux through the different decarboxylation enzymes.&quot;,
#  &quot;id&quot;: &quot;fig8s3&quot;,
#  &quot;label&quot;: &quot;Figure 8—figure supplement 3&quot;,
#  &quot;trusted&quot;: true
#}

#Create figure
fig_exp9 = go.Figure()

#Add trace for each decarboxylation enzyme
for i, r_id in enumerate(c4_mode_r_id_enzyme.keys()):
    
    #Create bar plot trace
    trace = go.Bar(
        y = df_result_exp9.loc[r_id],
        x = df_result_exp9.columns,
        name = c4_mode_r_id_enzyme[r_id]
    )
    
    #Add trace to figure
    fig_exp9.add_trace(trace)

#Update xaxes
fig_exp9.update_xaxes(
    title = dict(
        text = &#39;Max CO\u2082 Uptake Rate [µmol/(m\u00B2s)]&#39;,
        font = dict(size=18)
    ),
    tickfont = dict(size=16)  
)

#Update yaxes
fig_exp9.update_yaxes(
    range = [0, 40],
    title = dict(
        text = &#39;Flux [µmol/(m\u00B2s)]&#39;,
        font = dict(size=18)
    ),
    tickfont = dict(size=16)  
)

#Update layout
fig_exp9.update_layout(
    barmode=&#39;stack&#39;,
    width= 1000,
    height=500,
    legend=dict(
        font=dict(size=18),
    )
)

#Show figure
fig_exp9.show()</code></pre>
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          <figcaption>
            <h4 itemscope="" itemtype="http://schema.stenci.la/Heading"
              id="effect-of-co2-limitation-on-the-flux-through-the-different-decarboxylation-enzymes">
              Effect of CO<sub itemscope="" itemtype="http://schema.stenci.la/Subscript"><span
                  data-itemtype="http://schema.org/Number">2</span></sub> limitation on the flux
              through the different decarboxylation enzymes.</h4>
          </figcaption>
        </figure>
        <stencila-code-chunk itemscope="" itemtype="http://schema.stenci.la/CodeChunk"
          data-execution_count="44" data-programminglanguage="python">
          <pre class="language-python" itemscope="" itemtype="http://schema.stenci.la/CodeBlock"
            slot="text"><code>######################################################################################################################################
######################################################################################################################################
##############                    EXPERIMENT 10: Effect of Malate/Aspartate-Exchange Ratio on C4 Mode                   ##############
######################################################################################################################################
######################################################################################################################################

print(f&quot;Code Cell 46: Experiment 10 -- Effect of Malate/Aspartate-Exchange Ratio on C4 Mode&quot;)

#Create copy of c4 model
c4_model_exp10 = c4_model.copy() 

#Reaction variables
B_Ex_Suc = c4_model_exp10.reactions.get_by_id(&quot;[B]_Ex_Suc&quot;)
B_RBO = c4_model_exp10.reactions.get_by_id(&quot;[B]_RBO_h&quot;)
M_RBO = c4_model_exp10.reactions.get_by_id(&quot;[M]_RBO_h&quot;)

#Decarboxylation reactions ids and names of enzymes
c4_mode_r_id_enzyme = {&#39;[B]_MalDH2_m&#39;: &#39;NAD-ME&#39;,  &#39;[B]_PEPC1_c&#39;: &#39;PEP-CK&#39;, &#39;[B]_MalDH4_h&#39;: &#39;NADP-ME&#39;}

#Max exchange flux
ex_flux = 40

#Malate flux
mal_flux = np.arange(0,45,5)

#Initialise dataframes to hold results
df_result_exp10 = pd.DataFrame(index=c4_model.reactions.list_attr(&#39;id&#39;), columns=mal_flux, dtype=&#39;float64&#39;)

#Test all mal/asp exchange values
for mal_value in tqdm(df_result_exp10.columns):

    #Add Mal/Asp transport constraint 
    set_fixed_flux(&#39;[MB]_Mal_c&#39;,mal_value, c4_model_exp10)
    set_fixed_flux(&#39;[MB]_Asp_c&#39;,ex_flux - mal_value, c4_model_exp10)
        
    #Optimize/Maximize sucrose output
    B_Ex_Suc.objective_coefficient = 1.
    B_RBO.objective_coefficient = -1.
    M_RBO.objective_coefficient = -1.
    result_exp10_fba = c4_model_exp10.optimize(&#39;maximize&#39;)
    
    #Optimize/Minimize total flux
    if result_exp10_fba.status == &#39;optimal&#39;: 
        
        result_exp10_pfba = cobra.flux_analysis.parsimonious.pfba(c4_model_exp10)
        df_result_exp10[mal_value] = result_exp10_pfba.fluxes
        </code></pre>
          <figure slot="outputs">
            <pre class="language-text" itemscope="" itemtype="http://schema.stenci.la/CodeBlock"><code>Code Cell 46: Experiment 10 -- Effect of Malate/Aspartate-Exchange Ratio on C4 Mode
</code></pre>
            <pre class="language-text" itemscope=""
              itemtype="http://schema.stenci.la/CodeBlock"><code>  0%|          | 0/9 [00:00&lt;?, ?it/s]</code></pre>
          </figure>
        </stencila-code-chunk>
        <figure itemscope="" itemtype="http://schema.stenci.la/Figure" id="fig8s4"
          title="Figure 8—figure supplement 4"><label data-itemprop="label">Figure 8—figure
            supplement 4</label>
          <stencila-code-chunk itemscope="" itemtype="http://schema.stenci.la/CodeChunk"
            data-execution_count="45" data-programminglanguage="python">
            <pre class="language-python" itemscope="" itemtype="http://schema.stenci.la/CodeBlock"
              slot="text"><code>##################################################################################
##############              Figure 8—figure supplement 4            ##############
##################################################################################

#{
#  &quot;caption&quot;: &quot;### Effect of malate:aspartate transport ratio on the flux through the different decarboxylation enzymes.&quot;,
#  &quot;id&quot;: &quot;fig8s4&quot;,
#  &quot;label&quot;: &quot;Figure 8—figure supplement 4&quot;,
#  &quot;trusted&quot;: true
#}


#Create Figure
fig_exp10 = go.Figure()

#Add Trace for each decarboxylation enzyme
for i, r_id in enumerate(c4_mode_r_id_enzyme.keys()):
    
    #Create bar plot trace
    trace = go.Bar(
        y = df_result_exp10.loc[r_id],
        x = df_result_exp10.columns,
        name = c4_mode_r_id_enzyme[r_id]
    )
    
    #Add trace to figure
    fig_exp10.add_trace(trace)

#Update xaxes
fig_exp10.update_xaxes(
    tickvals = df_result_exp10.columns,
    ticktext = [f&#39;{mal_value} : {ex_flux - mal_value}&#39; for mal_value in df_result_exp10.columns],
    title = dict(
        text = &#39;Mal : Asp Exchange&#39;,
        font = dict(size=18)
    ),
    tickfont = dict(size=16)  
)

#Update yaxes
fig_exp10.update_yaxes(
    range = [0, 40],
    title = dict(
        text = &#39;Flux [µmol/(m\u00B2s)]&#39;,
        font = dict(size=18)
    ),
    tickfont = dict(size=16)  
)

#Update layout
fig_exp10.update_layout(
    barmode=&#39;stack&#39;,
    width= 1000,
    height=500,
    legend=dict(
        font=dict(size=18),
    )
)

#Show figure
fig_exp10.show()</code></pre>
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: 40","5 : 35","10 : 30","15 : 25","20 : 20","25 : 15","30 : 10","35 : 5","40 : 0"],"tickvals":[0,5,10,15,20,25,30,35,40],"title":{"font":{"size":18},"text":"Mal : Asp Exchange"}},"yaxis":{"range":[0,40],"tickfont":{"size":16},"title":{"font":{"size":18},"text":"Flux [µmol/(m²s)]"}}}}
                  </script><img src="index.html.media/12" alt="" itemscope=""
                    itemtype="http://schema.org/ImageObject">
                </picture>
              </stencila-image-plotly>
            </figure>
          </stencila-code-chunk>
          <figcaption>
            <h4 itemscope="" itemtype="http://schema.stenci.la/Heading"
              id="effect-of-malateaspartate-transport-ratio-on-the-flux-through-the-different-decarboxylation-enzymes">
              Effect of malate:aspartate transport ratio on the flux through the different
              decarboxylation enzymes.</h4>
          </figcaption>
        </figure>
        <p itemscope="" itemtype="http://schema.stenci.la/Paragraph">Finally, we assumed that
          intercellular transport capacity for charged metabolites might be different between
          species. Assuming a fixed transport ratio between aspartate and malate (<a href="#fig8s4"
            itemscope="" itemtype="http://schema.stenci.la/Link">Figure 8—figure supplement 4</a>)
          introduces a shift in the C4 state. Higher proportions of malate exchange foster the use
          of NADP-ME (<a href="#fig8s4" itemscope="" itemtype="http://schema.stenci.la/Link">Figure
            8—figure supplement 4</a>). In contrast, higher portions of aspartate exchange foster
          the use of PEP-CK (<a href="#fig8s4" itemscope=""
            itemtype="http://schema.stenci.la/Link">Figure 8—figure supplement 4</a>).</p>
        <h2 itemscope="" itemtype="http://schema.stenci.la/Heading" id="discussion">Discussion</h2>
        <p itemscope="" itemtype="http://schema.stenci.la/Paragraph">Evolutionary CBM can suggest
          the molecular outcomes of past evolutionary events if models are parametrised with
          objective functions representing possible selective pressures. In the case of C4
          photosynthesis, more than sixty independent evolutionary origins represent metabolic types
          characterised by their decarboxylation enzyme. The selective pressure which drives
          evolution towards one or the other flux are unknown and were tested using CBM.</p>
        <h3 itemscope="" itemtype="http://schema.stenci.la/Heading"
          id="one-cell-model-reflects-c3-plant-physiology"><em itemscope=""
            itemtype="http://schema.stenci.la/Emphasis">One-cell</em> model reflects C3 plant
          physiology</h3>
        <p itemscope="" itemtype="http://schema.stenci.la/Paragraph">To analyse evolution towards C4
          photosynthesis based on C3 metabolism, a CBM of C3 metabolism is required (<a href="#fig1"
            itemscope="" itemtype="http://schema.stenci.la/Link">Figure 1</a>). Design, simulation,
          validation cycles used current knowledge about plant biochemistry <cite itemscope=""
            itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a
              href="#bib32"><span>32</span><span>Heldt</span><span>2015</span></a></cite> to
          identify possible errors in the metabolic map required for modelling. Even after error
          correction (<a href="#table1" itemscope="" itemtype="http://schema.stenci.la/Link">Table
            1</a>), a significant problem remained, namely excessive fluxes to balance protons in
          all compartments. This observation leads to the realisation that the biochemical knowledge
          about transport reactions does not extend to the protonation state of the substrates,
          which affects all eukaryotic CBM efforts. In plants, predominantly export and vacuolar
          transport reactions are directly or indirectly coupled with proton gradients to energise
          transport <span itemscope="" itemtype="http://schema.stenci.la/CiteGroup"><cite
              itemscope="" itemtype="http://schema.stenci.la/Cite"><a
                href="#bib16"><span>16</span><span>Bush</span><span>1993</span></a></cite><cite
              itemscope="" itemtype="http://schema.stenci.la/Cite"><a
                href="#bib50"><span>50</span><span>Neuhaus</span><span>2007</span></a></cite></span>.
          For chloroplasts and mitochondria, proton-coupled transport reactions have been described
          but may couple different metabolite transporters together rather than energising them
          <cite itemscope="" itemtype="http://schema.stenci.la/Cite"
            data-citationmode="Narrative"><a href="#bib26"><span>26</span><span>Furumoto et
                al.</span><span>2011</span></a></cite>. Introducing proton sinks in all compartments
          solves the immediate modelling problem. However, intracellular transport reactions and
          their energetic costs are no longer correctly assessed by the model. Despite this band-aid
          fix which will be required for all eukaryotic constraint-based models which include
          proton-coupled transport reactions, the curated <em itemscope=""
            itemtype="http://schema.stenci.la/Emphasis">one-cell</em> model correctly predicts
          energy usage and its distribution (<a href="#fig1s3" itemscope=""
            itemtype="http://schema.stenci.la/Link">Figure 1—figure supplement 3</a>, <a
            href="#fig1s4" itemscope="" itemtype="http://schema.stenci.la/Link">Figure 1—figure
            supplement 4</a> and <cite itemscope="" itemtype="http://schema.stenci.la/Cite"
            data-citationmode="Narrative"><a href="#bib40"><span>40</span><span>Li et
                al.</span><span>2017</span></a></cite>). This indicates that in models which exclude
          vacuolar transport and energised export reactions, energy calculations remain likely
          within the correct order of magnitude. Overall, our <em itemscope=""
            itemtype="http://schema.stenci.la/Emphasis">one-cell</em> model operates within
          parameters expected for a C3 plant: The predicted PPFD lies within the range of light
          intensities used for normal growth condition of <em itemscope=""
            itemtype="http://schema.stenci.la/Emphasis">Arabidopsis thaliana</em>, which varies
          between 100 μmol/(m<sup itemscope="" itemtype="http://schema.stenci.la/Superscript"><span
              data-itemtype="http://schema.org/Number">2</span></sup>s)–200 μmol/(m<sup itemscope=""
            itemtype="http://schema.stenci.la/Superscript"><span
              data-itemtype="http://schema.org/Number">2</span></sup>s), see <a href="#table2"
            itemscope="" itemtype="http://schema.stenci.la/Link">Table 2</a>. The gross rate
          of O<sub itemscope="" itemtype="http://schema.stenci.la/Subscript"><span
              data-itemtype="http://schema.org/Number">2</span></sub> evolution for a PPFD of 200
          μmol/(m<sup itemscope="" itemtype="http://schema.stenci.la/Superscript"><span
              data-itemtype="http://schema.org/Number">2</span></sup>s) is estimated to be 16.5
          μmol/(m<sup itemscope="" itemtype="http://schema.stenci.la/Superscript"><span
              data-itemtype="http://schema.org/Number">2</span></sup>s) in the literature <cite
            itemscope="" itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a
              href="#bib75"><span>75</span><span>Sun et al.</span><span>1999</span></a></cite>,
          which is in close proximity to the predicted flux of the <em itemscope=""
            itemtype="http://schema.stenci.la/Emphasis">one-cell</em> model, see <a href="#table2"
            itemscope="" itemtype="http://schema.stenci.la/Link">Table 2</a>. For the amount of
          respiratory ATP that is used for maintenance, <cite itemscope=""
            itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a
              href="#bib40"><span>40</span><span>Li et al.</span><span>2017</span></a></cite>
          predicted an even lower proportion of energy 16%, see <a href="#fig1s4" itemscope=""
            itemtype="http://schema.stenci.la/Link">Figure 1—figure supplement 4(B)</a>. The model’s
          flux map is in accordance with known C3 plant physiology <cite itemscope=""
            itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a
              href="#bib32"><span>32</span><span>Heldt</span><span>2015</span></a></cite>, and its
          input and output parameters match expected values (<a href="#fig2" itemscope=""
            itemtype="http://schema.stenci.la/Link">Figure 2(B)</a>). The current model excludes
          specialised metabolism since the output function focuses solely on substances exported
          through the phloem in a mature leaf. If the model were to be used to study biotic
          interactions in the future, the addition of specialised metabolism in the metabolic map
          and a new output function would be required.</p>
        <h3 itemscope="" itemtype="http://schema.stenci.la/Heading"
          id="the-two-cell-model-predicts-a-c4-cycle-if-photorespiration-is-present">The two-cell
          model predicts a C4 cycle if photorespiration is present</h3>
        <p itemscope="" itemtype="http://schema.stenci.la/Paragraph">Most evolutionary concepts
          about C4 photosynthesis assume that selective pressure drives pathway evolution due to
          photorespiration and carbon limitation <cite itemscope=""
            itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a
              href="#bib31"><span>31</span><span>Heckmann et al.</span><span>2013</span></a></cite>.
          Most extant C4 species occupy dry and arid niches <cite itemscope=""
            itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a
              href="#bib23"><span>23</span><span>Edwards et al.</span><span>2010</span></a></cite>,
          even more, the period of C4 plant evolution was accompanied with an increased oxygen
          concentration in the atmosphere <cite itemscope="" itemtype="http://schema.stenci.la/Cite"
            data-citationmode="Narrative"><a
              href="#bib62"><span>62</span><span>Sage</span><span>2004</span></a></cite>. Therefore,
          it is frequently assumed that carbon limitation by excessive photorespiration drives the
          evolution of C4 photosynthesis. Yet, in most habitats plants are limited by nutrients
          other than carbon <span itemscope="" itemtype="http://schema.stenci.la/CiteGroup"><cite
              itemscope="" itemtype="http://schema.stenci.la/Cite"><a
                href="#bib1"><span>1</span><span>Agren et
                  al.</span><span>2012</span></a></cite><cite itemscope=""
              itemtype="http://schema.stenci.la/Cite"><a
                href="#bib36"><span>36</span><span>Körner</span><span>2015</span></a></cite></span>.
          Ecophysiological analyses also show that C4 can evolve in non-arid habitats <span
            itemscope="" itemtype="http://schema.stenci.la/CiteGroup"><cite itemscope=""
              itemtype="http://schema.stenci.la/Cite"><a href="#bib43"><span>43</span><span>Liu and
                  Osborne</span><span>2015</span></a></cite><cite itemscope=""
              itemtype="http://schema.stenci.la/Cite"><a href="#bib44"><span>44</span><span>Lundgren
                  and Christin</span><span>2017</span></a></cite><cite itemscope=""
              itemtype="http://schema.stenci.la/Cite"><a href="#bib53"><span>53</span><span>Osborne
                  and Freckleton</span><span>2009</span></a></cite></span>. To resolve this apparent
          contradiction, we tested whether resource limitation may also lead to the evolution of a
          C4 cycle. We optimised the model approximating resource limitation via an objective
          function for total minimal flux at different photorespiratory levels. Indeed, with
          increasing photorespiration, the optimisation for resource efficiency leads to the
          emergence of the C4 cycle as the optimal solution. Balancing the resource cost of
          photorespiration against the resource cost of the C4 cycle, the model predicts that N
          limitation may have facilitated C4 evolution given high levels of photorespiration. Other
          possible selective pressures such as biotic interactions can currently not be tested using
          the model since specialised metabolism is not included in the metabolic map or the output
          function. Extant C4 species have higher C : N ratios reflecting the N-savings the
          operational C4 cycle enables <cite itemscope="" itemtype="http://schema.stenci.la/Cite"
            data-citationmode="Narrative"><a href="#bib61"><span>61</span><span>Sage et
                al.</span><span>1987</span></a></cite>. The photorespiratory pump using glycine
          decarboxylase based CO<sub itemscope="" itemtype="http://schema.stenci.la/Subscript"><span
              data-itemtype="http://schema.org/Number">2</span></sub> enrichment also emerges from
          the model, showing that C2 photosynthesis is also predicted under simple resource
          limitation. Indeed N-savings have been reported from C2 plants compared with their C3
          sister lineages <cite itemscope="" itemtype="http://schema.stenci.la/Cite"
            data-citationmode="Narrative"><a href="#bib66"><span>66</span><span>Schlüter et
                al.</span><span>2016</span></a></cite>. Simply minimising photorespiration as the
          objective function also yields C4 photosynthesis as the optimal solution. Hence, two
          alternatively or parallelly acting selective pressures towards C4 photosynthesis,
          limitation in C and/or N, are identified by the model. In both cases, the model correctly
          predicts the C4 cycle of carboxylation and decarboxylation and the C2 photorespiratory
          pump as observed in extant plants. The evolution of C4 photosynthesis in response to
          multiple selective pressures underscores its adaptive value and potential for agriculture.
          Intermediacy also evolves indicating that it, too, is likely an added value trait which
          could be pursued by breeding and engineering efforts.</p>
        <p itemscope="" itemtype="http://schema.stenci.la/Paragraph">The optimal solutions for the
          metabolic flux patterns predict an intermediate stage in which CO<sub itemscope=""
            itemtype="http://schema.stenci.la/Subscript"><span
              data-itemtype="http://schema.org/Number">2</span></sub> transport via photorespiratory
          intermediates glycolate and glycerate (<a href="#fig4" itemscope=""
            itemtype="http://schema.stenci.la/Link">Figure 4</a>) and decarboxylation by glycine
          decarboxylase complex (<a href="#fig3" itemscope=""
            itemtype="http://schema.stenci.la/Link">Figure 3(B)</a>) is essential. All of the models
          of C4 evolution <span itemscope="" itemtype="http://schema.stenci.la/CiteGroup"><cite
              itemscope="" itemtype="http://schema.stenci.la/Cite"><a
                href="#bib48"><span>48</span><span>Monson</span><span>1999</span></a></cite><cite
              itemscope="" itemtype="http://schema.stenci.la/Cite"><a
                href="#bib6"><span>6</span><span>Bauwe</span><span>2010</span></a></cite><cite
              itemscope="" itemtype="http://schema.stenci.la/Cite"><a
                href="#bib63"><span>63</span><span>Sage et
                  al.</span><span>2012</span></a></cite><cite itemscope=""
              itemtype="http://schema.stenci.la/Cite"><a href="#bib31"><span>31</span><span>Heckmann
                  et al.</span><span>2013</span></a></cite><cite itemscope=""
              itemtype="http://schema.stenci.la/Cite"><a href="#bib86"><span>86</span><span>Williams
                  et al.</span><span>2013</span></a></cite></span> predict that the establishment of
          a photorespiratory CO<sub itemscope="" itemtype="http://schema.stenci.la/Subscript"><span
              data-itemtype="http://schema.org/Number">2</span></sub> pump is an essential
          intermediate step towards the C4 cycle. The photorespiratory CO<sub itemscope=""
            itemtype="http://schema.stenci.la/Subscript"><span
              data-itemtype="http://schema.org/Number">2</span></sub> pump, also known as C2
          photosynthesis, relocates the photorespiratory CO<sub itemscope=""
            itemtype="http://schema.stenci.la/Subscript"><span
              data-itemtype="http://schema.org/Number">2</span></sub> release to the bundle sheath
          cells. Plants using the photorespiratory CO<sub itemscope=""
            itemtype="http://schema.stenci.la/Subscript"><span
              data-itemtype="http://schema.org/Number">2</span></sub> pump are often termed C3-C4
          intermediates owing to their physiological properties <cite itemscope=""
            itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a
              href="#bib63"><span>63</span><span>Sage et al.</span><span>2012</span></a></cite>.
          Displaying the flux solution in <a href="#fig3" itemscope=""
            itemtype="http://schema.stenci.la/Link">Figure 3 (A) -(D)</a> and <a href="#fig4"
            itemscope="" itemtype="http://schema.stenci.la/Link">Figure 4</a> on a metabolic map in
          <a href="#fig3s1" itemscope="" itemtype="http://schema.stenci.la/Link">Figure 3—figure
            supplement 1</a> clearly illustrates that increasing photorespiratory flux through
          Rubisco drives the two-cell metabolic model from C3 to C4 metabolism by passing the C3-C4
          intermediate state. On the C3-C4 trajectory, the activity of Rubisco is shifted from the
          mesophyll to the bundle sheath, as well as from the constrained to the CCM-dependent
          Rubisco population as a consequence of the increased costs of photorespiration under
          increased <span itemscope="" itemtype="http://schema.stenci.la/MathFragment"><span
              class="mjx-chtml"><span class="mjx-math"
                aria-label="{p}_{{O}_{2}}:{p}_{C{O}_{2}}"><span class="mjx-mrow"
                  aria-hidden="true"><span class="mjx-msubsup"><span class="mjx-base"><span
                        class="mjx-texatom"><span class="mjx-mrow"><span class="mjx-mi"><span
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                      class="mjx-sub"
                      style="font-size: 70.7%; vertical-align: -0.36em; padding-right: 0.071em;"><span
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                                      style="padding-top: 0.372em; padding-bottom: 0.372em;">2</span></span></span></span></span></span></span></span></span></span><span
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                      class="mjx-sub"
                      style="font-size: 70.7%; vertical-align: -0.36em; padding-right: 0.071em;"><span
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                              style="padding-top: 0.519em; padding-bottom: 0.298em; padding-right: 0.045em;">C</span></span><span
                            class="mjx-msubsup"><span class="mjx-base"><span
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          ratio, see <a href="#equ5" itemscope="" itemtype="http://schema.stenci.la/Link">Equation
            5</a>. The increase of the oxygenation rate in the photorespiration constraint drives
          the reprogramming of the metabolism to avoid oxygenation by establishing the C4 cycle.
          Therefore, our analysis recovers the evolutionary C3-C4 trajectory and confirms the
          emergence of a photorespiratory CO<sub itemscope=""
            itemtype="http://schema.stenci.la/Subscript"><span
              data-itemtype="http://schema.org/Number">2</span></sub> pump as an essential step
          during the C4 evolution also under optimisation for resources <cite itemscope=""
            itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a
              href="#bib31"><span>31</span><span>Heckmann et al.</span><span>2013</span></a></cite>.
          The model may also provide a reason for why some plant species have halted their evolution
          in this intermediary phase <cite itemscope="" itemtype="http://schema.stenci.la/Cite"
            data-citationmode="Narrative"><a href="#bib65"><span>65</span><span>Scheben et
                al.</span><span>2017</span></a></cite>. Under the conditions of resource limitations
          and intermediate photorespiration, the model predicts intermediacy as the optimal
          solution. In a very narrow corridor of conditions, no further changes are required to
          reach optimality and the model thus predicts that a small number of species may remain
          intermediate.</p>
        <h3 itemscope="" itemtype="http://schema.stenci.la/Heading"
          id="two-cell-model-realises-different-c4-states"><em itemscope=""
            itemtype="http://schema.stenci.la/Emphasis">Two-cell</em> model realises different C4
          states</h3>
        <p itemscope="" itemtype="http://schema.stenci.la/Paragraph">Since the model predicts C4
          metabolism without specific constraints, different input and reaction constraints can be
          tested for their influence on the molecular nature of the C4 cycle. This approach may
          identify the selective pressure and boundaries limiting evolution. Initial optimisation
          without additional constraints or input limitations predict a C4 cycle based on
          decarboxylation by NADP-ME (<a href="#fig3" itemscope=""
            itemtype="http://schema.stenci.la/Link">Figure 3</a> and <a href="#fig3s1" itemscope=""
            itemtype="http://schema.stenci.la/Link">Figure 3—figure supplement 1(A)</a>). This
          prediction recapitulates intuition; the NADP-ME based C4 cycle is considered the &#39;most
          straight forward&#39; incarnation of C4 photosynthesis, it is always explained first in
          textbooks and is a major focus of research. The NADP-ME based cycle thus represents the
          stoichiometrically optimal solution when resource limitation or photorespiration are
          considered. Once NADP-ME is no longer available via constraint, PEP-CK and NAD-ME become
          optimal solutions albeit with a prediction of malate and pyruvate as the transfer acids
          (<a href="#fig8" itemscope="" itemtype="http://schema.stenci.la/Link">Figure 8</a>). The
          FVA identified aspartate and alanine as slightly less optimal solutions (<a href="#fig6"
            itemscope="" itemtype="http://schema.stenci.la/Link">Figure 6</a>). Since <em
            itemscope="" itemtype="http://schema.stenci.la/Emphasis">in vivo</em> this slightly less
          optimal solution has evolved in all NAD-ME origins tested to date, kinetic rather than
          stoichiometric reasons suggest themselves for the use of aspartate and alanine <cite
            itemscope="" itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a
              href="#bib13"><span>13</span><span>Bräutigam et
                al.</span><span>2018</span></a></cite>.</p>
        <h3 itemscope="" itemtype="http://schema.stenci.la/Heading"
          id="light-is-a-potential-evolutionary-driver-for-the-different-c4-states">Light is a
          potential evolutionary driver for the different C4 states</h3>
        <p itemscope="" itemtype="http://schema.stenci.la/Paragraph">Since all extant C3 species and
          therefore also the ancestors of all C4 species contain all decarboxylation enzymes <cite
            itemscope="" itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a
              href="#bib4"><span>4</span><span>Aubry et al.</span><span>2011</span></a></cite>, it
          is unlikely that unavailability of an enzyme is the reason for the evolution of different
          decarboxylation enzymes in different origins <cite itemscope=""
            itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a
              href="#bib62"><span>62</span><span>Sage</span><span>2004</span></a></cite>. Stochastic
          processes during evolution, that is up-regulation of particular enzyme concentrations via
          changes in expression and therefore elements <em itemscope=""
            itemtype="http://schema.stenci.la/Emphasis">cis</em> to the gene <cite itemscope=""
            itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a
              href="#bib14"><span>14</span><span>Bräutigam and
                Gowik</span><span>2016</span></a></cite>, may have played a role in determining
          which C4 cycle evolved. Alternatively, environmental determinants may have contributed to
          the evolution of different C4 cycles. Physiological experiments have pointed to a
          connection between nitrogen use efficiency and type of decarboxylation enzyme <cite
            itemscope="" itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a
              href="#bib58"><span>58</span><span>Pinto et al.</span><span>2016</span></a></cite>.
          Hence the variation in nitrogen input to the model was tested for their influence on
          optimal solutions with regard to decarboxylation enzymes. Input limitation of nitrogen,
          water as a metabolite, and CO<sub itemscope=""
            itemtype="http://schema.stenci.la/Subscript"><span
              data-itemtype="http://schema.org/Number">2</span></sub> limited the output of the
          system but did not change the optimal solution concerning decarboxylation, see <a
            href="#fig8s1" itemscope="" itemtype="http://schema.stenci.la/Link">Figure 8—figure
            supplement 1</a>, <a href="#fig8s2" itemscope=""
            itemtype="http://schema.stenci.la/Link">Figure 8—figure supplement 2</a>, and <a
            href="#fig8s3" itemscope="" itemtype="http://schema.stenci.la/Link">Figure 8—figure
            supplement 3</a>, making it an unlikely candidate as the cause. Differences in nitrogen
          use is possibly a consequence of decarboxylation type.</p>
        <p itemscope="" itemtype="http://schema.stenci.la/Paragraph">In some grasses, light
          penetrable cells overlay the vascular bundle leading to different light availability
          (summarised in <cite itemscope="" itemtype="http://schema.stenci.la/Cite"
            data-citationmode="Narrative"><a href="#bib8"><span>8</span><span>Bellasio and
                Lundgren</span><span>2016</span></a></cite> and <cite itemscope=""
            itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a
              href="#bib34"><span>34</span><span>Karabourniotis et
                al.</span><span>2000</span></a></cite>) and hence light availability and
          distribution were tested (<a href="#fig8" itemscope=""
            itemtype="http://schema.stenci.la/Link">Figure 8</a>). Changes in light input and
          distribution of light input between mesophyll and bundle sheath indeed altered the optimal
          solutions (<a href="#fig8" itemscope="" itemtype="http://schema.stenci.la/Link">Figure
            8</a>). The changes in the solution can be traced to the energy status of the plant
          cells. For very high light intensities, the alternative oxidases in the mitochondria are
          used to dissipate the energy and hence a path towards NAD-ME is paved. Under light
          limitation, the C4 cycle requires high efficiency and hence PEP-CK which, at least in part
          allows energy conservation by using PEP rather than pyruvate as the returning C4 acid, is
          favoured. Interestingly, the sensitivity of different species towards environmental
          changes in light is influenced by the decarboxylation enzyme present <cite itemscope=""
            itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a
              href="#bib72"><span>72</span><span>Sonawane et al.</span><span>2018</span></a></cite>.
          NADP-ME species are less compromised compared to NAD-ME species by shade possibly
          reflecting an evolutionary remnant as NAD-ME is predicted to emerge only in high light
          conditions. PEP-CK is more energy efficient compared to malic enzyme based decarboxylation
          which requires PEP recycling by PPDK at the cost of two molecules of ATP (<a href="#fig3"
            itemscope="" itemtype="http://schema.stenci.la/Link">Figure 3(D)</a>). Notably, two C4
          plants known to rely on PEP-CK <em itemscope=""
            itemtype="http://schema.stenci.la/Emphasis">P. maximum</em> and <em itemscope=""
            itemtype="http://schema.stenci.la/Emphasis">A. semialata</em> (African accessions) are
          shade plants which grow in the understory <cite itemscope=""
            itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a
              href="#bib44"><span>44</span><span>Lundgren and
                Christin</span><span>2017</span></a></cite>. PEP-CK can be co-active with NADP-ME
          and NAD-ME (<a href="#fig8" itemscope="" itemtype="http://schema.stenci.la/Link">Figure
            8</a>). This co-use of PEP-CK with a malic enzyme has been shown in C4 plants <span
            itemscope="" itemtype="http://schema.stenci.la/CiteGroup"><cite itemscope=""
              itemtype="http://schema.stenci.la/Cite"><a href="#bib57"><span>57</span><span>Pick et
                  al.</span><span>2011</span></a></cite><cite itemscope=""
              itemtype="http://schema.stenci.la/Cite"><a href="#bib87"><span>87</span><span>Wingler
                  et al.</span><span>1999</span></a></cite></span> and explained as an adaptation to
          different energy availability and changes in light conditions <span itemscope=""
            itemtype="http://schema.stenci.la/CiteGroup"><cite itemscope=""
              itemtype="http://schema.stenci.la/Cite"><a href="#bib57"><span>57</span><span>Pick et
                  al.</span><span>2011</span></a></cite><cite itemscope=""
              itemtype="http://schema.stenci.la/Cite"><a href="#bib7"><span>7</span><span>Bellasio
                  and Griffiths</span><span>2014</span></a></cite></span>. Dominant use of PEP-CK in
          the absence of malic enzyme activity as suggested (<a href="#fig3" itemscope=""
            itemtype="http://schema.stenci.la/Link">Figure 3(B)</a>, <a href="#fig3s1" itemscope=""
            itemtype="http://schema.stenci.la/Link">Figure 3—figure supplement 1</a> and <a
            href="#fig5" itemscope="" itemtype="http://schema.stenci.la/Link">Figure 5</a>) is rare
          <em itemscope="" itemtype="http://schema.stenci.la/Emphasis">in vivo</em><span
            data-itemtype="http://schema.org/Number">0</span><cite itemscope=""
            itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a
              href="#bib80"><span>80</span><span>Ueno and Sentoku</span><span>2006</span></a></cite>
          but observed in <em itemscope="" itemtype="http://schema.stenci.la/Emphasis">P.
            maximum</em> and in <em itemscope="" itemtype="http://schema.stenci.la/Emphasis">A.
            semialata</em>. While the model predictions are in line with ecological observations, we
          cannot exclude that kinetic constraints (i.e. <cite itemscope=""
            itemtype="http://schema.stenci.la/Cite"><a href="#bib13"><span>13</span><span>Bräutigam
                et al.</span><span>2018</span></a></cite>) may also explain why a stoichiometrically
          optimal solution such as the NADP-ME cycle is not favoured in nature where NADP-ME and
          NAD-ME species evolve in nearly equal proportions <cite itemscope=""
            itemtype="http://schema.stenci.la/Cite" data-citationmode="Narrative"><a
              href="#bib62"><span>62</span><span>Sage</span><span>2004</span></a></cite>.</p>
        <h3 itemscope="" itemtype="http://schema.stenci.la/Heading" id="conclusion">Conclusion</h3>
        <p itemscope="" itemtype="http://schema.stenci.la/Paragraph">CBM of photosynthetically
          active plant cells revealed a major knowledge gap impeding CBM, namely the unknown
          protonation state of most transport substrates during intracellular transport processes.
          When photoautotrophic metabolism was optimised in a single cell for minimal metabolic flux
          and therefore, optimal resource use, C3 photosynthetic metabolism was predicted as the
          optimal solution. Under low photorespiratory conditions, a two-celled model which contains
          a CCM-dependent Rubisco optimised for resource use, still predicts C3 photosynthesis.
          However, under medium to high photorespiratory conditions, a molecularly correct C4 cycle
          emerged as the optimal solution under resource limitation and photorespiration reduction
          as objective functions which points to resource limitation as an additional driver of C4
          evolution. Light and light distribution was the environmental variable governing the
          choice of decarboxylation enzymes. Modelling compartmented eukaryotic cells correctly
          predicts the evolutionary trajectories leading to extant C4 photosynthetic plant species.
        </p>
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