Download McFil: metabolic carbon flow in leaves

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Supporting Information for "An analytical model of non-photorespiratory CO2 release
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in the light and dark in leaves of C3 species based on stoichiometric flux balance" by
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Thomas N. Buckley and Mark A. Adams (PCE 10-246).
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Calculation of anabolic demand terms
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The simulations shown in this paper used values for the anabolic demand/supply terms (Vana,
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Vby, Bn, Bp and Bt) and the maintenance ATP demand term (M) estimated from simplified
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biosynthetic stoichiometries for amino acids, lignin, phospholipids and cellulose applied to
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published data on leaf composition. The procedures are outlined below.
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Leaf composition.
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First, we estimated proportions (on a dry weight basis) of non-labile anabolic products
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(protein, lignin & soluble phenolics, lipids, total structural and nonstructural carbohydrates,
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and organic acids), % C, specific leaf area and C:N ratio from regressions of these values
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against aboveground net primary productivity for 15 species across a range of habitats of
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differing productivity, as reported by Poorter and de Jong (1999). We interpolated values at
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500 g m-2 yr-1, approximately the middle of the reported range. We estimated carbohydrate
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content as 55.13% by combining total structural and nonstructural carbohydrates and organic
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acids; we assumed the latter to be most similar to carbohydrates in terms of energy and
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elemental content. Lipid content was 6.41%, lignin and soluble phenolics were 19.23% and
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protein was 19.23%. For protein biosynthesis, we derived an overall biosynthetic
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stoichiometry for eight amino acids (Glu, Gln, Asp, Ser, Ala, Thr, Phe and Asn) that
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comprised 90% of total leaf amino acids on a molar basis, in an average of five datasets for
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leaf amino acid contents (spinach, Riens et al., 1991; barley, Winter, Lohaus & Heldt, 1992;
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and wheat, Caputo & Barneix, 1997; tomato, Valle, Boggio & Heldt, 1998; potato, Karley,
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Douglas & Parker, 2002). The least abundant of these eight, phenylalanine, contributed
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4.13%, whereas the next most abundant amino acid, valine, contributed 1.93%. The
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proportions contributed by these amino acids in our simulations are given in Table S2.
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Calculation of supply/demand rates
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We converted leaf composition values to rates of anabolic demand as follows. For product y
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(e.g, protein) and co-factor x (ATP, CO2, etc.), net x demand was calculated as:
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 g y   C py mol C s
  
demand   f y

g

  1  s yc g y
 

   ry g    s yx mol x
  g d   mol C
s


 
g
   SLA 2dm

mleaf
 
  10 3 mol d 


  8.64 mol s 

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where fy is the dry matter fraction of y; Cpy is the C content of y per g; syc is the CO2
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production per mole of source C in the biosynthesis of y, which corrects Cpy to a source
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carbon basis; ry is the sum of leaf relative growth rate and the non-recycled turnover rate (ry =
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RGR + uy(1 - y), where uy is turnover rate and y is recycled fraction); syx is the consumption
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of x per source C used in the biosynthesis of y (the syx were derived from biosynthetic
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stoichiometries as described below); SLA is specific leaf area; and 103/8.64 (= 106/(24∙3600))
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corrects molar and time dimensions. These demand terms were then summed for protein,
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lipids, carbohydrates and phenolics for each of NADH, NADPH, ATP and CO2 to give the
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terms Bn, Bp, Bt and Vby, respectively, with units of mol m-2leaf s-1. The maintenance term, M,
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was calculated in similar fashion except that ry was just the recycled turnover rate (ry = uy∙y),
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and ion gradient maintenance was added, as discussed below. Product-specific components
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of Vana (source carbon flow into anabolic products) were also calculated in similar fashion,
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except that syx was omitted. Finally, for simplicity the stoichiometries described below were
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based on triose phosphate as a carbon source. To account for the additional ATP cost of
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using sucrose or starch as the carbon source, which consumes one ATP per three source C
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atoms, we added the quantity Vana/3 to the biosynthetic ATP demand term Bt.
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The calculations above were repeated for three different scenarios: (a) rapidly growing
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leaves, with a relative growth rate of 0.086 d-1 (this value was chosen to match the "young"
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leaves of H. arbutifolia studied by Villar et al. (1995)), (b) mature leaves, with an RGR of
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0.008 d-1 (matching the "mature" leaves of Villar et al. (1995), and (c) mature leaves as in (b),
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but with the protein component of anabolic demand from (a) added to represent amino acid
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synthesis for export to non-photosynthetic tissues. Scenario (c) models a plant with whole
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plant C:N and protein:N ratios equal to those of the leaves, and a leaf mass ratio of 0.5 and a
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relative growth rate for non-leaf tissues of 0.086 d-1. The three sets of values of anabolic
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supply/demand terms are shown in Table Error! Reference source not found.. Two values
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are given for the NADH and chloroplastic NADPH terms (Bn and Bp), reflecting
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stoichiometries for N assimilation from NO3- and NH4+. Carboxylation capacity, Vm, was
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estimated using the values for C:N ratio, specific leaf area and %C corresponding to our
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estimates of anabolic product contents from Poorter & de Jong (1999), combined with the
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overall regression of Vm vs leaf N content given by Meir et al. (2002) for ten tree species
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({Vm/[mol m-2 s-1]} = 22.6{N/[g m-2]} + 1.18).
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Biosynthetic stoichiometries
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We used simplified biosynthetic stoichiometries to derive the parameters syx (net consumption
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(or production) of energy carriers (ATP, etc) per carbon atom in anabolic products) for the
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anabolic demand calculations described above. These stoichiometries are outlined below
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Protein. Stoichiometries for amino acid biosynthesis pathways are outlined below
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and results are listed in Table S3. Two values were derived for NADH and NADPH, based
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on synthesis beginning from NO3- or NH4+, respectively. For NO3--based values, an
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additional demand of one NADH and 3 NADPH (representing 6 reduced ferredoxin in the
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light) was added to the total demands for the biosynthetic pathway for each NH4+ consumed
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in synthesis. Three values are given for ATP, representing (i) de novo biosynthesis of amino
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acids only, (ii) de novo biosynthesis plus protein assembly, which included an additional 3.5
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ATP per amino acid (the median of 3-4 ATP equivalents required per peptide bond), and (iii)
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protein degradation and reassembly only, which included 5.5 ATP per amino acid
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(representing the 3-4 for polymerisation, plus 2 ATP equivalents for hydrolysis of each
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peptide bond). Value (i) was used for the growth component of Bt in simulations for mature
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leaves exporting amino acids; value (ii) was used for the turnover component of Bt in all
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simulations, and for the growth component of Bt in young and mature non-exporting
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simulations; and value (iii) was used for the turnover repolymerisation component of M in all
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simulations. Cpy (C content per g) was 0.03334 mol C g-1; this is a proportion-weighted
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average of the eight amino acids.
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Phospholipid biosynthesis. The glycerol backbone of phospholipids is generated from
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triose phosphate (TP), oxidising one NADH. The two fatty acids each require 8 acetyl-CoA
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(generating 2 NADH and 2 ATP and releasing one CO2 if derived from TP) and seven
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polymerisation steps, each consuming 2 NADPH and one ATP. Synthesis of serine (taken
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here as the archetype for phosphate-linked R-group; see below for details of biosynthetic
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stoichiometry for Ser) consumes one more TP, an NH4+ and an NADPH, and yields two
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NADH; two ATP equivalents are used to activate the phosphatidate for R-group linkage.
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Thus, 18 TP and 29 NADPH are consumed, 34 NADH and 16 ATP are produced, and 16
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CO2 are released. The net consumption of co-products per source C atom (syx) are then:
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34/(183) = +0.630 NADH/C, -29/54 = -0.537 NADPH/C, 16/54 = +0.296 CO2/C, and 16/54
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= +0.296 ATP/C. If NH4+ consumed in Ser synthesis is generated from NO3- in leaves, one
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additional NADH and three NADPH equivalents are consumed, changing the totals to 0.611
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for NADH and -0.593 for NADPH. Cpy was 0.05238 mol C/g, based on phosphatidyl serine.
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Phenolic biosynthesis. We base these stoichiometries on lignin, assuming lignin
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represents the majority of phenolics in typical leaf tissues. Lignin biosynthesis is not very
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well understood, but most of the costs arise from synthesis of phenylalanine, which consumes
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7/3 TP, one ATP, 2 NADPH and one NH4+ and yields one NADH and one CO2 overall (see
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the Phe biosynthetic stoichiometry below). We acknowledge that these figures may
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underestimate the NADPH costs of lignin synthesis because NADPH-linked peroxidases are
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suspected to generate reactive oxygen species involved in activating subunits of lignin for
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cross-linking and polymerisation. These figures give net co-product consumption of +1/7
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NADH/C, -2/7 NADPH/C, -1/7 ATP/C and +1/7 CO2/C. If the NH4+ is generated from NO3-
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in the leaf, then one additional NADH and three NADPH equivalents are consumed,
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changing the totals to 0 NADH and -5/7 NADPH.
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Polysaccharide biosynthesis. We assume one ATP is consumed per six-carbon
monosaccharide in polysaccharide synthesis, including cellulose, starch and pectins.
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Terpenoid biosynthesis. Although terpenoids were absent from our leaf composition
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parameter set, we simulated the addition of anabolic demand for terpenoids in an experiment
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in the Discussion. For simplicity, we used the mevalonic acid pathway, in which three
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acetyl-CoA units, 2 NADPH and 3 ATP are consumed and one CO2 is released in the
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synthesis of isopentanyl diphosphate, which then isomerises and/or polymerises to form
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longer terpene chains without further energy or carbon flow. Synthesis of the three acetyl-
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CoA units from TP generates 6 NADH, 6 ATP and 3 CO2, so the total stoichiometry is -2/9
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NADPH/C, +3/9 ATP/C, +4/9 CO2/C and +6/9 NADH/C.
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Turnover and ion fluxes. Following de Vries (1975), we estimated protein turnover as
0.1059 day-1. This assumes 44% of leaf protein is in Rubisco and turns over 0.06 day-1, and
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that 56% is in other proteins and chlorophyll, which are assumed to turn over 0.15 day-1.
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Based on measurements of amino acid degradation by Trewavas (1972) (0.04 d-1 average for
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Arg, Lys, Glu and Leu) relative to protein degradation under the same conditions (0.10 d-1),
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we assume 60 percent amino acid recycling. Membrane lipid turnover is variable; we
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estimated 0.5 d-1 based on the turnover rates of the two largest components of membrane
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lipids in Cucurbita pepo leaves (Roughan, 1970), which is in the range of turnover rates cited
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by de Vries (1975) for non-leaf tissues and animal cells, and we also assumed 90% recycling
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of membrane lipids, following de Vries (1975). We assume negligible turnover for
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polysaccharides and phenolics. For total ATP costs for maintaining ion gradients, we applied
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de Vries' (1975) calculation of 8 mg glucose per gram of dry mass per day, which equates to
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1.6∙10-3 mol ATP per gram dry mass per day.
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Amino acids. Many of the following summary pathways are based partly on
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stoichiometries earlier in the list. All refer to carbon skeleton stoichiometries, which are
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given after the amino acids. Glu: Glutamate synthesis consumes one -ketoglutarate (-KG),
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one NADPH equivalent, one ATP and one NH4+. This assumes Fdx-GOGAT, not NADH-
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GOGAT, catalyses all glutamate synthesis. Asp: Aspartate synthesis consumes one molecule
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of oxaloacetic acid (OAA) and one Glu, and yields one -KG. Gln: Glutamine synthesis
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using GS consumes one Glu, one ATP and one NH4+. Asn: Asparagine synthesis using
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glutamine-dependent asparagine synthetase consumes one Asp, one Gln and two ATP
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equivalents, and yields one Glu. This assumes negligible activity of ammonium-dependent
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AS. Ala: Alanine synthesis consumes one pyruvate (Pyr) and one Glu and yields one -KG.
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Thr: Threonine synthesis consumes one Asp, 2 ATP and 2 NADPH. Phe: Phenylalanine
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synthesis consumes one chorismate and one Glu, and yields one -KG and one CO2;
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synthesis of the chorismate, in turn, consumes one phosphoenol pyruvate (PEP), one
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erythrose-4-phosphate (E4P), one NADPH and one ATP. Ser: The phosphorylated pathway
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for serine synthesis consumes one 3-phosphoglycerate (3-PGA) and one Glu and yields one
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NADH and one -KG. The glycolate pathway predominates in leaves. However, diverting
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one Ser from photorespiration eliminates the production of one 3-PGA and consumption of
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one ATP and NADH. It also eliminates a Ser amino donor for the synthesis of one Gly from
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glyoxylate via serine:glyoxylate aminotransferase, which requires that one glyoxylate instead
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be aminated with Glu, via glutamate:glyoxylate aminotransferase; this consumes one Glu and
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yields one -KG. Thus, the two pathways have identical net stoichiometries.
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Carbon skeletons. Many of the stoichiometries above are expressed in terms of
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carbon compounds. The stoichiometries we used to convert source carbohydrate to those
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carbon compounds are as follows. PEP and 3-PGA: Synthesis of either molecule in
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glycolysis consumes one TP and yields one NADH and one ATP. Pyr: Pyr synthesis in
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glycolysis consumes one PEP and generates one ATP. AcCoA. Acetyl CoA synthesis from
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Pyr decarboxylation consumes one Pyr and generates one NADH and one CO2. E4P: three
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molecules of E4P can be generated from four TP in the reversible reactions of the pentose
164
phosphate pathway; i.e., one E4P from 1.33 TP. OAA: we assume OAA synthesis for
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anabolic demands is formed by carboxylation of PEP, which consumes one CO2 and one
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PEP. -KG: we assume -ketoglutarate is formed in the TCA cycle from one OAA and one
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acetyl-CoA, generating one NADH and one CO2 directly.
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Summary. Stoichiometries are outlined in Table S1. Most individual amino acid
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stoichiometries outlined above must be combined with others in the list, and with carbon
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skeleton stoichiometries, to give results entirely in terms of TP, ATP, NADH, NADPH
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equivalents and CO2. The resultant net stoichiometries are summarised in Table S2. Finally,
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results were expressed on a source carbon atom basis (and, for protein, averaged for the eight
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amino acids used here, weighted by their proportions as given in Table S2). The results are
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given in Table S3. Alternative stoichiometries were calculated for ATP, NADH and NADPH
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as discussed above, and are also given in Table S3.
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Table S1. Gross stoichiometries for biosynthesis of eight amino acids and their carbon
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skeleton precursors. Numbers shown are the number of molecules of the compound in the
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column heading that are consumed in the synthesis of one molecule of the compound named
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in the first column. Positive numbers represent net yield rather than consumption. Net
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stoichiometries are given in Table S2.
PEP
3-PGA
Pyr
Ac
OAA
-KG
E4P
Glu
Gln
Asp
Ser
Ala
Thr
Phe
Asn
1
1
1
1
1
Asp
Gln
Glu
E4P
-KG
OAA
Acetyl-CoA
Pyr
3-PGA
PEP
NH4+
TP
CO2
NADPH
NADH
ATP
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-1
-1
-1
1
1
-1
1
1
-1
-1
-1
-1
-4/3
-1
-1
-1
-1
-1
-1
-1
1
-1
-1
-2
-1
-2
-2
-1
-1
-1
-1
-1
1
1
1
-1
1
-1
1
-1
-1
1
-1
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-1
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Table S2. Net stoichiometries for biosynthesis of eight amino acids, and the percent of total
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amino acids contributed by each in calculations of anabolic demand/supply terms.
Glu
Asp
Gln
Ser
Ala
Thr
Asn
Phe
mol%
29.6
20.2
17.8
10.5
9.1
5.5
4.2
3.1
ATP
2
0
1
0
1
-2
-3
-1
NADH
4
1
4
2
1
1
1
1
NADPH
-1
-1
-1
-1
-1
-3
-1
-2
CO2
1
-1
1
0
0
-1
-1
1
TP
-2
-1
-2
-1
-1
-1
-1
-7/3
NH4+
-1
-1
-2
-1
-1
-1
-2
-1
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Table S3. Net production of NADH, NADPH, ATP and CO2 per mole of source carbon in
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the biosynthesis and maintenance of five classes of compounds used to calculate anabolic
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demand in the current study. Dimensions are moles of NADH, NADPH, CO2 or ATP per
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mole of source carbon consumed in biosynthesis of products listed in the column headings.
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Two values are given for NADH and NADPH, based on nitrogen assimilation beginning
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from either nitrate or ammonium (N assimilation affects stoichiometries for phospholipid
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synthesis here because we used phosphatidyl serine as the archetype for phosphate-linked R
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group). Three values are given for ATP under the heading of protein. For biosynthesis, (1)
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represents de novo synthesis of amino acids only, and (2) represents both de novo synthesis
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and polymerisation of amino acids. For maintenance, (3) represents degradation of proteins
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and repolymerisation of existing amino acids only. Calculations are described above under
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"Supply/demand stoichiometries for biosynthesis and maintenance processes."
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NADH (from NH4+)
NADH (from NO3+)
NADPH (from NH4+)
NADPH (from NO3+)
CO2
ATP (biosynthesis)
ATP (maintenance)
protein
0.520 mol mol-1
0.229
-0.229
-1.165
-0.016
0.076(1), -0.793(2)
-1.365(3)
phospholipids
0.630
0.611
-0.537
-0.593
0.296
0.296
-
carbohydrate
0
0
0
0
0
-0.5
-
phenolics
0.143
0
-0.286
-0.714
0.143
-0.143
-
terpenoids
0.667
0.667
-0.222
-0.222
0.444
0.333
-
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References cited
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Caputo C. & Barneix A.J. (1997) Export of amino acids to the phloem in relation to N supply
in wheat. Physiologia Plantarum, 101, 853-860.
Karley A.J., Douglas A.E. & Parker W.E. (2002) Amino acid composition and nutritional
quality of potato leaf phloem sap for aphids. The Journal of Experimental Biology,
205, 3009-3018.
Meir P., Kruijt B., Broadmeadow M., Barbosa E., Kull O., Carswell F., Nobre A. & Jarvis
P.G. (2002) Acclimation of photosynthetic capacity to irradiance in tree canopies in
relation to leaf nitrogen concentration and leaf mass per unit area. Plant, Cell and
Environment, 25, 343-357.
Penning de Vries F.W.T. (1975) The cost of maintenance processes in plant cells. Annals of
Botany, 39, 77-92.
Poorter H. & de Jong R. (1999) A comparison of specific leaf area, chemical composition and
leaf construction costs of field plants from 15 habitats differing in productivity. New
Phytologist, 143, 163-176.
Riens B., Lohaus G., Heineke D. & Heldt H.W. (1991) Amino acid and sucrose content
determined in the cytosolid, chloroplastic, and vacuolar compartments and in the
phloem sap of spinach leaves. Plant Physiology, 97, 227-233.
Roughan P.G. (1970) Turnover of the glycerolipids of pumpkin leaves. The importance of
phosphatidylcholine. Biochemical Journal, 117, 1-8.
Trewavas A. (1972) Control of the protein turnover rates in Lemna minor. Plant Physiology,
49, 47-51.
Valle E.M., Boggio S.B. & Heldt H.W. (1998) Free amino acid composition of phloem sap
and growing fruit of Lycopersicon esculentum. Plant and Cell Physiology, 39, 458461.
Villar R., Held A.A. & Merino J. (1995) Dark leaf respiration in light and darkness of an
evergreen and a deciduous plant species. Plant Physiology, 107, 421-427.
Winter H., Lohaus G. & Heldt H.W. (1992) Phloem transport of amino acidds in relation to
the cytosolic levels in barley leaves. Plant Physiology, 99, 996-1004.
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