S1

RuBisCO improves the carbon efficiency of developing green seeds without the calvin cycle

Jörg Schwender, Fernando Goffman, John B. Ohlrogge & Yair Shachar-Hill

Supplementary Information

Supplementary information contents:

I. How much CO2 can be incorporated into seed protein? The contribution of carboxylation reactions that produce oxaloacetate to the total seed CO2 balance are described assuming that oxaloacetate is stored in seed protein biomass via amino acids of the aspartate family.

II. Carbon balance of Glycolysis vs. Bypass. A listing of stoichiometries of the enzymatic reactions and their sums for these two alternative metabolic routes for converting hexose into acetyl-CoA.

13 13 III. Incorporation of CO2 by RuBisCO. A detailed description of C-labeling experiments and the calculation of flux through the RuBisCO reaction based on label in its products.

IV. Elementary flux mode analysis of carbohydrate to oil conversion, including cofactor balances (ATP, NADPH). Input file for computer-aided flux mode analysis, listing of stoichiometries for each mode and tabulations for each mode of the cofactor balances, carbon efficiencies and the involvement of particular metabolic steps. S2

Used abbreviations for Metabolites and Enzyme reactions (in Fig. 1 of main text and in Supplement):

Metabolites: Ac-CoA Acetyl coenzyme-A DHAP Dihydroxyacetone 3-phosphate E4P Erythrose 4-phosphate Fru-6P Fructose 6-phosphate Fru-1,6-P2 Fructose 1,6-bisphosphate GAP Glyceraldehydes 3-phosphate Glc-6P Glucose 6-phosphate PEP Phosphoenol pyruvate PGA 3-phosphoglyceric acid 2-PGA 2-phosphoglyceric acid Pyr Pyruvic acid R-5P Ribose 5-phosphate Ru 1,5-P2 Ribulose 1,5-bisphosphate Ru-5P Ribulose 5-phosphate S-7P Sedoheptulose 7-phosphate Xu-5P Xylulose 5-phosphate. Enzymes: Aldo Fructose bisphosphate aldolase Eno 2-phosphoglycerate enolase FAS Fatty acid synthase GAPDH 3-phosphoglycerate dehydrogenase GPI Phosphoglucose isomerase HK Hexokinase PDH Pyruvate dehydrogenase PFK Phosphofructokinase PK Pyruvate kinase PGK Phosphoglycerate kinase PGL Phosphoglucono lactonase PGM Phosphoglyceromutase PRK Phosphoribulokinase RIso Ribose 5-phosphate isomerase TA Transaldolase TK Transketolase TPI Triose phosphate isomerase XEpi Xylulose 5-phosphate epimerase S3

I. How much CO2 can be incorporated into seed protein ?

We considered the possibility that the reduced CO2 emission in light was due to its re-fixation via phosphoenolpyruvate carboxylase or pyruvate carboxylase. The product, oxaloacetate, could be converted into amino acids and stored in proteins or possibly secreted from the embryo (e.g. in reduced form as malate). However, based on the amino acid composition of B. napus embryos, recovery of CO2 via oxaloacetate into seed protein can account for only about 4 % of the CO 2 released by PDH (see below). Also, by analysis of the growth medium no export of malate or other such fixation products was detected (NMR data not shown). The total carbon balance of a developing seed depends on all the biochemical reactions that consume and produce CO2. CO2 can be stored in seed protein via carboxylation of phosphoenol pyruvate to oxaloacetate which can be converted into aspartate, asparagine, threonine, isoleucine, methionine and lysine. To assess the amount of CO2 that could be incorporated into protein we first consider the different biomass fractions of Brassica napus seeds and express these as moles of carbon. The biomass composition of B. napus seeds is 45 % oil and 30 % protein (W / DW)1. The storage seed protein consists of 60 % cruciferin, 20 % napin and 20 % oleosin 2. Based on the amino acid compositions of these proteins2, 3, together with the molecular weights and the number of carbon atoms in each amino acid, the carbon fractions for all amino acids could be calculated. This yields a value of 0.66 millimoles of carbon per g total seed dry weight in the C-4 position (derived from CO 2) of aspartate family amino acids (Table S1). On a similar basis it was calculated that 37.3 millimoles of carbon per g DW are stored in fatty acids of seed oil (Table S1). To produce this oil requires incorporation of 18.7 millimoles of acetyl CoA units into fatty acids and thus 18.7 millimoles CO2 produced by pyruvate dehydrogenase. Thus only (100 x 0.66 / 18.7) = 3.5 % of the CO2 produced by pyruvate dehydrogenase could have been refixed into protein.

Table S1 Biomass composition of B. napus embryos mmoles carbon / g DW g gDW-1 MW1 mmoles carbons per All carbons C-4 of Asp g DW-1 molecule derived amino acids Protein L-Glu 0.013 129.13 0.100 5 0.498 L-Gln 0.041 128.15 0.322 5 1.608 L-Pro 0.014 97.13 0.144 5 0.719 L-Arg 0.024 156.20 0.156 5 0.781 L-Asp 0.015 115.11 0.128 4 0.514 0.128 L-Asn 0.014 114.12 0.125 4 0.501 0.125 L-Thr 0.013 101.12 0.131 4 0.526 0.131 L-Ile 0.014 113.18 0.126 6 0.757 0.126 L-Met 0.006 131.21 0.049 5 0.245 0.049 L-Lys 0.013 128.19 0.104 6 0.622 0.104 L-Ala 0.013 71.10 0.181 3 0.544 L-Val 0.017 99.15 0.174 5 0.868 L-Leu 0.025 113.18 0.224 6 1.342 L-Ser 0.017 87.09 0.192 3 0.577 L-Gly 0.015 57.07 0.260 2 0.519 L-Cys 0.004 103.16 0.043 3 0.129 L-Phe 0.016 147.19 0.111 9 1.000 L-Tyr 0.012 163.19 0.072 9 0.650 L-Trp 0.005 186.23 0.026 11 0.288 L-His 0.007 137.16 0.049 6 0.295 Total 0.30 12.983 0.664

Oil 0.54 826 0.654 57 37.264 1 molecular weight of amino acids refers to the polymer (M-H2O) S4

II. Carbon balance of Glycolysis versus Bypass The individual reactions and the net carbon efficiencies for the conversion of glucose to acetyl-CoA are compared for glycolysis and for the bypass. Carbon balances are presented without detailed accounting for cofactors. Cofactors are treated more comprehensively in the section on elementary flux mode analysis.

A) Carbon balance for plastidic glycolysis + FA synthesis Glycolysis to acetyl-CoA is balanced. Since light activation increases the affinity of plastidic GAPDH to NADP, GAPDH is included as NADPH producing. (1) 5 Glc + 5ATP  5Glc-6P (HK) (2) 5 glc-6P  5 Fru-6P (GPI)

(3) 5 fru-6-P + 5 ATP  5 Fru-1,6P2 (PFK)

(4) 5 Fru-1,6P2  10 GAP (Aldo, TPI) (5) 10 GAP  10 1,3PGA + 10 NAD(P)H (GAPDH) (6) 10 1,3PGA  10 3PGA + 10 ATP (PGA kin) (7) 10 3PGA  10 2PGA (PGM) (8) 10 2PGA  10 PEP (Eno) (9) 10 PEP  10 Pyr + 10 ATP (PK)

(10) 10 Pyr  10 Ac-CoA + 10 NADH + 10 CO2 (PDH) sum of equations (1) to (10):

(11) 5 Glc  10 Ac-CoA + 10 NAD(P)H + 10 NADH + 10 ATP + 10 CO2

5 Glucose molecules (30 C) are transformed to 20 C-atoms in acetyl-CoA with 10 CO2 released. Therefore fatty acid synthesis incorporates 66.7 % of the carbon provided as carbohydrate.

B) The reactions of the “glycolysis bypass” The transformation of glucose to fructose-6P and to RuBP is considered. Subsequently RuBP is transformed via RuBisCO to PGA and further to Ac-CoA. For incorporation into fatty acids additional cofactors have to be provided by photosynthesis.

Glc to Fru-6P and GAP: (12) 5 Glc + 5ATP  5 Glc-6P (HK) (13) 5 Glc-6P  5 Fru-6P (GPI)

(14) Fru-6P + ATP  Fru 1,6-P2 (PFK)

(15) Fru 1,6-P2  DHAP + GAP (Aldo) (16) DHAP  GAP (TPI) sum of (12) to (16): (17) 5 Glc + 6 ATP  4 Fru-6P + 2 GAP

Fru-6P and GAP to RuBP: (18) 2 Fru-6P + 2 GAP 2 Xu-5P + 2 E4P (TK) (19) 2 Fru-6P + 2 E4P  2 S-7P + 2 GAP (TA) (20) 2 S-7P + 2 GAP  2 Xu-5P + 2 R-5P (TK) (21) 4 Xu-5P  4 Ru-5P (XEpi) (22) 2 R-5P  2 Ru-5P (RIso)

(23) 6 Ru-5P + 6 ATP 6 Ru-1,5-P2 sum of (17) to (23): (24) 5 Glc + 12 ATP 6 RuBP

RuBP to Ac-CoA:

(25) 6 Ru-1,5-P2 + 6 CO2 12 3-PGA (RuBisCO) (26) 12 3-PGA  12 2-PGA (PGM) (27) 12 2-PGA  12 PEP (Eno) (28) 12 PEP  12 Pyr + 12 ATP (PK)

(29) 12 Pyr  12 Ac-CoA + 12 NADH + 12 CO2 (PDH) Sum of (24) to (29): S5

(30) 5 Glc  12 Ac-CoA + 12 NADH + 6 CO2

5 Glucose molecules (30 C) are transformed to 24 C-atoms in acetyl-CoA with 6 CO 2 being released. Therefore 80 % of the carbon provided as carbohydrate is incorporated into fatty acid by this novel route, compared to 66.7% by the conventional glycolytic route. Bypass of GAPDH and PGA kinase requires that ATP and reductant must be provided by light. S6

13 III. Incorporation of CO2 by RuBisCO

13 Incorporation of C-labeled CO2 13 As shown in Table 2 (main text), after labeling embryos with CO2, the labeling patterns in phenylalanine (Phe), valine (Val) and in fatty acids indicate that 13C-label is only present in C-1 of phosphoglyceric acid (PGA), while C-2 and C-3 are almost unlabeled. If CO 2 is fixed via the cyclic reductive pentose phosphate pathway (Calvin cycle), label would be distributed among all three carbon positions. The “bypass” scheme shown in figure S1 explains how this can occur. 13 13 After growing the embryos in an atmosphere containing 2 % (v/v) of CO2 (99 % C), we detected 2.5 % 13C in carbon one of Phe and a similar enrichment in carbon one of Val. PGA produced by

RuBisCO from fully labeled CO2 results in C-1 being labeled to 50 %. Hence a 2.5 % enrichment in 13 C-1 vs. a 99 % C-enrichment in CO2 would indicate only a 5 % contribution to PGA synthesis by 13 RuBisCO. However, the C-enrichment of CO2 inside the tissue is not 99%. The rapid production of unlabeled CO2 by plastidic PDH results in a high level of CO2 inside the developing embryo (ref. 4) and a limited exchange rate of the atmospheric labeled CO2 with the internal CO2 lowers the labeling in the internal CO2 (see Fig. S-1A).

Quantification of RuBisCO flux In additional experiments we took advantage of the fact that plastidic PDH is responsible for most of 5 13 the internal CO2 production . By introducing label via [1- C]-alanine, which is converted to plastidic pyruvate, C-1 of plastidic pyruvate is then released as CO2 by PDH (Fig. S1-B). This pyruvate is also the precursor for valine, whose 13C content in C-1 can be determined and used as a measurement of 13 the level of C in the internal CO2 that is released by plastidic PDH (Fig. S1-B). From this estimate of 13 the enrichment of internal CO2 and the C level in C-1 of phenylalanine (which is derived from PEP and PGA) the contribution of RuBisCO can be calculated as follows:

For C-1 of PGA the isotopic and metabolic steady state equations are (see Fig. 1b of main text ): dF PGA(1)  F V  F V  F V  F V  0 (S1) dt TP(1) GAPDH CO2 RuB Ru1,5BP(3) RuB PGA(1) PDH d[PGA] V  2V V  0 (S2) dt GAPDH RuB PDH

13 th Where FX ( N ) is the fractional C-enrichment in the N carbon position of metabolite X and where VE is the flux through enzyme E. GAPDH = glyceraldehyde 3 phosphate dehydrogenase, RuB =RuBisCO, Ru-1,5-BP = Ribulose 1,5 bisphosphate.

The C-1 of TP and C-3 of Ru-1,5-P2 are unlabeled so the first and third terms of equations S1 are zero, and by substitution of (S2) into (S1) one obtains:

FCO2(1)VRub  FPGA(1) 2VRub  VGAPDH   0 which can be written as:

V F Rub  PGA(1) (S3) 2VRub  VGAPDH  FCO2

Considering that the proportion of PGA made from RuBisCO is (see Fig. 1b of main text): S7

2V Rub , 2VRub VGAPDH  equation (S3) can be used to express the same proportion as:

2V 2F Pr oportion of PGA from RuBisCO  Rub  PGA(1) 2VRub  VGAPDH  FCO2 Resulting in: 2F Pr oportion of PGA from RuBisCO  PGA(1) (S4) FCO2

13 13 Using equation (S4) and the experimental data from [1- C]Ala and [U- C3]Ala (Table 2, main text) the contribution of RuBisCO to oil synthesis was calculated to be 36-50 %.

1 D PHE(1-2) A B Triose-P C Triose-P Triose-P Ru1,5-P Ru1,5-P2 Ru1,5-P2 2 PHE(1-9) PHE(2-9)

1 1 1 Phe

PGA PGA PGA 1 PGM, ENO PGM, ENO PGM, ENO Val (1-5) Val(2-5) 1 1 1 1 1 1 Val

PEP PEP OAA PEP PK Phe PK Phe PK Phe

1 1 1 1 1 1 1 1 1

[1-13C]Alanine Pyruvate [U-13C ]Alanine Pyruvate Alanine Pyruvate Val Val 3 Val PDH PDH PDH CO CO CO2 13 2 2 External CO2

Ac-CoA Ac-CoA Ac-CoA

Figure S1: Fate of labeled CO2 in the fatty acid biosynthesis pathway (plastidic compartment). The first location of CO2 fixed by RuBisCO is carbon one of PGA, which is processed via glycolysis to pyruvate, where 13 the same carbon is released as CO2 by pyruvate dehydrogenase (PDH). A) If CO2 is fed an unknown rate of 13 diffusion exchange allows labeled CO2 from the outside to enter the metabolic cycle. Amino acids derived from PEP and pyruvate, respectively can be used to measure the labeling pattern in their respective precursors. 13 13 13 B) + C) Alternatively, labeled CO2 is produced by feeding C-labeled alanine. B) By feeding [1- C]Ala pyruvate is labeled at carbon position C-1. Valine C-1 represents the label in CO 2 evolved by PDH while Phe C- 13 1 represents the label in PGA. C) By feeding [U- C3]Ala analogous measurements for CO2 and PGA can be made. The conversion of pyruvate back to PEP would entail PEP and Phe molecules labeled at multiple carbon positions. Accordingly the absence of mass isotopomers > m1 in mass spectra of Phe shows that the conversion of pyr to Phe does not exist, hence the label in C-1 of Phe can entirely be attributed to labeling of PGA via RubisCO. Similar experiments with labeled glutamine showed that labeled oxaloacetate (OAA) is not converted back to PEP. D) Mass spectrometry analysis of amino acids. Different fragments of valine and phenylalanine are shown, allowing in both cases a measure of 13C-enrichment in carbon one. 13C-label is shown in black. Dashed arrows with “X” denote metabolic conversions that were excluded by additional labeling experiments (data not shown). PGA = phosphoglyceric acid; PEP = phosphoenol pyruvate; OAA = oxaloacetate; VAL = valine; Phe = phenylalanine. PGM = phosphoglycerate mutase, ENO = enolase; PK = pyruvate kinase; PDH pyruvate dehydrogenase complex.

Does label in C-1 of phenylalanine and tyrosine quantitatively represent PGA? For calculation of RubisCO flux (equation S4) the 13C-enrichment in C-1 of PGA was assumed to be equal to the 13C-enrichment in C-1 of Phe and Tyr. The upper carbons of Phe and Tyr are derived from PGA via phosphoenolpyruvate (PEP) as shown in Fig. S1. However, PEP could also derive from pyruvate by pyruvate dikinase (EC 2.7.9.2) or from oxaloacetate (OAA) by PEP carboxykinase (EC 4.1.1.49) (figure S1c, dashed arrows). To exclude these possibilities, 13C-labeling experiments S8

13 with uniformly labeled substrates were performed: After feeding [U- C3]alanine, valine (derived 13 13 from plastidic pyruvate) was intensely labeled ( C3- and C2 mass isotopomers) but no such adjacently multiple labels were found in phenylalanine, suggesting that pyruvate is not converted back 13 to PEP (Fig. S1-C). In addition, after feeding [U- C5]glutamine mass spectra of aspartate, threonine 13 13 and phenylalanine revealed that the fractions of multiply labeled molecules ([ C3]- and [ C4]-) were abundant in both the oxaloacetate-derived amino acids while no such multiply labeled molecule species were found in the PEP-derived phenylalanine. Therefore we conclude that label in C-1 of Phe 13 derived from C-1 of PEP, can completely be attributed to the carboxylation of CO2 by RuBisCO.

Does the label in C-1 of Valine quantitatively represent label of internal CO2 ? 13 For calculation of RubisCO flux (equation S4) the C-enrichment for internal CO2 was assumed to be 13 equal to the C-enrichment in C-1 of Val. However, additional CO2 producing reactions (i. e. oxidative pentose-phosphate pathway, TCA cycle and malic enzyme, see ref. 5) could contribute 13 13 unlabeled CO2. In the case of [1- C]Ala labeling the resulting C-enrichment for CO2 was corrected using flux estimates for the additional CO2 producing reactions (ref. 5) together with label measurements representing C-1 of glucose (6-phosphogluconate dehydrogenase), C-6 of citrate (citrate dehydrogenase), C-1 of proline (ketoglutarate dehydrogenase) and C-4 of aspartate (malic 13 enzyme) (data not shown). By this correction the valine-derived estimates for C-enrichment in CO2 were reduced by about 15 % compared to the levels measured in C-1 of valine. Accordingly the simplifying assumptions in equation S4 cause an underestimation of the RuBisCO flux. S9

IV. Elementary flux modes analysis of carbohydrate to oil conversion

Computation of elementary flux modes Elementary flux modes analysis was performed using METATOOL6,7. By defining the stoichiometries of a metabolic network, all possible distinct routes by which substrates can flow through the network are described and all feasible metabolic conversions can be described by linear combinations of the flux vectors of the resulting elementary modes.

Elementary-modes analysis of oil synthesis was performed using a network of 26 reactions with 15 reversible (ENZREV, see below) and 11 irreversible steps (ENZIRREV) are implemented. The reactions can be taken from common biochemistry textbooks. The enzymes of glycolysis, OPPP, Calvin cycle and the synthesis of stearic acid (C18:0) were considered (see below, compare Fig. S2). Ambivalent enzyme functions are included and assumed to be present in B. napus plastids (both NADH- and NADPH dependent glyceraldehyde 3-phosphate dehydrogenase8-10 ; transaldolase together with sedoheptulose bisphosphate aldolase + sedoheptulose bisphosphatase). With the resulting model network (see below) consisting of 26 metabolic reactions with 22 internal metabolites (METINT) and 8 external metabolites (METEXT) all possible routes for conversion of glucose to stearic acid (C18:0) were considered. The external metabolites are glucose, CO2, C18:0, ADP/ATP, NADP/NADPH and inorganic phosphate (Pi). They can be consumed or produced by the network while all internal metabolites are to be balanced. We fixed NAD and NADH as balanced internal metabolites because there turned out to be a constant small imbalance between production and consumption of NAD for all flux modes. This is because the only reaction in the model generating acetyl-CoA is pyruvate dehydrogenase complex, producing equimolar amounts of acetyl-CoA and NADH. At the same time the fatty acid synthase complex needs both substrates in nearly the same ratio (9:8, considering synthesis of stearic acid = C18:0). Consequently, treating NAD/NADH as external metabolites always results in a constant and relative small NADH surplus (data not shown). Since in embryos of B. napus the plastidial glyceraldehyde-3-phosphate-dehydrogenase has both NAD- and NADP specific8-10 activity, both reactions were included in the model. This transhydrogenase reaction accounts for adjusting the small NADH surplus between pyruvate dehydrogenase complex and fatty acid synthetase.

Input file for METATOOL (Version 4.3 (25 October 2002) meta4.3_int.exe)4,5:

// Fatty acid synthesis // OPPP/glycolysis with // Phosphoribulokinase, RuBisCO // Aldolase (SH7P), Sedoheptulose bisphosphatase // two GAP-DH enzymes (NADP specific, NADPH-specific) GAP-DH reversible -ENZREV PGI Aldo TPI GAPDH1 GAPDH2 PGK PGM Eno PGL Riso Xepi TKI TKII TA SAldo -ENZIRREV HK PFK FBPase PK PDH G6PDH GND PRK RuBisCO SBPase FAS -METINT E4P S7P Xu5P Ru5P R5P GO6P GL6P RuBP DHAP FDP F6P G6P GAP PGA13 PGA3 PGA2 PEP R5P Pyr SBP AcCoA NAD NADH -METEXT CO2 Glc ATP ADP NADP NADPH C18 Pi -CAT HK : Glc + ATP = G6P + ADP . // hexokinase PGI : G6P = F6P . // Glucose-6P isomerase PFK : F6P + ATP = FDP + ADP . // Phosphofructokinase FBPase : FDP = F6P + Pi . // Fructose bisphosphatase Aldo : FDP = DHAP + GAP . // Aldolase (FruBP) TPI : DHAP = GAP . // Triosephosphate isomerase GAPDH1 : GAP + NADP + Pi = PGA13 + NADPH // NADP-GAP-dehydrogenase GAPDH2 : GAP + NAD + Pi = PGA13 + NADH . // NAD-GAP-dehydrogenase PGK : PGA13 + ADP = PGA3 + ATP . // phosphoglycerate kinase PGM : PGA3 = PGA2 . // phosphoglycerate mutase Eno : PGA2 = PEP . // enolase Pk : PEP + ADP = Pyr + ATP . // pyruvate kinase PDH : Pyr + NAD = AcCoA + CO2 + NADH . // pyruvate dehydrogenase S10

// Fatty acid synthesis (condensation + reduction): FAS : 9 AcCoA + 8 NADH + 8 NADPH + 8 ATP = C18 + 8 NAD + 8 NADP + 8 ADP + 8 Pi . G6PDH : G6P + NADP = GO6P + NADPH . // glucose-6-phosphate dehydrogenase PGL : GO6P = GL6P . // 6-phosphoglucono-lactonase GND : GL6P + NADP = Ru5P + NADPH + CO2 . // 6-phosphogluconate dehydrogenase Riso : Ru5P = R5P . // ribose-5P isomerase Xepi : Ru5P = Xu5P . // xylulose-5P epimerase TKI : Xu5P + R5P = GAP + S7P . // transketolase TKII : E4P + Xu5P = F6P + GAP . // transketolase TA : S7P + GAP = E4P + F6P . // transaldolase PRK : Ru5P + ATP = RuBP + ADP . // Phosphoribulokinase RuBisCO : RuBP + CO2 = 2 PGA3 . // RuBisCO SAldo : E4P + DHAP = SBP . // Aldolase (SBP) SBPase : SBP = S7P + Pi . // Sedoheptulose bisphosphatase Abbreviations of metabolites: Internal metabolites: E4P, erythrose-4-phosphate; S7P, sedoheptulose-7-phosphate; Xu5P, xylulose-5-phosphate; R5P, ribose-5- phosphate; Ru5P, ribulose-5-phosphate; GO6P, 6-phosphoglucono-lactone; GL6P, 6-phosphogluconate; RuBP, ribulose 1,5- bisphosphate; DHAP, dihydroxyacetone phosphate; FDP, Fructose 1,6-bisphosphate; F6P, fructose-6-phosphate; G6P, glucose-6- phosphate; GAP, glyceraldehyde-3-phosphate; PGA13, glycerate 1,3-bisphosphate; PGA3, 3-phosphoglycerate; PGA2, 2- phosphoglycerate; PEP, phosphoenol pyruvate; R5P, Ribulose-5-phosphate; Pyr, pyruvate; SBP, sedoheptulose 1,7-bisphosphate;

AcCoA, acetyl-Coenzyme A; NAD; NADH -External metabolites: CO2, carbon dioxide; Glc, glucose; ATP; ADP; NADP; NADPH; Pi; C18, octadecanoic acid.

Fatty acid producing elementary flux modes In total 28 elementary flux modes were obtained (see output below). 22 of these produce C18:0 (modes 7 to 28). Several of the fatty acid producing modes seem to produce more NADPH then needed for fatty acid synthesis, apparently a co-oxidation of glucose. These modes were sorted out in the following way: The synthesis of one mol C18:0 needs 8 moles NADPH. Mode 26 which employs only glycolysis for pyruvate formation has a small over-production of NADPH (surplus of 2 mol NADPH per mol C18:0). This mode will be considered because it is the “classic” pathway of oil synthesis. Modes with a higher NADPH surplus than 2 mol NADPH per mol C18:0 (modes 23 + 28) were excluded from the following considerations, leaving 20 modes that produce fatty acid by minimal oxidation of glucose (see below in bold, see Table S2). These 20 carbon use efficient flux modes fall into 4 principal types which are listed in table S2 and presented schematically in Fig. S2, together with their carbon economy and cofactor requirements.

Output generated by METATOOL: 1: ATP = ADP + Pi 2: ATP = ADP + Pi 3: Glc + ATP + 12 NADP = 6 CO2 + ADP + 12 NADPH + Pi 4: Glc + ATP + 12 NADP = 6 CO2 + ADP + 12 NADPH + Pi 5: ATP = ADP + Pi 6: ATP = ADP + Pi 7: 9 Glc + 16 ATP + 4 NADP = 18 CO2 + 16 ADP + 4 NADPH + 2 C18 + 16 Pi 8: 22 Glc + 47 ATP + 4 NADP = 42 CO2 + 47 ADP + 4 NADPH + 5 C18 + 47 Pi 9: 22 Glc + 47 ATP + 4 NADP = 42 CO2 + 47 ADP + 4 NADPH + 5 C18 + 47 Pi 10: 22 Glc + 47 ATP + 4 NADP = 42 CO2 + 47 ADP + 4 NADPH + 5 C18 + 47 Pi 11: 4 Glc + 9 ATP + 4 NADPH = 6 CO2 + 9 ADP + 4 NADP + C18 + 9 Pi 12: 13 Glc + 28 ATP = 24 CO2 + 28 ADP + 3 C18 + 28 Pi 13: 4 Glc + 10 ATP + 4 NADPH = 6 CO2 + 10 ADP + 4 NADP + C18 + 10 Pi 14: 9 Glc + 19 ATP + 4 NADP = 18 CO2 + 19 ADP + 4 NADPH + 2 C18 + 19 Pi 15: 9 Glc + 19 ATP + 4 NADP = 18 CO2 + 19 ADP + 4 NADPH + 2 C18 + 19 Pi 16: 15 Glc + 32 ATP + 28 NADPH = 18 CO2 + 32 ADP + 28 NADP + 4 C18 + 32 Pi 17: 11 Glc + 27 ATP + 24 NADPH = 12 CO2 + 27 ADP + 24 NADP + 3 C18 + 27 Pi 18: 15 Glc + 38 ATP + 28 NADPH = 18 CO2 + 38 ADP + 28 NADP + 4 C18 + 38 Pi 19: 11 Glc + 32 ATP + 24 NADPH = 12 CO2 + 32 ADP + 24 NADP + 3 C18 + 32 Pi 20: 18 CO2 + 71 ATP + 52 NADPH = 71 ADP + 52 NADP + C18 + 71 Pi 21: 18 CO2 + 71 ATP + 52 NADPH = 71 ADP + 52 NADP + C18 + 71 Pi 22: 18 CO2 + 71 ATP + 52 NADPH = 71 ADP + 52 NADP + C18 + 71 Pi 23: 9 Glc + ADP + 56 NADP + Pi = 36 CO2 + ATP + 56 NADPH + C18 24: 18 Glc + 49 ATP + 44 NADPH = 18 CO2 + 49 ADP + 44 NADP + 5 C18 + 49 Pi 25: 3 Glc + 20 ATP + 16 NADPH = 20 ADP + 16 NADP + C18 + 20 Pi 26: 9 Glc + 2 ADP + 4 NADP + 2 Pi = 18 CO2 + 2 ATP + 4 NADPH + 2 C18 27: 18 Glc + 58 ATP + 44 NADPH = 18 CO2 + 58 ADP + 44 NADP + 5 C18 + 58 Pi 28: 27 Glc + 5 ADP + 64 NADP + 5 Pi = 72 CO2 + 5 ATP + 64 NADPH + 5 C18 S11

Types of flux modes Mode A (Fig. S2) uses the conventional conversion of hexose to C18:0 (via glycolysis) resulting in nearly balanced cofactors. Hereby NADPH is provided by the NADP dependent activity of GAPDH. The modes of the “oxidative bypass” (Fig. S2 modes B) bypass most of glycolysis via the oxidative reactions of the pentose phosphate pathway and RuBisCO but require more ATP than glycolysis. Furthermore, if reductant to drive the RuBisCO bypass is provided by OPPP, as in modes B, then the

CO2 balance remains close to the glycolytic route (mode A) because the metabolic production of reductant requires the emission of an oxidized byproduct, CO2. The route outlined in Fig. 1a (main text) corresponds to the family of non-oxidative bypass modes (Fig. S2 modes C) that have higher efficiency in carbon conversion than modes A and B. With autotrophy (modes D) there is net CO2 uptake and fatty acid is made solely from CO2 but with great demand for ATP and reductant. Only 13 -2 -1 modes C can account for the C-labelling patterns and oil:CO2 ratios observed at 50 µmol m s . Although flux modes A, B, C and D each define distinct flux states, combining different proportions of the modes describes a continuous range of feasible carbon efficiencies. Increased carbon efficiency is achieved by increasing flux through RuBisCO, but comes at the cost of increasing cofactor requirement (Fig. S2).

A. Glycolysis B. Oxidative C. Non-oxidative D. Autotrophy bypass bypass

Hexose Hexose Hexose Hexose CO 1 2 CO2 HP 2 PP HP PP HP PP HP PP HP PP 3 PPP PPP PPP PPP PPP

TP TP TP TP TP 4 5 PGA PGA PGA PGA PGA CO CO 6 2 2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 Pyruvate Pyruvate Pyruvate Pyruvate Pyruvate 7 Ac-CoA Ac-CoA Ac-CoA Ac-CoA Ac-CoA 8 C18:0 C18:0 C18:0 C18:0 C18:0

Carbon in C18:0 / Carbon uptake 66.7 % 66.7 % 80 % ∞ (glucose)

ATP balance +1 -8 -8 -71

NADPH balance +2 +2 -7 -52

Number of modes 1 9 7 3 of this type Figure S2 Elementary flux-modes analysis of seed metabolism from glucose to fatty acids was performed using a network of 26 reactions. Carbon use efficiency (carbon stored in oil / carbon uptake as glucose), cofactor balances and 4 characteristic fluxes are shown relative to the formation of one mol C18:0. Of the 28 elementary modes, 20 produce stearic acid (C18:0). These modes fall into 4 categories (A to D) and for each category one representative mode is shown. Each category comprises modes that are very similar; with small differences in

CO2 and ATP balance due to ambivalent enzyme functions present in the model (e.g. transaldolase can replace sedoheptulose bisphosphate aldolase / sedoheptulose bisphosphatase). NADH is balanced in all cases (see methods, main text). Modes with negative ATP or NADPH balance require cofactor supply from photosynthetic light reactions. Numbers refer to enzymes: hexokinase (1); Glucose-6-phosphate dehydrogenase, 6-phosphoglucono-lactonase, 6-phosphogluconate dehydrogenase (2); Phosphofructokinase, fructose-1,6-bisphosphate aldolase (3); Phosphoribulokinase, Ribulose-1,5-bisphosphate carboxylase/oxygenase (4); NADH-glyceraldehyde-3-phosphate dehydrogenase, NADPH-glyceraldehyde-3-phosphate dehydrogenase, phosphoglycerate kinase (5); phosphoglycerate mutase, enolase, pyruvate kinase (6); pyruvate dehydrogenase complex (7); Synthesis of stearic acid by fatty acid synthethase complex (8); transketolase, transaldolase, ribose-5-phosphate isomerase, ribulose-5-phosphate epimerase (PPP) Metabolites: Ac-CoA, acetyl coenzyme-A; HP, hexose phosphates (glucose 6-phosphate, fructose 6-phosphate); PP, pentose phosphates (ribulose 5-phosphate, ribose 5-phosphate, xylulose 5- phosphate); TP, triose phosphates (dihydroxy acetone phosphate, glyceraldehyde 3-phosphate). S12

Relevance of the bypass for embryos developing in planta Growing embryos inside the green fruit structures of B. napus receive attenuated green light11, 12. It has been argued that green light is very ineffective in driving photosynthesis and that therefore photosynthesis may not contribute significantly to oil synthesis of B. napus seeds12. However, in contrary to this it has been shown that green light is as effective as blue or red light in driving photosynthesis and that photosynthesis in the inner cell layers of leaves is driven by green light13. Considering that 20-30 % of ambient daylight may penetrate the B. napus silique wall11,12 and considering our findings on the role of RuBisCO in developing oilseeds it remains to estimate whether the bypass can be driven under daylight conditions. The RubisCO bypass as described here is dependent on the availability of cofactors (ATP, NADPH) produced by photosynthesis. It has been reported for developing seeds of B. napus that net CO2 fixation is reached only under very high light (saturation at ~1000 µE m-2 s-1)11,12 which is normally not present within the fruit structures of Brassica. Modes D (Fig. S2) would describe this high-light autotrophy, with their relative cofactor demand representing the photosynthetic cofactor production. Comparing the cofactor demands of modes D and C (Fig. 2A) it appears that modes C (Bypass) need less than 15 % of ATP and NADPH of modes D. This means that with only a small fraction (<15%) of light required for autotrophy, RuBisCO in seeds carries all the metabolic flux. In fact, the range of light used in our experiments (< 100 µE m-2 s-1) is < 10 % the light intensity needed to drive maximal photosynthesis rates with B. napus developing seeds12. Accordingly, under the low light conditions that the seeds face in planta the bypass of conventional glycolysis represents the dominant metabolic flux in B. napus seeds. S13 Table S2: Detailed list of elementary flux modes. All balances are normalized to the production of 1 mol C18:0. Characteristic balances of outer metabolites are shown. The columns are distributed on two pages (S2a, S2b)

Table S2a

Description mode # ATP NADPH Glucose C18 CO2 net CO2 Carbon use uptake efficiency 100x(C into oil) / (C uptake as glucose) Autotrophy 20 -71.00 -52.00 0.00 1 -18.00 18.00 ∞ Autotrophy 21 -71.00 -52.00 0.00 1 -18.00 18.00 ∞ Autotrophy 22 -71.00 -52.00 0.00 1 -18.00 18.00 ∞ non-oxidative bypass 24 -9.80 -8.80 -3.60 1 3.60 0.00 83.3% non-oxidative bypass 27 -11.60 -8.80 -3.60 1 3.60 0.00 83.3% non-oxidative bypass 16 -8.00 -7.00 -3.75 1 4.50 0.00 80.0% non-oxidative bypass 18 -9.50 -7.00 -3.75 1 4.50 0.00 80.0% non-oxidative bypass 17 -9.00 -8.00 -3.67 1 4.00 0.00 81.8% non-oxidative bypass 19 -10.67 -8.00 -3.67 1 4.00 0.00 81.8% non-oxidative bypass 25 -20.00 -16.00 -3.00 1 0.00 0.00 100.0% oxidative bypass 11 -9.00 -4.00 -4.00 1 6.00 0.00 75.0% oxidative bypass 13 -10.00 -4.00 -4.00 1 6.00 0.00 75.0% oxidative bypass 12 -9.33 0.00 -4.33 1 8.00 0.00 69.2% oxidative bypass 8 -9.40 0.80 -4.40 1 8.40 0.00 68.2% oxidative bypass 9 -9.40 0.80 -4.40 1 8.40 0.00 68.2% oxidative bypass 10 -9.40 0.80 -4.40 1 8.40 0.00 68.2% oxidative bypass 7 -8.00 2.00 -4.50 1 9.00 0.00 66.7% oxidative bypass 14 -9.50 2.00 -4.50 1 9.00 0.00 66.7% oxidative bypass 15 -9.50 2.00 -4.50 1 9.00 0.00 66.7% glycolysis 26 1.00 2.00 -4.50 1 9.00 0.00 66.7% S14 Table S2b

Description mode PP from # PGA PPP % PGA from from NADH-GAPDH + NADPH / mol (2TKI + TP --> RuBisCO1 RuBisCO NADPH-GAPDH OPPP Aldolase acetyl-CoA TKII) PPP Autotrophy 20 600.0% 54.00 -45.00 0.00 0.00 -5.78 27 1.8 Autotrophy 21 600.0% 54.00 -45.00 0.00 -18.00 -5.78 27 5.4 Autotrophy 22 600.0% 54.00 -45.00 0.00 -9.00 -5.78 27 1.5 non-oxidative bypass 24 120.0% 10.80 -1.80 0.00 0.00 -0.98 5.4 4.5 non-oxidative bypass 27 120.0% 10.80 -1.80 0.00 1.80 -0.98 5.4 1.67 non-oxidative bypass 16 100.0% 9.00 0.00 0.00 0.75 -0.78 4.5 5 non-oxidative bypass 18 100.0% 9.00 0.00 0.00 2.25 -0.78 4.5 9 non-oxidative bypass 17 111.1% 10.00 -1.00 0.00 0.33 -0.89 5 1 non-oxidative bypass 19 111.1% 10.00 -1.00 0.00 2.00 -0.89 5 3 non-oxidative bypass 25 200.0% 18.00 -9.00 0.00 0.00 -1.78 9 1 oxidative bypass 11 111.1% 10.00 -1.00 2.00 0.00 -0.44 3 1 oxidative bypass 13 111.1% 10.00 -1.00 2.00 1.00 -0.44 3 0.2 oxidative bypass 12 111.1% 10.00 -1.00 4.00 0.00 0.00 1 0.6 oxidative bypass 8 111.1% 10.00 -1.00 4.40 0.00 0.09 0.6 0 oxidative bypass 9 111.1% 10.00 -1.00 4.40 -0.40 0.09 0.6 1 oxidative bypass 10 111.1% 10.00 -1.00 4.40 -0.20 0.09 0.6 0 oxidative bypass 7 100.0% 9.00 0.00 4.50 0.00 0.22 0 0 oxidative bypass 14 111.1% 10.00 -1.00 5.00 0.00 0.22 0 -9 oxidative bypass 15 111.1% 10.00 -1.00 5.00 -0.50 0.22 0 -1.8 glycolysis 26 0.0% 0.00 9.00 0.00 4.50 0.22 0 0 1: % PGA from RuBisCO = 2 VRub / (2 VRub + VGAPDH). See Fig. 1b of main text. S15

References (supplement)

1. Murphy, D. J. & Cummis, I. Biosynthesis of seed storage products during embryogenesis in rape seed, Brassica napus. J Plant Physiol. 135, 63-69 (1989). 2. Norton G. Nature and biosynthesis of storage proteins. In G Röbbelen, RK Downey, A Ashri, eds., Oil Crops of the World. McGraw Hill, NY, pp 165-191 (1989). 3. Murphy D.J., Keen J.N., Osullivan J.N., Au D.M.Y., Edwards E.W., Jackson P.J., Cummis I., Gibbons T., Shaw C.H. & Ryan A.J. A class of amphipathic proteins associated with lipid storage bodies in plants and possible similarities with animal serum apolipoproteins. Biochim Biophys Acta 1088: 86-94 ( 1991). 4. Goffman, F. D., Ruckle, M., Ohlrogge, J. B. & Shachar-Hill, Y. Carbon dioxide concentrations are very high in developing oil seeds. Plant Physiol. Biochem. 42, 703-708 (2004). 5. Schwender J., Ohlrogge, J. & Shachar-Hill, Y.: Understanding flux in plant metabolic networks. Curr. Opin. Plant Biol. 7, 309-317 (2004). 6. Schuster, S., Fell, D. & Dandekar, T. A general definition of metabolic pathways useful for systematic organization and analysis of complex metabolic networks. Nature Biotechnol. 18, 326-332 (2000). 7. Schuster, S., Dandekar, T. & Fell, D. A. Detection of elementary flux modes in biochemical networks: a promising tool for pathway analysis and metabolic engineering. Trends Biotechnol. 17, 53–60 (1999). 8. Kang, F. and Rawsthorne, S. Starch and fatty-acid synthesis in plastids from developing embryos of oilseed rape (Brassica napus L.) Plant Journal 6, 795-805 (1994). 9. Eastmond, P. J. & Rawsthorne, S. Coordinate changes in carbon partitioning and plastidial metabolism during the development of oilseed rape embryo. Plant Physiol. 122, 767-774 (2000). 10. Gupta, R. & Singh, R. Enzymes of glycolytic and pentose phosphate pathways in cytosolic and leucoplastic fractions of developing seeds of Brassica campestris. Indian J. Biochem. & Biophys. 34, 288-295 (1997).

11. King, S.P., Badger, M.R. & Furbank, R.T. CO2 refixation characteristics of developing canola seeds and silique wall. Aust. J. Plant Phys. 25, 377-386 (1998). 12. Eastmond, P., Kolacna, L., & Rawsthorne, S. Photosynthesis by developing embryos of oilseed rape (Brassica napus L). J. Exp. Bot. 47, 1763-1769 (1996).

13. Sun, J., Nishio, J.N. & Vogelmann, T.C. Green light drives CO2 fixation deep within leaves. Plant Cell Physiol. 39, 1020-1026 (1998).