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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.
III. Incorporation of 13CO2 by RuBisCO. A detailed description of 13C-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
DHAP
E4P
Fru-6P
Fru-1,6-P2
GAP
Glc-6P
PEP
PGA
2-PGA
Pyr
R-5P
Ru 1,5-P2
Ru-5P
S-7P
Xu-5P
Acetyl coenzyme-A
Dihydroxyacetone 3-phosphate
Erythrose 4-phosphate
Fructose 6-phosphate
Fructose 1,6-bisphosphate
Glyceraldehydes 3-phosphate
Glucose 6-phosphate
Phosphoenol pyruvate
3-phosphoglyceric acid
2-phosphoglyceric acid
Pyruvic acid
Ribose 5-phosphate
Ribulose 1,5-bisphosphate
Ribulose 5-phosphate
Sedoheptulose 7-phosphate
Xylulose 5-phosphate.
Enzymes:
Aldo
Eno
FAS
GAPDH
GPI
HK
PDH
PFK
PK
PGK
PGL
PGM
PRK
RIso
TA
TK
TPI
XEpi
Fructose bisphosphate aldolase
2-phosphoglycerate enolase
Fatty acid synthase
3-phosphoglycerate dehydrogenase
Phosphoglucose isomerase
Hexokinase
Pyruvate dehydrogenase
Phosphofructokinase
Pyruvate kinase
Phosphoglycerate kinase
Phosphoglucono lactonase
Phosphoglyceromutase
Phosphoribulokinase
Ribose 5-phosphate isomerase
Transaldolase
Transketolase
Triose phosphate isomerase
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 CO2 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 % oleosin2. 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 CO2)
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
g gDW -1
Protein
L-Glu
L-Gln
L-Pro
L-Arg
L-Asp
L-Asn
L-Thr
L-Ile
L-Met
L-Lys
L-Ala
L-Val
L-Leu
L-Ser
L-Gly
L-Cys
L-Phe
L-Tyr
L-Trp
L-His
Total
Oil
1
MW 1
mmoles
g DW -1
0.013
0.041
0.014
0.024
0.015
0.014
0.013
0.014
0.006
0.013
0.013
0.017
0.025
0.017
129.13
128.15
97.13
156.20
115.11
114.12
101.12
113.18
131.21
128.19
71.10
99.15
113.18
87.09
0.100
0.322
0.144
0.156
0.128
0.125
0.131
0.126
0.049
0.104
0.181
0.174
0.224
0.192
0.015
0.004
0.016
0.012
0.005
0.007
0.30
57.07
103.16
147.19
163.19
186.23
137.16
0.260
0.043
0.111
0.072
0.026
0.049
0.54
826
0.654
carbons per
molecule
mmoles carbon / g DW
All carbons
C-4 of Asp
derived amino
acids
5
5
5
5
4
4
4
6
5
6
3
5
6
3
2
3
9
9
11
6
57
molecular weight of amino acids refers to the polymer (M-H2O)
0.498
1.608
0.719
0.781
0.514
0.501
0.526
0.757
0.245
0.622
0.544
0.868
1.342
0.577
0.519
0.129
1.000
0.650
0.288
0.295
12.983
37.264
0.128
0.125
0.131
0.126
0.049
0.104
0.664
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
(13) 5 Glc-6P
(14) Fru-6P + ATP
(15) Fru 1,6-P2
(16) DHAP
sum of (12) to (16):
(17) 5 Glc + 6 ATP
 5 Glc-6P
 5 Fru-6P
 Fru 1,6-P2
 DHAP + GAP
 GAP
(HK)
(GPI)
(PFK)
(Aldo)
(TPI)
 4 Fru-6P + 2 GAP
Fru-6P and GAP to RuBP:
(18) 2 Fru-6P + 2 GAP 2 Xu-5P + 2 E4P
(19) 2 Fru-6P + 2 E4P  2 S-7P + 2 GAP
(20) 2 S-7P + 2 GAP  2 Xu-5P + 2 R-5P
(21) 4 Xu-5P  4 Ru-5P
(22) 2 R-5P  2 Ru-5P
(23) 6 Ru-5P + 6 ATP 6 Ru-1,5-P2
(TK)
(TA)
(TK)
(XEpi)
(RIso)
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
(26) 12 3-PGA
 12 2-PGA
(27) 12 2-PGA
 12 PEP
(28) 12 PEP
 12 Pyr + 12 ATP
(29) 12 Pyr
 12 Ac-CoA + 12 NADH + 12 CO2
Sum of (24) to (29):
(30) 5 Glc
 12 Ac-CoA + 12 NADH + 6 CO2
(RuBisCO)
(PGM)
(Eno)
(PK)
(PDH)
S5
5 Glucose molecules (30 C) are transformed to 24 C-atoms in acetyl-CoA with 6 CO2 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
III. Incorporation of 13CO2 by RuBisCO
Incorporation of 13C-labeled CO2
As shown in Table 2 (main text), after labeling embryos with 13CO2, 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 CO2 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.
After growing the embryos in an atmosphere containing 2 % (v/v) of 13CO2 (99 % 13C), 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
C-1 vs. a 99 % 13C-enrichment in CO2 would indicate only a 5 % contribution to PGA synthesis by
RuBisCO. However, the 13C-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
the internal CO2 production5. By introducing label via [1-13C]-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
the level of 13C in the internal CO2 that is released by plastidic PDH (Fig. S1-B). From this estimate of
the enrichment of internal CO2 and the 13C 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 ):
dFPGA(1)
 FTP (1)VGAPDH  FCO 2VRuB  FRu1,5 BP(3)VRuB  FPGA(1)VPDH  0
dt
d [ PGA]
 VGAPDH  2VRuB  VPDH  0
dt
(S1)
(S2)
Where FX ( N ) is the fractional 13C-enrichment in the Nth 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:
FCO 2(1)VRub  FPGA(1) 2VRub  VGAPDH   0
which can be written as:
FPGA(1)
VRub

2VRub  VGAPDH  FCO 2
(S3)
Considering that the proportion of PGA made from RuBisCO is (see Fig. 1b of main text):
S7
2VRub
,
2VRub  VGAPDH 
equation (S3) can be used to express the same proportion as:
Pr oportion of PGA from RuBisCO 
2 FPGA(1)
2VRub

2VRub  VGAPDH  FCO 2
Resulting in:
Pr oportion of PGA from RuBisCO 
2 FPGA(1)
(S4)
FCO 2
Using equation (S4) and the experimental data from [1-13C]Ala and [U-13C3]Ala (Table 2, main text)
the contribution of RuBisCO to oil synthesis was calculated to be 36-50 %.
A
Triose-P
b
Ru
O
is C
Ru1,5-P2
B
Triose-P
CO
Ru1,5-P2
C
Triose-P
1
1
1
1
1
Pyruvate
[1-13C]Alanine
Val
CO2
Ac-CoA
OAA
Phe
1
1
Pyruvate
Val
PDH
PDH
D
Ru1,5-P2
[U-13C3]Alanine
Ac-CoA
PHE(2-9)
Val(1-5) Val
(2-5)
Val
PEP
Phe
1
1
Pyruvate
Val
PDH
CO2
External 13CO2
PHE(1-2)
1
1
PK
1
1
PHE(1-9)
Phe
1
PEP
PK
Phe
CO
PGM, ENO
1
1
PEP
PK
bis
PGA
PGM, ENO
PGM, ENO
Ru
1
PGA
PGA
Alanine
bis
1
1
1
Ru
CO2
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
the same carbon is released as CO2 by pyruvate dehydrogenase (PDH). A) If 13CO2 is fed an unknown rate of
diffusion exchange allows labeled 13CO2 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.
B) + C) Alternatively, labeled 13CO2 is produced by feeding 13C-labeled alanine. B) By feeding [1-13C]Ala
pyruvate is labeled at carbon position C-1. Valine C-1 represents the label in CO2 evolved by PDH while Phe C1 represents the label in PGA. C) By feeding [U-13C3]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.
13
C-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
with uniformly labeled substrates were performed: After feeding [U-13C3]alanine, valine (derived
from plastidic pyruvate) was intensely labeled (13C3- and 13C2 mass isotopomers) but no such
adjacently multiple labels were found in phenylalanine, suggesting that pyruvate is not converted back
to PEP (Fig. S1-C). In addition, after feeding [U-13C5]glutamine mass spectra of aspartate, threonine
and phenylalanine revealed that the fractions of multiply labeled molecules ([13C3]- and [13C4]-) 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
derived from C-1 of PEP, can completely be attributed to the carboxylation of 13CO2 by RuBisCO.
Does the label in C-1 of Valine quantitatively represent label of internal CO2 ?
For calculation of RubisCO flux (equation S4) the 13C-enrichment for internal CO2 was assumed to be
equal to the 13C-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
unlabeled CO2. In the case of [1-13C]Ala labeling the resulting 13C-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
enzyme) (data not shown). By this correction the valine-derived estimates for 13C-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-5phosphate; Ru5P, ribulose-5-phosphate; GO6P, 6-phosphoglucono-lactone; GL6P, 6-phosphogluconate; RuBP, ribulose 1,5bisphosphate; DHAP, dihydroxyacetone phosphate; FDP, Fructose 1,6-bisphosphate; F6P, fructose-6-phosphate; G6P, glucose-6phosphate; GAP, glyceraldehyde-3-phosphate; PGA13, glycerate 1,3-bisphosphate; PGA3, 3-phosphoglycerate; PGA2, 2phosphoglycerate; 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 CO 2
uptake and fatty acid is made solely from CO2 but with great demand for ATP and reductant. Only
modes C can account for the 13C-labelling patterns and oil:CO2 ratios observed at 50 µmol m-2 s-1.
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).
PP
2
HP
3
TP
6
Pyruvate
CO2
TP
PGA
CO2
PP
CO2
TP
CO2
CO2
PPP
TP
C
Bis
Ru
PGA
PP
HP
PPP
C
Bis
Ru
PGA
PP
HP
PPP
TP
CO2
Hexose
CO2
HP
PPP
4
PGA
PP
HP
PPP
5
Hexose
D. Autotrophy
CO2
CO2
O
Hexose
CO2
1
C. Non-oxidative
bypass
O
Hexose
B. Oxidative
bypass
O
A. Glycolysis
C
Bis
Ru
PGA
CO2
Pyruvate
Pyruvate
Pyruvate
Pyruvate
Ac-CoA
Ac-CoA
Ac-CoA
Ac-CoA
C18:0
C18:0
C18:0
C18:0
CO2
7
Ac-CoA
8
C18:0
Carbon in C18:0 /
Carbon uptake
(glucose)
66.7 %
66.7 %
80 %
∞
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 5phosphate); 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
uptake
Autotrophy
Autotrophy
Autotrophy
non-oxidative bypass
non-oxidative bypass
non-oxidative bypass
non-oxidative bypass
non-oxidative bypass
non-oxidative bypass
non-oxidative bypass
oxidative bypass
oxidative bypass
oxidative bypass
oxidative bypass
oxidative bypass
oxidative bypass
oxidative bypass
oxidative bypass
oxidative bypass
glycolysis
20
21
22
24
27
16
18
17
19
25
11
13
12
8
9
10
7
14
15
26
-71.00
-71.00
-71.00
-9.80
-11.60
-8.00
-9.50
-9.00
-10.67
-20.00
-9.00
-10.00
-9.33
-9.40
-9.40
-9.40
-8.00
-9.50
-9.50
1.00
-52.00
-52.00
-52.00
-8.80
-8.80
-7.00
-7.00
-8.00
-8.00
-16.00
-4.00
-4.00
0.00
0.80
0.80
0.80
2.00
2.00
2.00
2.00
0.00
0.00
0.00
-3.60
-3.60
-3.75
-3.75
-3.67
-3.67
-3.00
-4.00
-4.00
-4.33
-4.40
-4.40
-4.40
-4.50
-4.50
-4.50
-4.50
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
-18.00
-18.00
-18.00
3.60
3.60
4.50
4.50
4.00
4.00
0.00
6.00
6.00
8.00
8.40
8.40
8.40
9.00
9.00
9.00
9.00
18.00
18.00
18.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
Carbon use
efficiency
100x(C into oil) /
(C uptake as
glucose)
∞
∞
∞
83.3%
83.3%
80.0%
80.0%
81.8%
81.8%
100.0%
75.0%
75.0%
69.2%
68.2%
68.2%
68.2%
66.7%
66.7%
66.7%
66.7%
S14
Table S2b
Description
Autotrophy
Autotrophy
Autotrophy
non-oxidative bypass
non-oxidative bypass
non-oxidative bypass
non-oxidative bypass
non-oxidative bypass
non-oxidative bypass
non-oxidative bypass
oxidative bypass
oxidative bypass
oxidative bypass
oxidative bypass
oxidative bypass
oxidative bypass
oxidative bypass
oxidative bypass
oxidative bypass
glycolysis
mode
#
20
21
22
24
27
16
18
17
19
25
11
13
12
8
9
10
7
14
15
26
% PGA from
RuBisCO1
600.0%
600.0%
600.0%
120.0%
120.0%
100.0%
100.0%
111.1%
111.1%
200.0%
111.1%
111.1%
111.1%
111.1%
111.1%
111.1%
100.0%
111.1%
111.1%
0.0%
PGA
from
RuBisCO
54.00
54.00
54.00
10.80
10.80
9.00
9.00
10.00
10.00
18.00
10.00
10.00
10.00
10.00
10.00
10.00
9.00
10.00
10.00
0.00
NADH-GAPDH +
NADPH-GAPDH
-45.00
-45.00
-45.00
-1.80
-1.80
0.00
0.00
-1.00
-1.00
-9.00
-1.00
-1.00
-1.00
-1.00
-1.00
-1.00
0.00
-1.00
-1.00
9.00
OPPP
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
2.00
2.00
4.00
4.40
4.40
4.40
4.50
5.00
5.00
0.00
1: % PGA from RuBisCO = 2 VRub / (2 VRub + VGAPDH). See Fig. 1b of main text.
Aldolase
0.00
-18.00
-9.00
0.00
1.80
0.75
2.25
0.33
2.00
0.00
0.00
1.00
0.00
0.00
-0.40
-0.20
0.00
0.00
-0.50
4.50
NADPH / mol
acetyl-CoA
-5.78
-5.78
-5.78
-0.98
-0.98
-0.78
-0.78
-0.89
-0.89
-1.78
-0.44
-0.44
0.00
0.09
0.09
0.09
0.22
0.22
0.22
0.22
PP from
PPP
(2TKI +
TKII)
27
27
27
5.4
5.4
4.5
4.5
5
5
9
3
3
1
0.6
0.6
0.6
0
0
0
0
TP -->
PPP
1.8
5.4
1.5
4.5
1.67
5
9
1
3
1
1
0.2
0.6
0
1
0
0
-9
-1.8
0
S15
References (supplement)
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