Download Pod photosynthesis and seed dark CO2 fixation support oil

J. Biosci., Vol 20, Number 1, January 1995, pp 49–58. © Printed in India.
Pod photosynthesis and seed dark CO2 fixation
support oil synthesis in developing Brassica seeds
H R SINGAL, GURMEET TALWAR, ANITA DUA and
RANDHIR SINGH*
Plant Biochemistry and Molecular Biology Laboratory, Department of Chemistry and
Biochemistry, CCS Haryana Agricultural University, Hisar 125 004, India
MS received 2 May 1994; revised 26 August 1994
Abstract. Rate of photosynthesis and activities of photosynthetic carbon reduction cycle
enzymes were determined in pods (siliqua), whereas rate of dark CO2 fixation, oil content
and activities of enzymes involved in dark CO2 metabolism were measured in seeds of
Brassica campestris L. cv. Toria at different stages of pod/seed development. The period
between 14 and 35 days after anthesis corresponded to active phase of seed development
during which period, seed dry weight and oil content increased sharply. Rate of pod
photosynthesis and activities of photosynthetic carbon reduction cycle enzymes were maximum
in younger pods but sufficiently high levels were retained up to 40 days after anthesis.
The rate of dark 14CO2 fixation in seeds increased up to 21 days after anthesis and declined
thereafter but maintaining sufficiently high rates till 35 days after anthesis. Similarly various
enzymes viz., phosphoenolpyruvate carboxylase, NAD+-malate dehydrogenase and
NADP+-malic enzyme, involved in dark CO2 metabolism retained sufficient activities during
the above period. These enzyme activities were more than adequate to maintain the desired
supply of malate which mainly arises from dark CO2 fixation in seeds and further translocated
to leucoplasts for onward synthesis of fatty acids. Enzyme localization experiments revealed
phosphoenolpyruvate carboxylase and enzymes of sucrose metabolism to be present only
in cytosol, whereas enzymes of glycolysis were present both in cytosolic and leucoplastic
fractions. These results indicated that oil synthesis in developing Brassica seeds is supported
by pod photosynthesis and dark CO2 fixation in seeds as the former serves as the source
of sucrose and the latter as a source of malate.
Keywords.
1.
Pod photosynthesis; dark CO2 fixation; oil synthesis; Brassica seeds.
Introduction
Oil (triacylglycerol) makes up about 29–54% of the dry weight of Brassica seeds
(Singh and Mehta 1992) and its synthesis from sucrose constitutes one of the major
metabolic activity of embryos during seed development (Perry and Harwood 1993).
Dennis (1989) presented a model depicting the probable pathway of triacylglycerol
synthesis from sucrose. The model is primarily based on enzyme compartmentation
studies (Smith et al 1992) and suggests that glycolytic intermediates cross the
leucoplast membrane and serve as carbon skeletons for fatty acid synthesis. Though
acetate incorporation into fatty acids has been used to study the cofactor requirements
for fatty acid biosynthesis, it is unlikely that it may serve as the in vivo carbon
precursor for fatty acid synthesis mainly because of its relatively low cellular
concentration and low efficiency of utilization for de novo fatty acid synthesis in
*Corresponding author.
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H R Singal et al
oil seeds (Pollard and Singh 1987). Smith et al (1992) examined the incorporation
of radio-labelled substrates into fatty acids by leucoplasts isolated from endosperm
of developing castor oil seeds. Compared to pyruvate and acetate, exogenous malate
was found to support very high rates of fatty acid synthesis in these preparations.
Furthermore, they could detect significant activity of NADP+-malic enzyme in
leucoplasts. Based on these results, they proposed that malate may be an important
alternative carbon substrate for fatty acid synthesis in vivo. It was further hypothesised
that phosphoenolpyruvate (PEP) carboxylase in concert with cytosolic malate
dehydrogenase, could convert PEP derived from imported sucrose via cytosolic
glycolysis into malate, which would then be imported into the leucoplast for fatty
acid biosynthesis. This has gained support from the observation that developing
seeds of oil crops contain very high activities of PEP carboxylase (Singal et al
1987; Sangwan et al 1992). The model proposed above (Smith et al 1992) entails
high rates of dark CO2 fixation during the active phase of storage oil accumulation
in developing seeds of these crops. Since in Brassica, the photosynthetic contribution
of pod towards seed yield is quite substantial, being as high as 70–100% (Sheoran
et al 1991; Singal et al 1992), the pod photosynthesis during seed development
vis-a-vis oil synthesis in these crops assumes greater significance. It was, therefore,
thought appropriate to determine rates of pod photosynthesis and seed dark CO2
fixation alongwith the activities and compartmentation of the enzymes involved in
these pathways, during the period of seed development in Brassica campestris L. (cv.
Toria).
2.
Materials and methods
2.1 Crop
B. campestris was raised in pots filled with farm soil following recommended
agronomic practices. Fully opened flowers were tagged on the day of anthesis
during the active flowering phase. The pods (siliqua) formed from such flowers
were sampled at various time intervals until full maturity. Fresh weight and dry
weight of seeds separated from pods were determined at each sampling.
2.2 Chemicals
All biochemicals and enzymes used in these investigations were purchased from
Sigma Chemical Co., St. Louis, Mo, USA. NaH14CO3 (specific activity 54mCi
mmol-1) was procured from Bhabha Atomic Research Centre, Bombay. All other
chemicals used were of analytical grade.
2.3 CO2 exchange rate
CO2 exchange rate, stomatal conductance, transpiration rate and CO2 concentration
of the intact pods were monitored with a portable leaf chamber analyser (ADC,
LCA-2, England) fitted with data logger as described by Sheoran et al (1991) . The
photosynthetic photon flux density and temperature during these measurements were
1000 µmol mm–2 s-1 and 25±3°C, respectively.
CO2 assimilation in developing Brassica seeds
51
2.4 Dark CO2 fixation
Rate of I4CO2 fixation in the dark by isolated seeds was measured as per the
method of Singal et al (1987).
2.5 Preparation of enzyme extract
Five hundred mg of each tissue was used and enzyme extracts prepared as described
previously (Singal et al 1986).
2.6 Preparation of leucoplast and cytosol fractions
Leucoplasts were isolated by following the method of Smith et al (1992). Five g
of seeds obtained from pods at 20 DAA were homogenized in 50 mM Hepes-KOH
(pH 7·5) containing 0·4 M sorbitol, 0·4 mM EDTA, 1 mM MgCl2, 1 mM DTT, 1%
BSA and 1% Ficoll. The homogenate was filtered through four layers of cheese
cloth and centrifuged at 500 g for 5 min. The resulting supernatant was further
centrifuged at 6000 g for 10 min. The pellet obtained was resuspended in 5 ml
homogenizing buffer and layered onto a discontinuous percoll gradient. The band
of leucoplasts at 22–35% percoll interface was collected and used for enzyme
analysis. The supernatant was centrifuged at 15,000 g for 10 min to get rid of
nuclei and mitochondira. The resulting supernatant was referred to as cytosolic
fraction. The intactness of the leucoplasts was checked by enolase assay.
2.7 Enzyme assays
Enzyme activities were determined spectrophotometrically at 340 nm by following
the oxidation of NAD(P)H or reduction of NAD(P). All assays were carried out
at 300C and completed within 4 h of extraction. Preliminary assays were done for
all the enzymes to determine their stability and optimum conditions where linear
reaction rates with respect to time and enzyme concentrations were obtained. The
activities of various PCR cycle enzymes viz., 3-PGA kinase (EC 2.7.2.3),
NAD+-glyceraldehyde-3-P dehydrogenase (EC 1.2.1.12), NADP+-glyceraldehyde-3-P
dehydrogenase (EC 1.2.1.13), aldolase (EC 4.2.1.11), FBPase (EC 3.1.3.11) and
Ru-5-P kinase (EC 2.7.1.19) were determined by following the standard assay
methods reported earlier (Singal et al 1985; Sheoran et al 1990). PEP carboxyylase
(EC 4.1.1.31), NAD+-malate dehydrogenase (EC 1.1.1.37) and NADP+-malic enzyme
(EC 1:1.1.40) in seed extracts were assayed as described by Singal et al (1987).
Hexokinase (EC 2.7.1.1), sucrose synthase (EC 2.4.2.13), invertase (EC 3.2.1.26),
UDPG-pyrophosphorylase (EC 2.7.7.9) were assayed by following the method of
Kumar and Singh (1984). Phosphofructokinase (EC 2.7.1.11) and hexose-P isomerase
(EC 5.3.1.9) were determined as per the methods of Kelly and Latzko (1977) and
MacDonald and apRees (1983), respectively. 3-PGA kinase (EC 2.7.2.3) and pyruvate
kinase (EC 2.7.1.40) were assayed by following the procedures of Latzko and
Gibbs (1968) and Gupta and Singh (1989), respectively.
2.8
Estimation of protein and oil content
Protein in the enzyme extracts was measured according to Lowry et al (1951). Oil
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H R Singal et al
content in the seed was estimated by the method of Gupta et al (1985) using
NMR (MK-111A, New Port Analyser) equipped with 2 ml sample coil assembly.
3.
Results and discussion
Three distinct phases of development can be seen in seeds of oil crops (Perry and
Harwood 1993). The first phase, which commences immediately after fertilization
involves rapid cell division with little deposition of storage material. In the second
phase, rapid synthesis and accumulation of storage material (lipids) occur and in
the third and final phase, known as dehydration phase, desiccation takes place.
After a very slow initial increase up to 14 days after anthesis (DAA), Brassica
seeds recorded sharp increase in dry weight in the period between 14 and 35 DAA
(table 1), during which period oil content also increased very sharply. Thereafter,
the fresh weight of seeds declined, but the dry weight and oil content further
registered slight increase. Thus for B. campestris grown under net house condition,
the first, second and third phase of seed development corresponded to the periods
from 0 to 14, 14 to 35 and 35 DAA onwards until maturity, respectively. Very
little increase in oil content in the first and third phase of seed development
confirms to the general developmental pattern typical of oil seed crops grown under
growth room and field conditions (Perry and Harwood 1993).
Table 1. Fresh weight, dry weight and oil content of Brassica seeds at
different days after anthesis.
Values are mean ± SE of 6 different observations.
The current photosynthesis of fruiting structures plays an important role in yield
buildup in crop plants in general and oilseed crops in particular (Singh 1993). In
Brassica, the photosynthetic contribution of the pod (siliqua) is quite substantial
being as high as 70 to 100% (Allen et al 1971; Sheoran et al 1991; Singal et al
1992). Furthermore, greater fixation of labelled I4CO2 by the external surface of
the podwall compared to internal feeding and rapid translocation of fixed carbon
from podwall to seeds (Sheoran et al 1991) confirm contribution of pod photosynthesis
to seed growth. Otherwise also, at the pod formation stage, the whole canopy in
this crop is covered by pods and barely any leaves are visible. This has been
confirmed by higher pod area index (4·91) compared to leaf area index (4.10)
(Singh et al 1986). All these observations suggest the photosynthetic contribution
of pods towards seed development in Brassica. Keeping in view the above, we
monitored here the rate of pod photosynthesis alongwith the activities of some
CO2 assimilation in developing Brassica seeds
53
PCR cycle enzymes to correlate active phase of pod photosynthesis with that of
oil deposition in developing seeds.
Rate of photosynthesis in the present study (table 2) increased with pod growth
up to 20 DAA and then declined until maturity (50 DAA). However, the decrease
during the period 20–40 DAA was not significant, implying that the rate of
Table 2. Photosynthesis rate, transpiration rate, stomatal conductance and
internal CO2 concentration in Brassica pods at different days after anthesis.
Values are mean ±+ SE of 6 different observations.
photosynthesis in Brassica pods follows a triphasic pattern. In the first phase lasting
up to 20 DAA, the rate of photosynthesis increases; remains more or less constant
in the second phase (20–40 DAA) and then decreases in the last phase (40–50
DAA). Stomatal conductance, which was minimum at day 10 after anthesis increased
up to 30 DAA and then decreased during the later intervals. Rate of transpiration
followed a pattern similar to that of stomatal conductance and was maximum at
day 30 after anthesis. Internal CO2 concentration remained almost unchanged around
290ppm till maturity. The decrease in photosynthesis during the maturity phase
could not be explained on the basis of decreased stomatal conductance as the
concentration of internal CO2 remained unaffected till the end of pod growth. In
leaf tissues also, conductance is known to be reduced with age (Friedrich and
Huffaker 1980; Wittenbach 1983), without having significant effect on the
concentration of internal CO2 (Schulze and Hall 1982). Similar observations have
been reported earlier also from this lab (Singal et al 1987, 1992).
The various PCR cycle enzymes viz., NAD+- and NADP+-glyceraIdehyde-3-P
dehydrogenases, 3-PGA kinase, FBPase, aldolase and Ru-5-P kinase when monitored
during various time intervals of pod development, were found to have maximum
activities in the younger pods (table 3). However, each of these enzymes retained
reasonably good activity even during the later stages until 40 DAA (enzyme activities
were sufficient to support the rates of CO2 assimilation during these intervals),
confirming the interval of 10–40 DAA as the phase of active photosynthesis. During
maturation phase, decrease in enzyme activities may be due to desiccation which
sets in somewhere around 35–40 DAA. This may also be the reason for overall
decrease in the rate of photosynthesis during this period.
According to the latest model proposed by Smith et al (1992) for fatty acid
synthesis in leucoplasts of developing endosperms of oil seeds, malate serves as
an important carbon substrate for fatty acid synthesis in vivo. They further hypothesised
that malate in cytoplasm may arise from the action of PEP carboxylase in concert
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H R Singal et al
Table 3. Activities of some PCR cycl enzymes in Brassica pods at different days after anthesis.
Values are mean ± SE of 6 different observations.
with malate dehydrogenase. These observations prompted us to measure rates of
dark CO2 fixation alongwith the activities and localization of the enzymes involved
in the above metabolism in developing seeds of Brassica. The rate of dark CO2
fixation expressed as µmol mg-1 protein h-1 increased with seed development up
to 21 DAA and then declined until the harvest of the seeds (table 4). However,
sufficient rates were maintained during the interval 7–35 DAA. Similarly the various
enzymes viz., PEP carboxylase, NAD+-malate dehydrogenase and NADP+-malic
enzyme involved in dark CO2 metabolism retained sufficient activities during the
above period, indicating malate to arise mainly from dark CO2 fixation of seeds.
Our previous experiments with 14CO2 had also indicated malate to be the major
product of 14CO2 assimilation in seeds (Singal et al 1987). Furthermore, the specific
activity of PEP carboxylase obtained here is in the same range reported earlier for
seeds of castor oil (Sangwan et al 1992) and is far in excess to support the
observed rates of dark CO2 fixation. Similarly, the specific activity of NADP+-malic
enzyme falls in the range of values reported earlier for the enzyme from developing
castor oil seeds (Smith et al 1992), where malate has been shown to be the most
active precursor for fatty acid synthesis. Therefore, the rate of dark CO2 fixation
and the localization of enzyme activities associated with this metabolism in the
seed appear to adequately maintain the desired supply of malate to leucoplasts for
onward synthesis of fatty acids.
Enzyme compartmentatron studies revealed PEP carboxylase and enzymes of
sucrose metabolism viz. invertase, sucrose synthase, UDPG-pyrophosphorylase and
Table 4. Rate of dark CO2 fixation and activities of enzymes involved in dark CO2
metabolism in Brassica seeds at different days after anthesis
Values are mean ± SE of 6 different observations.
ND, Not detectable.
CO2 assimilation in developing Brassica seeds
55
Table 5. Activities of enzymes of carbon metabolism in leucoplast and cytosol fractions
of 20 days old Brassica seeds.
Values are mean ± SE of 6 different observations.
hexokinase to be present only in cytosol (table 5). However, glycolytic enzymes
such as phosphofructokinase, phosphoglucoisomerase, glyceraldehyde-3-P-dehydrogenase, phosphoglycerate phosphokinase and pyruvate kinase; though showed higher
activity in cytosolic fraction, were present in both cytoplasmic and leucoplasitc
fractions. These results are consistent with the proposal that sucrose on entering
cytosol is hydrolysed to hexoses which then enter glycolysis and converted to PEP.
Conversion of PEP to malate through combined actions of PEP carboxylase and
NAD-malate dehydrogenase is solely localized in cytosolic fraction, as the enzyme
PEP carboxylase was found to be absent from leucoplast. The presence of glycolytic
complement in leucoplast takes care of carbon entering this fracation as triose-P.
The results presented here indicate that in B. campestris, the period between 14
and 35 DAA is the period of dry matter vis-a-vis oil accumulation and during this
period the pods maintain high rates of photosynthesis. In our earlier communication
(Singal et al 1993), we have shown that the early phase of pod development,
which is also the active phase of pod photosynthesis, is favourable for sucrose
synthesis and during later half, there is shift in metabolic path of carbon from
sucrose to starch, indicating that the sucrose synthesized as a product of pod
photosynthesis is translocated to developing seeds (Sheoran et al 1991). On reaching
embryos of developing seeds, sucrose is ultimately converted to PEP by known
reactions of glycolysis (Smith et al 1992). PEP can be metabolized further in two
ways. First, pyruvate kinase may catalyse the conversion of PEP to pyruvate; which
then crosses the leucoplast membrance. Second, PEP may be carboxylated via PEP
carboxylase to oxaloacetate and with the action of cytosolic malate dehydrogenase,
converted to malate which then crosses the leucoplast membrane. Results of the
enzyme compartmentation experiment (table 5) seem to support this point of view.
In leucoplast, malate is decarboxylated via NADP+-malic enzyme to pyruvate and
CO 2. Pyruvate dehydrogenase complex then converts pyruvate to acetyl CoA for
onward fatty acid synthesis (figure 1). The sequence of reaction depicted in figure
1 shows cytosolic and plastidic pool of PEP to make significant contributions
towards generation of carbon skeletons and co-factors required for fatty acid
biosynthesis in leucoplasts of developing seeds of oilseed crops. The reductant
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Η R Singal et al
Figure 1. Probable mechanism of carbon flow for fatty acid synthesis in leucoplasts of
developing Brassica seeds. PEPC, PEP carboxylase; MDH, malate dehydrogenase; ME,
NADP+-malic enzyme; PK, pyruvate kinase; PDC, pyruvate dehydrogenase complex.
required for incorporation of carbon into fatty acids is produced as a consequence
of the metabolism of malate to acetyl CoA through the leucoplast localized
NADP+-malic enzyme and pyruvate dehydrogenase complex (Smith et al 1992).
ATP requirement of acetyl CoA carboxylase is met by leucoplastic pyruvate kinase
reaction. However, the respective in vivo contributions of plastidic pyruvate kinase
versus the 'PEPC-malate dehydrogenase-malic enzyme' pathways for supporting oil
seed fatty acid synthesis requires further investigations. As suggested by Plaxton
et al (1992), the relative importance of each route may vary diurnally according
to the rate of photosynthate import from source tissues. In any case, the two
metabolic routes utilized by oilseeds for conversion of sucrose into fatty acids
exemplify the remarkable flexibility of plant carbohydrate metabolism, making it
an essential trait of the biochemical adaptations, which help the plants to respond
dynamically and appropriately to their ever changing and frequently stressful
environment throughout their life cycle.
Acknowledgements
This work was supported by a grant from USDA under the co-operative Agricultural
Research Grant Programme (PL-480 No. FG-IN-743). Thanks are due to Dr S Κ
Gupta for NMR facilities.
CO2 assimilation in developing Brassica seeds
57
References
Allen E J, Morgan D G and Ridgman W J 1971 A physiological analysis of the growth of oilseed
rape; J. Agric. Sci. 77 339-341
Deanis D T 1989 Fatty acid biosynthesis in plastids; in Physiology, biochemistry and genetics of
nongreen plastids (eds) C D Boyer, J C Shannon and R C Hardison (Rockville: American Society
of Plant Physiologists) pp 120-129
Friedrich J W and Huffaker R C 1980 Photosynthesis, leaf resistance and ribulose-l,5-bisphosphate
carboxylase degradation in senescing barley leaves; Plant Physiol. 65 1103-1107
Gupta S K, Dhawan K, and Yadava T P 1985 Estimation of oil content by wide-line NMR; Oil Crops
Newsl. 2 17-21
Gupta V K and Singh R 1989 Properties of pyruvate kinase from immature pod wall of chickpea; Plant
Pkysiol. Biochem. 27 703-711
Kelly G J and Latzko E 1977 Chloroplast Phosphofructokinase I. Proof of phosphofmctokinase activity
in chloroplasts; Plant Physiol. 60 290-294
Kumar R and Singh R 1984 Level of free sugars, intermediate metabolites and enzymes of sucrose-starch
conversion in developing wheat grains; J. Agric. Food Chem. 32 806-808
Latzok E and Gibbs M 1968 Distribution and activity of enzymes of reductive pentose phosphate cycle
in spinach leaves and in chloroplasts isolated by different methods; Z. Pflanzenphysiol. 59 184-194
Lowry O H, Rosebrough N J, Farr A L and Randall R J 1951 Protein measurement with folin phenol
reagent; J. Biol. Chem. 193 265-275
Mac Donald F D and apRees T 1983 Enzymic properties of amyloplasts from suspension cultures of
soybean; Biochem. Biophys. Ada 755 81-89
Perry H J and Harwood J L 1993 Changes in the lipid content of developing seeds of Brassica napus;
Phytochemisty 32 1411-1415
Plaxton W C, Sangwan R S, Singh N, Gauthier D A and Turpin D H 1992 Phosphoenolpyruvate
metabolism of developing oilseeds; Proceedings of Seed oil Modification Workshop, American Oil
Chemists Society, USA
Pollard M R and Singh S S 1987 Fatty acid synthesis in developing oilseeds; in The metabolism,
structure and function of plant lipids (eds) P K Stumpf, J B Mudd and W D Nes (New York:
Plenum Press) pp 455-463
Sangwan R S, Singh N and Plaxton W C 1992 Phosphoenolpyruvate carboxylase activity and
concentration in the endosperm of developing and germinating castor oil seeds; Plant Physiol. 99
445-449
Schulze E D and Hall A E 1982 Stomatal responses, water loss and CO2 assimilation rates of plants
in contrasting environments; in Encyclopedia of plant physiology (eds) O L Lange, P S Nobel, C E
Osmond and H Ziegler (Berlin; Springer-Verlag) pp 181-230
Sheoran I S, Singal H R and Singh R 1990 Effect of cadmium and nickel on photosynthesis and enzymes
of photosynthetic carbon reduction cycle in pigeonpea (Cajanus cajan L.); Photosyn. Res. 23 345-351
Sheoran I S, Sawhney V, Babbar S and Singh R 1991 In vivo Fixation of CO2 by attached pods of
Brassica campestris L; Ann. Bot. 67 425-428
Singal H R, Sheoran I S and Singh R 1985 Effect of water stress on photosynthesis and in vitro
activities of PCR cycle enzymes in pigeonpea (Cajanus cajan L.); Photosyn. Res. 7 69-76
Singal H R, Sheoran I S and Singh R 1986 In vitro enzyme activities and products of 14CO2 assimilation
in flagleaf and ear parts of wheat (Triticum aestivum L); Photosyn. Res. 8 113-122
Singal H R, Sheoran I S and Singh R 1987 Photosynthetic carbon fixation characteristics of fruiting
structures of Brassica campestris L.; Plant Physiol. 83 1043-1047
Singal H R, Sheoran I S and Singh R 1992 Photosynthetic contribution of pods towards seed yield in
Brassica; Proc. Indian Natl. Sci. Acad. B58 365-370
Singal H R, Laura J S and Singh R 1993 Photosynthetic carbon reduction cycle metabolites and enzymes
of sucrose and starch biosynthesis in developing Brassica pods; Indian J. Biochem. Biophys. 30
270-276
Singh D P, Singh P and Sharma H C 1986 Diurnal pattern of photosynthesis, evapotranspiration and
water use efficiency in mustard at different growth phases under field conditions; Photosynthetica 20
117-123
Singh R 1993 Photosynthetic characteristics of fruiting structures of cultivated crops; in Photosynthesis:
Photoreactions to plant productivity (eds) Y P Abrol, P Mohanty and Govindjee (New Delhi: Oxford
58
HR Singal et al
and IBΗ Publishing Co.) pp 451-469
Singh S Ρ and Mehta S L 1992 Manipulation of oil quantity and quality in annual oilseed crops; J.
Oilseed Res. 9 97-118
Smith R G, Gauthier D A, Dennis D Τ and Turpin D Η 1992 Malaie and pyruvate dependent fatty
acid synthesis in leucoplasts from developing castor endosperm; Plant Physiol. 98 1233-1238
Wittenbach V A 1983 Effect of pod removal on leaf photosynthesis and soluble protein composition of
field grown soybeans; Plant Physiol. 73 121-124