Reduction of dark chilling stress in N-fixing soybean by nitrate as indicated by chlorophyll a fluorescence kinetics

Sub-optimal night temperatures below 15 degrees C (dark chilling) frequently reduce soybean [Glycine max (L.) Merrill] production. Nitrate application is known to alleviate some of the negative effects of low root zone temperatures, probably by counteracting the inhibition caused by decreased symbiotic nitrogen fixation (SNF). Under field conditions, however, dark chilling is frequently not accompanied by low root zone temperatures. The possibility that nitrate might increase dark-chilling tolerance under these conditions is still largely unexplored. In addition to quantifying vegetative development by means of the plastochron index, O-J-I-P (O-I(1)-I(2)-P) chlorophyll a fluorescence transients were recorded in soybean genotypes of contrasting chilling tolerance during and following exposure to dark chilling in the absence of low root zone temperatures. Plants, inoculated with the N(2)-fixing bacteria, Bradyrhizobium japonicum, were grown with and without nitrate supplementation. The recorded O-J-I-P chlorophyll a fluorescence transients were analysed by the so-called JIP-test which translates stress-induced alterations in these transients to changes in biophysical parameters that quantifies the energy flow through photosystem II (PSII). One of these parameters, the performance index (PI(ABS)), combines the three main functional steps (light energy absorption, excitation energy trapping, and conversion of excitation energy to electron transport) of photosynthetic activity by a PSII reaction centre complex into a single multiparametric expression. By using the PI(ABS) we could convincingly show that nitrate supplementation considerably enhances dark-chilling tolerance and recovery capacity of plants in the absence of low root zone temperatures. This was especially true for the chilling-sensitive genotype ('Java 29'), suggesting that the response of SNF to dark chilling might be an important factor contributing towards genotypic differences in chilling tolerance. Our results corroborated previous reports about the superior chilling tolerance of 'Maple Arrow', a chilling-tolerant genotype. The results obtained indicated that the PI(ABS) is a far more sensitive indicator of dark-chilling stress than the maximum quantum yield of primary photochemistry (F(V)/F(M)).     

The role of low soil temperature in the inhibition of growth and PSII function during dark chilling in soybean genotypes of contrasting tolerance

Dark chilling affects growth and yield of warm-climate crops such as soybean [ Glycine max (L.) Merr.]. Several studies have investigated chilling-stress effects on photosynthesis and other aspects of metabolism, but none have compared effects of whole-plant chilling (WPC; shoots and roots) with that of aboveground chilling in legumes. This is important because low root temperatures might induce additional constraints, such as inhibition of N₂ fixation, thereby aggravating chilling-stress symptoms. Effects of dark chilling on PSII, shoot growth, leaf ureide content and photosynthetic capacity were studied in two soybean genotypes, Highveld Top (chilling tolerant) and PAN809 (chilling sensitive), in experiments comparing effects of WPC with that of shoot chilling (SC). Both treatments inhibited shoot growth in PAN809 but not Highveld Top. Also, WPC in PAN809 caused a decrease in leaf ureide content followed by severe chlorosis and alterations in O-J-I-P fluorescence-rise kinetics, distinct from SC. A noteworthy difference was the appearance of a ΔK peak in the O-J-I-P fluorescence rise in response to WPC. These genotypic and treatment differences also reflected in the degree of inhibition of CO₂ assimilation rates. The appearance of a ΔK peak, coupled with growth inhibition, reduced ureide content, chlorosis and lower CO₂ assimilation rates, provides mechanistic information about how WPC might have aggravated chilling-stress symptoms in PAN809. We introduce a model explaining how chilling soil temperatures might trigger N-limitation in sensitive genotypes and how characteristic changes in O-J-I-P fluorescence-rise kinetics are linked to changes in carbon and nitrogen metabolism.     

Dark chilling effects on soybean genotypes during vegetative development: parallel studies of CO2 assimilation,chlorophyll a fluorescence kinetics O-J-I-P and nitrogen fixation

The effects of dark chilling on CO2 assimilation, chlorophyll a fluorescence kinetics and nitrogen fixation were compared in two Glycine max (L.) Merr. genotypes. The aim was to elucidate the mechanisms by which photosynthesis was inhibited as well as identification of selection criteria for dark chilling tolerance. Seedlings were dark chilled (8 degrees C) for 9 consecutive nights but kept at normal day temperatures (28 degrees C). CO2 gas exchange analysis indicated that photosynthesis in Maple Arrow was inhibited largely as a result of stomatal limitation, while in Fiskeby V, it indicated inhibition of the mesophyll reactions. Increased intercellular CO2 concentration and decreased carboxylation efficiency suggested loss of Rubisco activity in Fiskeby V, although no effect on the KM (CO2) of Rubisco was observed. Quantification and deconvolution of the Chl a fluorescence transients into several phenomenological and biophysical parameters (JIP-test) revealed large genotypic differences in the response of PSII to dark chilling. These parameters differentially changed in the two genotypes during the progression of the chilling treatment. Among them, the performance index, reflecting several responses of the photochemical apparatus, provided the best preliminary overall assessment of the genotypes. In contrast, the quantum yield of primary photochemistry varphiPo (FV/FM) was quite insensitive. The recovery of most of the JIP-test parameters in Maple Arrow after 6 and 9 nights of dark chilling was a major genotypic difference. Genotypic differences were also observed with regard to the ureide response and N2 fixation appeared to be more sensitive to dark chilling than CO2 assimilation. The JIP-test provided information consistent with results derived from CO2 assimilation and N2 fixation studies suggesting that it can substitute the much more time-consuming methods for the detection of chilling stress and can well satisfy the requirements of a rapid and accurate screening method.     

Soybean cultivars 'Williams 82' and 'Maple Arrow' produce both urea and ammonia during ureide degradation

The ability of two soybean (Glycine max L. [Merrill]) cultivars, 'Williams 82' and 'Maple Arrow', which were reported to use different ureide degradation pathways, to degrade the ureides allantoin and allantoate was investigated. Protein fractions and total leaf homogenates from the fourth trifoliate leaves of both cultivars were examined for the ability to evolve either (14)CO(2) or [(14)C]urea from (14)C-labelled ureides in the presence of various inhibitors. (14)CO(2) evolution from [2,7-(14)C]allantoate was catalysed by 25-50% saturated ammonium sulphate fractions of both cultivars. This activity was inhibited by acetohydroxamate (AHA), which has been used to inhibit plant ureases, but not by phenylphosphorodiamidate (PPD), a more specific urease inhibitor. Thus, in both cultivars, allantoate may be metabolized by allantoate amidohydrolase. This activity was sensitive to EDTA, consistent with previous reports demonstrating that allantoate amidohydrolase requires manganese for full activity. Total leaf homogenates of both cultivars evolved both (14)CO(2) and [(14)C]urea from [2,7-(14)C] (ureido carbon labelled) allantoin, not previously reported in either 'Williams 82' or in 'Maple Arrow'. In situ leaf degradation of (14)C-labelled allantoin confirmed that both urea and CO(2)/NH(3) are direct products of ureide degradation. Growth of plants in the presence of PPD under fixing and non-fixing conditions caused urea accumulation in both cultivars, but did not have a significant impact on total seed nitrogen. Urea levels were higher in N-fixing plants of both cultivars. Contrary to previous reports, no significant biochemical difference was found in the ability of these two cultivars to degrade ureides under the conditions used.     

Limitation of photosynthetic carbon metabolism by dark chilling in temperate and tropical soybean genotypes.

In the experiments reported in this paper, we characterised the physiological and biochemical factors involved in the chilling-induced inhibition of photosynthetic carbon metabolism in soybean [Glycine max (L.) Merr.] genotypes of temperate and tropical adaptation. Plants of Maple Arrow (temperate genotype) and Java 29 (tropical genotype) were exposed to a single night at 8 degrees C. Dark chilling resulted in the inhibition of diurnal CO2 assimilation rate and decreased stomatal conductance in both genotypes. Further analysis, however, revealed a difference in the response of the two genotypes. Stomatal limitation was largely responsible for the inhibition of CO2 assimilation in Maple Arrow, whereas mesophyll limitation dominated the inhibition in Java 29. The results indicate that inhibition of stromal fructose-1,6-bisphosphatase (sFBPase; EC 3.1.3.11) activity and impaired electron transport capacity were responsible for the decrease in ribulose-1,5-bisphosphate (RuBP) regeneration capacity in Java 29. Sucrose-phosphate synthase (SPS; EC 2.4.1.14) activity was progressively inhibited during the light period in this genotype and might impose an additional constraint on photosynthesis. Maple Arrow appears to possess, at least with respect to photosynthetic carbon metabolism, physiological and biochemical characteristics that contribute towards its superior dark chilling tolerance.     

Separately and simultaneously induced dark chilling and drought stress effects on photosynthesis, proline accumulation and antioxidant metabolism in soybean

The effects of separately or simultaneously induced dark chilling and drought stress were evaluated in two Glycine max (L.) Merrill cultivars. For the separately induced dark chilling treatment (C), plants were incubated at 8 °C during 9 consecutive dark periods. During the days, plants were kept at normal growth temperatures. For the separately induced drought treatment (D), plants were maintained at normal growth temperatures without irrigation. For the simultaneously induced dark chilling and drought stress treatment (CD), plants were dark chilled without irrigation. All treatments caused similar decreases in pre-dawn leaf water potential, but resulted in distinct physiological and biochemical effects on photosynthesis. In Maple Arrow, where C had the smallest effect on photosynthesis, prolonged CD caused less inhibition of photosynthesis compared to D. Compared to Fiskeby V, the photosynthetic apparatus of Maple Arrow appears to possess superior dark chilling tolerance, a property which probably also conveyed enhanced protection against CD. Proline accumulation was prevented by CD at the ψ_PD where D already resulted in considerable accumulation. The superior capacity for proline accumulation in Maple Arrow would seem to be an important factor in its stress tolerance. Antioxidant activity evoked by CD and D was higher than for C alone. In Fiskeby V, the small increase in ascorbate peroxidase (EC 1.11.1.7) activity, which was in most cases not accompanied by increased gluthatione reductase (EC 1.6.4.2) activity, could impact negatively on its stress tolerance. These results demonstrate large genotypic differences in response to chilling and drought stress, even between soybean cultivars regarded as chilling tolerant.     

Regulation of respiration and the oxygen diffusion barrier in soybean protect symbiotic nitrogen fixation from chilling-induced inhibition and shoots from premature senescence

Symbiotic nitrogen fixation is sensitive to dark chilling (7 degrees C-15 degrees C)-induced inhibition in soybean (Glycine max). To characterize the mechanisms that cause the stress-induced loss of nodule function, we examined nodule structure, carbon-nitrogen interactions, and respiration in two soybean genotypes that differ in chilling sensitivity: PAN809 (PAN), which is chilling sensitive, and Highveld Top (HT), which is more chilling resistant. Nodule numbers were unaffected by dark chilling, as was the abundance of the nitrogenase and leghemoglobin proteins. However, dark chilling decreased nodule respiration rates, nitrogenase activities, and NifH and NifK mRNAs and increased nodule starch, sucrose, and glucose in both genotypes. Ureide and fructose contents decreased only in PAN nodules. While the chilling-induced decreases in nodule respiration persisted in PAN even after return to optimal temperatures, respiration started to recover in HT by the end of the chilling period. The area of the intercellular spaces in the nodule cortex and infected zone was greatly decreased in HT after three nights of chilling, an acclimatory response that was absent from PAN. These data show that HT nodules are able to regulate both respiration and the area of the intercellular spaces during chilling and in this way control the oxygen diffusion barrier, which is a key component of the nodule stress response. We conclude that chilling-induced loss of symbiotic nitrogen fixation in PAN is caused by the inhibition of respiration coupled to the failure to regulate the oxygen diffusion barrier effectively. The resultant limitations on nitrogen availability contribute to the greater chilling-induced inhibition of photosynthesis in PAN than in HT.     

Comparison of inhibition of N2 fixation and ureide accumulation under water deficit in four common bean genotypes of contrasting drought tolerance

Background and Aims

Drought is the principal constraint on world production of legume crops. There is considerable variability among genotypes in sensitivity of nitrogen fixation to drought, which has been related to accumulation of ureides in soybean. The aim of this study was to search for genotypic differences in drought sensitivity and ureide accumulation in common bean (Phaseolus vulgaris) germplasm that may be useful in the improvement of tolerance to water deficit in common bean.

Methods

Changes in response to water deficit of nitrogen fixation rates, ureide content and the expression and activity of key enzymes for ureide metabolism were measured in four P. vulgaris genotypes differing in drought tolerance.

Key Results

A variable degree of drought-induced nitrogen fixation inhibition was found among the bean genotypes. In addition to inhibition of nitrogen fixation, there was accumulation of ureides in stems and leaves of sensitive and tolerant genotypes, although this was higher in the leaves of the most sensitive ones. In contrast, there was no accumulation of ureides in the nodules or roots of stressed plants. In addition, the level of ureides in the most sensitive genotype increased after inhibition of nitrogen fixation, suggesting that ureides originate in vegetative tissues as a response to water stress, probably mediated by the induction of allantoinase.

Conclusions

Variability of drought-induced inhibition of nitrogen fixation among the P. vulgaris genotypes was accompanied by subsequent accumulation of ureides in stems and leaves, but not in nodules. The results indicate that shoot ureide accumulation after prolonged exposure to drought could not be the cause of inhibition of nitrogen fixation, as has been suggested in soybean. Instead, ureides seem to be produced as part of a general response to stress, and therefore higher accumulation might correspond to higher sensitivity to the stressful conditions.     

Effects of decreasing source/sink ratio in soybeans on photosynthesis, photorespiration, transpiration and yield

Abstract. Shading of all side leaflets of a determinate soybean cultivar during pod filling significantly increased rates of photosynthesis in the unshaded centre leaflets, compared to centre leaflets of controls. Higher rates were associated with both higher stomatal and mesophyll conductances, and were reversible within 2 days when shades were removed. These higher rates of photosynthesis were not associated with decreased percentage enhancement by low oxygen, indicating that treatment effects were probably not associated with changes in photorespiration relative to photosynthesis. Percentage enhancement did, however increase as the plants approached physiological maturity, chiefly because of a decrease in photosynthesis.
In spite of these increases in rates of photosynthesis seed weight per plant was decreased by 37% in plants with side leaflets shaded for the entire pod-filling period and by 28% in plants shaded for only the second half of the period. In plants where shades were removed during the second half of pod filling seed yield was reduced by only 19% because shade removal delayed leaf senescence. The four treatments reduced yield by different mechanisms. Plants shaded continuously during pod filling produced fewer seeds than controls, but the weight per seed was similar. When shading was applied during the second half of pod fillings seed number was unchanged but weight per seed was significantly reduced. In contrast when shades were removed for the second half of pod filling, seed number remained similar to that of continuously shaded plants, but seed weight increased.
Although all shading treatments reduced yield, the reduction was not proportional to the 63% reduction in leaf area available for photosynthesis. This was because (1) photosynthetic rates in the centre leaflet of shaded plants were higher than rates in controls, (2) stem and lower surface photosynthesis in shaded leaf-lets contributed to whole leaflet photosynthesis.     

Growth and Development of Soybean (Glycine max [L.] Merr.) Pods: CO(2) Exchange and Enzyme Studies

The rates of CO₂ exchange and ¹⁴CO₂ incorporation in the light and dark and the activities of several photosynthetic, photorespiratory, and respiratory enzymes of soybean (Glycine max [L.] Merr. cv. Wye) reproductive structures were determined at weekly intervals from anthesis to pod maturity. At all stages of pod development soybean reproductive structures were found to be incapable of net photosynthesis under the experimental conditions employed, but capable of gross photosynthesis and light-induced ¹⁴CO₂ uptake. Consistent with the lack of net photosynthesis throughout the development of the reproductive structure, the maximum in vitro activity of ribulose 1,5-bisphosphate carboxylase (EC 4.1.1.39) in pod tissue was only 3% of that in leaf extracts when expressed on a fresh weight basis. We concluded that the major role of the reproductive structure of the soybean with respect to photosynthetic carbon metabolism is the reassimilation of its respiratory CO₂.     

Foliar application of fungicide-opera alleviates negative impact of water stress in soybean plants

The modulatory effect of opera was investigated on the physiological and morphological aspects in soybean thriving in water stress environment. The data procured from current investigation indicated that water stress significantly declined the plant growth, leaf area in addition to photosynthetic efficiency, nitrate reductase activity and crop yield at various stages of growth such as vegetative (VS), flowering (FS) and pod filling stage (PFS). However, foliar application of opera (0.15%) was effective to enhance the the leaf area (42%), rate of photosynthesis (194%), and nitrate reductase activity (68%) at FS stage while the maximum enhancement in biomass accumulation (92%) and yield (119%) was observed at PFS stage as compared to their control plants. The opera is applied as foliar spray in field experiments to augment the assimilation of nitrogen and carbon in soybean which contributes to increased crop development and productivity under water stress conditions.     

Drought enhances maize chilling tolerance. II. Photosynthetic traits and protective mechanisms against oxidative stress

In the present research we studied the photosynthetic traits and protective mechanisms against oxidative stress in two maize ( Zea mays L.) genotypes differing in chilling sensitivity (Z7, tolerant and Penjalinan, sensitive) subjected to 5°C for 5 days, with or without pretreatment by drought. The drought pretreatment decreased the symptoms of chilling injury in Penjalinan plants estimated as necrotic leaf area and maximum quantum yield of photosystem II. Furthermore, drought pretreatment diminished the level of lipid peroxidation caused by chilling in Penjalinan plants. After one day of recovery from chilling the Z7 and drought-pretreated Penjalinan plants showed higher net photosynthesis rates than the non-drought-pretreated Penjalinan plants, thereby decreasing the probability of generating reactive oxygen species. The greater net photosynthesis was correlated with the greater NADP-malate dehydrogenase activity. No differences in either the de-epoxidation state of the xanthophyll cycle or the antioxidant enzyme activities were found among the chilled groups of plants. However, a drastic decrease in ascorbate content was observed in chilled Penjalinan plants without drought pretreatment. As we found an increase of H₂O₂ content after drought pretreatment, we suggest its involvement as a signal in the drought-enhanced chilling tolerance of maize.     

Leaf ureide degradation and N(2) fixation tolerance to water deficit in soybean

Accumulation of ureides in leaves is associated with the sensitivity of N(2) fixation in soybean to soil water deficit. Consequently, ureide degradation in leaves may be a key to increasing soybean tolerance to dry soils. Previous research indicated that allantoic acid degradation is catalysed by different enzymes in cultivars Maple Arrow and Williams. The enzyme found in Williams requires manganese as a cofactor. The first objective of this study was to determine if the two degradation pathways were associated with differences in N(2) sensitivity to soil water deficits. N(2) fixation of Williams grown on low-Mn soil was sensitive to stress, but it was relatively tolerant when grown on soil amended with Mn. N(2) fixation in Maple Arrow was relatively tolerant of soil drying regardless of the Mn treatment. The second objective of this study was to expand the study of the degradation pathway to nine additional genotypes. Based on ureide degradation in the presence and absence of Mn, these genotypes also segregated for the two degradation pathways. Those genotypes with the Mn-dependent pathway tended to have drought-sensitive N(2) fixation, but there was one exception. The genotypes not requiring Mn for ureide degradation were drought-tolerant except for one genotype. These results demonstrated the possibility for increasing N(2) fixation tolerance to soil water deficits in soybean by selection of lines with high ureide degradation rates, which were commonly associated with the Mn-independent pathway.     

Yield, growth and physiological responses of mung bean [Vigna radiata (L.) Wilczek] genotypes to waterlogging at vegetative stage

A study was conducted to examine the physiological response of contrasting mung bean (Vigna radiata) genotypes viz., T 44 & MH–96–1 (tolerant) and Pusa Baisakhi & MH–1K–24 (sensitive) under waterlogging conditions. Plants were waterlogged at vegetative stage (30 days after sowing) for 3, 6 and 9 days. Waterlogging resulted in decreased leaf area, crop growth rate, root growth and nodules number, membrane stability index, photosynthesis rate, chlorophyll and carotenoid contents, flowering rate, pod setting, yield and altered dry matter partitioning. Sensitive genotypes showed large reductions in aforementioned physiological traits and slow recovery in photosynthesis rate. On the other hand, tolerant genotypes maintained higher photosynthetic rate, chlorophylls and carotenoids, growth rate, membrane stability and fast photosynthetic recovery under waterlogging. After 9 days of exposure to waterlogging, photosynthetic rate and yield losses in most sensitive genotype (MH-1K-24) were 83 and 85 %, respectively. On an average, photosynthetic loss at 3, 6 and 9 days of waterlogging was 43, 51, and 63 %, respectively, while grain yield loss was 20, 34 and 52 % respectively.     

A Comparative Study of PEPC Activity of Green Organs in C3 Plant

PEPC and relevant enzymes in different photosynthetic organs of wheat and soybean were studied. Almost all green organs examined have been found to contain PEPC. PEPC activity in pod hull and seed coat of soybean as well as in paleae of wheat is higher than that in leaves. 14CO2 can be fixed in different green organs either in light or in dark, however in dark, 14CO2 fixation in pnl hull and seed coat of soybean as well as in paleae of wheat is higher than in leaves. Similarly, N AD-malic enzyme and NAD-malate dehydrogenase are also higher in those organs than in leaves. It was shown that active β-carboxylation of PEPC took place in the fruit organs. The above results indicate that PEPC is important not only for CO2 fixation during photosynthesis but also for recapturing CO2 released from respiration.     

A Comparison of the Effects of Chilling on Leaf Gas Exchange in Pea (Pisum sativum L.) and Cucumber (Cucumis sativus L.)

The effects of chilling on the photosynthesis of a chilling-resistant species, pea (Pisum sativum L. cv Alaska) and a chilling-sensitive species, cucumber (Cucumis sativus L. cv Ashley) were compared in order to determine the differences in the photosynthetic chilling sensitivity of these two species. For these experiments, plants were chilled (5°C) for different lengths of time in the dark or light. Following a 1 hour recovery period at 25°C, photosynthetic activity was measured by gas exchange (CO₂ uptake and H₂O release), quantum yield, and induced chlorophyll fluorescence. The results show that pea photosynthesis was largely unaffected by two consecutive nights of chilling in the dark, or by chilling during a complete light and dark cycle (15 hours/9 hours). Cucumber gas exchange was reduced by one night of chilling, but its quantum yield and variable fluorescence were unaffected by dark chilling. However, chilling cucumber in the light led to reduced CO₂ fixation, increased internal leaf CO₂ concentration, decreased quantum yield, and loss of variable fluorescence. These results indicate that chilling temperatures in conjunction with light damaged the light reactions of photosynthesis, while chilling in the dark did not.     

低温对杂交水稻乳熟期剑叶光合作用和光合产物运输的影响

为了解寒露风引起晚稻减产的生理原因,本试验以两个不同抗冷力的杂交水稻组合为材料,研究了低温对水稻乳熟期剑叶光合作用与果糖１,６－二磷酸酯酶（ＦＢＰａｓｅ）活性的影响。结果表明：低温引起离体剑叶光合效率与ＦＢＰａｓｅ 活性下降,不抗冷的“汕优６３”及其亲本的光合效率与ＦＢＰａｓｅ 活性比抗冷的“秀优５７”及其亲本下降幅度较大。乳熟期是籽粒灌浆的高峰期,自然低温（寒露风）造成晚造水稻减产与乳熟期连体剑叶的光合作用下降和ＦＢＰａｓｅ 对低温的适应和调节能力有关     

Evaluation of high yielding soybean germplasm under water limitation

Limited information is available for soybean root traits and their plasticity under drought stress. To date, no studies have focused on examining diverse soybean germplasm for regulation of shoot and root response under water limited conditions across varying soil types. In this study, 17 genetically diverse soybean germplasm lines were selected to study root response to water limited conditions in clay (trial 1) and sandy soil (trial 2) in two target environments. Physiological data on shoot traits was measured at multiple crop stages ranging from early vegetative to pod filling. The phenotypic root traits, and biomass accumulation data are collected at pod filling stage. In trial 1, the number of lateral roots and forks were positively correlated with plot yield under water limitation and in trial 2, lateral root thickness was positively correlated with the hill plot yield. Plant Introduction (PI) 578477A and 088444 were found to have higher later root number and forks in clay soil with higher yield under water limitation. In sandy soil, PI458020 was found to have a thicker lateral root system and higher yield under water limitation. The genotypes identified in this study could be used to enhance drought tolerance of elite soybean cultivars through improved root traits specific to target environments.     

Pod photosynthesis and seed dark CO2 fixation support oil synthesis in developingBrassica seeds

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 ofBrassica 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 dark¹⁴CO₂ 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 developingBrassica 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