Effect of pod removal on leaf photosynthesis and soluble protein composition of field-grown soybeans

Well nodulated, field-grown soybeans (Glycine max [L.] Merr. var Williams) were depodded just prior to seed development and near mid pod-fill. Both treatments caused a considerable increase in leaf dry weight, suggesting continued photosynthate production following pod removal. Moreover, depodding had a marked effect on leaf soluble protein without affecting total proteolytic activity. Early depodding caused a 50% increase in leaf protein, and both early and late depodding caused the retention of protein for several weeks following the decline in control leaves. But despite this retention of protein, leaves of depodded plants showed no difference in the onset of the irreversible decline in photosynthesis. Therefore, although depodding delayed the loss of leaf chlorophyll and protein, it did not delay the onset of functional leaf senescence and in fact, actually appeared to enhance the rate of decline in photosynthesis. There was a good correlation between the irreversible decline in ribulose bisphosphate carboxylase (activity and amount) and that of photosynthesis. In contrast, the correlation did not seem as good between stomatal closure and the onset of the irreversible decline in photosynthesis. The reason total soluble protein remained high following depodding while carboxylase, which normally comprised 40% of the soluble protein, declined was because several polypeptides increased in amounts sufficient to offset the loss of carboxylase. This change in leaf protein composition indicates a change in leaf function; this is discussed in terms of other recent findings.     

Purification and characterization of a soybean leaf storage glycoprotein

Removing the pods from soybean (Glycine max [L.] Merr. cv Wye) plants induces a change in leaf function which is characterized by a change in the leaf soluble protein pattern. The synthesis of at least four polypeptides (~27, 29, 54, and 80 kilodaltons) is enhanced, and these polypeptides accumulate to levels comprising over 50% of the soluble protein. Heat girdling the petiole also causes the accumulation of these polypeptides, suggesting that the signal for changing leaf function may be associated with inhibition of phloem transport. The 27 and 29 kilodalton polypeptides are glycosylated and have been purified to greater than 90% by (NH₄)₂SO₄ fractionation, concanavilin A affinity, and gel filtration chromatography. These peptides appear to comprise a single protein. Mouse antiserum has been prepared against this glycoprotein and has been used to check for cross-reactivity with seed proteins and to quantitate changes with leaf development. No cross-reactivity was observed with seed soluble proteins from several stages of development. Quantitation showed the highest content in podded plants at, and shortly following, flowering, with levels subsequently declining in conjunction with seed growth. In depodded plants, the level of glycoprotein continued to increase following flowering and accounted for 45% of the soluble leaf protein by 4 weeks after depodding.     

Paraveinal Mesophyll of Soybean Leaves in Relation to Assimilate Transfer and Compartmentation : III. Immunohistochemical Localization of Specific Glycopeptides in the Vacuole after Depodding

Immunohistochemical staining was used to determine the cellular distribution of two glycosylated polypeptides (molecular weights of 27 and 29 kilodaltons) which are normally present at low levels in soybean (Glycine max var `Wye') leaves but which markedly accumulate after depodding. These polypeptides, which comprise a substantial portion of the total leaf soluble protein of depodded plants, were exclusively located in the vacuoles of paraveinal mesophyll and associated bundle sheath cells. These results support the unique role of the soybean leaf paraveinal mesophyll in the transport and spatial compartmentation of nitrogen reserves in relation to seed filling.     

Sink removal and leaf senescence in soybean : cultivar effects

Three cultivars of soybean (Glycine max [L.] Merr. cvs Harper, McCall, and Maple Amber) were grown in the field and kept continuously deflowered throughout the normal seedfill period. For all cultivars, deflowering led to delayed leaf abscission and a slower rate of chlorophyll loss. Compared to control plants, photosynthesis and ribulose 1,5-bis-phosphate carboxylase/oxygenase (Rubisco) level declined slightly faster for deflowered Harper, but for both McCall and Maple Amber, leaves of deflowered plants maintained approximately 20% of maximum photosynthesis and Rubisco level 1 month after control plants had senesced. Deflowering led to decreased leaf N remobilization and increased starch accumulation for all cultivars, but cultivars differed in that for McCall and Maple Amber, N and starch concentrations slowly but steadily declined over time whereas for Harper, N and starch concentrations remained essentially constant over time. SDS-PAGE of leaf proteins indicated that for all cultivars, deflowering led to accumulation of four polypeptides (80, 54, 29, and 27 kilodaltons). Western analysis using antisera prepared against the 29 and 27 kilodalton polypeptides verified that these polypeptides were the glycoproteins previously reported to accumulate in vacuoles of paraveinal mesophyll cells of depodded soybean plants. The results indicated that depending on the cultivar, sink removal can lead to either slightly faster or markedly slower loss of photosynthesis and Rubisco. This difference, however, was not associated with the ability to synthesize leaf storage proteins. For any particular cultivar, declines in chlorophyll, photosynthesis, and Rubisco were initiated at approximately the same time for control and deflowered plants. Thus, even though cultivars differed in rate of decay of photosynthetic rate and Rubisco level in response to sink removal, the initiation of leaf senescence was not influenced by presence or absence of developing fruits.     

Cysteine proteinases regulate chloroplast protein content and composition in tobacco leaves: a model for dynamic interactions with ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) vesicular bodies

The roles of cysteine proteinases (CP) in leaf protein accumulation and composition were investigated in transgenic tobacco (Nicotiana tabacum L.) plants expressing the rice cystatin, OC-1. The OC-1 protein was present in the cytosol, chloroplasts, and vacuole of the leaves of OC-1 expressing (OCE) plants. Changes in leaf protein composition and turnover caused by OC-1-dependent inhibition of CP activity were assessed in 8-week-old plants using proteomic analysis. Seven hundred and sixty-five soluble proteins were detected in the controls compared to 860 proteins in the OCE leaves. A cyclophilin, a histone, a peptidyl-prolyl cis-trans isomerase, and two ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) activase isoforms were markedly altered in abundance in the OCE leaves. The senescence-related decline in photosynthesis and Rubisco activity was delayed in the OCE leaves. Similarly, OCE leaves maintained higher leaf Rubisco activities and protein than controls following dark chilling. Immunogold labelling studies with specific antibodies showed that Rubisco was present in Rubisco vesicular bodies (RVB) as well as in the chloroplasts of leaves from 8-week-old control and OCE plants. Western blot analysis of plants at 14 weeks after both genotypes had flowered revealed large increases in the amount of Rubisco protein in the OCE leaves compared to controls. These results demonstrate that CPs are involved in Rubisco turnover in leaves under optimal and stress conditions and that extra-plastidic RVB bodies are present even in young source leaves. Furthermore, these data form the basis for a new model of Rubisco protein turnover involving CPs and RVBs.     

Fruit removal in soybean induces the formation of an insoluble form of ribulose-1,5-bisphosphate carboxylase/oxygenase in leaf extracts*

In some soybean (Glycine max (L.) Merr.) cultivars, fruit removal does not delay the apparent loss of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco, EC 4.1.1.39) activity and abundance or the decline in photosynthesis. Analysis of leaf extracts from defruited plants indicated a time-dependent increase in both Rubisco activity and abundance in a 30000 · g pellet fraction in cultivars which had been reported to lose all Rubisco protein from the supernatant fraction. Attempts to solubilize the pelleted Rubisco by increasing the buffer volume/tissue ratio or by adding alkylphenoxypolyethoxyethanol (Triton X-100), ethylenediaminetetraacetic acid (EDTA), or NaCl were unsuccessful. However, treatment of the pellets with denaturants such as 8 M urea or 5% (w/v) sodium dodecyl sulfate (SDS) did release Rubisco from the pellet. Redistribution of protein to the pellet fraction appeared to be specific for Rubisco since the amount of ribulose-5-phosphate kinase (EC 2.7.1.19) found in the pellet fraction of leaf extracts of control and defruited plants was small and constant over time. The loss of soluble Rubisco, and the concomitant increase in insoluble Rubisco, in response to fruit removal varied with genotype and was reproducible in both field and greenhouse environments. In addition, the effect was influenced by node position and light; lower and-or shaded leaves exhibited less Rubisco in the pellet fraction than leaves from the top of the plant that was fully exposed to sunlight. When isolated by sucrose-density-gradient centrifugation, the insoluble Rubisco was found to co-purify with a 30-kDa (kilodalton) polypeptide. These results indicate that alteration of the source/sink ratio by removing fruits results in the formation of an insoluble form of Rubisco in leaf extracts of soybean. Whether or not Rubisco exists as an insoluble complex with the 30-kDa polypeptide in intact leaves of defruited plants remains to be determined.Abbreviations kDa kilodalton - PGA kinase 3-phosphoglyceric acid kinase (EC 2.7.2.3) - Rubisco ribulose-1,5-bisphosphate car-boxylase/oxygenase (EC 4.1.1.39) - Ru5P kinase ribulose-5-phosphate kinase (EC 2.7.1.19) - SDS-PAGE sodium dodecyl sulfatepolyacrylamide gel electrophoresis     

Reduction of ribulose-1,5-bisphosphate carboxylase/oxygenase content by antisense RNA reduces photosynthesis in transgenic tobacco plants

A complementary DNA for the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) was cloned from tobacco (Nicotiana tabacum) and fused in the antisense orientation to the cauliflower mosaic virus 35S promoter. This antisense gene was introduced into the tobacco genome, and the resulting transgenic plants were analyzed to assess the effect of the antisense RNA on Rubisco activity and photosynthesis. The mean content of extractable Rubisco activity from the leaves of 10 antisense plants was 18% of the mean level of activity of control plants. The soluble protein content of the leaves of anti-small subunit plants was reduced by the amount equivalent to the reduction in Rubisco. There was little change in phosphoribulokinase activity, electron transport, and chlorophyll content, indicating that the loss of Rubisco did not affect these other components of photosynthesis. However, there was a significant reduction in carbonic anhydrase activity. The rate of CO₂ assimilation measured at 1000 micromoles quanta per square meter per second, 350 microbars CO₂, and 25°C was reduced by 63% (mean value) in the antisense plants and was limited by Rubisco activity over a wide range of intercellular CO₂ partial pressures (p_i). In control leaves, Rubisco activity only limited the rate of CO₂ assimilation below a p_i of 400 microbars. Despite the decrease in photosynthesis, there was no reduction in stomatal conductance in the antisense plants, and the stomata still responded to changes in p_i. The unchanged conductance and lower CO₂ assimilation resulted in a higher p_i, which was reflected in greater carbon isotope discrimination in the leaves of the antisense plants. These results suggest that stomatal function is independent of total leaf Rubisco activity.     

DEVELOPMENTAL CHANGES IN ENERGY (ATP AND ADP) AND NUTRIENT LEVELS PRIOR TO SENESCENCE IN PODDED AND DEPODDED PEA PLANTS

Energy (ATP and ADP) levels in stem apices of depodded pea plants (Pisum sativum L. cv. Little Marvel) were significantly higher than those of podded plants during the pod-filling stage before whole plant senescence. This difference in energy content appeared before decreases in leaf chlorophyll and soluble proteins occurred in plants of both treatments. In contrast, the mineral nutrient levels (N, P, K, Ca, Mg, Fe, and Mn) in stem apices of plants from both treatments were similar. Energy levels in reproductive leaves from podded and depodded plants were similar. The mineral nutrients in leaves with fruits in their axils and similar leaves of depodded plants were comparable except for nitrogen. Plant growth measurements—dry weight, leaf area, leaf dry weight, root/shoot ratio—were significantly higher in depodded than podded plants. Whole plant senescence occurred significantly earlier in podded than in depodded pea plants.     

Protein compositions of mesophyll and paraveinal mesophyll of soybean leaves at various developmental stages

Mesophyll and paraveinal mesophyll protoplasts (PVMP) were isolated from leaves of soybean (Glycine max) at various stages of physiological development, and protein compositions of the two protoplast types were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting. Polypeptides of 27, 29 (previously shown to be storage proteins), and 94 kilodaltons were found to be PVMP-specific proteins and were present in both nodulated and nonnodulated plants. The 27 and 94 kilodalton polypeptides were major PVMP constituents. All three polypeptides accumulate as early as one-quarter leaf expansion. Immunoblotting and immunocytochemical studies using antibodies against the 27/29 kilodalton proteins confirmed that they are specific to the paraveinal mesophyll (PVM) and that they are localized in the PVM vacuole. The 27 kilodalton polypeptide increased significantly by two weeks depodding, and this accumulation was restricted to the PVM vacuole. Radiolabeling experiments showed that the difference in relative amounts of the 27 and 29 kilodalton polypeptides was due to a greater rate of synthesis of the 27 kilodalton polypeptide. The 94 kilodalton polypeptide accumulated to a maximum at anthesis, but was absent at 2 weeks postanthesis in both depodded and podded nodulated plants, probably because they were nitrogen limited. In nonnodulated plants, it was present through 2 weeks postanthesis. The results confirm that the 27 and 29 kilodalton proteins of soybean leaf are stored in the PVM vacuole and show that they are accumulated early during leaf development while they are still strong sinks for nitrogen. The 94 kilodalton protein, previously found to accumulate in leaves after depodding, is also a PVM protein and is likely a third vegetative storage protein, although its accumulation appears to be more dependent on excess nitrogen availability. The results further support the hypothesis that the PVM is a specialized leaf tissue that functions in synthesis and compartmentation of storage proteins.     

Photosynthesis and its related physiological variables in the leaves of Brassica genotypes as influenced by sulphur fertilization

In the present investigation, we examined the effect of sulphur fertilization on photosynthesis (Pn) and its related physiological variables in the leaves of field grown Brassica genotypes ( Brassica juncea [L.] Czern. and Coss. cv. Pusa Jai Kisan and Brassica campestris L. cv. Pusa Gold) over a whole growing season. Sulphur fertilization significantly ( P <0.05) increased the Pn rate on leaf area basis at all the growth stages over −S treatment. The photosynthesis related variables such as soluble protein and Rubisco (ribulose-1,5-bisphosphate carboxylase/oxygenase) protein were significantly higher in the leaves of plants grown with +S treatment, when compared to −S treatment. Sulphur fertilization also improved the chlorophyll, N and S content in the leaves of +S treated plants over −S treatment. Leaf-S content was linearly correlated with Pn rate, N-content and Rubisco protein in the leaves of both genotypes. An interesting relationship between N-content and Pn rate in the leaves of −S and +S treated plants was observed. In −S plants, the relationship between Pn rate and N-content per unit area of fully matured leaves became non-linear when leaf-N exceeded 1.5 g m⁻², while in +S plants the same remained linear. Rubisco protein was linearly related to Pn rate and leaf-N content. The ratio of Rubisco/soluble protein was lesser in the leaves of −S treated plants than +S treated plants. The effect of sulphur fertilization on Pn is discussed in relation to improved nitrogen utilization efficiency of the plants that leads to incorporation of reduced-N into the protein, especially in Rubisco protein rather than the non-protein compounds.     

Root-zone CO2 and root-zone temperature effects on photosynthesis and nitrogen metabolism of aeroponically grown lettuce (Lactuca sativa L.) in the tropics

Effects of elevated root-zone (RZ) CO₂ concentration (RZ [CO₂]) and RZ temperature (RZT) on photosynthesis, productivity, nitrate (NO₃ ^?), total reduced nitrogen (TRN), total leaf soluble and Rubisco proteins were studied in aeroponically grown lettuce plants in a tropical greenhouse. Three weeks after transplanting, four different RZ [CO₂] concentrations (ambient, 360 ppm, and elevated concentrations of 2,000; 10,000; and 50,000 ppm) were imposed on plants at 20°C-RZT or ambient(A)-RZT (24–38°C). Elevated RZ [CO₂] resulted in significantly higher light-saturated net photosynthetic rate, but lower light-saturated stomatal conductance. Higher elevated RZ [CO₂] also protected plants from both chronic and dynamic photoinhibition (measured by chlorophyll fluorescence Fv/Fm ratio) and reduced leaf water loss. Under each RZ [CO₂], all these variables were significantly higher in 20°C-RZT plants than in A-RZT plants. All plants accumulated more biomass at elevated RZ [CO₂] than at ambient RZ [CO₂]. Greater increases of biomass in roots than in shoots were manifested by lower shoot/root ratios at elevated RZ [CO₂]. Although the total biomass was higher at 20°C-RZT, the increase in biomass under elevated RZ [CO₂] was greater at A-RZT. Shoot NO₃ ^? and TRN concentrations, total leaf soluble and Rubisco protein concentrations were higher in all elevated RZ [CO₂] plants than in plants under ambient RZ [CO₂] at both RZTs. Under each RZ [CO₂], total leaf soluble and Rubisco protein concentrations were significantly higher at 20°C-RZT than at A-RZT. Our results demonstrated that increased P _Nmax and productivity under elevated [CO₂] was partially due to the alleviation of midday water loss, both dynamic and chronic photoinhibition as well as higher turnover of Calvin cycle with higher Rubisco proteins.     

Trends in leaf photosynthesis in historical rice varieties developed in the Philippines since 1966

Crop improvement in terms of yield is rarely linked to leaf photosynthesis. However, in certain crop plants such as rice, it is predicted that an increase in photosynthetic rate will be required to support future grain yield potential. In order to understand the relationships between yield improvement and leaf photosynthesis, controlled environment conditions were used to grow 10 varieties which were released from the International Rice Research Institute (IRRI) between 1966 and 1995 and one newly developed line. Two growth light intensities were used: high light (1500 micromol m(-2) s(-1)) and low light (300 micromol m(-2) s(-1)). Gas exchange, leaf protein, chlorophyll, and leaf morphology were measured in the ninth leaf on the main stem. A high level of variation was observed among high light-grown plants for light-saturated photosynthetic rate per unit leaf area (P(max)), stomatal conductance (g), content of ribulose bisphosphate carboxylase-oxygenase (Rubisco), and total leaf protein content. Notably, between 1966 and 1980 there was a decline in P(max), g, leaf protein, chlorophyll, and Rubisco content. Values recovered in those varieties released after 1980. This striking trend coincides with a previous published observation that grain yield in IRRI varieties released prior to 1980 correlated with harvest index whereas that for those released after 1980 correlated with biomass. P(max) showed significant correlations with both g and Rubisco content. Large differences were observed between high light- and low light-grown plants (photoacclimation). The photoacclimation 'range' for P(max) correlated with P(max) in high light-grown plants. It is concluded that (i) leaf photosynthesis may be systematically affected by breeding strategy; (ii) P(max) is a useful target for yield improvements where yield is limited by biomass production rather than partitioning; and (iii) the capacity for photoacclimation is related to high P(max) values.     

Sugar repression of photosynthesis: the role of carbohydrates in signalling nitrogen deficiency through source:sink imbalance

The aim of this work was to examine whether carbohydrates are involved in signalling N deficiency through source:sink imbalance. Photosynthetic metabolism in tobacco was studied over 8 d during the withdrawal of N from previously N-sufficient plants in which the source:sink ratio was manipulated by shading leaves on some of the plants. In N-sufficient plants over this time-scale, there was a small decline in photosynthetic rate, Rubisco protein and amino acid content, with a larger decrease in carbohydrate content. Withdrawal of N from the growing medium induced a large decrease in the rate of photosynthesis (35% reduction after 8 d under the growing conditions, with a reduction also apparent at high and low measuring CO₂), which was caused by a large decrease in the amount of Rubisco protein (62% after 8 d) and Rubisco activity. Higher amounts; of hexoses preceded the loss of photosynthetic activity and sucrose and starch accumulation. Reduction of the sourcersink ratio by shading prevented the loss of photosynthetic activity and the increase in hexoses and other carbohydrates. These data indicate that the reduction of photosynthesis that accompanies N deficiency in intact plants has the characteristics of sugar repression of photosynthesis observed in model systems, but that the accumulation of hexose prior to the decline in photosynthesis is small. The possibility that sugar repression of photosynthesis under physiological conditions depends more crucially on the C:N status of leaves than the carbohydrate status alone is discussed.     

Growth and photosynthesis under high and low irradiance of Arabidopsis thaliana antisense mutants with reduced ribulose-1,5-bisphosphate carboxylase/oxygenase activase content.

Photosynthesis and growth to maturity of antisense ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) activase Arabidopsis thaliana with reduced concentrations of activase relative to wild-type (Wt) plants were measured under low (200 mumol m-2 s-1) and high (600 mumol m-2 s-1) photosynthetic photon flux density growing conditions. Both growth and photosynthesis were significantly reduced in an Arabidopsis clone (R100) with 30 to 40% Wt activase, an effect that was more pronounced in high light. The aboveground biomass of the antisense clone R100 reached 80% of Wt under low light and 65% of Wt under high light. Decreased growth in the antisense plants was attributed to reduced relative rates of growth and leaf area expansion early in development; all plants attained similar values of relative rates of growth and leaf elongation by 21 d after planting. Reductions in photosynthesis were attributed to decreased Rubisco activation in the antisense plants. Rubisco constituted about 40% of total soluble protein in both Wt and clone R100 under both light regimes. Activase content was 5% and 1.4% of total soluble protein in Wt and clone R100, respectively, and also was unaffected by growth irradiance. The stoichiometry of Rubisco to activase was estimated at 20 Rubisco active sites per activase tetramer in Wt Arabidopsis and 60 to 80 in the transgenic clone R100. We conclude that Wt Arabidopsis does not contain Rubisco activase in great excess of the amount required for optimal growth.     

Regulation of photosynthesis in developing leaves of soybean chlorophyll-deficient mutants

In this report we examine the factors that regulate photosynthesis during leaf ontogeny in y3y3 and Y11y11, two chlorophyll-deficient mutants of soybean. Photosynthetic rates were similar during wild type and Y11y11 leaf development, but the senescence decline in photosynthesis was accelerated in y3y3. Photosynthetic rates fell more rapidly than chlorophyll concentrations during senescence in wild type leaves, indicating that light harvesting is not strongly limiting for photosynthesis during this phase of leaf development. Chlorophyll concentrations in Y11y11, though significantly lower than normal, were able to support normal photosynthetic rates throughout leaf ontogeny. Chlorophyll a/b ratios were constant during leaf development in the wild type, but in the mutants they progressively increased (y3y3) or decreased (Y11y11). In all three sets of plants, photosynthetic rates were directly proportional to Rubisco contents and activities, suggesting that Rubisco plays a dominant role in regulating photosynthesis throughout leaf ontogeny in these plants. The expression of some photosynthetic proteins, such as Rubisco activase, was coordinately regulated with that of Rubisco in all three genotypes, i.e. an early increase, coincident with leaf expansion, followed by a senescence decline in the fully-expanded leaf. On the other hand, the light harvesting chlorophyll a/b-binding proteins of PS II (the CAB proteins), while they showed a profile similar to that of Rubisco in the wild type and y3y3, progressively increased in amount during Y11y11 leaf development. We conclude that Y11y11 may be defective in the accumulation of a component required for LHC II assembly or function, while y3y3 has more global effects and may be a regulatory factor that controls the duration of senescence.     

Biochemical and molecular basis for impairment of photosynthetic potential

Ozone induces reductions in net photosynthesis in a large number of plant species. A primary mechanism by which photosynthesis is reduced is through impact on carbon dioxide fixation. Ozone induces loss in Rubisco activity associated with loss in concentration of the protein. Evidence is presented that ozone may induce oxidative modification of Rubisco leading to subsequent proteolysis. In addition, plants exposed to ozone sustain reduction in rbcS, the mRNA for the small subunit of Rubisco. This loss in rbcS mRNA may lead to a reduced potential for synthesis of the protein. The regulation of O₃-induced loss of Rubisco, and implications of the decline in this protein in relation to accelerated senescence are discussed.     

Co-overproducing Rubisco and Rubisco activase enhances photosynthesis in the optimal temperature range in rice

Rubisco limits C₃ photosynthesis under some conditions and is therefore a potential target for improving photosynthetic efficiency. The overproduction of Rubisco is often accompanied by a decline in Rubisco activation, and the protein ratio of Rubisco activase (RCA) to Rubisco (RCA/Rubisco) greatly decreases in Rubisco-overproducing plants (RBCS-ox). Here, we produced transgenic rice (Oryza sativa) plants co-overproducing both Rubisco and RCA (RBCS-RCA-ox). Rubisco content in RBCS-RCA-ox plants increased by 23%–44%, and RCA/Rubisco levels were similar or higher than those of wild-type plants. However, although the activation state of Rubisco in RBCS-RCA-ox plants was enhanced, the rates of CO₂ assimilation at 25°C in RBCS-RCA-ox plants did not differ from that of wild-type plants. Alternatively, at a moderately high temperature (optimal range of 32°C–36°C), the rates of CO₂ assimilation in RBCS-ox and RBCS-RCA-ox plants were higher than in wild-type plants under conditions equal to or lower than current atmospheric CO₂ levels. The activation state of Rubisco in RBCS-RCA-ox remained higher than that of RBCS-ox plants, and activated Rubisco content in RCA overproducing, RBCS-ox, RBCS-RCA-ox, and wild-type plants was highly correlated with the initial slope of CO₂ assimilation against intercellular CO₂ pressures (A:Ci) at 36°C. Thus, a simultaneous increase in Rubisco and RCA contents leads to enhanced photosynthesis within the optimal temperature range.

A simultaneous increase in Rubisco and RCA contents in transgenic rice leads to an enhancement of photosynthesis at moderately high temperatures within the optimal temperature range.     

Metabolome analysis of photosynthesis and the related primary metabolites in the leaves of transgenic rice plants with increased or decreased Rubisco content

Because the comprehensive effects on metabolism by genetic manipulation of leaf Rubisco content are unknown, metabolome analysis was carried out on transgenic rice plants with increased or decreased Rubisco content using the capillary electrophoresis-time-of-flight mass spectrometry (CE-TOFMS) technique. In RBCS-sense plants, an increase in Rubisco content did not improve light-saturated photosynthesis. Glyceraldehyde 3-phosphate and sedoheputulose 7-phosphate levels increased, but ribulose bisphosphate (RuBP), ATP and ADP levels were not affected. It is considered from these results that RuBP regeneration independent of ATP supply became a bottleneck for photosynthesis. In RBCS-antisense plants, a decline in Rubisco content decreased photosynthesis with a substantial accumulation of RuBP. ATP and ADP levels also increased and were associated with increases in the diphosphate and triphosphate compounds of other nucleosides. These results imply that a decline in Rubisco content slowed down the Calvin cycle and that the resultant excess energy of ATP was transferred to other nucleoside diphosphates and triphosphates. The levels of amino acids tended to decline in RBCS-sense plants and increase in RBCS-antisense plants, probably reflecting the demand for Rubisco synthesis. Starch and carbohydrate levels decreased only in RBCS-antisense plants. Thus, genetic manipulation of Rubisco contents widely affected C and N metabolism in rice.