Activase region on chloroplast ribulose-1,5-bisphosphate carboxylase/oxygenase. Nonconservative substitution in the large subunit alters species specificity of protein interaction.
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In the active form of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco, EC ), a carbamate at lysine 201 binds Mg(2+), which then interacts with the carboxylation transition state. Rubisco activase facilitates this spontaneous carbamylation/metal-binding process by removing phosphorylated inhibitors from the Rubisco active site. Activase from Solanaceae plants (e.g. tobacco) fails to activate Rubisco from non-Solanaceae plants (e.g. spinach and Chlamydomonas reinhardtii), and non-Solanaceae activase fails to activate Solanaceae Rubisco. Directed mutagenesis and chloroplast transformation previously showed that a proline 89 to arginine substitution on the surface of the large subunit of Chlamydomonas Rubisco switched its specificity from non-Solanaceae to Solanaceae activase activation. To define the size and function of this putative activase binding region, substitutions were created at positions flanking residue 89. As in the past, these substitutions changed the identities of Chlamydomonas residues to those of tobacco. Whereas an aspartate 86 to arginine substitution had little effect, aspartate 94 to lysine Rubisco was only partially activated by spinach activase but now fully activated by tobacco activase. In an attempt to eliminate the activase/Rubisco interaction, proline 89 was changed to alanine, which is not present in either non-Solanaceae or Solanaceae Rubisco. This substitution also caused reversal of activase specificity, indicating that amino acid identity alone does not determine the specificity of the interaction. (+info)
Alteration of the adenine nucleotide response and increased Rubisco activation activity of Arabidopsis rubisco activase by site-directed mutagenesis.
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Arabidopsis Rubisco was activated in vitro at rates 2- to 3-fold greater by recombinant Arabidopsis 43-kD Rubisco activase with the amino acid replacements Q111E and Q111D in a phosphate-binding loop, G-G-K-G-Q-G-K-S. However, these two mutant enzymes had only slightly greater rates of ATP hydrolysis. Activities of the Q111D enzyme were much less sensitive and those of Q111E were somewhat less sensitive to inhibition by ADP. Both mutant enzymes exhibited higher Rubisco activation activities over the physiological range of ADP to ATP ratios. Enzymes with non-polar, polar, and basic residues substituted at position Gln-111 exhibited rates of Rubisco activation less than the wild-type enzyme. Estimates of the relative affinity of the wild type and the Q111D, Q111E, and Q111S enzymes for adenosine nucleotides by a variety of methods revealed that the nucleotide affinities were the most diminished in the Q111D enzyme. The temperature stability of the Q111D and Q111E enzymes did not differ markedly from that of the 43-kD recombinant wild-type enzyme, which is somewhat thermolabile. The Q111D and Q111E enzymes, expressed in planta, may provide a means to better define the role of the ADP to ATP ratio in the regulation of Rubisco activation and photosynthesis rate. (+info)
Elevated CO(2) induces biochemical and ultrastructural changes in leaves of the C(4) cereal sorghum.
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We analyzed the impact of growth at either 350 (ambient) or 700 (elevated) microL L(-1) CO(2) on key elements of the C(4) pathway (photosynthesis, carbon isotope discrimination, and leaf anatomy) using the C(4) cereal sorghum (Sorghum bicolor L. Moench.). Gas-exchange analysis of the CO(2) response of photosynthesis indicated that both carboxylation efficiency and the CO(2) saturated rate of photosynthesis were lower in plants grown at elevated relative to ambient CO(2). This was accompanied by a 49% reduction in the phosphoenolpyruvate carboxylase content of leaves (area basis) in the elevated CO(2)-grown plants, but no change in Rubisco content. Despite the lower phosphoenolpyruvate carboxylase content, there was a 3-fold increase in C isotope discrimination in leaves of plants grown at elevated CO(2) and bundle sheath leakiness was estimated to be 24% and 33%, respectively, for the ambient and elevated CO(2)-grown plants. However, we could detect no difference in quantum yield. The ratio of quantum yield of CO(2) fixation to PSII efficiency was lower in plants grown at elevated CO(2), but only when leaf internal was below 50 microL L(-1). This suggests a reduction in the efficiency of the C(4) cycle when [CO(2)] is low, and also implies increased electron transport to acceptors other than CO(2). Analysis of leaf sections using a transmission electron microscope indicated a 2-fold decrease in the thickness of the bundle sheath cell walls in plants grown at elevated relative to ambient CO(2). These results suggest that significant acclimation to increased CO(2) concentrations occurs in sorghum. (+info)
Effects of the Calvin cycle on nicotinamide adenine dinucleotide concentrations and redox balances of Xanthobacter flavus.
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The levels of reduced and oxidized nicotinamide adenine dinucleotides were determined in Xanthobacter flavus during a transition from heterotrophic to autotrophic growth. Excess reducing equivalents are rapidly dissipated following induction of the Calvin cycle, indicating that the Calvin cycle serves as a sink for excess reducing equivalents. The physiological data support the conclusion previously derived from molecular studies in that expression of the Calvin cycle genes is controlled by the intracellular concentration of NADPH. (+info)
Weather and nodule mediated variations in delta 13C and delta 15N values in field-grown soybean (Glycine max L.) with special interest in the analyses of xylem fluids.
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The nodulating soybean (Enrei) and its non-nodulating mutant (EN 1282) were grown in outdoor plots for 2 years (1994: extraordinary dry, high temperature, 1995: ordinary year). Carbon and nitrogen accumulation, delta 13C and delta 15N values in plant parts and xylem fluids and delta 15N values in the water-extractable soil N were analysed throughout the growing period. Plant growth in 1994 was rapid during the early growth stages, but no pods were produced. In 1995, plant growth was normal and pods were formed. The delta 13C values of the leaves were less negative in 1994 than in 1995 and the nodulated plants showed less negative delta 13C values than non-nodulated plants in both years. The delta 13C values of the leaves during the vegetative phase were positively correlated to the leaf N concentrations. Leaf N concentrations in their turn were influenced by nodulation and weather conditions and/or soil available N. The delta 15N values in the plants and xylem fluids were lower in the nodulated soybean than in non-nodulated soybean in both years, and estimates of the contribution of N2 fixation in nodulated plants based on plant top delta 15N values were 7-14% in 1994 and 37-63% in 1995. The delta 13C values of xylem fluids did not differ between nodulated and non-nodulated plants. Thus, the expected contribution by phosphopenolpyruvate carboxylase-mediated CO2 fixation in the root nodules to plant C-incorporation could not have been significant. (+info)
Modelling photosynthesis and its control.
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The dynamic and steady-state behaviour of a computer simulation of the Calvin cycle reactions of the chloroplast, including starch synthesis and degradation, and triose phosphate export have been investigated. A major difference compared with previous models is that none of the reversible reactions are assumed to be at equilibrium. The model can exhibit alternate steady states of low or high carbon assimilation flux, with hysteresis in the transitions between the steady states induced by environmental factors such as phosphate and light intensity. The enzymes which have the greatest influence on the flux have been investigated by calculation of their flux control coefficients. Different patterns of control are exhibited over the assimilation flux, the flux to starch and the flux to cytosolic triose phosphate. The assimilation flux is mostly sensitive to sedoheptulose bisphosphatase and Rubisco, with the exact distribution depending on their relative activities. Other enzymes, particularly the triose phosphate translocator, become more influential when other fluxes are considered. These results are shown to be broadly consistent with observations on transgenic plants. (+info)
Photosynthetic electron sinks in transgenic tobacco with reduced amounts of Rubisco: little evidence for significant Mehler reaction.
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Transgenic tobacco (Nicotiana tabacum L. cv. W38) plants with an antisense gene directed against the mRNA of the small subunit of Rubisco were used to investigate the role of O2 as an electron acceptor during photosynthesis. The reduction in Rubisco has reduced the capacity for CO2-fixation in these plants without a similar reduction in electron transport capacity. Concurrent measurements of chlorophyll fluorescence and CO2 assimilation at different CO2 and O2 partial pressures showed close linear relationships between chloroplast electron transport rates calculated from chlorophyll fluorescence and those calculated from CO2-fixation. These relationships were similar for wild-type and transgenic plants, indicating that the reduced capacity for CO2 fixation in the transgenic plants did not result in extra electron transport not associated with the photosynthetic carbon reduction (PCR) or photorespiratory carbon oxidation (PCO) cycle. This was further investigated with mass spectrometric measurements of 16O2 and 18O2 exchange made concurrently with measurements of chlorophyll fluorescence. In all tobacco lines the rates of 18O2 uptake in the dark were similar to the 18O2 uptake rates at very high CO2 partial pressures in the light. Rates of oxygenase activity calculated from 18O2 uptake at the compensation point were linearly related to the Rubisco content of leaves. The ratios of oxygenase to carboxylase rates were calculated from measurements of 16O2 evolution and 18O2 uptake at the compensation point. These ratios were lower in the transgenic plants, consistent with their higher CO2 compensation points. It is concluded that although there may be some electron transport to O2 to balance conflicting demands of NADPH to ATP requirements, this flux must decrease in proportion with the reduced demand for ATP and NADPH consumption in the transgenic lines. The altered balance between electron transport and Rubisco capacity, however, does not result in rampant electron transport to O2 or other electron transport acceptors in the absence of PCR and PCO cycle activity. (+info)
Photosynthesis, plant growth and N allocation in transgenic rice plants with decreased Rubisco under CO2 enrichment.
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Ribulose-1,5-bisphosphate carboxylase (Rubisco) efficiency for CO2-saturated photosynthesis was examined in leaves of rice (Oryza sativa L.). The amount of Rubisco in a leaf was calculated to be 30-55% in excess for the light-saturated rate of photosynthesis at 100 Pa CO2. Long-term exposure to CO2 enrichment decreased the amount of Rubisco protein. However, N was not reallocated from decreased Rubisco to other components limiting photosynthesis, and the decrease in Rubisco was simply due to a decrease in total leaf-N content by CO2 enrichment. Thus, rice plants did not optimize N allocation into Rubisco at elevated CO2. Transgenic rice plants with decreased Rubisco were obtained by transformation with the rbcS antisense gene. The transformant with 65% wild-type Rubisco was selected as a plant with optimal Rubisco content for CO2-saturated photosynthesis at the level of a single leaf. This selected transgenic plant had 20% lower rates of photosynthesis at normal CO2 (36 Pa), but 5-15% higher rates of photosynthesis at elevated CO2 (100 Pa) for a given leaf N content. However, such transgenic plants did not necessarily show greater production of biomass even under conditions of CO2 enrichment. Although they had a higher N-use efficiency for plant growth under such conditions during the middle stage of growth, the growth rate was lower during the early stage of growth. Thus, improvement of N-use efficiency by a single leaf did not necessarily lead to greater production of biomass by the whole plant. (+info)