Visualization of Cavitated Vessels in Winter and Refilled Vessels in Spring in Diffuse-Porous Trees by Cryo-Scanning Electron Microscopy

The regulation of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) activity by 2-carboxyarabinitol 1-phosphate (CA1P) was investigated using gas-exchange analysis of antisense tobacco (Nicotiana tabacum) plants containing reduced levels of Rubisco activase. When an increase in light flux from darkness to 1200 μmol quanta m⁻² s⁻¹ was followed, the slow increase in CO₂ assimilation by antisense leaves contained two phases: one represented the activation of the noncarbamylated form of Rubisco, which was described previously, and the other represented the activation of the CA1P-inhibited form of Rubisco. We present evidence supporting this conclusion, including the observation that this second phase, like CA1P, is only present following darkness or very low light flux. In addition, the second phase of CO₂ assimilation was correlated with leaf CA1P content. When this novel phase was resolved from the CO₂ assimilation trace, most of it was found to have kinetics similar to the activation of the noncarbamylated form of Rubisco. Additionally, kinetics of the novel phase indicated that the activation of the CA1P-inhibited form of Rubisco proceeds faster than the degradation of CA1P by CA1P phosphatase. These results may be significant with respect to current models of the regulation of Rubisco activity by Rubisco activase.     

SDS-dependent proteases induced by ABA and its relation to Rubisco and Rubisco activase contents in rice leaves

Protease activities and its relation to the contents of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) and Rubisco activase were investigated in detached leaves of rice (Oryza sativa L.) floated on the solutions containing abscisic acid (ABA) or benzyladenine (BA). Rubisco and Rubisco activase contents were decreased during the time course and the decreases were enhanced by ABA and suppressed by BA. The decrease in Rubisco activase was faster than that in Rubisco. SDS-dependent protease activities at 50–70 kDa (rice SDS-dependent protease: RSP) analyzed by the gelatin containing PAGE were significantly enhanced by ABA. RSPs were also increased in attached leaves during senescence. RSPs had the pH optimum of 5.5, suggesting that RSPs are vacuolar protease. Both decrease in Rubisco and Rubisco activase contents and increase in RSPs activities were suppressed by cycloheximide. These findings indicate that the activities of RSPs are well correlated with the decrease in these protein contents. Immunoblotting analysis showed that Rubisco in the leaf extracts was completely degraded by 5 h at pH 5.5 with SDS where it was optimal condition for RSPs. However, the degradation of Rubisco did not proceed at pH 7.5 without SDS where it is near physiological condition for stromal proteins. Rubisco activase was degraded at similar rate under both conditions. These results suggest that RSPs can functions in a senescence related degradation system of chloroplast protein in rice leaves. Rubisco activase would be more susceptible to proteolysis than Rubisco under physiological condition and this could affect the contents of these proteins in leaves.     

Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase Content and Activity in Wheat, Rye and Triticale

Photosynthetic parameters were measured in triticale and its parents wheat and rye. Soluble protein content in leaves, ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) content per fresh mass, total chlorophyll content, biomass yield, leaf area, leaf mass and specific leaf mass were higher but Rubisco content expressed as percentage of soluble protein, carboxylase activity, photosynthetic rate and stomatal conductance were significantly lower in rye than in wheat. Native-PAGE of Rubisco revealed that rye carboxylase was different from that of wheat. The difference was not related to either the small or large subunit of Rubisco but, may be, to the ionic and/or other properties of the Rubisco protein moiety. Triticale Rubisco was similar to wheat. For most of the studied physiological parameters, triticale showed much more similarity with wheat than with rye.     

The drelationship between CO2-assimilation rate,Rubisco carbamylation and Rubisco activase content in activase-deficient transgenic tobacco suggests a simple model of activase action

Transgenic tobacco (Nicotiana tabacum L. cv. W38) plants with an antisense gene directed against the mRNA of ribulose-1,5-bisphosphate carboxylase/ oxygenase (Rubisco) activase were used to examine the relationship between CO₂-assimilation rate, Rubisco carbamylation and activase content. Plants used were those members of the r₁ progeny of a primary transformant with two independent T-DNA inserts that could be grown without CO₂ supplementation. These plants had from < 1% to 20% of the activase content of control plants. Severe suppression of activase to amounts below 5% of those present in the controls was required before reductions in CO₂-assimilation rate and Rubisco carbamylation were observed, indicating that one activase tetramer is able to service as many as 200 Rubisco hexadecamers and maintain wild-type carbamylation levels in vivo. The reduction in CO₂-assimilation rate was correlated with the reduction in Rubisco carbamylation. The anti-activase plants had similar ribulose-1,5-bisphosphate pool sizes but reduced 3-phosphoglycerate pool sizes compared to those of control plants. Stomatal conductance was not affected by reduced activase content or CO₂-assimilation rate. A mathematical model of activase action is used to explain the observed hyperbolic dependence of Rubisco carbamylation on activase content.Abbreviations CA1P 2-carboxyarabinitol-1-phosphate - P_ip_a intercellular, ambient partial pressure of CO₂ - PGA 3-phospho-glycerate - Rubisco ribulose-1,5-bisphosphate carboxylase/oxygenase - RuBP ribulose-1,5-bisphosphate - SSU small subunit of Rubisco     

Inhibition and Acclimation of Photosynthesis to Heat Stress Is Closely Correlated with Activation of Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase

Increasing the leaf temperature of intact cotton (Gossypium hirsutum L.) and wheat (Triticum aestivum L.) plants caused a progressive decline in the light-saturated CO₂-exchange rate (CER). CER was more sensitive to increased leaf temperature in wheat than in cotton, and both species demonstrated photosynthetic acclimation when leaf temperature was increased gradually. Inhibition of CER was not a consequence of stomatal closure, as indicated by a positive relationship between leaf temperature and transpiration. The activation state of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), which is regulated by Rubisco activase, was closely correlated with temperature-induced changes in CER. Nonphotochemical chlorophyll fluorescence quenching increased with leaf temperature in a manner consistent with inhibited CER and Rubisco activation. Both nonphotochemical fluorescence quenching and Rubisco activation were more sensitive to heat stress than the maximum quantum yield of photochemistry of photosystem II. Heat stress led to decreased 3-phosphoglyceric acid content and increased ribulose-1,5-bisphosphate content, which is indicative of inhibited metabolite flow through Rubisco. We conclude that heat stress inhibited CER primarily by decreasing the activation state of Rubisco via inhibition of Rubisco activase. Although Rubisco activation was more closely correlated with CER than the maximum quantum yield of photochemistry of photosystem II, both processes could be acclimated to heat stress by gradually increasing the leaf temperature.     

2-Carboxy-D-arabinitol 1-phosphate (CA1P) phosphatase: evidence for a wider role in plant Rubisco regulation

The genes for CA1Pase (2-carboxy-D-arabinitol-1-bisphosphate phosphatase) from French bean, wheat, Arabidopsis and tobacco were identified and cloned. The deduced protein sequence included an N-terminal motif identical with the PGM (phosphoglycerate mutase) active site sequence [LIVM]-x-R-H-G-[EQ]-x-x-[WN]. The corresponding gene from wheat coded for an enzyme with the properties published for CA1Pase. The expressed protein lacked PGM activity but rapidly dephosphorylated 2,3-DPG (2,3-diphosphoglycerate) to 2-phosphoglycerate. DTT (dithiothreitol) activation and GSSG inactivation of this enzyme was pH-sensitive, the greatest difference being apparent at pH 8. The presence of the expressed protein during in vitro measurement of Rubisco (ribulose-1,5-bisphosphate carboxylase/oxygenase) activity prevented a progressive decline in Rubisco turnover. This was due to the removal of an inhibitory bisphosphate that was present in the RuBP (ribulose-1,5-bisphosphate) preparation, and was found to be PDBP (D-glycero-2,3-pentodiulose-1,5-bisphosphate). The substrate specificity of the expressed protein indicates a role for CA1Pase in the removal of 'misfire' products of Rubisco.     

Rubisco Activase Activity in Spinach Leaf Extracts

Rubisco activase is a chloroplast stromal protein that catalyzesthe activation of ribulose-1,5- bisphosphate carboxylase/oxygenase(rubisco) in vivo. Activation must occur before rubisco cancatalyze the photosynthetic assimilation of CO₂. In leaves,photosynthesis and rubisco activation increase with increasinglight intensity. Techniques are described that allow the activityof rubisco activase to be measured in extracts of spinach (Spinaceaoleracea L.) leaf tissue. In this context, rubisco activaseactivity is defined as the ability to promote activation ofthe inactive ribulose-1,5- bisphosphate-bound rubisco in anATP-dependent reaction. Determination of rubisco activase activityin extracts of dark and light treated leaf tissue revealed thatthe activation state of rubisco activase was independent oflight intensity. ¹Present address: Department of Biological Sciences, 213 Carson-TaylorHall, Louisiana Tech University, Ruston, Louisiana 71272, U.S.A.     

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     

Covalent modification of a highly reactive and essential lysine residue of ribulose-1,5-bisphosphate carboxylase/oxygenase activase.

Chemical modification of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) activase with water-soluble N-hydroxysuccinimide esters was used to identify a reactive lysyl residue that is essential for activity. Incubation of Rubisco activase with sulfosuccinimidyl-7-amino-4-methylcoumarin-3-acetate (AMCA-sulfo-NHS) or sulfosuccinimidyl-acetate (sulfo-NHS-acetate) caused progressive inactivation of ATPase activity and concomitant loss of the ability to activate Rubisco. AMCA-sulfo-NHS was the more potent inactivator of Rubisco activase, exhibiting a second-order rate constant for inactivation of 239 M-1 s-1 compared to 21 M-1 s-1 for sulfo-NHS-acetate. Inactivation of enzyme activity by AMCA-sulfo-NHS correlated with the incorporation of 1.9 mol of AMCA per mol of 42-kD Rubisco activase monomer. ADP, a competitive inhibitor of Rubisco activase, afforded considerable protection against inactivation of Rubisco activase and decreased the amount of AMCA incorporated into the Rubisco activase monomer. Sequence analysis of the major labeled peptide from AMCA-sulfo-NHS-modified enzyme showed that the primary site of modification was lysine-247 (K247) in the tetrapeptide methionine-glutamic acid-lysine-phenylalanine. Upon complete inactivation of ATPase activity, modification of K247 accounted for 1 mol of AMCA incorporated per mol of Rubisco activase monomer. Photoaffinity labeling of AMCA-sulfo-NHS- and sulfo-NHS-acetate-modified Rubisco activase with ATP analogs derivatized on either the adenine base or on the gamma-phosphate showed that K247 is not essential for the binding of adenine nucleotides per se. Instead, the data indicated that the essentiality of K247 is probably due to an involvement of this highly reactive, species-invariant residue in an obligatory interaction that occurs between the protein and the nucleotide phosphate during catalysis.     

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.     

Heat stress effects on ribulose-1,5-bisphosphate carboxylase/oxygenase,Rubisco binding protein and Rubisco activase in wheat leaves

Changes in chlorophyll content, ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) binding protein (RBP), Rubisco activase (RA), Rubisco large (LS) and small (SS) subunits, and electrolyte leakage were investigated in wheat leaf segments during heat stress (HS) for 1 h and for 24 h at 40 °C in darkness or in light, as well as after recovery from heat stress (HSR) for 24 h at 25 °C in light. The 24-h HS treatment in darkness decreased irreversibly photosynthetic pigments, soluble proteins, RBP, RA, Rubisco LS and SS. An increase in RA and RBP protein contents was observed under 24-h HS and HSR in light. This increase was in accordance with their role as chaperones and the function of RBP as a heat shock protein.This work was partially supported by Swiss National Science Foundation (Project 31-55289.98).     

Reduction of ribulose biphosphate carboxylase activase levels in tobacco (Nicotiana tabacum) by antisense RNA reduces ribulose biphosphate carboxylase carbamylation and impairs photosynthesis.

The in vivo activity of ribulose-1,5-biphosphate carboxylase/oxygenase (Rubisco) is modulated in response to light intensity by carbamylation of the active site and by the binding of sugar phosphate inhibitors such as 2'-carboxyarabinitol-1-phosphate (CA 1P). These changes are influenced by the regulatory protein Rubisco activase, which facilitates the release of sugar phosphates from Rubisco's catalytic site. Activase levels in Nicotiana tabacum were reduced by transformation with an antisense gene directed against the mRNA for Rubisco activase. Activase-deficient plants were photosynthetically impaired, and their Rubisco carbamylation levels declined upon illumination. Such plants needed high CO2 concentrations to sustain reasonable growth rates, but the level of carbamylation was not increased by high CO2. The antisense plants had, on average, approximately twice as much Rubisco as the control plants. The maximum catalytic turnover rate (k cat) of Rubisco decreases in darkened tobacco leaves because of the binding of CA 1P. The dark-to-light increase in k cat that accompanies CA 1P release occurred to similar extents in antisense and control plants, indicating that normal levels of activase were not essential for CA 1P release from Rubisco in the antisense plants. However, CA 1P was released in the antisense plants at less than one-quarter of the rate that it was released in the control plants, indicating a role for activase in accelerating the release of CA 1P.     

A non-radioactive method for measuring Rubisco activase activity in the presence of variable ATP: ADP ratios,including modifications for measuring the activity and activation state of Rubisco

Rubisco (ribulose-1,5-bisphosphate carboxylase/oxygenase) catalyzes carboxylation of ribulose-1,5-bisphosphate, the first in a series of reactions leading to the incorporation of atmospheric CO₂ into biomass. Rubisco requires Rubisco activase (RCA), an AAA+ ATPase that reactivates Rubisco by remodelling the conformation of inhibitor-bound sites. RCA is regulated by the ratio of ADP:ATP, with the precise response potentiated by redox regulation of the alpha-isoform. Measuring the effects of ADP on the activation of Rubisco by RCA using the well-established photometric assay is problematic because of the adenine nucleotide requirement of 3-phosphoglycerate (3-PGA) kinase. Described here is a novel assay for measuring RCA activity in the presence of variable ratios of ADP:ATP. The assay couples the formation of 3-PGA from ribulose 1,5-bisphosphate and CO₂ to NADH oxidation through cofactor-dependent phosphoglycerate mutase, enolase, PEP carboxylase and malate dehydrogenase. The assay was used to determine the effects of Rubisco and RCA concentration and ADP:ATP ratio on RCA activity, and to measure the activation of a modified Rubisco by RCA. Variations of the basic assay were used to measure the activation state of Rubisco in leaf extracts and the activity of purified Rubisco. The assay can be automated for high-throughput processing by conducting the reactions in two stages.     

Light-dependent changes in ribulose bisphosphate carboxylase activase activity in leaves

An assay for the activity of ribulose bisphosphate carboxylase (Rubisco) activase in crude leaf extracts was developed. The assay is based on a spectrophotometric assay of Rubisco, and activase activity (in nanomoles activated Rubisco per minute per milligram chlorophyll) was calculated from the rate of increase in Rubisco activity over time. Activase activity measurements were made using samples from spinach (Spinacia oleracea) leaves undergoing (a) steady-state photosynthesis at various photon flux density (PFD) values and (b) nonsteady-state photosynthesis following an increase from darkness to a high PFD. Analysis of these samples showed that steady-state Rubisco activase activity was relatively low in darkness, increased with PFD, and saturated below 300 micromoles per square meter per second. Rubisco activity (measured spectrophotometrically) was also found to be low in darkness and to increase with PFD, but it saturated at much higher PFD values (approximately 1000 micromoles per square meter per second) along with the rate of photosynthesis. Following an increase in PFD from darkness to 650 micromoles per square meter per second, activase activity increased more or less linearly over a period of 5 to 6 minutes, after which it was constant. Rubisco activity, however, increased more slowly. The light-dependence of Rubisco activase is consistent with previous gas-exchange data showing two interdependent processes in the activation of Rubisco following an increase in PFD.     

Moderately High Temperatures Inhibit Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase (Rubisco) Activase-Mediated Activation of Rubisco

We tested the hypothesis that light activation of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) is inhibited by moderately elevated temperature through an effect on Rubisco activase. When cotton (Gossypium hirsutum L.) or wheat (Triticum aestivum L.) leaf tissue was exposed to increasing temperatures in the light, activation of Rubisco was inhibited above 35 and 30°C, respectively, and the relative inhibition was greater for wheat than for cotton. The temperature-induced inhibition of Rubisco activation was fully reversible at temperatures below 40°C. In contrast to activation state, total Rubisco activity was not affected by temperatures as high as 45°C. Nonphotochemical fluorescence quenching increased at temperatures that inhibited Rubisco activation, consistent with inhibition of Calvin cycle activity. Initial and maximal chlorophyll fluorescence were not significantly altered until temperatures exceeded 40°C. Thus, electron transport, as measured by Chl fluorescence, appeared to be more stable to moderately elevated temperatures than Rubisco activation. Western-blot analysis revealed the formation of high-molecular-weight aggregates of activase at temperatures above 40°C for both wheat and cotton when inhibition of Rubisco activation was irreversible. Physical perturbation of other soluble stromal enzymes, including Rubisco, phosphoribulokinase, and glutamine synthetase, was not detected at the elevated temperatures. Our evidence indicates that moderately elevated temperatures inhibit light activation of Rubisco via a direct effect on Rubisco activase.     

Development of leaf photosynthetic parameters in Betula pendula Roth leaves: correlations with photosystem I density

The global modelling of photosynthesis is based on exact knowledge of the leaf photosynthetic machinery. The capacities of partial reactions of leaf photosynthesis develop at different rates, but it is not clear how the development of photoreactions and the Calvin cycle are co-ordinated. We investigated the development of foliar photosynthesis in the temperate deciduous tree Betula pendula Roth. using a unique integrated optical/gas exchange methodology that allows simultaneous estimation of photosystem I and II (PS I and PS II) densities per leaf area, interphotosystem electron transport activities, and ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) kinetic properties. We combined these measurements with in vitro determinations of Rubisco, soluble protein and chlorophyll contents. We observed a strong increase in leaf photosynthetic capacity in developing leaves per leaf area, as well as per dry mass, that was paralleled by accumulation of leaf Rubisco. Enhanced mesophyll conductance was the outcome of increased carboxylation capacity and increased CO(2) diffusion conductance. However, Rubisco was only partly activated in the leaves, according to in vivo measurements of Rubisco kinetics. The amount of active Rubisco increased in proportion with development of PS I, probably through a direct link between Rubisco activase and PS I electron transport. Since the kinetics for post-illumination P700 re-reduction did not change, the synthesis of cytochrome b(6)f complex was also proportional to PS I. The synthesis of PS II began later and continued for several days after reaching the full PS I activity, but leaf chlorophyll was shared equally between the photosystems. Due to this, the antenna of PS II was very large and not optimally organized, leading to greater losses of excitation and lower quantum yields in young leaves. We conclude that co-ordinated development of leaf photosynthesis is regulated at the level of PS I with subordinated changes in PS II content and Rubisco activation.     

Effect of heat stress on the inhibition and recovery of the ribulose-1,5-bisphosphate carboxylase/oxygenase activation state

Experiments were conducted to determine the relative contributions of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco; EC 4.1.1.39) activation state vis-à-vis Rubisco activase and metabolite levels to the inhibition of cotton (Gossypium hirsutum L.) photosynthesis by heat stress. Exposure of leaf tissue in the light to temperatures of 40 or 45 °C decreased the activation state of Rubisco to levels that were 65 or 10%, respectively, of the 28 °C control. Ribulose-1,5-bisphosphate (RuBP) levels increased in heat-stressed leaves, whereas the 3-phosphoglyceric acid pool was depleted. Heat stress did not affect Rubisco per se, as full activity could be restored by incubation with CO₂ and Mg²⁺. Inhibition and recovery of Rubisco activation state and carbon dioxide exchange rate (CER) were closely related under moderate heat stress (up to 42.5 °C). Moderate heat stress had negligible effect on Fv/Fm, the maximal quantum yield of photosystem II. In contrast, severe heat stress (45 °C) caused significant and irreversible damage to Rubisco activation, CER, and Fv/Fm. The rate of Rubisco activation after alleviating moderate heat stress was comparable to that of controls, indicating rapid reversibility of the process. However, moderate heat stress decreased both the rate and final extent of CER activation during dark-to-light transition. Treatment of cotton leaves with methyl viologen or an oxygen-enriched atmosphere reduced the effect of heat stress on Rubisco inactivation. Both treatments also reduced tissue RuBP levels, indicating that the amount of RuBP present during heat stress may influence the degree of Rubisco inactivation. Under both photorespiratory and non-photorespiratory conditions, the inhibition of the CER during heat stress could be completely reversed by increasing the internal partial pressure of CO₂ (Ci). However, the inhibition of the CER by nigericin, a K⁺ ionophore, was not reversible when the Ci was increased at ambient or high temperature. Our results indicate that inhibition of photosynthesis by moderate heat stress is not caused by inhibition of the capacity for RuBP regeneration. We conclude that heat stress inhibits Rubisco activation via a rapid and direct effect on Rubisco activase, possibly by perturbing Rubisco activase subunit interactions with each other or with Rubisco. Received: 25 February 2000 / Accepted: 13 May 2000     

Direct Measurement of ATP-Dependent Proton Concentration Changes and Characterization of a K+-Stimulated ATPase in Pea Chloroplast Inner Envelope Vesicles

An important question concerning the role of carboxyarabinitol 1-phosphate (CA1P) metabolism in the light-dependent regulation of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) activity is the extent to which CA1P is bound to Rubisco in vivo. We report here the development of an extraction procedure using ammonium sulfate that stabilizes CA1P bound to Rubisco. This procedure exploits the ability of sulfate to bind at the catalytic site of Rubisco and to competitively balance the binding and release of CA1P from Rubisco. In darkened bean leaves about 75% of the Rubisco catalytic sites were found to be bound with CA1P. This confirms previous indirect estimates from gas exchange measurements. We have used this extraction procedure to examine CA1P-Rubisco interactions in bean during a natural transition from darkness to light. With increasing light intensity following sunrise, CA1P degradation proceeded in two distinct phases: first, a majority of the unbound CA1P pool was degraded at very low light levels ([less than or equal to]30 [mu]mol quanta m-2 s-1); second, CA1P initially bound to Rubisco was then degraded at increasing light levels (>30 [mu]mol quanta m-2 s-1). These results indicate that there is a low-fluence activation of CA1P phosphatase that can occur prior to CA1P release by Rubisco activase. This activation may be mediated by NADPH. During sunrise in bean, the level of the catalytically competent form of Rubisco was regulated by CA1P metabolism.