Midday depression in photosynthesis: Effect on sucrose-phosphate synthase and ribulose-1,5-bisphosphate carboxylase in leaves of Prosopis juliflora (Swartz) DC

The midday depression in net photosynthetic rate (PN) and stomatal conductance (gs) in Prosopis juliflora was studied in relation to two key enzymes of carbon metabolism. Diurnal gas exchange measurements carried out in autumn on P. juliflora showed a pronounced depression in PN and gs along with a decrease in apparent carboxylation efficiency (CE*) during midday. The activities of sucrose-phosphate synthase (SPS) and ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBPCO) also showed large diurnal fluctuations. Initial RuBPCO activity (that present in vivo) and total activity (fully carbamylated activity) increased gradually with increase in irradiance, in the morning reaching a maximum by 08:00 h. The RuBPCO activity declined sharply during midday due to decrease in initial activity. The drop in the % activation of RuBPCO indicated that the deactivation of RuBPCO was achieved via a decarbamylation mechanism. There was a marked similarity in the diurnal patterns of SPS activity and the PN. During a diurnal rhythm, SPS activity increased after irradiation, reaching a maximum at 08:00 h and then declined during midday. Diurnal fluctuations in SPS activity could be due to the changes in the amount of protein (changes in Vmax) as well as to the changes in kinetic properties (changes in Vlim). Hence the midday decline in photosynthesis is closely associated with the regulation of RuBPCO and SPS activities.     

Free subunits of ribulose-1,5-bisphosphate carboxylase in pea leaves

Pea leaves, supplied with [³⁵S]methionine, were homogenized and a crude hypotonic soluble fraction was centrifuged on sucrose gradients to separate fully assembled ribulose-1,5-biphosphate (RuBP) carboxylase from any free or partially assembled carboxylase subunits. Slowly sedimenting subunits of the enzyme were identified in upper fractions of the sucrose gradient, using polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS), isoelectric focussing, and immune precipitation. The presence of these subunits in low molecular weight form was shown not to be due to artefactual dissociation of the enzyme. It is suggested that these subunits are related to the assembly of RuBP carboxylase.     

Dependence of photosynthesis of sunflower and maize leaves on phosphate supply, ribulose-1,5-bisphosphate carboxylase/oxygenase activity, and ribulose-1,5-bisphosphate pool size

Sunflower (Helianthus annuus L. cv Asmer) and maize (Zea mays L. cv Eta) plants were grown under controlled environmental conditions with a nutrient solution containing 0, 0.5, or 10 millimolar inorganic phosphate. Phosphate-deficient leaves had lower photosynthetic rates at ambient and saturating CO₂ and much smaller carboxylation efficiencies than those of plants grown with ample phosphate. In addition, phosphate-deficient leaves contained smaller quantities of total soluble proteins and ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) per unit area, although the relative proportions of these components remained unchanged. The specific activity of Rubisco (estimated in the crude extracts of leaves) was significantly reduced by phosphate deficiency in sunflower but not in maize. Thus, there was a strong dependence of carboxylation efficiency and CO₂-saturated photosynthetic rate on Rubisco activity only in sunflower. Phosphate deficiency decreased the 3-phosphoglycerate and ribulose-1,5-bisphosphate (RuBP) contents of the leaf in both species. The ratio of 3-phosphoglycerate to RuBP decreased in sunflower but increased in maize with phosphate deficiency. The calculated concentrations of RuBP and RuBP-binding sites in the chloroplast stroma decreased markedly with phosphate deficiency. The ratio of the stromal concentration of RuBP to that of RuBP-binding sites decreased in sunflower but was not affected in maize with phosphate deficiency. We suggest that a decrease in this ratio made the RuBP-binding sites more vulnerable to blockage or inactivation by tight-binding metabolites/inhibitors, causing a decrease in the initial specific activity of Rubisco in the crude extract from phosphate-deficient sunflower leaves. However, the decrease in Rubisco specific activity was much less than the decrease in the RuBP content in the leaf and its concentration in the stroma. A large ratio of RuBP to RuBP-binding sites may have maintained the Rubisco-specific activity in phosphate-deficient maize leaves. We conclude that the effect of phosphate deficiency is more on RuBP regeneration than on Rubisco activity in both sunflower and maize.     

The state of activation of ribulose-1,5-bisphosphate carboxylase in wheat leaves

In light and in darkness, exposure of leaf segments to CO₂-free atmospheres caused a marked reduction in extractable RuBP carboxylase activity. By contrast, darkness caused a relatively small decrease in carboxylase activity in extracts from leaf segments kept in air containing CO₂. Recovery of carboxylase activity in leaves during illumination in air after exposure to CO₂-free conditions paralleled recovery of capacity for photosynthesis; in darkness recovery of carboxylase activity in leaves was slower than in the light. Extracts from leaves exposed to CO₂-free conditions recovered activity when provided with CO₂ and Mg²⁺; there were clearly, however, substances in the extracts that modified the activity achieved and caused anomalous decreases and increases with time after extraction. Studies of the effect of orthophosphate on the activity of purified wheat carboxylase in vitro were consistent with the view that many of the effects observed on the activity of crude leaf extracts were due to orthophosphate content.     

Properties of ribulose-1,5-bisphosphate carboxylase from dark- and light-exposed soybean leaves

The low activity of ribulose bisphosphate carboxylase from darkened soybean (Glycine max [L.] Merr. cv. Bragg) leaves was not raised to the level of that from leaves in the light by CO₂ and Mg²⁺, even after a 4-h incubation. The extract of darkened leaves, unlike the extract from illuminated leaves, was not fully CO₂/Mg²⁺-activatable after Sephadex gel filtration in the absence of Mg²⁺. (NH₄)₂SO₄ fractionation eliminated the inhibition effect found in the dark extracts resulting in similar rates for the extracts obtained from leaves in the dark and light. Although the V_max values of the gel-filtered extracts from dark and light leaves differed by 3-fold, the K_m(CO₂)-values were the same (12.7 μM), as were the K_m(RuBP)-values (250 μM). These data support the hypothesis that for soybean leaves in the dark a tightly-binding inhibitor renders much of the ribulose bisphosphate carboxylase enzyme catalytically non-functional.     

Dissociation of ribulose-1,5-bisphosphate bound to ribulose-1,5-bisphosphate carboxylase/oxygenase and its enhancement by ribulose-1,5-bisphosphate carboxylase/oxygenase activase-mediated hydrolysis of ATP

Ribulose bisphosphate (RuBP), a substrate of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), is an inhibitor of Rubisco activation by carbamylation if bound to the inactive, noncarbamylated form of the enzyme. The effect of Rubisco activase on the dissociation kinetics of RuBP bound to this form of the enzyme was examined and characterized with the use of ³H-labeled RuBP and proteins purified from spinach (Spinacia oleracea L.) In the absence of Rubisco activase and in the presence of a large excess of unlabeled RuBP, the dissociation rate of bound [1-³H]RuBP was much faster after a short (30 second) incubation than after an extended incubation (1 hour). After 1 hour of incubation, the dissociation rate constant (K_off) of the bound RuBP was 4.8 × 10⁻⁴ per second, equal to a half-time of about 35 minutes, whereas the rate after only 30 seconds was too fast to be accurately measured. This time-dependent change in the dissociation rate was reflected in the subsequent activation kinetics of Rubisco in the presence of RuBP, CO₂, and Mg²⁺, and in both the absence or presence of Rubisco activase. However, the activation of Rubisco also proceeded relatively rapidly without Rubisco activase if the RuBP level decreased below the estimated catalytic site concentration. High pH (pH 8.5) and the presence of Mg²⁺ in the medium also enhanced the dissociation of the bound RuBP from Rubisco in the presence of RuBP. In the presence of Rubisco activase, Mg²⁺, ATP (but not the nonhydrolyzable analog, adenosine-5′-O-[3-thiotriphosphate]), excess RuBP, and an ATP-regenerating system, the dissociation of [1-³H]RuBP from Rubisco was increased in proportion to the amount of Rubisco activase added. This result indicates that Rubisco activase-mediated hydrolysis of ATP is required for promotion of the enhanced dissociation of the bound RuBP from Rubisco. Furthermore, product analysis by ion-exchange chromatography demonstrated that the release of the bound RuBP, in an unchanged form, was considerably faster than the observed increase in Rubisco activity. Thus, RuBP dissociation was experimentally separated from activation and precedes the subsequent formation of active, carbamylated Rubisco during activation of Rubisco by Rubisco activase.     

Inhibition of ribulose-1,5-bisphosphate carboxylase/oxygenase by ribulose-1,5-bisphosphate epimerization and degradation products

Xylulose-1,5-bisphosphate in preparations of ribulose-1,5-bisphosphate (ribulose-P₂) arises from non-enzymic epimerization and inhibits the enzyme. Another inhibitor, a diketo degradation product from ribulose-P₂, is also present. Both compounds simulate the substrate inhibition of ribulose-P₂ carboxylase/oxygenase previously reported for ribulose-P₂. Freshly prepared ribulose-P₂ had little inhibitory activity. The instability of ribulose-P₂ may be one reason for a high level of ribulose-P₂ carboxylase in chloroplasts where the molarity of active sites exceeds that of ribulose-P₂. Because the K_D of the enzyme/substrate complex is ≤1 μM, all ribulose-P₂ generated in situ may be stored as this complex to prevent decomposition.     

A point mutation in the N-terminus of ribulose-1,5-bisphosphate carboxylase affects ribulose-1,5-bisphosphate binding

Mutagenesis in vitro of the gene encoding the large subunit of ribulose-1,5-bisphosphate carboxylase/ oxygenase (EC 4.1.1.39) from Anacystis nidulans was used to generate novel enzymes. Two conserved residues, threonine 4 and lysine 11 in the N-terminus were changed. The substitution of threonine 4 with serine or valine had little effect on the kinetic parameters. The substitution of lysine 11 with leucine, which is non-polar, increased the K _m for ribulose-1,5-bisphosphate from 82 to 190 M but its replacement with glutamine, which has polar properties, had no appreciable effect.Abbreviations Rubisco ribulose-1,5-bisphosphate carboxylase/oxygenase - RuBP ribulose-1,5-bisphosphate - LSU large sub-unit of Rubisco - SSU small subunit of Rubisco We thank Dr. S. Gutteridge (DuPont, Wilmington, USA) for structural information and for his comments on the results described. The technical assistance of Mr. A. Cowland and Mr. I. Major was invaluable.     

Solubilization of ribulose-1,5-bisphosphate carboxylase from the membrane fraction of pea leaves

The solubilization of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) from the membrane fraction was studied in whole leaf extracts and chloroplasts from pea. The amount of membrane-bound Rubisco was dependent on the pH of the chloroplastic lysate buffer. Maximum binding was found at pH 8.0, with about 8% of total leaf Rubisco being bound. The binding of Rubisco to the membranes was strong, and it was not released by repeated washing with hypotonic buffer or by changing ionic strength. Detergents such as Triton X-100, Tween 20, deoxycholate and dodecylsulfate were effective in solubilizing the membrane-bound Rubisco. Triton X-100 was most effective in the range of 0.04% to 0.2% and it solubilized Rubisco from the membrane without any decrease in enzyme activity.Abbreviations BSA bovine serum albumin - CABP carboxyarabinitol-1,5-bisphosphate - DTT dithiothreitol - LDS lithium dodecylsulfate - LHC light-harvesting chlorophyll protein complex - RuBP ribulose-1,5-bisphosphate - Rubisco RuBP carboxylase/oxygenase - SDS sodium dodecylsulfate - SDS-PAGE SDS-polyacrylamide gel electrophoresis     

Purification and characterization of maize ribulose-1,5-bisphosphate carboxylase

The ribulose-1,5-bisphosphate carboxylase/oxygenase purified from maize (a C₄ monocot) to homogeneity has a MW of532 000 and sedimentation coeffici     

Effects of ectopically expressed hyperthermophilic archaeon (Pyrococcus furiosus) ribulose-1,5-bisphosphate carboxylase/oxygenase on tobacco photosynthesis

Pyrococcus furiosus is a hyperthermophilic archaeon. Its ribulose-1,5-bisphosphate carboxylase/oxygenase (PfRubisco) has only large subunit (L). PfRubisco has a novel (L₂)₅, decameric structure and it possesses higher carboxylase activity and thermotolerance. To assess the potential functionality of PfRubisco in higher plants under high-temperature stress, PfRubisco coding sequence was transiently expressed in Nicotiana benthamiana by Pea early browning virus mediated ectopic expression. The transgenic PfRubisco plants produced chlorotic yellow stripes in their leaves. Relative to the control leaves, those with yellow stripes exhibited decreased net photosynthetic rate and chlorophyll content, altered chloroplast ultrastructure, and more severe photoinhibition of both photosystem I and II. We concluded that the ectopic expression of PfRubisco might disrupt the chloroplast development and function in N. benthamiana. The potential cause of the disruption was discussed.     

Photosynthesis, leaf resistances, and ribulose-1,5-bisphosphate carboxylase degradation in senescing barley leaves

The relationship between loss of ribulose-1,5-bisphosphate carboxylase (RuBPCase) and the decline in photosynthesis during the senescence of barley primary leaves was assessed. Loss of RuBPCase accounted for about 85% of the decrease in soluble protein. RuBPCase was highly correlated with in vitro RuBPCase activity (r = 0.95) and gross photosynthesis (r = 0.96). However, the rate of photosynthesis per milligram RuBPCase increased during the early stages of leaf senescence. The concentration of nonreducing sugars was negatively correlated (1% level) with photosynthesis. Free α-amino N, in contrast to nonreducing sugars, declined markedly during senescence. A decrease in chlorophyll and an increase in in vitro protease activity was observed, but these changes did not appear to be closely related to the decline in photosynthesis and RuBPCase. Mesophyll resistance increased at the same rate that photosynthesis and RuBPCase declined. Stomatal resistance increased more rapidly than mesophyll resistance and accounted for about 24% of the total increase in resistance to CO₂ diffusion. The concentration of CO₂ in the intercellular air spaces decreased during the last stage of senescence. Although loss of RuBPCase probably is the primary event responsible for the decline in photosynthesis during leaf senescence, other factors such as in vivo regulation and stomatal aperture must also be considered.     

The kinetics of ribulose-1,5-bisphosphate carboxylase/oxygenase in vivo inferred from measurements of photosynthesis in leaves of transgenic tobacco

Transgenic tobacco (Nicotiana tabacum L. cv. W38) with an antisense gene directed against the mRNA of the ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) small subunit was used to determine the kinetic properties of Rubisco in vivo. The leaves of these plants contained only 34% as much Rubisco as those of the wild type, but other photosynthetic components were not significantly affected. Consequently, the rate of CO₂ assimilation by the antisense plants was limited by Rubisco activity over a wide range of CO₂ partial pressures. Unlike in the wild-type leaves, where the rate of regeneration of ribulose bisphosphate limited CO₂ assimilation at intercellular partial pressures above 400 ubar, photosynthesis in the leaves of the antisense plants responded hyperbolically to CO₂, allowing the kinetic parameters of Rubisco in vivo to be inferred. We calculated a maximal catalytic turnover rate, k_cat, of 3.5+0.2 mol CO₂·(mol sites)^–1·s^–1 at 25° C in vivo. By comparison, we measured a value of 2.9 mol CO₂·(mol sites)^–1·^–1 in vitro with leaf extracts. To estimate the Michaelis-Menten constants for CO₂ and O₂, the rate of CO₂ assimilation was measured at 25° C at different intercellular partial pressures of CO₂ and O₂. These measurements were combined with carbon-isotope analysis (¹³C/¹²C) of CO₂ in the air passing over the leaf to estimate the conductance for transfer of CO₂ from the substomatal cavities to the sites of carboxylation (0.3 mol·m^–2·s^–1·bar^–1) and thus the partial pressure of CO₂ at the sites of carboxylation. The calculated Michaelis-Menten constants for CO₂ and O₂ were 259 ±57 bar (8.6±1.9M) and 179 mbar (226 M), respectively, and the effective Michaelis-Menten constant for CO₂ in 200 mbar O₂ was 549 bar (18.3 M). From measurements of the photocompensation point (_* = 38.6 ubar) we estimated Rubisco's relative specificity for CO₂, as opposed to O₂ to be 97.5 in vivo. These values were dependent on the size of the estimated CO₂-transfer conductance.Abbreviations and Symbols A CO₂-assimilation rate - g_w conductance for CO₂ transfer from the substomatal cavities to the sites of carboxylation - K_c, K_o Michaelis-Menten constants for carboxylation, oxygenation of Rubisco - k_cat V_cmax/[active site] - O partial pressure of O₂ at the site of carboxylation - p_c partial pressure of CO₂ at the site of carboxylation - p_i intercellular CO₂ partial pressure - R_d day respiration (non-photorespiratory CO₂ evolution) - Rubisco ribulose 1,5-bisphosphate carboxylase/oxygenase - RuBP ribulose-1,5-bisphosphate - S_c/o relative specificity factor for Rubisco - SSu small subunit of Rubisco - V_cmax, V_omax maximum rates of Rubisco carboxylation, oxygenation - _* partial pressure of CO₂ in the chloroplast at which photorespiratory CO₂ evolution equals the rate of carboxylation     

Biosynthesis of ribulose-1,5-bisphosphate carboxylase in spinach leaf protoplasts

Spinach leaf (Spinacia oleracea L. var. Kyoho) protoplasts sustain protein-synthesizing activity as measured by the incorporation of [¹⁴C]-leucine into the protein fraction both in the light and in the dark. By the immunoprecipitation of ribulose-1,5-bisphosphate (RuP₂) carboxylase with rabbit antibody raised against the purified spinach enzyme preparation, it was found that approximately 7% of the total radiocarbon incorporated into the protein fraction in the light was in the carboxylase molecules. However, there was no measurable net increase observed in the content of the enzyme protein in the experimental conditions employed. It was found that both chloramphenicol and cycloheximide inhibited the incorporation of [¹⁴C]leucine into RuP₂ carboxylase and its constituent subunits, as measured by the immunoprecipitation of the enzyme molecule and its subunits, A and B.     

Degradation of ribulose-1,5-bisphosphate carboxylase by proteolytic enzymes from crude extracts of wheat leaves

In crude extracts from the primary leaf of wheat seedlings, Triticum aestivum L., cv. Olympic, maximum proteinase activity, as determined by measuring the rate of release of amino nitrogen from ribulose-bisphosphate carboxylase (RuBPCase), was found to be obtained only when EDTA and L-cysteine were included in the extraction buffer. Highest proteinase activity was obtained by grinding at pH 6.8, although the level of activity was similar in the pH range 5.6 to 8.0; this range also coincided with maximum extractability of protein. The lower amount of RuBPCase degrading proteinase extracted at low pH was not due to an effect of pH on enzyme stability. The optimum temperature of reaction was 50° C and reaction rates were linear for at least 120 min at this temperature. In the absence of substrate the proteinase was found to be very sensitive to temperatures above 30° C, with even short exposures causing rapid loss of activity. The relation between assay pH and RuBPCase degradation indicated that degradation was restricted to the acid proteinase group of enzymes, with a pH optimum of 4.8, and no detectable activity at a pH greater than 6.4. The levels of extractable RuBPCase proteinase exhibited a distinct diurnal variation, with activity increasing during the latter part of the light period and then declining once the lights were turned off. The effect of leaf age on the level of RuBPCase, RuBPCase proteinase and total soluble protein was investigated. Maximum RuBPCase activity occurred 9 days after sowing as did soluble protein. After the maximum level was obtained, the pattern of total soluble protein was shown to be characterised by three distinct periods of protein loss: I (day 9–13) 125 ng leaf^-1 day^-1; II (day 15–27) 11 ng leaf^-1 day^-1; III (day 29–49) 22 ng leaf^-1 day^-1. Comparison of the pattern of RuBPCase activity and total protein suggest that the loss of RuBPCase may be largely responsible for the high rate of protein loss during period I. Proteinase activity increased sharply during the period of most rapid loss of RuBPCase activity, and because the specific activity of RuBPCase also declined, we concluded that RuBPCase was being degraded more rapidly than the other proteins. Once the majority of the RuBPCase was lost, there did not appear to be a direct relation between RuBPCase proteinase activity and rate of total soluble protein loss, since the proteinase exhibited maximum activity during the slowest period of protein loss (II), and was declining in activity while the rate of protein loss remained stable during the third and final period of total protein loss.Abbreviations RuBPCase ribulose-1,5-bisphosphate carboxylase (EC 4.1.1.39) - TCA trichloroacetic acid Supported by the Wheat Industry Research Council of Australia and the Australian Research Grants Committee D2 74/15052     

Electron microscopy of the complexes of ribulose-1,5-bisphosphate carboxylase (Rubisco) and Rubisco subunit-binding protein from pea leaves

The structure of ribulose-1,5-bisphosphate carboxylase (Rubisco) subunit-binding protein and its interaction with pea leaf chloroplast Rubisco were studied by electron microscopy and image analysis. Electron-microscopic evidence for the association of Rubisco subunit-binding protein, consisting of 14 subunits arranged with 72 point group symmetry, and oligomeric (L8S8) Rubisco was obtained.     

Activities of chlorophyllase, phosphoenolpyruvate carboxylase and ribulose-1,5-bisphosphate carboxylase in the primary leaves of soybean during senescence and drought

The effects of senescence and drought on the levels and activities of chlorophyllase (EC 3.1.1.14), phosphoenolpyruvate carboxylase (PEPC, EC 4.1.1.31) and ribulose-1,5-bisphosphate carboxylase (Rubisco, EC 4.1.1.39) in the intact primary leaves of soybean ( Glycine max L. cv. Jackson) were monitored. Plants were grown either (1) for 2 to 8 weeks and the primary leaves harvested every week or (2) for 2 weeks and the plants subjected to drought stress and compared to control plants that were watered daily. In the senescence experiment, chlorophyllase activity changed in parallel with water content, leaf chlorophyll and total protein per unit dry weight of leaf tissue, with all factors increasing in concert during expansion of the primary leaves in the first 4 to 5 weeks of seedling development. Thereafter, all factors, including chlorophyllase activity, declined reaching markedly reduced values at weeks 7 and 8 when the primary leaves were yellow and ready to abscise. PEPC and Rubisco activities peaked in the third week, i.e. well before full leaf expansion, and then declined. In contrast to its response during senescence, chlorophyllase activity per unit leaf dry weight did not change during drought stress, but the specific activity of the enzyme rose and showed an inverse relationship to total leaf chlorophyll and protein content. Rubisco activity was highly sensitive to drought, with decrements observed in the activity and in levels of the large subunit within 2 days of withholding water and before significant changes in leaf water content were detected.     

The ribulose-1,5-bisphosphate carboxylase/oxygenase gene cluster of Methylococcus capsulatus (Bath)

The genes encoding the ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) from Methylococcus capsulatus (Bath) were localised to an 8.3-kb EcoRI fragment of the genome. Genes encoding the large subunit ( cbbL), small subunit ( cbbS) and putative regulatory gene ( cbbQ) were shown to be located on one cluster. Surprisingly, cbbO, a second putative regulatory gene, was not located in the remaining 1.2-kb downstream (3') of cbbQ. However, probing of the M. capsulatus (Bath) genome with cbbO from Nitrosomonas europaea demonstrated that a cbbO homologue was contained within a separate 3.0-kb EcoRI fragment. Instead of a cbbR ORF being located upstream (5') of cbbL, there was a moxR-like ORF that was transcribed in the opposite direction to cbbL. There were three additional ORFs within the large 8.3-kb EcoRI fragment: a pyrE-like ORF, an rnr-like ORF and an incomplete ORF with no sequence similarity to any known protein. Phylogenetic analysis of cbbL from M. capsulatus (Bath) placed it within clade A of the green-type Form 1 Rubisco. cbbL was expressed in M. capsulatus (Bath) when grown with methane as a sole carbon and energy source under both copper-replete and copper-limited conditions. M. capsulatus (Bath) was capable of autotrophic growth on solid medium but not in liquid medium. Preliminarily investigations suggested that other methanotrophs may also be capable of autotrophic growth. Rubisco genes were also identified, by PCR, in Methylococcus-like strains and Methylocaldum species; however, no Rubisco genes were found in Methylomicrobium album BG8, Methylomonas methanica S1, Methylomonas rubra, Methylosinus trichosporium OB3b or Methylocystis parvus OBBP.