A biochemical model of photosynthetic CO2 assimilation in leaves of C3 species

Various aspects of the biochemistry of photosynthetic carbon assimilation in C₃ plants are integrated into a form compatible with studies of gas exchange in leaves. These aspects include the kinetic properties of ribulose bisphosphate carboxylase-oxygenase; the requirements of the photosynthetic carbon reduction and photorespiratory carbon oxidation cycles for reduced pyridine nucleotides; the dependence of electron transport on photon flux and the presence of a temperature dependent upper limit to electron transport. The measurements of gas exchange with which the model outputs may be compared include those of the temperature and partial pressure of CO₂(p(CO₂)) dependencies of quantum yield, the variation of compensation point with temperature and partial pressure of O₂(p(O₂)), the dependence of net CO₂ assimilation rate on p(CO₂) and irradiance, and the influence of p(CO₂) and irradiance on the temperature dependence of assimilation rate.Abbreviations RuP₂ ribulose bisphosphate - PGA 3-phosphoglycerate - C=p(CO₂) partial pressure of CO₂ - O=p(O₂) partial pressure of O₂ - PCR photosynthetic carbon reduction - PCO photorespiratory carbon oxidation     

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     

Induction and regulation of ribulose bisphosphate carboxylase activity in Rhizobium japonicum during formate-dependent growth

Rhizobium japonicum CJ1 was capable of growing using formate as the sole source of carbon and energy. During aerobic growth on formate a cytoplasmic NAD⁺-dependent formate dehydrogenase and ribulose bisphosphate carboxylase activity was demonstrated in cell-free extracts, but hydrogenase enzyme activity could not be detected. Under microaerobic growth conditions either formate or hydrogen metabolism could separately or together support ribulose bisphosphate carboxylase-dependent CO₂ fixation. A number of R. japonicum strains defective in hydrogen uptake activity were shown to metabolise formate and induce ribulose bisphosphate carboxylase activity. The induction and regulation of ribulose bisphosphate carboxylase is discussed.Abbreviations hup hydrogen uptake - MOPS 3-(N-morpholino)-propanesulphonate - TSA tryptone soya agar - RuBP ribulose 1,5-bisphosphate - FDH formate dehydrogenase     

Photorespiration-induced reduction of ribulose bisphosphate carboxylase activation level

Leaf photosynthesis and ribulose bisphosphate carboxylase activation level were inhibited in several mutants of the C₃ crucifer Arabidopsis thaliana which possess lesions in the photorespiratory pathway. This inhibition occurred when leaves were illuminated under a photorespiratory atmosphere (50% O₂, 350 microliters per liter CO₂, balance N₂), but not in nonphotorespiratory conditions (2% O₂, 350 microliters per liter CO₂, balance N₂). Inhibition of carboxylase activation level was observed in strains with deficient glycine decarboxylase, serine transhydroxymethylase, serine-glyoxylate aminotransferase, glutamate synthase, and chloroplast dicarboxylate transport activities, but inhibition did not occur in a glycolate-P phosphatase-deficient strain. Also, the photorespiration inhibitor aminoacetonitrile produced a decline in leaf and protoplast ribulose bisphosphate carboxylase activation level, but was without effect on intact chloroplasts. Fructose bisphosphatase, a light-activated enzyme which is strongly dependent on stromal pH and Mg²⁺ for regulation, was unaffected by conditions which caused inhibition of ribulose bisphosphate carboxylase. Thus, the mechanism of inhibition does not appear to involve changes in stromal Mg²⁺ and pH but rather is associated with metabolite flux through the photorespiratory pathway.     

Ribulose bisphosphate carboxylase activity in halophilic Archaebacteria

Among the several strains of halobacteria grown heterotrophically, ribulose bisphosphate carboxylase activity was detected in those which accumulate poly (-hydroxybutyrate), viz. Haloferax mediterranei, Haloferax volcanii and Halobacterium marismortui. In H. mediterranei, the activity was present in cell extracts prepared after growth on a variety of carbohydrates. The ribulose bisphosphate carboxylase activity in H. mediterranei was inhibited by carboxyarabinitol bisphosphate, and the enzyme cross-reacted with antibodies raised against the spinach enzyme. CO₂ fixation by cell extract was stimulated by the addition of ATP and NADH. Preliminary data suggested that hydrogen could be a possible reductant.Abbreviations RuBP ribulose bisphosphate - Ru5P ribulose 5-phosphate - R5P ribose 5-phosphate - CABP carboxyarabinitol bisphosphate - PHB poly (-hydroxybutyrate) - DTT dithiothreitol     

The Regulation of Photosynthesis in Leaves of Field-Grown Spring Wheat (Triticum aestivum L., cv Albis) at Different Levels of Ozone in Ambient Air

Wheat (Triticum aestivum L. cv Albis) was grown in open-top chambers in the field and fumigated daily with charcoal-filtered air (0.015 microliters per liter O₃), nonfiltered air (0.03 microliters per liter O₃), and air enriched with either 0.07 or 0.10 microliters per liter ozone (seasonal 8 hour/day [9 am-5 pm] mean ozone concentration from June 1 until July 10, 1987). Photosynthetic ¹⁴CO₂ uptake was measured in situ. Net photosynthesis, dark respiration, and CO₂ compensation concentration at 2 and 21% O₂ were measured in the laboratory. Leaf segments were freeze-clamped in situ for the determination of the steady state levels of ribulose 1,5-bisphosphate, 3-phosphoglycerate, triose-phosphate, ATP, ADP, AMP, and activity of ribulose, 1,5-bisphosphate carboxylase/oxygenase. Photosynthesis of flag leaves was highest in filtered air and decreased in response to increasing mean ozone concentration. CO₂ compensation concentration and the ratio of dark respiration to net photosynthesis increased with ozone concentration. The decrease in photosynthesis was associated with a decrease in chlorophyll, soluble protein, ribulose bisphosphate carboxylase/oxygenase activity, ribulose bisphosphate, and adenylates. No decrease was found for triose-phosphate and 3-phosphoglycerate. The ratio of ATP to ADP and of triosephosphate to 3-phosphoglycerate were increased suggesting that photosynthesis was limited by pentose phosphate reductive cycle activity. No limitation occurred due to decreased access of CO₂ to photosynthetic cells since the decrease in stomatal conductance with increasing ozone concentration did not account for the decrease in photosynthesis. Ozonestressed leaves showed an increased degree of activation of ribulose bisphosphate carboxylase/oxygenase and a decreased ratio of ribulose bisphosphate to initial activity of ribulose bisphosphate carboxylase/oxygenase. Nevertheless, it is suggested that photosynthesis in ozone stressed leaves is limited by ribulose bisphosphate carboxylation possibly due to an effect of ozone on the catalysis by ribulose bisphosphate carboxylase/oxygenase.     

Specific reduction of chloroplast glyceraldehyde-3-phosphate dehydrogenase activity by antisense RNA reduces CO2 assimilation via a reduction in ribulose bisphosphate regeneration in transgenic tobacco plants

The reduction of 3-phosphoglycerate (PGA) to triose phosphate is a key step in photosynthesis linking the photochemical events of the thylakoid membranes with the carbon metabolism of the photosynthetic carbon-reduction (PCR) cycle in the stroma. Glyceraldehyde-3-phosphate dehydrogenase: NADP oxidoreductase (GAPDH) is one of the two chloroplast enzymes which catalyse this reversible conversion. We report on the engineering of an antisense RNA construct directed against the tobacco (Nicotiana tabacum L.) chloroplastlocated GAPDH (A subunit). The construct was integrated into the tobacco genome by Agrobacterium-mediated transformation of leaf discs. Of the resulting transformants, five plants were recovered with reduced GAPDH activities ranging from 11 to 24% of wild-type (WT) activities. Segregation analysis of the kanamycin-resistance character in self-pollinated T₁ seed from each of the five transformants revealed that one plant (GAP-R) had two active DNA inserts and the others had one insert. T₁ progeny from GAP-R was used to generate plants with GAPDH activities ranging from WT levels to around 7% of WT levels. These were used to study the effect of variable GAPDH activities on metabolite pools for ribulose1,5-bisphosphate (RuBP) and PGA, and the accompanying effects on the rate of CO₂ assimilation and other gasexchange parameters. The RuBP pool size was linearly related to GAPDH activity once GAPDH activity dropped below the range for WT plants, but the rate of CO₂ assimilation was not affected until RuBP levels dropped to 30–40% of WT levels. That is, the CO₂ assimilation rate fell when RuBP per ribulose-1,5-biphosphate carboxylase-oxygenase (Rubisco) site fell below 2 mol·(mol site)^–1 while the ratio for WT plants was 4–5 mol·m(mol site)^–1. Leaf conductance was not reduced in leaves with reduced GAPDH activities, resulting in an increase in the ratio of intercellular to ambient CO₂ partial pressure. Conductance in plants with reduced GAPDH activities was still sensitive to CO₂ and showed a normal decline with increases in CO₂ partial pressure. Although PGA levels did not fluctuate greatly, the effect of reduced GAPDH activity on RuBP-pool size and assimilation rate can be interpreted as being due to a blockage in the regeneration of RuBP. Concomitant gas-ex change and chlorophyll a fluorescence measurements indicated that photosynthesis changed from being Rubisco-limited to being RuBP-regeneration-limited at a lower CO₂ partial pressure in the antisense plants than in WT plants. Photosynthetic electron transport was down-regulated by the build-up of a large proton gradient and the electron-transport chain did not become over-reduced due to a shortage of NADP. Plants with severely reduced GAPDH activity were not photoinhibited despite the continuous presence of a large thylakoid proton gradient in the light. Along with plant size, Rubisco activity, leaf soluble protein and chlorophyll content were reduced in plants with the lowest GAPDH activities. We conclude that chloroplastic GAPDH activity does not appear to limit steady-state photosynthetic CO₂ assimilation at ambient CO₂. This is because WT leaves maintain the ratio of RuBP per Rubisco site about twofold higher than the level required to achieve a maximal rate of CO₂ assimilation.Abbreviations and Symbols bp base pairs - DHAP dihydroxy-acetone phosphate - GAPDH glyceraldehyde-3-phosphate dehy-drogenase - PCR photosynthetic carbon reduction - PGA 3-phosphoglycerate - p_i intercellular CO₂ partial pressure - q_NP non-photochemical fluorescence quenching - q_Q photochemicalfluorescence quenching - PSII quantum efficiency of electronflow through PSII - Rubisco ribulose-1,5-bisphosphate carboxy-lase-oxygenase - RuBP ribulose-1,5-bisphosphate - WT wild type We thank Karin Harrison, Prue Kell, Anne Gallagher and Barbara Setchell for excellent technical assistance. G.D.P. and S.V.C. acknowledge support from QE II Research Fellowships (Australian Research Council).     

Construction of a Synechocystic PCC6803 mutant suitable for the study of variant hexadecameric ribulose bisphosphate carboxylase/oxygenase enzymes

The cyanobacterium Synechocystis PCC6803 was chosen as a target organism for construction of a suitable photosynthetic host to enable selection of variant plant-like ribulose bisphosphate carboxylase/oxygenase (Rubisco) enzymes. The DNA region containing the operon encoding Rubisco (rbc) was cloned, sequenced and used for the construction of a transformation vector bearing flanking sequences to the rbc genes. This vector was utilized for the construction of a cyanobacterial rbc null mutant in which the entire sequence comprising both rbc genes, was replaced by the Rhodospirillum rubrum rbcL gene linked to a chloramphenicol resistance gene. Chloramphenicol-resistant colonies, Syn6803rbc, were detected within 8 days when grown under 5% CO₂ in air. These transformants were unable to grow in air (0.03% CO₂). Analysis of their genome and Rubisco protein confirmed the site of the mutation at the rbc locus, and indicated that the mutation had segregated throughout all of the chromosome copies, consequently producing only the bacterial type of the enzyme. In addition, no carboxysome structures could be detected in the new mutant. Successful restoration of the wild-type rbc locus, using vectors bearing the rbc operon flanked by additional sequences at both termini, could only be achieved upon incubating the transformed cells under 5% CO₂ in air prior to their transferring to air. The yield of restored transformants was proportionally related to the length of those sequences flanking the rbc operon which participate in the homologous recombination. The Syn6803rbc mutant is amenable for the introduction of in vitro mutagenized rbc genes into the rbc locus, aiming at the genetic modification of the hexadecameric type Rubisco.Abbreviations Cm^r chloramphenicol resistance - Km^r kanamycin resistance - HCR high CO₂ requirer - Rubisco ribulose 1,5-bisphosphate carboxylase/oxygenase - SSC sodium chloride and sodium citrate - wt wild-type     

Prospects for manipulating the substrate specificity of ribulose bisphosphate carboxylase/oxygenase

Pierce, J. 1988. Prospects for manipulating the substrate specificity of ribulose bisphosphate carboxylase/oxygenase. - Physiol. Plant. 72: 690–698.
The idea of enhancing plant productivity by minimizing the apparently wasteful process of photorespiration has been an enduring one. Since the relative fluxes of carbon through the competing pathways of photosynthesis and photorespiration are determined by the kinetic properties of a single enzyme, ribulose bisphosphate carboxylase/oxygenase, it has been conjectured that genetic modification of this protein could provide more productive plants. Recent advances in techniques for studying ribulose bisphosphate carboxylase/oxygenase hold promise for determining whether such modifications will prove possible.     

Ribulose bisphosphate carboxylase/oxygenase content determined with [C]carboxypentitol bisphosphate in plants and algae

As is the case with spinach ribulose bisphosphate carboxylase/oxygenase (Rubisco), [¹⁴C]carboxyarabinitol bisphosphate (CABP) bound to purified Chlorella Rubisco with a molar ratio of unity to large subunit of the enzyme. The concentration of binding sites in extracts of photosynthetic organisms was determined by reacting the extracts with [¹⁴C]-carboxypentitol bisphosphate (CPBP) and precipitating the resultant Rubisco-[¹⁴C]CABP complex with a combination of polyethylene glycol-4000 and MgCl₂. Plots of the relationship between concentrations of [¹⁴C] CPBP in the reaction mixture and the precipitated [¹⁴C]CPBP gave a straight line and the concentration of binding sites were estimated by extrapolation to zero [¹⁴C]CPBP since the dissociation constant of CABP with Rubisco is 10⁻¹¹ molar. Spinach, pea, and soybean leaves contained 6.4 to 6.8 milligrams Rubisco per milligram chlorophyll, corresponding to 92 to 97 ribulose bisphosphate-binding sites per milligram chlorophyll. The Rubisco content of sunflower and wheat leaves was 5.3 to 5.5 milligrams per milligram chlorophyll. The concentrations in C₄ plants were not uniform and corn and Panicum miliaceum leaves contained 3 and 7 milligrams Rubisco per milligram chlorophyll. The Rubisco content of green algae was one-fifth to one-sixth that of C₃ plant leaves and was affected by the CO₂ concentration during growth. The content of Euglena and blue-green algae is also reported.     

Protection and enhancement of ribulose 1,5 bisphosphate carboxylase activity by exogenous proteins

When assayedin vitro, the activity of the photosynthetic enzyme ribulose 1,5 bisphosphate carboxylase/oxygenase is both enhanced and protected from spontaneous decay by exogenous proteins such as hemoglobin, serum albumin, and aldolase. Other proteins and amino acids tested are either ineffective (lysozyme, ferritin, lysine, and cysteine) or afford only partial protection (catalase, glycine, and phenylalanine). Protective proteins do not bind to, or exchange disulfides with, ribulose 1,5 bisphosphate carboxylase/oxygenase. Since their effect can be mimicked by reductively treated detergents such as Triton X-100, it appears that proteins protect from decay by quenching the spontaneous oxidative degradation and inhibiting surface adsorption which could lead to enzyme unfolding. Release of adsorbed molecules from the container surface is likely to be the cause of carboxylase activity enhancement.     

A facile method to determine the CO2/O2 specificity factor for ribulose bisphosphate carboxylase/oxygenase

A rapid method to determine the CO₂/O₂ specificity factor of ribulose 1,5-bisphosphate carboxylase/oxygenase is presented. The assay measures the amount of CO₂ and O₂ fixation at varying CO₂/O₂ ratios to determine the relative rates of each reaction. CO₂ fixation is measured by the incorporation of the moles of¹⁴CO₂ into 3-phosphoglycerate, while O₂ fixation is determined by subtraction of the moles of CO₂ fixed from the moles of RuBP consumed in each reaction. By analyzing the inorganic phosphate specifically hydrolyzed from RuBP under alkaline conditions, the amount of RuBP present before and after catalysis by rubisco can be determined.     

Optimal acclimation of the C3 photosynthetic system under enhanced CO2

A range of studies of C₃ plants have shown that there is a change in both the carbon flux and the pattern of nitrogen allocation when plants are grown under enhanced CO₂. This paper examines evidence that allocation of nitrogen both to and within the photosynthetic system is optimised with respect to the carbon flux. A model is developed which predicts the optimal relative allocation of nitrogen to key enzymes of the photosynthetic system as a function of CO₂ concentration. It is shown that evidence from flux control analysis is broadly consistent with this model, although at high nitrogen and under certain conditions at low nitrogen experimental data are not consistent with the model. Acclimation to enhanced CO₂ is also assessed in terms of resource allocation between photosynthate sources and sinks. A means of assessing the optimisation of this source-sink allocation is proposed, and several studies are examined within this framework. It is concluded that C₃ plants probably possess the genetic feedback mechanisms required to efficiently smooth out any imbalance within the photosynthetic system caused by a rise in atmospheric CO₂.Abbreviations A net rate of CO₂ assimilation - c _i intercellular CO₂ concentration - C_R ^A flux control coefficient for Rubisco with respect to flux A - FBPase fructose 1,6-bisphosphatase - k_app apparent catalytic rate constant - PCO photorespiratory carbon oxidation - PCR photosynthetic carbon reduction - PPFD photosynthetically active photon flux density - Rubisco ribulose 1,5-bisphosphate carboxylase/oxygenase - RuBP ribulose 1,5-bisphosphate - Ru5P ribulose 5-phosphate - SBPase sedoheptulose 1,7-bisphosphatase     

Effect of dissolved inorganic carbon on the expression of carboxysomes,localization of Rubisco and the mode of inorganic carbon transport in cells of the cyanobacterium Synechococcus UTEX 625

In the cyanobacterium Synechococcus UTEX 625, the extent of expression of carboxysomes appeared dependent on the level of inorganic carbon (CO₂+HCO _inf3 ^sup- ) in the growth medium. In cells grown under 5% CO₂ and in those bubbled with air, carboxysomes were present in low numbers (<2 · longitudinal section^-1) and were distributed in an apparently random manner throughout the centroplasm. In contrast, cells grown in standing culture and those bubbled with 30 l CO₂ · 1^-1 possessed many carboxysomes (>8 · longitudinal section^-1). Moreover, carboxysomes in these cells were usually positioned near the cell periphery, aligned along the interface between the centroplasm and the photosynthetic thylakoids. This arrangement of carboxysomes coincided with the full induction of the HCO _inf3 ^sup- transport system that is involved in concentrating inorganic carbon within the cells for subsequent use in photosynthesis. Immunolocalization studies indicate that the Calvin cycle enzyme ribulose bisphosphate carboxylase was predominantly carboxysome-localized, regardless of the inorganic carbon concentration of the growth medium, while phosphoribulokinase was confined to the thylakoid region. It is postulated that the peripheral arrangement of carboxysomes may provide for more efficient photosynthetic utilization of the internal inorganic carbon pool in cells from cultures where carbon resources are limiting.Abbreviations Chl chlorophyll - DIC dissolved inorganic carbon (CO₂+HCO _inf3 ^sup- +CO _inf3 ^sup2- ) - PRK phosphoribulokinase - RuBP ribulose 1,5-bisphosphate - Rubisco LS large subunit of ribulose 1,5-bisphosphate carboxylase/oxygenase     

Immunological evidence for the presence of ribulose bisphosphate carboxylase in guard cell chloroplasts

Ribulose bisphosphate carboxylase (Rubisco) has been found in Vicia faba L. guard cell chloroplasts by two immunological methods, using antibodies raised against highly purified subunits of ribulose bisphosphate carboxylase. Indirect cytoimmunofluorescence revealed binding of antibodies against both the small and the large subunits of ribulose bisphosphate carboxylase. Binding was observed only after partial digestion of guard cell walls by 4% Cellulysin to facilitate antibody penetration. After electrophoresis of a homogenate of guard cell protoplasts, the presence of both subunits was also revealed by immunolabeling technique. Positive response required the inhibition of proteolysis which appeared to be active upon homogenization.     

Some relationships between the biochemistry of photosynthesis and the gas exchange of leaves

A series of experiments is presented investigating short term and long term changes of the nature of the response of rate of CO₂ assimilation to intercellular p(CO₂). The relationships between CO₂ assimilation rate and biochemical components of leaf photosynthesis, such as ribulose-bisphosphate (RuP₂) carboxylase-oxygenase activity and electron transport capacity are examined and related to current theory of CO₂ assimilation in leaves of C₃ species. It was found that the response of the rate of CO₂ assimilation to irradiance, partial pressure of O₂, p(O₂), and temperature was different at low and high intercellular p(CO₂), suggesting that CO₂ assimilation rate is governed by different processes at low and high intercellular p(CO₂). In longer term changes in CO₂ assimilation rate, induced by different growth conditions, the initial slope of the response of CO₂ assimilation rate to intercellular p(CO₂) could be correlated to in vitro measurements of RuP₂ carboxylase activity. Also, CO₂ assimilation rate at high p(CO₂) could be correlated to in vitro measurements of electron transport rate. These results are consistent with the hypothesis that CO₂ assimilation rate is limited by the RuP₂ saturated rate of the RuP₂ carboxylase-oxygenase at low intercellular p(CO₂) and by the rate allowed by RuP₂ regeneration capacity at high intercellular p(CO₂).     

A sensitive,simultaneous analysis of ribulose 1,5-bisphosphate carboxylase/oxygenase efficiencies: Graphical determination of the CO2/O2 specificity factor

A simple approach to determine CO₂/O₂ specificity factor () of ribulose 1,5-bisphosphate carboxylase/oxygenase is described. The assay measures the amount of CO₂ fixation at varying [CO₂]/[O₂] ratios after complete consumption of ribulose 1,5-bisphosphate (RuBP). Carbon dioxide fixation catalyzed by the carboxylase was monitored by directly measuring the moles of ¹⁴CO₂ incorporated into 3-phosphoglycerate (PGA). This measurement at different [CO₂]/[O₂] ratios is used to determine graphically by several different linear plots the total RuBP consumed by the two activities and the CO₂/O₂ specificity factor. The assay can be used to measure the amounts of products of the carboxylase and oxygenase reactions and to determine the concentration of the substrate RuBP converted to an endpoint amount of PGA and phosphoglycolate. The assay was found to be suitable for all [CO₂]/[O₂] ratios examined, ranging from 14 to 215 micromolar CO₂ (provided as 1–16 mM NaHCO₃) and 614 micromolar O₂ provided as 50% O₂. The procedure described is extremely rapid and sensitive. Specificity factors for enzymes of highly divergent values are in good agreement with previously published data.Abbreviations HEPPS N-(2-hydroxyethyl)piperazine-N-(3-propanesulfonic acid) - L large subunit of rubisco - PGA 3-phosphoglyceric acid - rubisco ribulose 1,5-bisphosphate carboxylase/oxygenase - RuBP d-ribulose 1,5-bisphosphate - S small subunit of rubisco - XuBP d-xylulose 1,5-bisphosphate     

Nitrogen and Photosynthesis in the Flag Leaf of Wheat (Triticum aestivum L.)

Wheat (Triticum aestivum L. cv Yecora 70) plants were grown with various concentrations of nitrate nitrogen available to the roots. Sampling of flag leaves began after they had reached full expansion and continued throughout senescence. Rates of gas exchange, ribulose-1,5-bisphosphate (RuP₂) carboxylase activity, and the amounts of chlorophyll, soluble protein, nitrogen, and phosphorus were determined for each flag leaf. Rate of CO₂ assimilation was uniquely related to total leaf nitrogen irrespective of nutrient treatment, season, and leaf age. Assimilation rate increased with leaf nitrogen, but the slope of the relationship declined markedly when leaf nitrogen exceeded 125 millimoles nitrogen per square meter. Chlorophyll content and RuP₂ carboxylase activity were approximately proportional to leaf nitrogen content. As leaves aged, RuP₂ carboxylase activity and calculated Hill activity declined in parallel. With normal ambient partial pressure of CO₂, the intercellular partial pressure of CO₂ was always such that rate of assimilation appeared colimited by RuP₂ carboxylation and RuP₂ regeneration capacity.

The initial slope of rate of CO₂ assimilation against intercellular partial pressure of CO₂ varied nonlinearly with carboxylase activity. It is suggested that this was due to a finite conductance to CO₂ diffusion in the wall and liquid phase which causes a drop in CO₂ partial pressure between the intercellular spaces and the site of carboxylation. A double reciprocal plot was used to obtain an estimate of the transfer conductance.

     

Activation of Rubisco controls CO<Subscript>2</Subscript> assimilation in light: a perspective on its discovery

CO₂ fixation during photosynthesis is regulated by the activity of ribulose bisphosphate carboxylase (Rubisco). This conclusion became more apparent to me after CO₂-fixation experiments using isolated spinach chloroplasts and protoplasts, purified Rubisco enzyme, and intact leaves. Ribulose bisphosphate (RuBP) pools and activation of Rubisco were measured and compared to ¹⁴CO₂ fixation in light. The rates of ¹⁴CO ₂ assimilation best followed the changes in Rubisco activation under moderate to high light intensities. RuBP pool sizes regulated ¹⁴ ₂ assimilation only in very high CO₂ levels, low light and in darkness. Activation of Rubisco involves two separate processes: carbamylation of the protein and removal of inhibitors blocking carbamylation or blocking RuBP binding to carbamylated sites before reaction with CO₂ or O₂. This revised version was published online in June 2006 with corrections to the Cover Date.     

RuBPCO kinetics and the mechanism of CO2 entry in C3 plants

Abstract. The CO₂ partial pressure in the chloroplasts of intact photosynthetic C₃ leaves is thought to be less than the intercellular CO₂ partial pressure. The intercellular CO₂ partial pressure can be calculated from CO₂ and H₂O gas exchange measurements, whereas the CO₂ partial pressure in the chloroplasts is unknown. The conductance of CO₂ from the intercellular space to the chloroplast stroma and the CO₂ partial pressure in the chloroplast stroma can be calculated if the properties of photosynthetic gas exchange are compared with the kinetics of the enzyme ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBPCO). A discrepancy between gas exchange and RuBPCO kinetics can be attributed to a deviation of CO₂ partial pressure in the chloroplast stroma from that calculated in the intercellular space. This paper is concerned with the following: estimation of the kinetic constants of RuBPCO and their comparison with the CO₂ compensation concentration; their comparison with differential uptake of ¹⁴CO₂ and ¹²CO₂; and their comparison with O₂ dependence of net CO₂ uptake of photosynthetic leaves. Discrepancy between RuBPCO kinetics and gas exchange was found at a temperature of 12.5 °C, a photosynthetic photon flux density (PPFD) of 550 μmol quanta m^?2 s^?1, and an ambient CO₂ partial pressure of 40 Pa. Consistency between RuBPCO kinetics and gas exchange was found if CO₂ partial pressure was decreased, temperature incresed and PPFD decreased. The results suggest that a discrepancy between RuBPCO kinetics and gas exchange is due to a diffusion resistance for CO₂ across the chloroplast envelope which decreases with increasing temperature. At low CO₂ partial pressure, the diffusion resistance appears to be counterbalanced by active CO₂ (or HCO₃) transport with high affinity and low maximum velocity. At low PPFD, CO₂ partial pressure in the chloroplast stroma appears to be in equilibrium with that in the intercellular space due to low CO₂ flux.