Photoreductive path of carbon fixation in green plant photosynthesis. Reaction pathway of six-carbon ribulose 1,5-bisphosphate carboxylation adduct intermediate

In this paper we examine the six-carbon intermediate pathway of ribulose 1,5-bisphosphate (RuBP) carboxylation reaction in photosynthesis. Based on the observed reactions of purified RuBP carboxylase, mechanisms are described for carbon dioxide assimilation leading to the hydrolytic splitting of the six-carbon intermediate to two enzyme-bound glycerate-3-P (3-PGA) molecules. It is concluded that, under photosynthetic conditions, the reduction of enzyme-bound NADP+ by the chlorophyll is responsible for the rapid carboxylase turnover rate given by the lifetime, tau L = 0.4 s, which is nearly two orders of magnitude shorter than the corresponding value, tau D = 11 +/- 3 s, for the dark decay of enzyme-bound RuBP. The nocturnal inhibition and photoactivation of RuBP carboxylation are described in terms of the reversible light-dark cycles of the NADP+/NADPH redox couple and endogenous changes that accompany the 2-carboxy-D-arabinitol-1-phosphate binding to the enzyme active site.     

Photoinhibition under Atmospheric O2, the Activation State of RuBP Carboxylase and the Content of Photosynthetic Intermediates in Soybean and Wheat

Ward, D. A. and Drake, B. G. 1987. Photoinhibition under atmosphericO₂, the activation state of RuBP carboxylase and the contentof photosynthetic intermediates in soybean and wheat.—J.exp. Bot. 38: 1937–1948. Associations between photosynthesis, the activation state ofRuBP carboxylase and the contents of photosynthetic intermediateswere compared in soybean and wheat leaves before and after exposureto photoinhibitory treatments in the presence of atmosphericO₂. Exposing attached leaves to a supra-saturating irradiance(3 800 µmol quanta m^{– 2} s^–1) for 2 h in CO_2-freeair decreased carboxylation efficiency and the light-saturatedphotosynthetic rate in air by approximately 50%. Exposure tothe photoinhibitory treatment for periods in excess of 2 h didnot cause a further decrease of photosynthesis in soybean. Althoughphotosynthesis was reduced, the initial and total (fully-activated)activities of ribulose 1,5-bisphosphate carboxylase (RuBPCase)in leaf extracts were unaltered in each species by the photoinhibitorytreatment. This was true for leaves sampled under both air andat a rate-limiting intercellular CO₂ partial pressure (C_i) of75 µPa Pa^–1. The contents of ribulose l,5-bisphosphate(RuBP) and 3-phosphoglyceric acid (3-PGA) were reduced by thephotoinhibitory treatment in soybean leaves sampled in air andat a rate-limiting C_i, although the RuBP/3-PGA ratio was unaffected.The relative reduction of RuBP content in soybean leaves atrate-limiting C₁ was similar to the corresponding reductionof carboxylation efficiency. For wheat,the relative reductionof RuBP content at rate-limiting C_i (–19%) caused by thephotoinhibitory treatment was considerably less than the correspondingdecrease of carboxylation efficiency (–49%).The RuBP/3-PGAratio of wheat was also increased significantly by the photoinhibitorytreatment The significance of these observations to the regulationof CO₂-limited photosynthesis in leaves experiencing photoinhibitionunder atmospheric oxygen is discussed. Consideration is alsogiven to the previous contention that contemporary measurementsof initial activity in crude extracts may provide a spuriousindication of the amount of the enzyme-CO₂-Mg_{2 +} form of RuBPcarboxylase present in the leaf. Key words: Carboxylation efficiency, RuBP carboxylase, photoinhibition, RuBP, 3-PGA     

Cascade of bioreactors in series for conversion of 3-phospho-D-glycerate into D-ribulose-1,5-bisphosphate: kinetic parameters of enzymes and operation variables

A novel scheme employing enzymatic catalysts is described enabling conversion of D-ribulose-1,5-bisphosphate (RuBP) from 3-phospho-D-glycerate (3-PGA) without loss of carbon. Bioreactors harboring immobilized enzymes namely, phosphoglycerate kinase (PGK), glycerate phosphate dehydrogenase, triose phosphate isomerase (TIM), aldolase, transketolase (TKL), phosphatase (PTASE/FP), epimerase (EMR) and phosphoribulokinase (PRK), in accordance with this novel scheme were employed. These reactors were designed and constructed based on simulations carried out to study their performance under various operational conditions and allowed production of about 56 +/- 3% RuBP from 3-PGA. This method of synthesis of RuBP from 3-PGA employing immobilized enzyme bioreactors may be used for continuous regeneration of RuBP in biocatalytic carbon dioxide fixation processes from emissions where RuBP acts as acceptor of carbon dioxide to produce 3-PGA, rendering the fixation process continuous.     

RuBP carboxylase determination by enzymic estimation of D-3-PGA formed

RuBPcarboxylase activity was measured in extracts of barley (Hordeum Vulgare L., cv. HOP) seedlings both with the standard radiometric method and by measuring D-3-phosphoglyceric acid formed enzymically in a two stage assay. In the different conditions used, characterized by different NaHCO₃ concentrations, different pH and the presence and absence of oxygen, essentially the same ratio of D-3-PGA formed per ¹⁴CO₂ fixed was obtained. This ratio respected the known stoichiometry of two molecules of D-3-PGA formed per CO₂ fixed.It is suggested that measurement of D-3-PGA enzymically in a two stage assay can be routinely used for the determination of RuBP case activity instead of the radiometric method. The advantages and the validity of the method are discussed.Abbreviations Bicine N, N-bis-(2-hydroxyethyl)-glycine - NADH nicotinamide adenine dinucleotide, reduced - PGA phosphoglyceric acid - RuBP ribulose-1-5-bisphosphate     

A nonradioactive assay method for determination of enzymatic activity of D-ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco)

A sensitive and nonradioactive assay method for activity determination of Rubisco is described. The method is based on thin-layer chromatographic separation of 3-phosphoglycerate (3-PGA) and D-ribulose-1,5-bisphosphate (RuBP). This assay method allows the quantitative determination of Rubisco activity. Rates of carbon dioxide fixation on RuBP determined by this method were comparable to those obtained independently by other methods. This assay method is reproducible and relatively free from interference.     

RuBP carboxylase determination by enzymic estimation of D-3-PGA formed

RuBPcarboxylase activity was measured in extracts of barley (Hordeum Vulgare L., cv. HOP) seedlings both with the standard radiometric method and by measuring D-3-phosphoglyceric acid formed enzymically in a two stage assay. In the different conditions used, characterized by different NaHCO₃ concentrations, different pH and the presence and absence of oxygen, essentially the same ratio of D-3-PGA formed per ¹⁴CO₂ fixed was obtained. This ratio respected the known stoichiometry of two molecules of D-3-PGA formed per CO₂ fixed. It is suggested that measurement of D-3-PGA enzymically in a two stage assay can be routinely used for the determination of RuBP case activity instead of the radiometric method. The advantages and the validity of the method are discussed.     

Changes in leaf photosynthesis of transgenic rice with silenced <Emphasis Type="Italic">OsBP-73</Emphasis> gene

In comparison with its wild type (WT), the transgenic (TG) rice with silenced OsBP-73 gene had significantly lower plant height, grain number per panicle, and leaf net photosynthetic rate (P _N). Also, the TG rice showed significantly lower chlorophyll (Chl), ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBPCO), RuBPCO activase, and RuBP contents, photosystem 2 (PS2) photochemical efficiency (F_v/F_m and ΔF/F_m′), apparent quantum yield of carbon assimilation (Φ_c), carboxylation efficiency (CE), photosynthetic electron transport and photophosphorylation rates as well as sucrose phosphate synthase activity, but higher intercellular CO₂ concentration, sucrose, fructose, and glycerate 3-phosphate contents, and non-photochemical quenching of Chl fluorescence (NPQ). Thus the decreased P _N in the TG rice leaves is related to both RuBP carboxylation and RuBP regeneration limitations, and the latter is a predominant limitation to photosynthesis.     

Photosynthesis by sugar-cane leaves: A new carboxylation reaction and the pathway of sugar formation

1. Radioactive products in detached leaf segments were examined after periods of steady-state photosynthesis in (14)CO(2). 2. After exposure to (14)CO(2) for approx. 1sec. more than 93% of the fixed radioactivity was located in malate, aspartate and oxaloacetate. After longer periods large proportions of the radioactivity appeared in 3-phosphoglycerate, hexose monophosphates and sucrose. Similar results were obtained with leaves still attached to the plant. 3. Radioactivity appeared first in C-4 of the dicarboxylic acids and C-1 of 3-phosphoglycerate. The labelling pattern in hexoses was consistent with their formation from 3-phosphoglycerate. 4. The reaction giving rise to C(4) dicarboxylic acid appears to be the only quantitatively significant carboxylation reaction. 5. Evidence is provided that the radioactivity incorporated into the C(4) dicarboxylic acid pool is transferred to sugars via 3-phosphoglycerate. A scheme is proposed to account for these observations.     

Short-term feedback inhibition of photosynthesis in wheat leaves supplied with sucrose and glycerol at two temperatures

The inhibition of photosynthesis by reduced sink demand or low rates of end product synthesis was investigated by supplying detached wheat (Triticum aestivum L. cv. Tauro) leaves with 50 mM sucrose, 50 mM glycerol or water through the transpiration stream for 2 h, either at 23 or 12 °C. Lowering the temperature and sucrose and glycerol feeding decreased photosynthetic oxygen evolution at high irradiance and saturating CO₂. The decrease in temperature reduced the pools of sucrose and starch, and the ratio glucose 6-phosphate (G6P)/fructose 6-phosphate (F6P), while it increased the concentrations of G6P and F6P (hexose phosphates). Sucrose feeding, in contrast to glycerol feeding, increased sucrose, glucose and fructose contents and the G6P/F6P ratio. Sucrose and glycerol incubations at 23 °C, as well as decreasing the temperature in leaves incubated in water, increased the concentration of triose-phosphates (glyceraldehyde 3-phosphate and dihydroxyacetone phosphate, TP) and decreased the glycerate 3-phosphate (PGA) content, thus increasing the TP/PGA ratio; they also tended to increase the ribulose 1,5-bisphosphate (RuBP) content and the RuBP/PGA ratio. Sucrose and glycerol feeding at 12 °C and the decrease in temperature of leaves incubated in these solutions decreased TP and RuBP contents and the TP/PGA and RuBP/PGA ratios. The results suggest that the phosphate limitation caused by accumulation of end products, restriction of their synthesis and sequestration of cytosolic phosphate can inhibit photosynthesis through decreased carboxylation of RuBP or, with increased phosphate limitation, through lowered supply of ATP. This revised version was published online in August 2006 with corrections to the Cover Date.     

Effects of chronic ozone and elevated atmospheric CO2 concentrations on ribulose-1,5-bisphosphate in soybean (Glycine max)

Ribulose-1,5-bisphosphate (RuBP) pool size was determined at regular intervals during the growing season to understand the effects of tropospheric ozone concentrations, elevated atmospheric carbon dioxide concentrations and their interactions on the photosynthetic limitation by RuBP regeneration. Soybean (Glycine max [L.] Merr. cv. Essex) was grown from seed to maturity in open-top field chambers in charcoal-filtered air (CF) either without (22 nmol O₃ mol^?1) or with added O₃ (83 nmol mol^?1) at ambient (AA, 369 μmol CO₂ mol^?1) or elevated CO₂ (710 μmol mol^?1). The RuBP pool size generally declined with plant age in all treatments when expressed on a unit leaf area and in all treatments but CF-AA when expressed per unit ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco; EC 4.1.1.39) binding site. Although O₃ in ambient CO₂ generally reduced the RuBP pool per unit leaf area, it did not change the RuBP pool per unit Rubisco binding site. Elevated CO₂, in CF or O₃-fumigated air, generally had no significant effect on RuBP pool size, thus mitigating the negative O₃ effect. The RuBP pools were below 2 mol mol^?1 binding site in all treatments for most of the season, indicating limiting RuBP regeneration capacity. These low RuBP pools resulted in increased RuBP regeneration via faster RuBP turnover, but only in CF air and during vegetative and flowering stages at elevated CO₂. Also, the low RuBP pool sizes did not always reflect RuBP consumption rates or the RuBP regeneration limitation relative to potential carboxylation (%RuBP). Rather, %RuBP increased linearly with decrease in the RuBP pool turnover time. These data suggest that amelioration of damage from O₃ by elevated atmospheric CO₂ to the RuBP regeneration may be in response to changes in the Rubisco carboxylation.     

Balancing carboxylation and regeneration of ribulose-1,5- bisphosphate in leaf photosynthesis: temperature acclimation of an evergreen tree, Quercus myrsinaefolia

Changes in the temperature dependence of the photosynthetic rate depending on growth temperature were investigated for a temperate evergreen tree, Quercus myrsinaefolia . Plants were grown at 250 μ mol quanta m^–2 s^–1 under two temperature conditions, 15 and 30 °C. The optimal temperature that maximizes the light-saturated rate of photosynthesis at 350 μ L L^–1 CO₂ was found to be 20–25 and 30–35 °C for leaves grown at 15 and 30 °C, respectively. We focused on two processes, carboxylation and regeneration of ribulose-1,5-bisphosphate (RuBP), which potentially limit photosynthetic rates. Because the former process is known to limit photosynthesis at lower CO₂ concentrations while the latter limits it at higher CO₂ concentrations, we determined the temperature dependence of the photosynthetic rate at 200 and 1000 μ L L^–1 CO₂ under saturated light. It was revealed that the temperature dependence of both processes varied depending on the growth temperature. Using a biochemical model, we estimated the capacity of the two processes at various temperatures under ambient CO₂ concentration. It was suggested that, in leaves grown at low temperature (15 °C), the photosynthetic rate was limited solely by RuBP carboxylation under any temperature. On the other hand, it was suggested that, in leaves grown at high temperature (30 °C), the photosynthetic rate was limited by RuBP regeneration below 22 °C, but limited by RuBP carboxylation above 22 °C. We concluded that: (1) the changes in the temperature dependence of carboxylation and regeneration of RuBP and (2) the changes in the balance of these two processes altered the temperature dependence of the photosynthetic rate.     

A mechanistic model for photosynthesis based on the multisubstrate ordered reaction of ribulose 1,5 bisphosphate carboxylase

Abstract. A mechanistic model of photosynthesis is developed based on the characteristics of ribulose 1,5-bisphosphate (RuBP) carboxylase and the assimilation of CO₂ as an ordered reaction with RuBP binding before CO₂. An equation is derived which considers the effects of light (for regeneration of RuBP) and CO₂. Taking values for the maximum turnover of RuBP carboxylase at substrate saturation, the maximum carboxylation efficiency (maximum increase in rate per increase in CO₂ concentration) and the minimum quantum requirement for the C₃ pathway, photosynthesis in the absence of photorespiration is simulated. In the model, at varying concentrations of CO₂, the efficiency of light utilization approaches a maximum value as photon flux density decreases. Similarly, with a given maximum carboxyation capacity, at varying photon flux densities the carboxylation efficiency approaches a constant maximum value (equal to V _max/ K _{m CO2}) as CO₂ is decreased. However, a decrease in the state of activation of RuBP carboxylase under low light results in a lower carboxylation efficiency. Limits on the rate of photosynthesis, as the maximum capacity for regeneration of RuBP is reduced relative to carboxylation potential, or as the maximum capacity of the carboxylase varies, are considered.     

Partitioning of the Leaf CO2 Exchange into Components Using CO2 Exchange and Fluorescence Measurements

Photorespiration was calculated from chlorophyll fluorescence and ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) kinetics and compared with CO2 evolution rate in the light, measured by three gas-exchange methods in mature sunflower (Helianthus annuus L.) leaves. The gas-exchange methods were (a) postillumination CO2 burst at unchanged CO2 concentration, (b) postillumination CO2 burst with simultaneous transfer into CO2-free air, and (c) extrapolation of the CO2 uptake to zero CO2 concentration at Rubisco active sites. The steady-state CO2 compensation point was proportional to O2 concentration, revealing the Rubisco specificity coefficient (Ksp) of 86. Electron transport rate (ETR) was calculated from fluorescence, and photorespiration rate was calculated from ETR using CO2 and O2 concentrations, Ksp, and diffusion resistances. The values of the best-fit mesophyll diffusion resistance for CO2 ranged between 0.3 and 0.8 s cm-1. Comparison of the gas-exchange and fluorescence data showed that only ribulose-1,5-bisphosphate (RuBP) carboxylation and photorespiratory CO2 evolution were present at limiting CO2 concentrations. Carboxylation of a substrate other than RuBP, in addition to RuBP carboxylation, was detected at high CO2 concentrations. A simultaneous decarboxylation process not related to RuBP oxygenation was also detected at high CO2 concentrations in the light. We propose that these processes reflect carboxylation of phosphoenolpyruvate, formed from phosphoglyceric acid and the subsequent decarboxylation of malate.     

Interactive effects of elevated carbon dioxide and growth temperature on photosynthesis in cotton leaves

Cotton (Gossypium hirsutum L., cv DPL 5415) plants were grown in naturally lit environment chambers at day/night temperature regimes of 26/18 (T-26/18), 31/23 (T-31/23) and 36/28 °C (T-36/28) and CO2 concentrations of 350 (C-350), 450 (C-450) and 700 L L-1 (C-700). Net photosynthesis rates, stomatal conductance, transpiration, RuBP carboxylase activity and the foliar contents of starch and sucrose were measured during different growth stages. Net CO2 assimilation rates increased with increasing CO2 and temperature regimes. The enhancement of photosynthesis was from 24 mol CO2 m-2 s-1 (with C-350 and T-26/18) to 41 mol m-2 s-1 (with C-700 and T-36/28). Stomatal conductance decreased with increasing CO2 while it increased up to T-31/23 and then declined. The interactive effects of CO2 and temperature resulted in a 30% decrease in transpiration. Although the leaves grown in elevated CO2 had high starch and sucrose concentrations, their content decreased with increasing temperature. Increasing temperature from T-26/18 to 36/28 increased RuBP carboxylase activity in the order of 121, 172 and 190 mol mg-1 chl h-1 at C-350, C-450 and C-700 respectively. Our data suggest that leaf photosynthesis in cotton benefited more from CO_2 enrichment at warm temperatures than at low growth temperature regimes.     

Simultaneous kinetic analysis of ribulose 1,5-bisphosphate carboxylase/oxygenase activities

An assay was developed for simultaneous kinetic analysis of the activities of the bifunctional plant enzyme ribulose 1,5-bisphosphate carboxylase/oxygenase [EC 4.1.1.39]. [1-¹⁴C,5-³H]Ribulose 1,5-bisphosphate (RuBP) was used as the labeled substrate. Tritium enrichment of the doubly labeled 3-phosphoglycerate (3-PGA) product, common to both enzyme activities, may be used to calculate V_c/V_o ratios from the expression A/(B-A) where A and B represent the ³H/¹⁴C isotope ratios of doubly labeled RuBP and 3-PGA, and V_c and V_o represent the activities of carboxylase and oxygenase, respectively. Doubly labeled substrate was synthesized from [2-¹⁴C]glucose and [6-³H]glucose using the enzymes of the pentose phosphate pathway coupled with phosphoribulokinase.     

Determining RuBisCO activation kinetics and other rate and equilibrium constants by simultaneous multiple non-linear regression of a kinetic model

The forward and reverse rate constants involved in carbamylation, activation, carboxylation, and inhibition of D-ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) have been estimated by a new technique of simultaneous non-linear regression of a differential equation kinetic model to multiple experimental data. Parameters predicted by the model fitted to data from purified spinach enzyme in vitro included binding affinity constants for non-substrate CO2 and Mg2+ of 200+/-80 microM and 700+/-200 microM, respectively, as well as a turnover number (k(cat)) of 3.3+/-0.5 s(-1), a Michaelis half-saturation constant for carboxylation (K(M,C)) of 10+/-4 microM and a Michaelis constant for RuBP binding (K(M,RuBP)) of 1.5+/-0.5 microM. These and other constants agree well with previously measured values where they exist. The model is then used to show that slow inactivation of RuBisCO (fallover) in oxygen-free conditions at low concentrations of CO2 and Mg2+ is due to decarbamylation and binding of RuBP to uncarbamylated enzyme. In spite of RuBP binding more tightly to uncarbamylated enzyme than to the activated form, RuBisCO is activated at high concentrations of CO2 and Mg2+. This apparent paradox is resolved by considering activation kinetics and the fact that while RuBP binds tightly but slowly to uncarbamylated enzyme, it binds fast and loosely to activated enzyme. This modelling technique is presented as a new method for determining multiple kinetic data simultaneously from a limited experimental data set. The method can be used to compare the properties of RuBisCO from different species quickly and easily.     

Potential use of sugar binding proteins in reactors for regeneration of CO<Subscript>2</Subscript> fixation acceptor D-Ribulose-1,5-bisphosphate

Sugar binding proteins and binders of intermediate sugar metabolites derived from microbes are increasingly being used as reagents in new and expanding areas of biotechnology. The fixation of carbon dioxide at emission source has recently emerged as a technology with potentially significant implications for environmental biotechnology. Carbon dioxide is fixed onto a five carbon sugar D-ribulose-1,5-bisphosphate. We present a review of enzymatic and non-enzymatic binding proteins, for 3-phosphoglycerate (3PGA), 3-phosphoglyceraldehyde (3PGAL), dihydroxyacetone phosphate (DHAP), xylulose-5-phosphate (X5P) and ribulose-1,5-bisphosphate (RuBP) which could be potentially used in reactors regenerating RuBP from 3PGA. A series of reactors combined in a linear fashion has been previously shown to convert 3-PGA, (the product of fixed CO2 on RuBP as starting material) into RuBP (Bhattacharya et al., 2004; Bhattacharya, 2001). This was the basis for designing reactors harboring enzyme complexes/mixtures instead of linear combination of single-enzyme reactors for conversion of 3PGA into RuBP. Specific sugars in such enzyme-complex harboring reactors requires removal at key steps and fed to different reactors necessitating reversible sugar binders. In this review we present an account of existing microbial sugar binding proteins and their potential utility in these operations.     

Cotton bracts are adapted to a microenvironment of concentrated CO2 produced by rapid fruit respiration

Background and Aims

Elucidation of the mechanisms by which plants adapt to elevated CO₂ is needed; however, most studies of the mechanisms investigated the response of plants adapted to current atmospheric CO₂. The rapid respiration rate of cotton (Gossypium hirsutum) fruits (bolls) produces a concentrated CO₂ microenvironment around the bolls and bracts. It has been observed that the intercellular CO₂ concentration of a whole fruit (bract and boll) ranges from 500 to 1300 µmol mol⁻¹ depending on the irradiance, even in ambient air. Arguably, this CO₂ microenvironment has existed for at least 1·1 million years since the appearance of tetraploid cotton. Therefore, it was hypothesized that the mechanisms by which cotton bracts have adapted to elevated CO₂ will indicate how plants will adapt to future increased atmospheric CO₂ concentration. Specifically, it is hypothesized that with elevated CO₂ the capacity to regenerate ribulose-1,5-bisphosphate (RuBP) will increase relative to RuBP carboxylation.

Methods

To test this hypothesis, the morphological and physiological traits of bracts and leaves of cotton were measured, including stomatal density, gas exchange and protein contents.

Key results

Compared with leaves, bracts showed significantly lower stomatal conductance which resulted in a significantly higher water use efficiency. Both gas exchange and protein content showed a significantly greater RuBP regeneration/RuBP carboxylation capacity ratio (J_max/V_cmax) in bracts than in leaves.

Conclusions

These results agree with the theoretical prediction that adaptation of photosynthesis to elevated CO₂ requires increased RuBP regeneration. Cotton bracts are readily available material for studying adaption to elevated CO₂.     

Modelling photosynthetic responses to temperature of grapevine (Vitis vinifera cv. Semillon) leaves on vines grown in a hot climate

Field measurements of photosynthesis of Vitis vinifera cv. Semillon leaves in relation to a hot climate, and responses to photon flux densities (PFDs) and internal CO(2) concentrations (c(i) ) at leaf temperatures from 20 to 40 °C were undertaken. Average rates of photosynthesis measured in situ decreased with increasing temperature and were 60% inhibited at 45 °C compared with 25 °C. This reduction in photosynthesis was attributed to 15-30% stomatal closure. Light response curves at different temperatures revealed light-saturated photosynthesis optimal at 30 °C but also PFDs saturating photosynthesis increased from 550 to 1200 μmol (photons) m(-2)s(-1) as temperatures increased. Photosynthesis under saturating CO(2) concentrations was optimal at 36 °C while maximum rates of ribulose 1,5-bisphosphate (RuBP) carboxylation (V(cmax)) and potential maximum electron transport rates (J(max)) were also optimal at 39 and 36 °C, respectively. Furthermore, the high temperature-induced reduction in photosynthesis at ambient CO(2) was largely eliminated. The chloroplast CO(2) concentration at the transition from RuBP regeneration to RuBP carboxylation-limited assimilation increased steeply with an increase in leaf temperature. Semillon assimilation in situ was limited by RuBP regeneration below 30 °C and above limited by RuBP carboxylation, suggesting high temperatures are detrimental to carbon fixation in this species.     

A fluorimetric study of substrate and effector binding of D-ribulose-1,5-biphosphate carboxylase/oxygenase from spinach

2-p-toluidino-naphthalene-6-sulfonate (TNS) is a sensitive fluorescent reporter group for the detection of the events at the reaction centres of the ribulose biphosphate carboxylase/oxygenase from spinach. The formation of binary complexes of the carboxylase with substrates and effectors is associated with significant changes (ΔF) of the fluorescence emission of the enzyme-TNS-complex. This indicates substrate and effector induced conformational changes of the enzyme. From the concentration dependence of ΔF the following dissociation constants for ribulose biphosphate (RuBP) and Mg²⁺ were determined: K(RuBP) = 0,5 μM and K(Mg²⁺) = 1 mM. Sugar phosphates, e.g. 6-phosphogluconate, which show regulatory effects in the carboxylation and oxygenation of RuBP, function antagonistically to RuBP, presumably by competition with RuBP for its allosteric binding site.