Molecular biology of the C3 photosynthetic carbon reduction cycle

In recent years the enzymes of the C₃ photosynthetic carbon reduction (PCR) cycle have been studied using the techniques of molecular biology. In this review we discuss the primary protein sequences and structural predictions that have been made for a number of these enzymes, which, with the input of crystallographic analysis, gives the opportunity to understand the mechanisms of enzyme activity.The genome organisation and gene structure of the PCR enzymes is another area which has recently expanded, and we discuss the regulation of the genes encoding these enzymes and the complex interaction of various factors which influence their expression.     

Metabolic regulation in C4 photosynthesis: Phosphoenol pyruvate carboxylase and 3C intermediates of the photosynthetic carbon reduction cycle

Summary Gibberellins and auxins were extracted from embryos and suspensors of Phaseolus coccineus L. at two stages of development: A) heart-shaped embryo and B) cotyledonary embryo with suspensor in the initial stage of degeneration. The time interval between the two stages was 5–6 days.In both embryos and suspensors, gibberellin (GA)-like activity was found in three fractions: F-1 (ethyl acetate fraction at pH 8.0), F-2 (free GAs) and F-3 (bound GAs). At stage A, the total GA activity in the suspensor was about 30 times greater than in the embryo and the bound GAs contributed by about 90% to the total GA content. A dramatic decrease in level of bound GA-like substances was found in suspensors at stage B, when the level of total GAs in the embryo had increased to 10 times that at stage A. This might suggest a transport of GAs from the suspensor to the embryo. In both embryo and suspensor, qualitative changes in GAs with shift in activity of the fractions tested occurred at the two developmental stages.The methanolic extracts of stage A suspensors showed two inhibitors, one much more active than the other, and two large peaks of growth promoting activity at Rf 0.4–0.7; in stage A embryos, the general activity of the extracts was lower and the promoting effect was spread over Rf 0.3–0.9.The present results seem to support the view that the suspensor plays a role in embryogenesis by acting as a site of synthesis of growth regulators needed by the embryo.Abbreviations F-1 ethyl acetate fraction at pH 8.0 - F-2 free gibberellins - F-3 bound gibberellins - GA gibberellic acid - Stage A heart-shaped embryo - stage B cotyledonary embryo with suspensor in the initial stage of degeneration     

The metabolic significance of octulose phosphates in the photosynthetic carbon reduction cycle in spinach

¹⁴C-Labelled octulose phosphates were formed during photosynthetic ¹⁴CO₂ fixation and were measured in spinach leaves and chloroplasts. Because mono- and bisphosphates of d-glycero- d-ido-octulose are the active 8-carbon ketosugar intermediates of the L-type pentose pathway, it was proposed that they may also be reactants in a modified Calvin–Benson–Bassham pathway reaction scheme. This investigation therefore initially focussed only on the ido-epimer of the octulose phosphates even though ¹⁴C-labelled d-glycero- d-altro-octulose mono- and bisphosphates were also identified in chloroplasts and leaves. ¹⁴CO₂ predominantly labelled positions 5 and 6 of d-glycero- d-ido-octulose 1,8-P₂ consistent with labelling predictions of the modified scheme. The kinetics of ¹⁴CO₂ incorporation into ido-octulose was similar to its incorporation into some traditional intermediates of the path of carbon, while subsequent exposure to ¹²CO₂ rapidly displaced the ¹⁴C isotope label from octulose with the same kinetics of label loss as some of the confirmed Calvin pathway intermediates. This is consistent with octulose phosphates having the role of cyclic intermediates rather than synthesized storage products. (Storage products don’t rapidly exchange isotopically labelled carbons with unlabelled CO₂.) A spinach chloroplast extract, designated stromal enzyme preparation (SEP), catalysed and was used to measure rates of CO₂ assimilation with Calvin cycle intermediates and octulose and arabinose phosphates. Only pentose (but not arabinose) phosphates and sedoheptulose 7-phosphate supported CO₂ fixation at rates in excess of 120 μmol h⁻¹ mg⁻¹ Chl. Rates for octulose, sedoheptulose and fructose bisphosphates, octulose, hexose and triose monophosphates were all notably less than the above rate and arabinose 5-phosphate was inactive. Altro-octulose phosphates were more active than phosphate esters of the ido-epimer. The modified scheme proposed a specific phosphotransferase and SEP unequivocally catalysed reversible phosphate transfer between sedoheptulose bisphosphate and d-glycero- d-ido-octulose 8-phosphate. It was also initially hypothesized that arabinose 5-phosphate, an L-Type pentose pathway reactant, may have a role in a modified Calvin pathway. Arabinose 5-phosphate is present in spinach chloroplasts and leaves. Radiochromatography showed that ¹⁴C-arabinose 5-phosphate with SEP, but only in the presence of an excess of unlabelled ribose 5-phosphate, lightly labelled ribulose 5-phosphate and more heavily labelled hexose and sedoheptulose mono- and bisphosphates. However, failure to demonstrate any CO₂ fixation by arabinose 5-phosphate as sole substrate suggested that the above labelling may have no metabolic significance. Despite this arabinose and ribose 5-phosphates are shown to exhibit active roles as enzyme co-factors in transaldolase and aldolase exchange reactions that catalyse the epimeric interconversions of the phosphate esters of ido- and altro-octulose. Arabinose 5-phosphate is presented as playing this role in a New Reaction Scheme for the path of carbon, where it is concluded that slow reacting ido-octulose 1,8 bisphosphate has no role. The more reactive altro-octulose phosphates, which are independent of the need for phosphotransferase processing, are presented as intermediates in the new scheme. Moreover, using the estimates of phosphotransferase activity with altro-octulose monophosphate as substrate allowed calculation of the contributions of the new scheme, that ranged from 11% based on the intact chloroplast carboxylation rate to 80% using the carboxylation rate required for the support of octulose phosphate synthesis and its role in the phosphotransferase reaction.     

Aluminium modulation of the photosynthetic carbon reduction cycle in Zea mays

Two-weeks-old maize (Zea mays L. cv. XL-72.3) plants were submitted to Al concentrations of 0-81 g m-3 for 20 d, after which the A1 concentration-dependent effects on CO2 uptake by the mesophyll tissue and subsequent CO2 assimilation in the photosynthetic carbon reduction cycle of bundle sheath cells were investigated. The net photosynthetic rate (PN) and stomatal conductance (gs) increased continuously up to 27 g m-3 Al, whereas the intercellular CO2 concentration showed minimum values with the 27 g m-3 Al treatment. Moreover, the starch and saccharide concentrations, and fructose-1,6-bisphosphatase did not change significantly with increasing Al concentrations. The photosynthetic electron transport rates along with photosystems 2 and 1 started falling from 9 g m-3 Al onwards, while thylakoid acyl lipid composition did not show a clear pattern. With the Al concentration at 81 g m-3, NADP-malate dehydrogenase activity decreased to minimum values, whereas the opposite occurred with those of pyruvate dikinase, NADP-malic enzyme, and phosphoenolpyruvate carboxylase. Thus in vivo Al concentrations modulate the photosynthetic reduction cycle, possibly by interacting with the carbon flow rate exported to the cytosol. Although the inhibition of NADP-malate dehydrogenase activity might limit pyruvate dikinase, NADP-malic enzyme, and phosphoenolpyruvate carboxylase activities, in vivo the balance between phosphoenolpyruvate production and its carboxylation remains unaffected.     

Aluminium modulation of the photosynthetic carbon reduction cycle in Zea mays

Two-weeks-old maize (Zea mays L. cv. XL-72.3) plants were submitted to Al concentrations of 0-81 g m-3 for 20 d, after which the A1 concentration-dependent effects on CO2 uptake by the mesophyll tissue and subsequent CO2 assimilation in the photosynthetic carbon reduction cycle of bundle sheath cells were investigated. The net photosynthetic rate (PN) and stomatal conductance (gs) increased continuously up to 27 g m-3 Al, whereas the intercellular CO2 concentration showed minimum values with the 27 g m-3 Al treatment. Moreover, the starch and saccharide concentrations, and fructose-1,6-bisphosphatase did not change significantly with increasing Al concentrations. The photosynthetic electron transport rates along with photosystems 2 and 1 started falling from 9 g m-3 Al onwards, while thylakoid acyl lipid composition did not show a clear pattern. With the Al concentration at 81 g m-3, NADP-malate dehydrogenase activity decreased to minimum values, whereas the opposite occurred with those of pyruvate dikinase, NADP-malic enzyme, and phosphoenolpyruvate carboxylase. Thus in vivo Al concentrations modulate the photosynthetic reduction cycle, possibly by interacting with the carbon flow rate exported to the cytosol. Although the inhibition of NADP-malate dehydrogenase activity might limit pyruvate dikinase, NADP-malic enzyme, and phosphoenolpyruvate carboxylase activities, in vivo the balance between phosphoenolpyruvate production and its carboxylation remains unaffected.     

Enzyme activities of the carbon reduction cycle in some photosynthetic organisms

Profile analyses of the enzymes comprising the photosynthetic carbon reduction cycle have been performed in extracts of dark grown and greening Euglena gracilis var. bacillaris. Chlorella pyrenoidosa grown photoautotrophically, in the light with glucose or in the dark with glucose, Tolypothrix tenuis, Chromatium and leaves of spinach. Amounts of activity are compared with the level of photosynthetic CO₂ fixation. Only in Chromatium were all enzyme activities sufficient to support the in vivo rate of CO₂ fixation. In organisms other than Chromatium, some enzymes and particularly fructose 1,6-phosphatase and ribulose 1.5-diphosphate carboxylase appeared to be present in insufficient amounts to support the photosynthetic rate of the intact cell. Developmental studies with Euglena and growth studies with Chlorella led to the conclusion that these enzymes were associated with the cycle. Suppression of CO₂ fixation in heterotrophically grown Chlorella was accompanied by a striking decrease in the same enzymes whose activities increased in greening Euglena.     

Regulation of photosynthetic carbon reduction cycle by ribulose bisphosphate and phosphoglyceric Acid

The activation states of a number of chloroplastic enzymes of the photosynthetic carbon reduction cycle and levels of related metabolites were measured in leaves of sugar beet (Beta vulgaris L., Klein E-type multigerm) under slowly changing irradiance during a day. The activation states of both phosphoribulokinase and NADP⁺-glyceraldehyde-3-phosphate dehydrogenase increased early in the light period and remained constant during the middle of the day. Initial ribulose 1,5-bisphosphate carboxylase activity was already about one third of the midday level, did not change for the first 2 hours, but then increased in parallel with the rate of carbon fixation. Because the activation states increased by turns, first phosphoribulokinase and NADP⁺-glyceraldehyde-3-phosphate dehydrogenase and later ribulose 1,5-bisphosphate carboxylase, the ratios of the activation states changed remarkably. Levels of ribulose bisphosphate and phosphoglycerate, which were high enough to affect enzyme reaction rates and changed in concert with activation state, indicate that these metabolites are involved in feedback/feedforward regulation of enzymes of carbon assimilation. This regulatory sequence is able to explain how the reaction rates for the enzymes of carbon assimilation are adjusted to maintain their activities in balance with each other and with the flux of carbon fixation.     

The importance and place of the photosynthetic carbon sequestration in the organic branch of its global cycle

Results of comparative analysis of turnover times and the capacity of major global pools of organic carbon are presented; the place of photosynthetic carbon sequestration is defined; concept of its catalytic role in the regulation of the organic branch of the global carbon cycle is ground. Concept of reservoir-flux model of photosynthetic carbon sequestration and of the net photosynthetic production at the territory of Northern Eurasia is suggested.Translated from Fiziologiya Rastenii, Vol. 52, No. 1, 2005, pp. 81–89.Original Russian Text Copyright © 2005 by Voronin, Black.     

Modelling C3 photosynthesis: A sensitivity analysis of the photosynthetic carbon-reduction cycle

A model of the C ₃ photosynthetic system is developed which describes the sensitivity of the steadystate rate of carbon dioxide assimilation to changes in the activity of several enzymes of the system. The model requires measurements of the steady-state rate of carbon dioxide assimilation, the concentrations of several intermediates in the photosynthetic system, and the concentration of the active site of ribulose 1,5-bisphosphate carboxyalse/oxygenase (Rubisco). It is shown that in sunflowers (Helianthus annuus L.) at photon flux densities that are largely saturating for the rate of photosynthesis, the steady-stete rate of carbon dioxide assimilation is most sensitive to Rubisco activity and, to a lesser degree, to the activities of the stromal fructose, 6-bisphosphatase and the enzymes catalysing sucrose synthesis. The activities of sedoheptulose 1,7-bisphosphatase, ribulose 5-phosphate kinase, ATP synthase and the ADP-glucose pyrophosphorylase are calculated to have a negligible effect on the flux under the high-light conditions. The utility of this analysis in developing simpler models of photosynthesis is also discussed.Abbreviations c _i intercellular CO₂ concentration - C _infP ^supJ control coefficient for enzyme P with respect to flux J - DHAP dihydroxyacetonephosphate - E4P erythrose 4-phosphate - F6P fructose 6-phosphate - FBP fructose 1,6-bisphosphate - FBPase fructose 1,6-bisphosphatase - G3P glyceraldehyde 3-phosphate - G1P glucose 1-phosphate - G6P glucose 6-phosphate - Pi inorganic phosphate - PCR photosynthetic carbon reduction - PGA 3-phosphoglyceric acid - PPFD photosynthetically active photon flux density - R _n ^J response coefficient for effector n with respect to flux J - R5P ribose 5-phosphate - Rubisco ribulose 1,5-bisphosphate carboxylase/oxygenase - Ru5P ribulose 5-phosphate - RuBP ribulose 1,5-bisphosphate - S7P sedoheptulose 7-phosphate - SBP sedoheptulose 1,7-bisphosphate - SBPase sedoheptulose 1,7-bisphosphatase - SPS sucrose-phosphate synthase - Xu5P xylulose 5-phosphate - _n ^P elasticity coefficient for effector n with respect to the catalytic velocity of enzyme P This research was funded by an Australian Research Council grant to I.E.W. and was undertaken during a visity by K.A.M. to the James Cook University of North Queensland. The expert help of Glenys Hanley and Mick Kelly is greatly appreciated.     

Fitting photosynthetic carbon dioxide response curves for C(3) leaves

Photosynthetic responses to carbon dioxide concentration can provide data on a number of important parameters related to leaf physiology. Methods for fitting a model to such data are briefly described. The method will fit the following parameters: V(cmax), J, TPU, R(d) and g(m)[maximum carboxylation rate allowed by ribulose 1.5-bisphosphate carboxylase/oxygenase (Rubisco), rate of photosynthetic electron transport (based on NADPH requirement), triose phosphate use, day respiration and mesophyll conductance, respectively]. The method requires at least five data pairs of net CO(2) assimilation (A) and [CO(2)] in the intercellular airspaces of the leaf (C(i)) and requires users to indicate the presumed limiting factor. The output is (1) calculated CO(2) partial pressure at the sites of carboxylation, C(c), (2) values for the five parameters at the measurement temperature and (3) values adjusted to 25 degrees C to facilitate comparisons. Fitting this model is a way of exploring leaf level photosynthesis. However, interpreting leaf level photosynthesis in terms of underlying biochemistry and biophysics is subject to assumptions that hold to a greater or lesser degree, a major assumption being that all parts of the leaf are behaving in the same way at each instant.     

Inactivation of the photosynthetic carbon reduction cycle in isolated mesophyll protoplasts subjected to freezing stress

Isolated mesophyll protoplasts from Valerianella locusta L. were subjected to freeze-thaw cycles. Subsequently, steady-state pool sizes of ¹⁴C-labeled intermediates of the photosynthetic carbon reduction cycle were determined by high performance liquid chromatography. Protoplasts in which CO₂ fixation was inhibited by preceding freezing stress, showed a strong increase in the proportion of fructose-1,6-bisphosphate, sedoheptulose-1,7-bisphosphate and triose phosphates. These results indicate an inhibition of the activities of stromal fructose-1,6-bisphosphatase and sedoheptulose-1,7-bisphosphatase. Furthermore, freezing stress caused a slight increase in the proportion of labeled ribulose-1,5-bisphosphate, which may be based on an inhibition or ribulose bisphosphate carboxylase activity. It was shown earlier (Rumich-Bayer and Krause 1986) that freezing-thawing readily affects photosynthetic CO₂ assimilation independently of thylakoid inactivation. The present results are interpreted in terms of an inhibition of the light-activation system of the photosynthetic carbon reduction cycle, caused by freezing stress.Abbreviations FBP Fructose-1,6-bisphosphate - HMP Hexose Monophosphates - PGA 3-phosphoglycerate - PMP Pentose Monophosphates - RBP Ribulose-1,5-bisphosphate - SBP Sedoheptulose-1,7-bisphosphate - TP Triose Phosphates     

CO2 and O2 dependence of PS II activity in C4 plants having genetically produced deficiencies in the C3 or C4 cycle

The CO2 dependence of rates of CO2 fixation (A) and photochemistry of PS II at 5, 15 and 30% O2 were analyzed in the C4 plant Amaranthus edulis having a C4 cycle deficiency [phosphoenolpyruvate carboxylase (PEPC) mutants], and in the C4 plant Flaveria bidentis having a C3 cycle deficiency [Rubisco small subunit antisense (SSU)]. In the wild type (WT) A. edulis and its heterozygous mutant having less than 50% WT PEPC activity there was a similar dependence of A and PS II photochemistry on varying CO2, although the CO2 saturated rates were 25% lower in heterozygous plants. The homozygous plants having less than 2% PEPC of the WT had significant levels of photorespiration at ambient levels of CO2 and required about 30 times ambient levels for maximum rates of A. Despite variation in the capacity of the C4 cycle, more than 91% of PS II activity was linearly associated with A under varying CO2 at 5, 15 and 30% O2. However, the WT plant had a higher PS II activity per CO2 fixed under saturating CO2 than the homozygous mutant, which is suggested to be due to elimination of the C4 cycle and its associated requirement for ATP from a Mehler reaction. In the SSU F. bidentis plants, a decreased rate of A (35%) and PS II activity (33%) accompanied a decrease in Rubisco capacity. There was some increase in alternative electron sinks at high CO2 when the C3 cycle was constrained, which may be due to increased flux through the C4 cycle via an ATP generating Mehler reaction. Nevertheless, even with constraints on the function of the C4 or C3 cycle by genetic modifications, analyses of CO2 response curves under varying levels of O2 indicate that CO2 assimilation is the main determinant of PS II activity in C4 plants.