Leaf phosphate status, photosynthesis, and carbon partitioning in sugar beet: I. Changes in growth, gas exchange, and calvin cycle enzymes

Sugar beets (Beta vulgaris L. cv F58-554H1) were cultured hydroponically for 2 weeks in growth chambers with two levels of orthophosphate (Pi) supplied in half strength Hoagland solution. Low-P plants were supplied with 1/20th of the Pi supplied to control plants. With low-P treatment, the acid soluble leaf phosphate and total leaf P decreased by about 88%. Low-P treatment had a much greater effect on leaf area than on photosynthesis. Low-P decreased total leaf area by 76%, dry weight per plant by 60%, and the rate of photosynthesis per area at light saturation by 35%. Low-P treatment significantly decreased the total extractable activity of phosphoglycerate kinase (by 18%) and NADP-glyceraldehyde-3-phosphate dehydrogenase (by 16%), but did not decrease the total activities of ribulose-1,5-bisphosphate (RuBP) carboxylase (RuBPCase) and ribulose-5-phosphate kinase. Low-P treatment decreased the initial activities of three rate-limiting Calvin cycle enzymes, but had no effect on the initial activity of RuBPCase. Furthermore, low-P treatment significantly increased the total extractable activities of fructose-1,6-bisphosphatase (by 61%), fructose-1,6-bisphosphate aldolase (by 53%), and transketolase (by 46%). The results suggest that low-P treatment affected photosynthetic rate through an effect on RuBP regeneration rather than through RuBPCase activity and that the changes in Calvin cycle enzymes with low-P resulted in an increased flow of carbon to starch.     

Leaf Phosphate Status, Photosynthesis and Carbon Partitioning in Sugar Beet: II. Diurnal Changes in Sugar Phosphates, Adenylates, and Nicotinamide Nucleotides

Sugar Beets (Beta vulgaris L. cv F58-554H1) were cultured hydroponically in growth chambers. Leaf orthophosphate (Pi) levels were varied nutritionally. The effect of decreased leaf phosphate (low-P) status was determined on the diurnal changes in the pool sizes of leaf ribulose 1,5-bisphosphate (RuBP), 3-phosphoglycerate (PGA), triose phosphate, fructose 1,6-bisphosphate, fructose-6-phosphate, glucose-6-phosphate, adenylates, nicotinamide nucleotides, and Pi. Except for triose phosphate, low-P treatment caused a marked reduction in the levels of leaf sugar phosphates (on a leaf area basis) throughout the diurnal cycle. Low-P treatment decreased the average leaf RuBP levels by 60 to 69% of control values during the light period. Low-P increased NADPH levels and NADPH/NADP⁺ ratio but decreased ATP; the ATP/ADP ratio was unaffected. Low P treatment caused a marked reduction in RuBP regeneration (RuBP levels were half the RuBP carboxylase binding site concentration) but did not depress PGA reduction to triose phosphate. These results indicate that photosynthesis in low-P leaves was limited by RuBP regeneration and that RuBP formation in low-P leaves was not limited by the supply of ATP and NADPH. We suggest that RuBP regeneration was limited by the supply of fixed carbon, an increased proportion of which was diverted to starch synthesis.     

The relationship between carbon-dioxide-limited photosynthetic rate and ribulose-1,5-bisphosphate-carboxylase content in two nuclear-cytoplasm substitution lines of wheat,and the coordination of ribulose-bisphosphate-carboxylation and electron-transport capacities

Photosynthesis in two cultivars of Triticum aestivum was compared with photosynthesis in two lines having the same nuclear genomes but with cytoplasms derived from T. boeoticum. The in-vitro specific activity of ribulose-1,5-bisphosphate carboxylase (RuBPCase; EC 4.1.1.39) isolated from lines with T. boeoticum cytoplasm was only 71% of that of normal T. aestivum. By contrast, the RuBPCase activities calculated from the CO₂-assimilation rate at low partial pressures of CO₂, p(CO₂), were the same for all lines for a given RuBPCase content. This indicates that both types of RuBPCase have the same turnover numbers in-vivo of 27.5 mol CO₂·(mol enzyme)^–1·s^–1 (23°). The rate of CO₂ assimilation measured at normal p(CO₂), p _a =340 bar, and high irradiance could be quantitatively predicted from the amount of RuBPCase protein. The maximum rate of RuBP regeneration could also predict the rate of CO₂ assimilation at normal ambient conditions. Therefore, the maximum capacities for RuBP carboxylation and RuBP regeneration appear to be well-balanced for normal ambient conditions. As photosynthetic capacity declined with increasing leaf age, the capacities for RuBP carboxylation and RuBP regeneration declined in parallel.Abbreviations PAR photosynthetically active radiation - RuBP(Case) ribulose-1,5-bisphosphate (carboxylase)     

Leaf Phosphate Status,Photosynthesis, and Carbon Partitioning in Sugar Beet (IV. Changes with Time Following Increased Supply of Phosphate to Low-Phosphate Plants)

Changes in photosynthesis, carbon partitioning, and growth following resupply of orthophosphate (Pi) to moderately P-deficient plants (low-P) were determined for sugar beets (Beta vulgaris L. cv F58-554H1) cultured hydroponically in growth chambers. One set of plants was supplied with 1.0 mM Pi in half-strength Hoagland solution (control plants), and a second set (low-P plants) was supplied with 0.05 mM Pi. At the end of 2 weeks, the low-P plants were resupplied with 1.0 mM Pi. Low-P plants rapidly accumulated large amounts of Pi, and the photosynthesis rate increased to control values within 4 to 6 h. The rate of photosynthesis appeared to be controlled by ribulose-1,5-bisphosphate (RuBP); low P reduced photosynthesis and RuBP levels, and P resupply increased photosynthesis and RuBP in a manner parallel with time. Low-P treatment reduced adenylate levels substantially but not nicotinamide nucleotides; adenylate levels recovered to control values over 3 to 6 h. With low P, more photosynthate is allocated to non-P carbon compounds (e.g. starch, sucrose) than to sugar phosphates. When P is resupplied, sugar phosphates increase as starch and sucrose pools decrease; this increase in leaf (chloroplast) sugar phosphates was most likely responsible for the increases in RuBP and photosynthesis and may have increased adenylate levels (through enhanced levels of ribose-5-phosphate).     

Effect of growth conditions on carboxylating enzymes of Zea mays plants

Phosphoenolpyruvate (PEP) carboxylase (EC 4.1.1.31) and ribulose-1,5-bisphospate (RuBP) carboxylase (EC 4.1.1.39) activities in leaves of different maize hybrids grown under field conditions (high light intensity) and in a growth chamber (low light intensity) were determined. Light intensity and leaf age affected PEP carboxylase activity, whereas RuBP carboxylase was affected by leaf age only at low light intensity. PEP carboxylase/RuBP carboxylase activity ratio decreased according to light intensity and leaf age. Results demonstrate that Zea mays grown under field conditions is a typical C₄ species in all leaves independently from their position on the stem, whereas it may be a C₃ plant when it is grown in a growth chamber at low light intensityAbbreviations PEP phosphoenolpyruvate - RuBP ribulose-1,5-bisphosphate     

Metabolite levels in the stroma of spinach chloroplasts exposed to osmotic stress: Effects of the pH of the medium and exogenous dihydroxyacetone phosphate

The levels of stromal photosynthetic intermediates were measured in isolated intact spinach (Spinacia oleracea L.) chloroplasts exposed to reduced osmotic potentials. Stressed chloroplasts showed slower rates of metabolite accumulation upon illumination than controls. Relative to other metabolites sedoheptulose-1,7-bisphosphate (SBP) and fructose-1,6-bisphosphate (FBP) accumulated in the stroma in the stressed treatments. Under these conditions 3-phosphoglycerate (3-PGA) efflux to the medium was restricted. Chloroplasts previously incubated with [³²P]KH₂PO₄ and [³²P]dihydroxyacetone phosphate ([³²P]DAP) in the dark were characterized by very high FBP and SBP levels prior to illumination. Metabolism of these pools upon illumination increased with increasing pH of the medium but was consistently inhibited in osmotically stressed chloroplasts. The responses of stromal FBP and SBP pools under hypertonic conditions are discussed in terms of both inhibited light activation of fructose-1,6-bisphosphatase (EC 3.1.3.11) and sedoheptulose-1,7-bisphosphatase (EC 3.1.3.37), and likely increases in stromal ribulose-1,5-bisphosphate carboxylase (EC 4.1.1.39) active-site concentrations.Abbreviations and symbols DAP dihydroxyacetone phosphate - FBP fructose-1,6-bisphosphate - PGA 3-phosphoglycerate - RuBP ribulose-1,5-bisphosphate - SBP sedoheptulose-1,7-bisphosphate - _s osmotic potential     

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

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

Low concentration of bisulfite enhances photosynthesis in tea tree by promoting carboxylation efficiency in leaves

Tea tree (Melaleuca alternifolia) canopy was sprayed with low concentration of NaHSO₃ or mixture of NaHSO₃+ KH₂PO₄. The treatments significantly enhanced net photosynthetic rate (P _N), carboxylation efficiency (CE), and the maximum response of P _N to intercellular CO₂ concentration. The enhancement of P _N by foliar application of low concentrations of bisulfite was due to increasing CE relevant to ribulose-1,5-bisphosphate (RuBP) carboxylase/oxygenase activity and regeneration rate of RuBP depending on ATP formation.     

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).     

Influence of Etherel and Gibberellic Acid on Carbon Metabolism,Growth, and Essential Oil Accumulation in Spearmint (Mentha Spicata)

Controlled environment chamber and glasshouse studies were conducted on six herbaceous annual species grown at 350 (AC) and 700 (EC) mol(CO₂) mol^-1 to determine whether growth at EC resulted in acclimation of the apparent quantum yield of photosynthesis (QY) measured at limiting photosynthetic photon flux density (PPFD), or in acclimation of net photosynthetic rate (P _N) measured at saturating PPFD. It was also determined whether acclimation in P _N at limiting PPFD was correlated with acclimation of carboxylation efficiency or ribulose-1,5-bisphosphate (RuBP) regeneration rate measured at saturating PPFD. Growth at EC reduced both the QY and P _N at limiting PPFD in three of the six species. The occurrence of photosynthetic acclimation measured at a rate limiting PPFD was independent of whether photosynthetic acclimation was apparent at saturating measurement PPFD. At saturating measurement PPFD, acclimation to EC in the apparent carboxylation efficiency and RuBP regeneration capacity also occurred independently. Thus at least three components of the photosynthetic system may adjust independently when leaves are grown at EC. Estimates of photosynthetic acclimation at both high and low PPFD are necessary to accurately predict photosynthesis at the whole plant or canopy level as [CO₂] increases.     

Characterization of the pyrenoid isolated from unicellular green algaChlamydomonas reinhardtii: Particulate form of RuBisCO protein

Summary The pyrenoid is a protein complex in the chloroplast stroma of eukaryotic algae. After the treatment with mercury chloride, pyrenoids were isolated by sucrose density gradient centrifugation from cell-wall less mutant cells, CW-15, as well as wild type cells, C-9, of unicellular green algaChlamydomonas reinhardtii. Pyrenoids were characterized as a fraction whose protein/chlorophyll ratio was very high, and also examined by Nomarski differential interference microscopy. Most of the components consisted of 55 kDa and 16 kDa polypeptides (11) which were immunologically identified as the large and small subunit of RuBisCO (ribulose-1,5-bisphosphate carboxylase/oxygenase) protein, respectively. Some minor polypeptides were also detected. Substantial amount of RuBisCO protein is present as a particulate form in the pyrenoid in addition to the soluble form in algal chloroplast stroma.Abbreviations BPB bromophenol blue - DAB 3,3-diaminobenzidine - DTT dithiothreitol - ELISA enzyme-linked immunosorbent assay - High-CO₂ cells cells grown under air enriched with 4% CO₂ - Low-CO₂ cells cells grown under ordinary air (containing 0.04% CO₂) - NP-40 nonionic detergent (Nonidet) P-40 - PAGE polyacrylamide gel electrophoresis - PAP peroxidase-antiperoxidase conjugate - RuBisCO ribulose-1,5-bisphosphate carboxylase/oxygenase - RuBP ribulose-1,5-bisphosphate - SDS sodium dodecylsulfate     

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.     

Mutations in loop six of the large subunit of ribulose-1,5-bisphosphate carboxylase affect substrate specificity

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 Synechococcus PCC 6301) was used to generate novel enzymes in Escherichia coli. Residues in C-terminal loop 6 of the / barrel structure of the large subunit were changed. Replacement of valine 331 with alanine caused a 90% reduction in V _max but did not alter the enzyme's relative specificity towards either of its gaseous substrates, CO₂ and O₂. However replacement of alanine 340 with glutamate decreased the enzyme's specificity for CO₂ but had no significant effect on either the K _m for ribulose-1,5-bisphosphate or CO₂ or on V _max. In contrast replacing a small cassette of residues 338-341 produced a small increase in the specificity factor.Abbreviations Rubisco ribulose-1,5-bisphosphate carboxylase/oxygenase - RuBP ribulose-1,5-bisphosphate - CABP 2-carbox-yarabinitol-1,5-bisphosphate We thank Karen Moore for the statistical analysis of the specificity factors. We acknowledge helpful discussions with Jim Pitts and Richard Pickersgill. This work was aided by the invaluable technical assistance of Iain Major.     

Effects of temperature on the regulation of photosynthetic carbon assimilation in leaves of maize and barley

The aim of this work was to examine the effect of temperature in the range 5 to 30 ° C upon the regulation of photosynthetic carbon assimilation in leaves of the C₄ plant maize (Zea mays L.) and the C₃ plant barley (Hordeum vulgare L.). Measurements of the CO₂-assimilation rate in relation to the temperature were made at high (735 bar) and low (143 bar) intercellular CO₂ pressure in barley and in air in maize. The results show that, as the temperature was decreased, (i) in barley, pools of phosphorylated metabolites, particularly hexose-phosphate, ribulose 1,5-bisphosphate and fructose 1,6-bisphosphate, increased in high and low CO₂; (ii) in maize, pools of glycerate 3-phosphate, triose-phosphate, pyruvate and phosphoenolpyruvate decreased, reflecting their role in, and dependence on, intercellular transport processes, while pools of hexose-phosphate, ribulose 1,5-bis phosphate and fructose 1,6-bisphosphate remained approximately constant; (iii) the redox state of the primary electron acceptor of photosystem II (Q_A) increased slightly in barley, but rose abruptly below 12° C in maize. Non-photochemical quenching of chlorophyll fluorescence increased slightly in barley and increased to high values below 20 ° C in maize. The data from barley are consistent with the development of a limitation by phosphate status at low temperatures in high CO₂, and indicate an increasing regulatory importance for regeneration of ribulose 1,5-bisphosphate within the Calvin cycle at low temperatures in low CO₂. The data from maize do not show that any steps of the C₄ cycle are particularly cold-sensitive, but do indicate that a restriction in electron transport occurs at low temperature. In both plants the data indicate that regulation of product synthesis results in the maintenance of pools of Calvin-cycle intermediates at low temperatures.Abbreviations Glc6P glucose-6-phosphate - Fru6P fructase-6-phosphate - Frul,6bisP fructose-1,6-bisphosphate - PGA glycerate-3-phosphate - p _i intercellular partial pressure of CO₂ - RuBP ribulose-1,5-bisphosphate - triose-P sum of glyceraldehyde-3-phosphate and dihydroxyacetone phosphate We thank the Agricultural and Food Research Council, UK (Research grant PG50/67) and the Science and Engineering Research Council, UK for financial support. C.A.L. was supported by the British Council, by the Conselho Nacional de Desenvolvimento Cientiflco e Tecnologico (CNPq), Brazil and by an Overseas Research Student Award. We also thank Mark Stitt (Bayreuth, FRG) and Debbie Rees for helpful discussions.     

Effects of salt stress on the growth,ion content,stomatal behaviour and photosynthetic capacity of a salt-sensitive species,Phaseolus vulgaris L.

Phaseolus vulgaris (cv. Hawkesbury Wonder) was grown over a range of NaCl concentrations (0–150 mM), and the effects on growth, ion relations and photosynthetic performance were examined. Dry and fresh weight decreased with increasing external NaCl concentration while the root/shoot ratio increased. The Cl^- concentration of leaf tissue increased linearly with increasing external NaCl concentration, as did K⁺ concentration, although to a lesser degree. Increases in leaf Na⁺ concentration occurred only at the higher external NaCl concentrations (100 mM). Increases in leaf Cl^- were primarily balanced by increases in K⁺ and Na⁺. X-ray microanalysis of leaf cells from salinized plants showed that Cl^- concentration was high in both the cell vacuole and chloroplast-cytoplasm (250–300 mM in both compartments for the most stressed plants), indicating a lack of effective intracellular ion compartmentation in this species. Salinity had little effect on the total nitrogen and ribulose-1,5-bisphosphate (RuBP) carboxylase (EC 4.1.1.39) content per unit leaf area. Chlorophyll per unit leaf area was reduced considerably by salt stress, however. Stomatal conductance declined substantially with salt stress such that the intercellular CO₂ concentration (C _i) was reduced by up to 30%. Salinization of plants was found to alter the ¹³C value of leaves of Phaseolus by up to 5 and this change agreed quantitatively with that predicted by the theory relating carbon-isotope fractionation to the corresponding measured intercellular CO₂ concentration. Salt stress also brought about a reduction in photosynthetic CO₂ fixation independent of altered diffusional limitations. The initial slope of the photosynthesis versus C _i response declined with salinity stress, indicating that the apparent in-vivo activity of RuBP carboxylase was decreased by up to 40% at high leaf Cl^- concentrations. The quantum yield for net CO₂ uptake was also reduced by salt stress.Abbreviations and symbols A net CO₂ assimilation rate - C _a ambient CO₂ concentration - C _i intercellular CO₂ concentration - RuBP ribulose-1,5-bisphosphate - ¹³C ratio of ¹³C to ¹²C relative to standard limestone     

Ribulose-1,5-bisphosphate regeneration limitation in rice leaf photosynthetic acclimation to elevated CO2

Our previous study has demonstrated that both RuBP carboxylation limitation and RuBP regeneration limitation exist simultaneously in rice grown under free-air CO₂ enrichment (FACE, about 200 μmol mol⁻¹ above the ambient air CO₂ concentration) conditions [G.-Y. Chen, Z.-H. Yong, Y. Liao, D.-Y. Zhang, Y. Chen, H.-B. Zhang, J. Chen, J.-G. Zhu, D.-Q. Xu, Photosynthetic acclimation in rice leaves to free-air CO₂ enrichment related to both ribulose-1,5-bisphosphate carboxylase limitation and ribulose-1,5-bisphosphate regeneration limitation. Plant Cell Physiol. 46 (2005) 1036–1045]. To explore the mechanism for forming of RuBP regeneration limitation, we conducted the gas exchange measurements and some biochemical analyses in FACE-treated and ambient rice plants. Net CO₂ assimilation rate (A_net) in FACE leaves was remarkably lower than that in ambient leaves when measured at the same CO₂ concentration, indicating that photosynthetic acclimation to elevated CO₂ occurred. In the meantime the maximum electron transport rate (ETR) (J_max), maximum carboxylation rate (V_cmax) in vivo, and RuBP contents decreased significantly in FACE leaves. The whole chain electron transport rate and photophosphorylation rate reduced significantly while ETR of photosystem II (PSII) did not significantly decrease and ETR of photosystem I (PSI) was significantly increased in the chloroplasts from FACE leaves. Further, the amount of cytochrome (Cyt) f protein, a key component localized between PSII and PSI, was remarkably declined in FACE leaves. It appears that during photosynthetic acclimation the decline in the Cyt f amount is an important cause for the decreased RuBP regeneration capacity by decreasing the whole chain electron transport in FACE leaves.     

Pyrenoid protein from the brown alga Pilayella littoralis

Characterization by peptide mapping and amino acid analysis of the two major pyrenoid polypeptides from the brown alga Pilayella littoralis shows that they are very similar to the subunits of ribulose-1,5-bisphosphate carboxylase (EC 4.1.1.39) from this alga. The observed similarities are discussed in relation to previous pyrenoid protein characterization from members of the Chlorophyceae.Abbreviations DTT dithiothreitol - EDTA Na₂ ethylenediamine tetraacetic acid (disodium salt) - PMFS phenylmethylsul-phonylfluoride - PVPP polyvinylpyrrolidone - RuBP ribulose-1,5-bisphosphate - RuBPCase ribulose-1,5-bisphosphate carboxylase - SDS sodium dodecyl sulphate - SDS-PAGE sodium dodecyl sulphate polyacrylamide gel electrophoresis - TRIS 2-amino-2-(hydroxymethyl) propane-1,3-diol - TPCK L-1-tosylamido-2-phenylethylchoromethyl ketone     

Photosynthetic acclimation to CO2 enrichment related to ribulose-1,5-bisphosphate carboxylation limitation in wheat

Net photosynthetic rate (P _N) measured at the same CO₂ concentration, the maximum in vivo carboxylation rate, and contents of ribulose-1,5-bisphosphate (RuBP) carboxylase/oxygenase (RuBPCO) and RuBPCO activase were significantly decreased, but the maximum in vivo electron transport rate and RuBP content had no significant change in CO₂-enriched [EC, about 200 μmol mol⁻¹ above the ambient CO₂ concentration (AC)] wheat leaves compared with those in AC grown wheat leaves. Hence photosynthetic acclimation in wheat leaves to EC is largely due to RuBP carboxylation limitation.     

Formation of the tight-binding inhibitor, 3-ketoarabinitol-1,5-bisphosphate by ribulose-1,5-bisphosphate carboxylase/oxygenase is O2-dependent

Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) (EC 4.1.1.39) not only catalyzes carboxylation and oxygenation of ribulose-1,5-bisphosphate (RuBP), but it can also act either as an epimerase or isomerase converting RuBP into xylulose-1,5-bisphosphate (XuBP) or 3-ketoarabinitol-1,5-bisphosphate (KABP), respectively, a process called misfire. XuBP is formed as a result of misprotonation at C3 of the RuBP-enediol. It is released from Rubisco active sites and accumulates in the reaction mixture. Increasing the amounts of CO₂ or O₂ decreases XuBP production. However, KABP synthesis, which has been proposed to be only a product due to C2 misprotonation of the RuBP-endiol, is dependent upon the presence of O₂. KABP remains tightly bound to Rubisco active sites after its formation, causing the loss of Rubisco activity (fallover). The results suggest that the non-stabilized form of the peroxy-intermediate in the oxygenase reaction can be converted in a backreaction to KABP and molecular oxygen. The stabilization of the peroxy-intermediate due to the presence of Mn²⁺ instead of Mg²⁺ eliminates the formation of KABP.     

The occurrence of both C3 and C4 photosynthetic characteristics in a single Zea mays plant

The activities of the carboxylating enzymes ribulose-1,5-biphosphate (RuBP) carboxylase and phosphoenolpyruvate (PEP) carboxylase in leaves of three-week old Zea mays plants grown under phytotron conditions were found to vary according to leaf position. In the lower leaves the activity of PEP carboxylase was lower than that of RuBP carboxylase, while the upper leaves exhibited high levels of PEP carboxylase. Carbon dioxide compensation points and net photosynthetic rates also differed in the lower and upper leaves. Differences in the fine structure of the lowermost and uppermost leaves are shown. The existence of both the C₃ and C₄ photosynthetic pathways in the same plant, in this and other species, is discussed.Abbreviations PEP phosphoenolpyruvate - RuBP ribulose-1,5-biphosphate