Enzyme co-localization in pea leaf chloroplasts: glyceraldehyde-3-P dehydrogenase,triose-P isomerase,aldolase and sedoheptulose bisphosphatase

Nearest neighbor analysis of immunocytolocalization experiments indicates that the enzymes glyceraldehyde-3-P dehydrogenase, triose-P isomerase and aldolase are located close to one another in the pea leaf chloroplast stroma, and that aldolase is located close to sedoheptulose bisphosphatase. Direct transfer of the triose phosphates between glyceraldehyde-3-P dehydrogenase and triose-P isomerase, and from glyceraldehyde-3-P dehydrogenase and triose-P isomerase to aldolase, is then a possibility, as is direct transfer of sedoheptulose bisphosphate from aldolase to sedoheptulose bisphosphatase. Spatial organization of these enzymes may be important for efficient CO₂ fixation in photosynthetic organisms. In contrast, there is no indication that fructose bisphosphatase is co-localized with aldolase, and direct transfer of fructose bisphosphate from aldolase to fructose bisphosphatase seems unlikely.     

Rubisco Activase mRNA Expression in Spinach: Modulation by Nanoanatase Treatment

Characterized by a photocatalysis property, nanoanatase is closely related to the photosynthesis of spinach. It could not only improve light absorbance, transformation from light energy to electron energy, and active chemical energy, but also promote carbon dioxide (CO₂) assimilation of spinach. However, the molecular mechanism of carbon reaction promoted by nanoanatase remains largely unclear. In this study, we report that the amounts of Rubisco activase (rca) mRNA in the nanoanatase-treated spinach were increased by about 51%, whereas bulk-TiO₂ treatment produced an increase of only 5%. Accordingly, the protein level of Rubisco activase from the nanoanatase-treated spinach was increased by 42% compared with the control; however, bulk-TiO₂ treatment resulted in a 5% improvement. Further analysis indicated that the activity of Rubisco activase in the nanoanatase-treated spinach was significantly higher than the control by up to 2.75 times, and bulk-TiO₂ treatment had no such significant effects. Together, one of the molecular mechanisms of carbon reaction promoted by nanoanatase is that the nanoanatase treatment results in the enhancement of rca mRNA expressions, protein levels, and activities of Rubisco activase, thereby leading to the improvement of Rubisco carboxylation and the high rate of photosynthetic carbon reaction.     

The activation state of Rubisco directly limits photosynthesis at low CO2 and low O2 partial pressures

Using gas exchange, enzyme assays, and theoretical modeling of photosynthetic responses to light and CO₂, we investigated whether decarbamylation of the active site of Rubisco at low CO₂ and low light leads to a condition where the activation state of Rubisco directly limits the rate of net CO₂ assimilation. Photosynthetic limitation by a reduction in the activation state of Rubisco would be indicated as a decline in the initial slope of the photosynthetic CO₂ response relative to what is predicted using theoretical models. In bean (Phaseolus vulgaris) and oat (Avena sativa), we saw no discrepancy between predicted and observed initial slope values at 200 and 400 mbar O₂, indicating no limitation by the carbamylation state of Rubisco. At 30 mbar O₂ and light saturation, we also saw no discrepancy between predicted and observed initial slope values; however, at subsaturating light intensity, our observed initial slope values were less than the modeled initial slope values that corresponded to an RuBP regeneration limitation. Moreover, significant reduction of the Rubisco activation state occurred in both species at 30 mbar O₂ and 30 μbar CO₂. When the model was reprogrammed to account for observed levels of Rubisco deactivation, the predicted and measured initial slope values at low O₂ and low PPFD were similar, indicating the reduction in carbamylation state accounted for the discrepancy. We interpret this as evidence for a direct limitation of the carbamylation state of Rubisco, probably because of a CO₂ limitation for carbamate formation. This limitation was only observed at intercellular CO₂ levels below what is encountered in vivo. At physiologically relevant CO₂ levels in situ, the leaves maintained sufficient Rubisco activity to avoid cabamylation state limitations in the steady state. This revised version was published online in June 2006 with corrections to the Cover Date.     

Carbonic Anhydrase Activities in Pea Thylakoids

Pea thylakoids with high carbonic anhydrase (CA) activity (average rates of 5000 µmol H⁺ (mg Chl)^–1 h^–1 at pH 7.0) were prepared. Western blot analysis using antibodies raised against the soluble stromal -CA from spinach clearly showed that this activity is not a result of contamination of the thylakoids with the stromal CA but is derived from a thylakoid membrane-associated CA. Increase of the CA activity after partial membrane disintegration by detergent treatment, freezing or sonication implies the location of the CA in the thylakoid interior. Salt treatment of thylakoids demonstrated that while one part of the initial enzyme activity is easily soluble, the rest of it appears to be tightly associated with the membrane. CA activity being measured as HCO₃ ^– dehydration (dehydrase activity) in Photosystem II particles (BBY) was variable and usually low. The highest and most reproducible activities (approximately 2000 µmol H⁺ (mg Chl)^–1 h^–1) were observed in the presence of detergents (Triton X-100 or n-octyl--D-glucopyranoside) in low concentrations. The dehydrase CA activity of BBY particles was more sensitive to the lipophilic CA inhibitor, ethoxyzolamide, than to the hydrophilic CA inhibitor, acetazolamide. CA activity was detected in PS II core complexes with average rate of 13,000 µmol H⁺ (mg Chl)^–1 h^–1 which was comparable to CA activity in BBY particles normalized on a PS II reaction center basis.This revised version was published online in October 2005 with corrections to the Cover Date.     

Inactivation of the monocistronic rca gene in Anabaena variabilis suggests a physiological ribulose bisphosphate carboxylase/oxygenase activase-like function in heterocystous cyanobacteria

There was no discernible effect after incubating recombinant Anabaena Rubisco and carboxyarabinitol 1-phosphate with the product of the Anabaena rca gene. Since the unactivated cyanobacterial Rubisco is not readily inhibited by ribulose 1,5-bisphosphate and fallover is not observed, a genetic basis for the function of the Rubisco activase-like gene (rca) was sought. The monocistronic rca gene was inactivated in vivo and resulting mutant strains of A. variabilis were found to be incapable of synthesizing immunologically detected RCA protein. The requirement for the product of the rca gene in the light was further examined by measuring Rubisco activity in permeabilized whole cells of wild-type and rca mutant strains at different light intensities. In a 1% CO₂-air atmosphere, inactivation of rca reduced the ability of A. variabilis to elevate Rubisco activity under high light (73 mol quanta m^–2 s^–1), but had little effect under low light (8 mol m^–2 s^–1). For air-grown cultures, differences in the rates exhibited by the wild-type and rca mutant to fully activate Rubisco during a whole-cell assay were enhanced by increases in light intensity. The significance of the rca mutation was underlined by effects on growth as, unlike the wild-type, growth rates did not increase after cells transferred from low to high light intensities. Higher exogenous CO₂ concentrations (1%) were required to sustain a normal growth rate for the A. variabilis rca mutant. When grown in air levels of CO₂, the rca mutant not only needed longer times to double in cell density but also exhibited greatly diminished Rubisco activity compared with the wild-type strain. Despite the unusual properties of cyanobacterial Rubisco, these results suggest a physiological role for the product of the rca gene in maximizing the activity of Rubisco in heterocystous cyanobacteria.     

Effects of Carbonic Anhydrase Inhibitors on Proton Exchange and Photosynthesis in Pea Protoplasts

At concentrations of 100–200 M, ethoxyzolamide, a lipophilic inhibitor of carbonic anhydrase, considerably (by 60%) inhibited light-induced CO₂-dependent oxygen evolution in pea protoplasts at the optimum concentration of inorganic carbon (100 M CO₂) in the medium. At the same concentrations of the inhibitor, electron transport in isolated pea thylakoids was inhibited only by 6–9%. Acetazolamide, a water-soluble inhibitor of carbonic anhydrase, affected neither the rate of CO₂-dependent O₂evolution in protoplasts nor electron transport in thylakoid membranes. A light-dependent proton uptake by protoplasts was demonstrated. At pH 7.2, the induction kinetics and the rate of proton uptake were similar to those for CO₂-dependent O₂evolution. The rate of proton uptake was decreased twofold by 1 mM acetazolamide. This fact agrees with the notion that a membrane-bound carbonic anhydrase is operative in the plasma membrane of higher plant cells. A mechanism of its functioning is suggested. Possible functions of carbonic anhydrases in the cells of C₃-plants are discussed.     

The growth of soybean under free air [CO<Subscript>2</Subscript>] enrichment (FACE) stimulates photosynthesis while decreasing in vivo Rubisco capacity

Down-regulation of light-saturated photosynthesis (A_sat) at elevated atmospheric CO₂ concentration, [CO₂], has been demonstrated for many C₃ species and is often associated with inability to utilize additional photosynthate and/or nitrogen limitation. In soybean, a nitrogen-fixing species, both limitations are less likely than in crops lacking an N-fixing symbiont. Prior studies have used controlled environment or field enclosures where the artificial environment can modify responses to [CO₂]. A soybean free air [CO₂] enrichment (FACE) facility has provided the first opportunity to analyze the effects of elevated [CO₂] on photosynthesis under fully open-air conditions. Potential ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) carboxylation (V_c,max) and electron transport through photosystem II (J_max) were determined from the responses of A_sat to intercellular [CO₂] (C_i) throughout two growing seasons. Mesophyll conductance to CO₂ (g_m) was determined from the responses of A_sat and whole chain electron transport (J) to light. Elevated [CO₂] increased A_sat by 15–20% even though there was a small, statistically significant, decrease in V_c,max. This differs from previous studies in that V_c,max/J_max decreased, inferring a shift in resource investment away from Rubisco. This raised the C_i at which the transition from Rubisco-limited to ribulose-1,5-bisphosphate regeneration-limited photosynthesis occurred. The decrease in V_c,max was not the result of a change in g_m, which was unchanged by elevated [CO₂]. This first analysis of limitations to soybean photosynthesis under fully open-air conditions reveals important differences to prior studies that have used enclosures to elevate [CO₂], most significantly a smaller response of A_sat and an apparent shift in resources away from Rubisco relative to capacity for electron transport.Abbreviations FACE Free air [CO₂] enrichment - Rubisco Ribulose-1,5-bisphosphate carboxylase/oxygenase - RuBP Ribulose-1,5-bisphosphate - SoyFACE Soybean free air [CO₂] enrichment - VPD Vapor pressure deficit     

Relationship of photosynthetic acclimation to changes of Rubisco activity in field-grown winter wheat and barley during growth in elevated carbon dioxide

The responses of photosynthesis, Rubisco activity, Rubisco protein, leaf carbohydrates and total soluble protein to three carbon dioxide treatments were studied in winter wheat [Triticum aestivum (L.)] and barley [Hordeum vulgare (L.)]. Barley and wheat plants were grown in small field plots during 1995 and 1996 in clear, acrylic chambers (1.2–2.4 m²) and were provided with continuous carbon dioxide fertilization at concentrations of 350, 525 and 700 mol mol^–1. Photosynthetic rates of barley penultimate leaves and wheat flag leaves measured at growth carbon dioxide concentrations decreased with leaf age in all three CO₂ treatments during 1995 and 1996. Photosynthetic acclimation to elevated CO₂ was observed on seven of eight measurement dates for barley and ten of eleven measurement dates for wheat over both years. Initial Rubisco activity, total soluble protein and Rubisco protein in barley penultimate leaves and wheat flag leaves also decreased with leaf age. Total Rubisco activity was not used because of enzyme degradation. There was a significant CO₂ treatment effect on initial Rubisco activity, total soluble protein and Rubisco protein for wheat in 1995 and 1996 and for barley in 1995. Responses of barley penultimate leaf Rubisco activity and leaf protein concentrations to elevated carbon dioxide were nonsignificant in 1996. A significant CO₂ treatment effect also was detected when means of Rubisco activity, soluble protein and Rubisco protein for wheat flag leaves were combined over harvests and years. These three flag leaf parameters were not significantly different in the 350 and 525 mol mol^–1 CO₂ treatments but were decreased during growth in 700 mol mol^–1 CO₂ relative to the other two CO₂ treatments. Ratios of photosynthesis at 700 and 350 mol mol^–1 were compared to ratios of Rubisco activity at 700 and 350 mol mol^–1 using wheat flag leaf data from 1995 and 1996. Regression analysis of these data were linear [y = 0.586 + 1.103t x (r² = 0.432)] and were significant at P 0.05. This result indicated that photosynthetic acclimation was positively correlated with changes of initial Rubisco activity in wheat flag leaves in response to CO₂ enrichment. Effects of elevated CO₂ on wheat leaf proteins during 1995 and 1996 and on barley during 1995 were consistent with an acceleration of senescence.     

Carbohydrates of the freshwater alga Tribonema aequale. II. Preliminary photosynthetic studies with 14C

Tribonema aequale Pascher was cultured for 30 min and 2 h in Na₂ ¹⁴CO₃. An aliquot of the 2 h material was grown for a further 45 h in culture medium devoid of ¹⁴C. The alga was sequentially extracted and the respective radioactivities of the extracts were measured. Evidence is presented that not only mannitol and glucose, but also the water-soluble glucose-containing polysaccharides, are active metabolites. The polysaccharides soluble in 4% aqueous alkali appear to be continuously synthesised and that soluble in 18% alkali is apparently incorporated into the insoluble non-extractable polysaccharide during growth.     

The Effect of Abscisic Acid and Methyl Jasmonate on Carbonic Anhydrase Activity in Pea

Short-term (2 h) treatment with 10 μM abscisic acid decreased stomatal conductance and net photosynthetic rate, and increased carbonic anhydrase activity in pea seedlings. The treatment with 10 μM methyl jasmonate did not significantly affect these parameters. This revised version was published online in June 2006 with corrections to the Cover Date.     

Effect of surplus glucose on physiological and biochemical characteristics of sugar beet leaves in relation to the age of the leaf and the whole plant

Effect of surplus glucose on physiological and biochemical parameters of leaves of different age was investigated in sugar beet (Beta vulgaris L., subsp. saccharifera) plants in the stages of vegetative growth (SVG). Early and late SVG were differentiated by the ratio between the weights of roots and aboveground organs (0.10 and 0.35, respectively). The excess of Glu was produced by incubation of the disks excised from detached leaves in water or 0.1 M Glu at radiant flux density of 250 μmol/(m² s) with the light regime pattern described as night/day/night/light (8/16/8/3 h). In all the leaf disks incubated in water and glucose solution, the content of Glu and other soluble carbohydrates considerably increased as compared with their content in the leaves they were taken from. After disk incubation in water and glucose solution, the content of chlorophyll (a + b) rose as compared with its level in respective leaves in early SVG; in late SVG, it declined. In early SVG, the rate of the O₂ photosynthetic evolution (Ph) in the ageing leaves under saturating concentration of NaHCO₃ after incubation in water and Glu solution declined more considerably than in young leaves. In late SVG, incubation of leaf disks in water and Glu solution weakly affected P _n. The rate of O₂ dark consumption in the leaf disks of all the types of treatment increased after incubation in water and especially in Glu solution. Activity of soluble carbonic anhydrase (sCA) in the extracts from young leaves in early SVG after their incubation in water and Glu solution was essentially the same, but after the incubation of ageing leaves in Glu solution, it reliably decreased. In late SVG, sCA activity sharply decreased after incubation in water and Glu solution irrespective of the leaf age. In late SVG, activity of Rubisco in the young leaves did not change after their incubation in water but decreased after incubation of the leaves of the three ages in Glu solution. In early SVG, nonphotochemical fluorescence quenching (NPQ) in the young intact leaf was lower than in the ageing leaf, and after leaf incubation in water and Glu solution, it rose. In late SVG, the value of NPQ was greater than in early SVG and, in contrast to the leaves of early SVG, it declined after leaf incubation; in water, this decline was more pronounced than in the Glu solution. In early SVG, efficient quantum yield of photosystem II (PSII) was much greater than in late SVG and it declined in the leaves incubated with Glu. It was concluded that surplus Glu can maintain biosynthetic processes in the young leaves of young sugar beet plants (trophic function). A decline in the level of chlorophyll and the activities of sCA and Rubisco in the course of leaf development and senescence is considered as a symptom of the suppression of biosynthesis of proteins of chlorophyll-protein complexes and the enzymes (Rubisco and sCA).     

Inhibition by ethoxyzolamide of a photoacoustic uptake signal in leaves: Evidence for carbonic anhydrase catalyzed CO2-solubilisation

A photoacoustic pulse-modulation technique is applied for the study of a CO₂-stimulated gas uptake signal in leaves (Reising and Schreiber, Photosynthe Res 31: 227–238, 1992). It is shown that this uptake signal can be substantially suppressed by application of the carbonic anhydrase inhibitor, ethoxyzolamide, to leaf discs. This inhibitor does not affect the O₂-evolution signal in air or the chlorophyll fluorescence induction pattern at high CO₂, when non-saturating light intensities are used. On the basis of these findings it is concluded that at least a major part of the CO₂-stimulated photoacoustic uptake signal results from light-modulated CO₂-solubilisation catalysed by carbonic anhydrase. Modulated CO₂-solubilisation appears likely to be induced by light driven H⁺-translocation from the stroma into the thylakoid lumen. Comparison of the induction patterns of chlorophyll fluorescence quenching and the uptake signal suggests a correlation between membrane energisation and CO₂-uptake. The importance of O₂-dependent electron flow as a major cause of membrane energisation is discussed. It is proposed that in the absence of CO₂ the combination of Mehler- and ascorbate peroxidase reactions does not result in a photobaric signal, as O₂-uptake and O₂-evolution components cancel each other. Two main conclusions, which are of considerable importance for future practical applications of the photoacoustic method, are drawn from these findings: (1) When high CO₂ is applied to leaves, the photobaric uptake component may provide a unique means of monitoring the function of stromal carbonic anhydrase in vivo. (2) Brief flushing of the photoacoustic cell with air may prevent the occurrence of an uptake signal, thus allowing a straight-forward deconvolution into photothermal and O₂-evolution components.     

Diversity in forms and functions of carbonic anhydrase in terrestrial higher plants

The review summarizes current data on the existence in terrestrial higher plants of several carbonic anhydrase forms differing in their properties, molecular structure, and intracellular localization. Possible functions of these carbonic anhydrases are discussed as well as specific features of carbon-concentrating mechanisms in phototrophic tissues of plants with C₃ and C₄ pathways of photosynthesis.     

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.     

Activity of 1-Aminocyclopropane Carboxylic Acid Synthase in Two Mustard (Brassica juncea L.) Cultivars Differing in Photosynthetic Capacity

The pattern of activity of 1-aminocyclopropane carboxylic acid synthase (ACS) was similar to photosynthetic and growth traits observed at 30, 45, and 60 d after sowing in mustard (Brassica juncea L.) cultivars Varuna and RH 30 differing in photosynthetic capacity. Higher activity of ACS and therefore ethylene release in Varuna than RH 30 increased stomatal conductance, intercellular CO₂ concentration, carboxylation rate (carbonic anhydrase and intrinsic water use efficiency), and thus net photosynthetic rate (P _N) and leaf and plant dry masses (DM) at all sampling times. Moreover, Varuna also had larger leaf area which contributed to higher P _N and DM. A positive correlation between ACS activity and P _N and leaf area was found in both the cultivars. Thus ACS activity may affect P _N through ethylene-induced changes on foliar gas exchange and leaf growth.     

The roles of carbonic anhydrases in photosynthetic CO2 concentrating mechanisms

Cyanobacteria, algae, aquatic angiosperms and higher plants have all developed their own unique versions of photosynthetic CO₂ concentrating mechanisms (CCMs) to aid Rubisco in efficient CO₂ capture. An important aspect of all CCMs is the critical roles that the specialised location and function that various carbonic anhydrase enzymes play in the overall process, participating the interconversion of CO₂ and HCO₃ ⁻ species both inside and outside the cell. This review examines what we currently understand about the nature of the carbonic anhydrase enzymes, their localisation and roles in the various CCMs that have been studied in detail. This revised version was published online in August 2006 with corrections to the Cover Date.     

Leaf Photosynthesis in Eight Almond Tree Cultivars

Response of gas exchange traits to irradiance were studied in eight almond tree (Prunus amygdalus) cultivars: Desmayo Largueta, Falsa Barese, Garrigues, Lauranne, Marcona, Masbovera, Nonpareil and Ramillete, grafted on a hybrid rootstock almond × peach GF-677. From these responses cultivars can be classified from the best to the worst photosynthetic performance as follows: Falsa Barese, Masbovera, Marcona, Nonpareil, Ramillete, Desmayo Largueta, Lauranne and Garrigues. The highest net photosynthetic rate was 20.3 μmol m⁻² s⁻¹ in Falsa Barese. In the absence of water stress, photosynthetic rate was not limited by stomatal conductance. Consequently, non-stomatal limitations prevailed under such conditions. This revised version was published online in July 2006 with corrections to the Cover Date.     

Effect of O2 and CO2 on net CO2 exchange in a high-CO2-requiring mutant of Chlamydomonas reinhardtii during dark-light-dark transitions

Net CO₂ exchange was monitored through a dark-light-dark transition, under 2% and 21% O₂ in the presence and absence of CO₂, in Chlamydomonas reinhardtii wild type and the high-CO₂-requiring mutant ca-1-12-1C. Upon illumination at 350 l/l CO₂, ca-1-12-1C cell exhibited a large decrease in net CO₂ uptake following an initial surge of CO₂ uptake. Net CO₂ uptake subsequently attained a steady-state rate substantially lower than the maximum. A large, O₂-enchanced post-illumination burst of CO₂ efflux was observed after a 10-min illumination period, corresponding to a minimum in the net CO₂ uptake rate. A smaller, but O₂-insensitive post-illumination burst was observed following a 30-min illumination period, when net CO₂ uptake was at a steady-state rate. These post-illumination bursts appeared to reflect the release of an intracellular pool of inorganic carbon, which was much larger following the initial surge of net CO₂ uptake than during the subsequent steady-state CO₂ uptake period.With the mutant in CO₂-free gas, O₂-stimulated, net CO₂ efflux was observed in the light, and a small, O₂-dependent post-illumination burst was observed. With wild-type cells no CO₂ efflux was observed in the light in CO₂-free gas under either 2% or 21% O₂, but a small, O₂-dependent post-illumination burst was observed. These results were interpreted as indicating that photorespiratory rates were similar in the mutant and wild-type cells in the absence of CO₂, but that the wild-type cells were better able to scavenge the photorespiratory CO₂.     

岩溶区土壤微生物驱动的自养固碳过程与机制研究进展

自养微生物能够利用无机碳作为碳源合成自身营养物质,具有很强的环境适应能力,在富钙偏碱的岩溶区土壤的碳固定过程中扮演重要角色.本文综述了土壤微生物驱动的自养固碳过程、岩溶区土壤固碳功能微生物、自养固碳的分子机制及其产生的生态环境效应,并提出了亟待解决的关键科学问题.为深入研究和认识岩溶区土壤生态系统自养微生物驱动的固碳过...