Ribulose Bisphosphate Research Articles

Ab Initio Molecular Dynamics Simulation and Energetics of the Ribulose-1,5-biphosphate Carboxylation Reaction Catalyzed by Rubisco: Toward Elucidating the Stereospecific Protonation Mechanism.

In the carboxylation reaction catalyzed by ribulose 1,5-bisphosphate (RuBP) carboxylase-oxygenase (Rubisco), which is fundamental to photosynthesis, scission of a C-C bond in the six-carbon gemdiolate intermediate forms a carbanion that must be protonated stereospecifically to form product. It is thought that a conserved lysine side chain (LYS175 in spinach Rubisco), in the immediate vicinity of the carbanion, provides the necessary proton. Here, we endeavor to determine from the electronic-structure calculations whether protonation via this route is energetically possible. The two-dimensional energy surface was mapped to determine the minimum energy path (MEP) using density functional theory (B3LYP) and incorporating basis set superposition and classical (London) dispersion corrections. The potential of mean force (free energy) was then calculated from ab initio molecular dynamics simulations with umbrella sampling in the vicinity of the MEP on the scission-protonation reaction coordinate. MEP calculations were also carried out to evaluate the possibility of an active-site water near the phosphate (P1) of RuBP, with an excess proton positioned at P1, as an alternative facilitator of stereospecific protonation via a classical Grotthuss mechanism. In both cases, the C-C bond scission in the six-carbon intermediate and proton transfer from the donor was found to be concerted and highly asynchronous, without a stable carbanion intermediate. However, the free energy change was unfavorable for direct protonation by the LYS175 side chain. In contrast, the Grotthuss mechanism yielded stable products and an activation energy in good agreement with experiment. It also provides a plausible mechanism for alternative product formed in enzyme mutations at the LYS175 position and is consistent with the observed deuterium isotope effects.

Identification of drought stress-responsive proteins in common bean

Proteomics has emerged as a vital tool for high throughput systematic analysis of protein expression which helps to unlock various biochemical pathways. Keeping in view the relevance of differential expression studies at the post transcriptional level, we attempted to explore the candidate proteins that are triggered under low water stress conditions at vegetative and reproductive stages in common bean. The evaluation of drought-responsive proteins was performed by resolving the quantified protein extracted from leaf samples using SDS PAGE for detection of differential bands followed by trypsin digestion and MALDI-MS analysis. We report the presence of seven significant proteins where five proteins (Glucan endo-1.3 beta-glucosidase, Auxin-responsive protein, Endochitinase, Endochitinase CH5B and L-type lectin domain containing receptor kinase IV.3) were upregulated and two proteins (Ribulose bisphosphate carboxylase/oxygenase activase, ADP-ribosylation factor-like protein 8a) were down regulated. The study conducted herein indicates the strong interaction and varied activation of proteins at both the developmental stages. The identified proteins are primarily localized in nucleus, cell membrane, cytoskeleton, vacuole and chloroplast and play significant roles in plant defense, photosynthesis, and various molecular regulatory processes.

Mapping the \u2018early salinity response\u2019 triggered proteome adaptation in contrasting rice genotypes using iTRAQ approach

BackgroundTo delineate the adaptive mechanisms operative under salinity stress, it is essential to study plant responses at the very early stages of stress which are very crucial for governing plant survival and adaptation. We believe that it is the initial perception and response phase which sets the foundation for stress adaptation in rice seedlings where plants can be considered to be in a state of osmotic shock and ion buildup.ResultsAn isobaric Tags for Relative and Absolute Quantitation (iTRAQ) approach was used to analyze the pre-existing differences as well as the very early salt shock responsive changes in the proteome of seedlings of contrasting rice genotypes, viz salt-sensitive IR64 and salt-tolerant Pokkali. In response to a quick salt shock, shoots of IR64 exhibited hyperaccumulation of Na+, whereas in Pokkali, these ions accumulated more in roots. Interestingly, we could find 86 proteins to be differentially expressed in shoots of Pokkali seedlings under non-stress conditions whereas under stress, 63 proteins were differentially expressed in Pokkali shoots in comparison to IR64. However, only, 40 proteins under non-stress and eight proteins under stress were differentially expressed in Pokkali roots. A higher abundance of proteins involved in photosynthesis (such as, oxygen evolving enhancer proteins OEE1 & OEE3, PsbP) and stress tolerance (such as, ascorbate peroxidase, superoxide dismutase, peptidyl-prolyl cis-trans isomerases and glyoxalase II), was observed in shoots of Pokkali in comparison to IR64. In response to salinity, selected proteins such as, ribulose bisphosphate carboxylase/oxygenase activase, remained elevated in Pokkali shoots. Glutamate dehydrogenase - an enzyme which serves as an important link between Krebs cycle and metabolism of amino acids was found to be highly induced in Pokkali in response to stress. Similarly, other enzymes such as peroxidases and triose phosphate isomerase (TPI) were also altered in roots in response to stress.ConclusionWe conclude that Pokkali rice seedlings are primed to face stress conditions where the proteins otherwise induced under stress in IR64, are naturally expressed in high abundance. Through specific alterations in its proteome, this proactive stress machinery contributes towards the observed salinity tolerance in this wild rice germplasm.

Molecular characterization and in vitro interaction analysis of Op14-3-3 μ protein from Opuntia ficus-indica: identification of a new client protein from shikimate pathway

In plants, 14-3-3 proteins are important modulators of protein-protein interactions in response to environmental stresses. The aim of the present work was to characterize one Opuntia ficus-indica 14-3-3 and get information about its client proteins. To achieve this goal, O. ficus-indica 14-3-3 cDNA, named as Op14-3-3 μ, was amplified by 3’-RACE methodology. Op14-3-3 μ contains an Open Reading Frame of 786 bp encoding a 261 amino acids protein. Op14-3-3 μ cDNA was cloned into a bacterial expression system and recombinant protein was purified. Differential Scanning Fluorimetry, Dynamic Light Scattering, and Ion Mobility-Mass Spectrometry were used for Op14-3-3 μ protein characterization, and Affinity-Purification-Mass Spectrometry analysis approach was used to obtain information about their potential client proteins. Pyrophosphate-fructose 6-phosphate 1-phosphotransferase, ribulose bisphosphate carboxylase large subunit, and vacuolar-type H+-ATPase were identified. Interestingly chorismate mutase p-prephenate dehydratase was also identified. Op14-3-3 μ down-regulation was observed in Opuntia calluses when they were induced with Jasmonic Acid, while increased accumulation of Op14-3-3 μ protein was observed. The putative interaction of 14-3-3 μ with chorismate mutase, which have not been reported before, suggest that Op14-3-3 μ could be an important regulator of metabolites biosynthesis and responses to stress in Opuntia spp. SignificanceOpuntia species are important crops in arid and semiarid areas worldwide, but despite its relevance, little information about their tolerance mechanism to cope with harsh environmental conditions is reported. 14-3-3 proteins have gained attention due to its participation as protein-protein regulators and have been linked with primary metabolism and hormones responses. Here we present the characterization of the first Opuntia ficus-indica 14-3-3 (Op14-3-3) protein using affinity purification-mass spectrometry (AP-MS) strategy. Op14-3-3 has high homology with other 14-3-3 from Caryophyllales. A novel Op14-3-3 client protein has been identified; the chorismate mutase p-prephenate dehydratase, key enzyme that links the primary with secondary metabolism. The present results open new questions about the Opuntia spp. pathways mechanisms in response to environmental stress and the importance of 14-3-3 proteins in betalains biosynthesis.

Early events following phosphorus restriction involve changes in proteome and affects nitric oxide metabolism in soybean leaves

Phosphorus (P) is a macronutrient with structural and regulatory functions, essential for energy transfer. Under limited P availability, plant cells respond to internal signals, adjusting their metabolic pathways in order to reorganize physiological priorities. This work was conducted with the aim to explore the initial changes following P deprivation, before neither growth nor photosynthesis was strongly affected. The first unifoliate leaves of nine-day-old soybean plants (Glycine max, cv. Williams 82) were analyzed. Plants grown hydroponically under control conditions (+P, nutrient solution with 500 μM H2PO4−) were compared with those restricted in phosphate (-P, without H2PO4− in the nutrient solution). No visible symptoms of P-deficiency, such as changes in pigment intensity and leaf number or size, were observed up to 48 h of P deprivation. Neither fresh weight nor shoot/root FW ratio was affected in plants as a consequence of early P-restriction. Shotgun proteomic analyses of leaves from plants exposed to 24 h of P-deprivation, revealed that a total of 202 proteins were exclusively detected and 232 increased their relative abundance under –P conditions. The proteins affected belong mainly to the catalytic activity group according to Gene Ontology Consortium including proteins like 6-phosphogluconate dehydrogenase (from pentose phosphate pathway), pyrophosphate-fructose 6-phosphate 1-phosphotransferase subunit alpha, alpha-1,4 glucan phosphorylases (from carbohydrate metabolic processes), and other enzymes involved in metabolic processes such as glyceraldehyde-3-phosphate dehydrogenase, pyruvate kinase and fructose-bisphosphate aldolase (from gycolisis), and nitrate reductase (from nitrogen assimilation). Early events in leaves also involve higher levels of the bioactive molecule nitric oxide (NO), detected employing confocal laser microscopy, accompanied with a parallel increase in nitrate reductase activity. Furthermore, the presence of nitrated proteins was detected using shotgun liquid chromatography–mass spectrometry (LCMS/MS). This post-translational protein modification, affected proteins mainly related to photosynthesis (chlorophyll a–b binding protein, ribulose bisphosphate carboxylase), and some other metabolic processes (glutamine synthetase, methionine synthase, lipoxygenase). This methodology allowed the confirmation of nitration sites in proteins previously described as putatively nitrated. Taken together the data presented suggest important metabolic changes, including nitric oxide metabolism, during the first hours of P deprivation.

Role of Structural Features in Oligomerization, Active-Site Integrity and Ligand Binding of Ribose-1,5-Bisphosphate Isomerase

Pentose bisphosphate pathway, exclusively found in archaea, is similar to the pentose phosphate pathway present in bacteria and eukarya. In pentose bisphosphate pathway, the conversion of ribose moieties of nucleosides into 3-phosphoglycerate (3-PGA) involves multiple steps; one of them being the conversion of ribose-1,5-bisphosphate (R15P) to ribulose-1,5-bisphosphate (RuBP) catalyzed by an enzyme ribose-1,5-bisphosphate isomerase (R15Pi). The availability of the three-dimensional structure of R15Pi had facilitated the understanding of various structural and functional aspects of the enzyme. Nevertheless, the structure of R15Pi also left several significant questions unanswered that would aid in understanding the structure-function relationship of the enzyme. Thus, we have taken up a computational approach to further understand the role of various structural features of the enzyme R15Pi. Results obtained from molecular dynamics (MD) simulations aided in understanding the obligation of the enzyme R15Pi to oligomerize and also in deciphering the role of catalytic residue(s) in structural stability. Identification of invariant water molecules of the enzyme R15Pi helped in discerning their significance at the active-site pocket and structurally important regions. Further, molecular docking studies allowed the identification of the amino acid residues essential for holding the substrate (R15P) or product (RuBP) in the vicinity of the active site of the enzyme R15Pi. Interestingly, results of the molecular docking studies assisted in the identification of an “alternate binding site” for the substrate, R15P. Finally, based on these results, we propose a mechanism of “substrate sliding” for the enzyme R15Pi.

Combining gene network, metabolic and leaf-level models shows means to future-proof soybean photosynthesis under rising CO2

AbstractGlobal population increase coupled with rising urbanization underlies the predicted need for 60% more food by 2050, but produced on the same amount of land as today. Improving photosynthetic efficiency is a largely untapped approach to addressing this problem. Here, we scale modelling processes from gene expression through photosynthetic metabolism to predict leaf physiology in evaluating acclimation of photosynthesis to rising atmospheric concentrations of CO2 ([CO2]). Model integration with the yggdrasil interface enabled asynchronous message passing between models. The multiscale model of soybean (Glycine max) photosynthesis calibrated to physiological measures at ambient [CO2] successfully predicted the acclimatory changes in the photosynthetic apparatus that were observed at 550 ppm [CO2] in the field. We hypothesized that genetic alteration is necessary to achieve optimal photosynthetic efficiency under global change. Flux control analysis in the metabolic system under elevated [CO2] identified enzymes requiring the greatest change to adapt optimally to the new conditions. This predicted that Rubisco was less limiting under elevated [CO2] and should be down-regulated allowing re-allocation of resource to enzymes controlling the rate of regeneration of ribulose-1,5-bisphosphate (RuBP). By linking the Gene Regulatory Network through protein concentration to the metabolic model, it was possible to identify transcription factors (TFs) that matched the up- and down-regulation of genes needed to improve photosynthesis. Most striking was TF Gm-GATA2, which down-regulated genes for Rubisco synthesis while up-regulating key genes controlling RuBP regeneration and starch synthesis. The changes predicted for this TF most closely matched the physiological ideotype that the modelling predicted as optimal for the future elevated [CO2] world.

Phylogenetic analysis of Aquilaria Lam. (Thymelaeaceae) based on DNA barcoding

Abstract Aquilaria Lam. is an important group of trees that produce agarwood, which is widely used for manufacturing medicine, perfumes and incense. The members of the genus Aquilaria are close to being extinct due to illegal harvesting and are now protected in many countries. In this study, five DNA barcodes [internal transcribed spacer (ITS), maturase K (matK), psbA-trnH, ribulose bisphosphate carboxylase (rbcL) and trnL-trnF] and their combinations were evaluated for the discrimination of the major (16 out of 21) species of Aquilaria based on three criteria: sequence variation, genetic distances and the discrimination ability. In addition, we attempted to determine the phylogenetic relationship between Aquilaria and Gyrinops species using three phylogenetic analysis methods. We observed that the combination barcode ITS+trnL-trnF had a good discrimination ability based on the best match and best close match methods, provided more genetic information, and clearly indicated the comprehensive phylogenetic relationship between most Aquilaria and Gyrinops species. Considering the overall performance of these barcodes, the ITS+trnL-trnF is a suitable barcode for the identification of Aquilaria species.

Knockout of SlSBPASE Suppresses Carbon Assimilation and Alters Nitrogen Metabolism in Tomato Plants.

Sedoheptulose-1,7-bisphosphatase (SBPase) is an enzyme in the Calvin–Benson cycle and has been documented to be important in carbon assimilation, growth and stress tolerance in plants. However, information on the impact of SBPase on carbon assimilation and nitrogen metabolism in tomato plants (Solanum lycopersicum) is rather limited. In the present study, we investigated the role of SBPase in carbon assimilation and nitrogen metabolism in tomato plants by knocking out SBPase gene SlSBPASE using clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) gene editing technology. Compared with wild-type plants, slsbpase mutant plants displayed severe growth retardation. Further analyses showed that knockout of SlSBPASE led to a substantial reduction in SBPase activity and as a consequence, ribulose-1,5-bisphosphate (RuBP) regeneration and carbon assimilation rate were dramatically inhibited in slsbpase mutant plants. It was further observed that much lower levels of sucrose and starch were accumulated in slsbpase mutant plants than their wild-type counterparts during the photoperiod. Intriguingly, mutation in SlSBPASE altered nitrogen metabolism as demonstrated by changes in levels of protein and amino acids and activities of nitrogen metabolic enzymes. Collectively, our data suggest that SlSBPASE is required for optimal growth, carbon assimilation and nitrogen metabolism in tomato plants.

Elevated CO2 Improves Photosynthesis Under High Temperature by Attenuating the Functional Limitations to Energy Fluxes, Electron Transport and Redox Homeostasis in Tomato Leaves.

Elevated atmospheric CO2 improves leaf photosynthesis and plant tolerance to heat stress, however, the underlying mechanisms remain unclear. In this study, we exposed tomato plants to elevated CO2 (800 μmol mol-1) and/or high temperature (42°C for 24 h), and examined a range of photosynthetic and chlorophyll fluorescence parameters as well as cellular redox state to better understand the response of photosystem II (PSII) and PSI to elevated CO2 and heat stress. The results showed that, while the heat stress drastically decreased the net photosynthetic rate (Pn), maximum carboxylation rate (Vcmax), maximum ribulose-1,5-bis-phosphate (RuBP) regeneration rate (Jmax) and maximal photochemical efficiency of PSII (Fv/Fm), the elevated CO2 improved those parameters under heat stress and at a 24 h recovery. Furthermore, the heat stress decreased the absorption flux, trapped energy flux, electron transport, energy dissipation per PSII cross section, while the elevated CO2 had the opposing effects that eventually decreased photoinhibition, damage to photosystems and reactive oxygen species accumulation. Similarly, the elevated CO2 helped the plants to maintain a reduced redox state as evidenced by the increased ratios of ASA:DHA and GSH:GSSG under heat stress and at recovery. Furthermore, the concentration of NADP+ and ratio of NADP+ to NADPH were induced by elevated CO2 at recovery. This study unraveled the crucial mechanisms of elevated CO2-mediated changes in energy fluxes, electron transport and redox homeostasis under heat stress, and shed new light on the responses of tomato plants to combined heat and elevated CO2.

High plant density inhibits vegetative branching in cotton by altering hormone contents and photosynthetic production

Manual removal of vegetative branches (VB) is a traditional cultural practice widely adopted in Chinese cotton production, but the increasing labor cost in the country has reduced its cost-effectiveness. It is well known that high plant density inhibits the VB growth of cotton and can be a potential alternative to the traditional practice, but the underlying mechanisms are poorly understood. This study mainly aimed to determine how the VB growth of cotton could be inhibited by increasing plant density. Cotton was planted on 76-cm row widths at low (3 plants m−2), moderate (6 plants m−2) and high (9 plants m−2) plant densities. The VB growth and development, as well as the underlying agronomic, physiological and molecular events were examined. The high plant density decreased the number of VB by 67.3% and the ratio of VB biomass to total biomass by 95.0% at 125 days after seeding (DAS) compared with the low plant density. The net photosynthetic (Pn) rate, chlorophyll content and ribulose-1, 5-bisphosphate (RuBP) carboxylase activity in VB leaves were considerably decreased under high plant density, followed by reduced soluble sugar and starch contents, suggesting a suppressed photosynthetic production in the leaves. This decreased photosynthesis resulting from reduced RuBP carboxylase activity and chlorophyll content was one of the reasons for the poor VB growth under high plant density. High plant density increased the auxin (IAA) content and promoted auxin polar transport by increasing the expression of the auxin biosynthesis gene, GhYUC5 and auxin polar transport gene, GhPIN1. It also increased the content of cytokinins (CKs) by increasing the expression of the CKs biosynthesis gene, GhIPT3 in the main-stem tips, and decreasing the IAA and CKs contents as well as auxin polar transport by reducing the expression of GhYUC5, GhPIN1, GhPIN5 and GhIPT3 in the VB tips. The GhPhyB gene was down-regulated by high plant density, but the strigolactones (SLs) content was increased by up-regulating the SL receptor gene, GhD14 in the VB tips. The decreased IAA, CKs, GA and BR contents plus the increased content of SLs due to differential expression of hormone-related genes in the VB tips was another reason for the inhibition of vegetative branching. Although high plant density did not increase seedcotton yield, it improved the fiber quality by decreasing the number of VB-sourced bolls. Our results suggested that high plant density can be a substitute for the traditional plant pruning.

Optimizing photorespiration for improved crop productivity.

In C3 plants, photorespiration is an energy-expensive process, including the oxygenation of ribulose-1,5-bisphosphate (RuBP) by ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) and the ensuing multi-organellar photorespiratory pathway required to recycle the toxic byproducts and recapture a portion of the fixed carbon. Photorespiration significantly impacts crop productivity through reducing yields in C3 crops by as much as 50% under severe conditions. Thus, reducing the flux through, or improving the efficiency of photorespiration has the potential of large improvements in C3 crop productivity. Here, we review an array of approaches intended to engineer photorespiration in a range of plant systems with the goal of increasing crop productivity. Approaches include optimizing flux through the native photorespiratory pathway, installing non-native alternative photorespiratory pathways, and lowering or even eliminating Rubisco-catalyzed oxygenation of RuBP to reduce substrate entrance into the photorespiratory cycle. Some proposed designs have been successful at the proof of concept level. A plant systems-engineering approach, based on new opportunities available from synthetic biology to implement in silico designs, holds promise for further progress toward delivering more productive crops to farmer's fields.

Taxonomic reassessment of Ulva prolifera (Ulvophyceae, Chlorophyta) based on specimens from the type locality and Yellow Sea green tides

:Since 2008, the green seaweed Ulva prolifera has caused the world's largest green tide in the Yellow Sea, China. It has subsequently attracted considerable research interest. However, species identification is an essential step for advancing this research. Based on phylogenetic analyses using molecular sequences such as internal transcribed spacer (ITS) or ribulose bisphosphate carboxylase large chain (rbcL), specimens of U. prolifera collected worldwide were separated into a European clade and the Ulva linza–procera–prolifera (LPP) complex clade that included the Chinese bloom-forming strains and Japanese brackish strains. This has resulted in considerable controversy as to the identity of U. prolifera and the bloom-forming species in the Yellow Sea. To resolve this issue, we examined populations of U. prolifera from the type locality at Lolland Island, Denmark, and globally significant sites including sites from Japan and China using morphological, developmental, molecular and crossing studies. We found that almost all the Danish strains agree with the description of the type specimen and were included in the LPP clade. They had a branched morphology in culture and an obligate asexual life history with quadriflagellate zoosporoids. We conclude that this taxon in the LPP clade is true U. prolifera. Based on culture morphology, mating compatibility and the 5S rDNA spacer sequences, the Chinese bloom-forming strains were distinct, and the new subspecies U. prolifera subsp. qingdaoensis subsp. nov. is described. Strains of the European clade showing gamete incompatibility with the sexual members of the LPP clade were assigned to the species Ulva splitiana.

Plant-feeding phlebotomine sand flies, vectors of leishmaniasis, prefer Cannabis sativa

Blood-sucking phlebotomine sand flies (Diptera: Psychodidae) transmit leishmaniasis as well as arboviral diseases and bartonellosis. Sand fly females become infected with Leishmania parasites and transmit them while imbibing vertebrates' blood, required as a source of protein for maturation of eggs. In addition, both females and males consume plant-derived sugar meals as a source of energy. Plant meals may comprise sugary solutions such as nectar or honeydew (secreted by plant-sucking homopteran insects), as well as phloem sap that sand flies obtain by piercing leaves and stems with their needle-like mouthparts. Hence, the structure of plant communities can influence the distribution and epidemiology of leishmaniasis. We designed a next-generation sequencing (NGS)-based assay for determining the source of sand fly plant meals, based upon the chloroplast DNA gene ribulose bisphosphate carboxylase large chain (rbcL). Here, we report on the predilection of several sand fly species, vectors of leishmaniasis in different parts of the world, for feeding on Cannabis sativa We infer this preference based on the substantial percentage of sand flies that had fed on C. sativa plants despite the apparent "absence" of these plants from most of the field sites. We discuss the conceivable implications of the affinity of sand flies for C. sativa on their vectorial capacity for Leishmania and the putative exploitation of their attraction to C. sativa for the control of sand fly-borne diseases.

Proteomic Identification of Differentially Altered Proteins During Regeneration from Nodular Cluster Cultures in Vriesea reitzii (Bromeliaceae)

Tissue culture techniques have been employed for bromeliad mass propagation by means of the morphogenetic route of nodular cluster cultures (NC), which exhibit high regenerative potential. This study aimed to identify proteins differentially altered during the regeneration of shoots from NC in the bromeliad Vriesea reitzii. Proteomic analysis was performed by two-dimensional electrophoresis and MALDI-TOF mass spectrometry. Image analysis from NC, microshoots and shoots resulted in the detection of 345, 482, and 209 protein spots, respectively. Proteins related to the stress response (catalase, probable l-ascorbate peroxidase 3 and heat shock protein 81-1) and amino acid metabolism (glutamine synthetase, 4-hydroxy-tetrahydrodipicolinate reductase 1, serine hydroxymethyltransferase 1 and 14-3-3-like protein GF14-F) and other proteins (elongation factor Ts, probable signal peptidase complex subunit 2, calcium-dependent protein kinase 23, and ubiquitin-like protein-NEDD8-like protein RUB3) exhibited increased abundance in the early stages of development. On the other hand, proteins related to carbohydrate metabolism (ribulose bisphosphate carboxylase/oxygenase activase, ribulose bisphosphate carboxylase, and phosphoglycerate kinase 1) and ATP synthesis (ATP synthase subunit alpha and ATP synthase subunit beta) exhibited increased abundance in later stages. Changes in proteins related to cellular metabolic processes, the assembly of the photosynthetic mechanism, the response to medium stress and the dynamics of the cell cytoskeleton contribute to a high regenerative potential, which characterizes NC. To our knowledge, this is the first proteomic analysis performed during the regeneration process from NC in bromeliads. This study may provide important insights into proteins and cellular events involved in the micropropagation of this group of plants.

Effects of co-overproduction of sedoheptulose-1,7-bisphosphatase and Rubisco on photosynthesis in rice

ABSTRACT Overproduction of Rubisco did not proportionately increase the rate of CO2 assimilation (A) in rice probably because the capacity of regeneration of the substrate of Rubisco, ribulose-1,5-bisphosphate (RuBP), could not afford the increased Rubisco capacity. Since sedoheptulose-1,7-bisphosphatase (SBPase) is thought to be one of the limiting factors of RuBP regeneration capacity, SBPase and Rubisco were co-overproduced in rice in order to improve photosynthesis. Although SBPase and Rubisco contents increased by 82–102% and 20–30%, respectively, A did not increase under the conditions of high irradiance and different [CO2] partial pressures. Thus, co-overproduction of SBPase and Rubisco did not improve photosynthesis in rice. Overproduction and antisense suppression of SBPase did not greatly affect A at high [CO2], although it is thought to be determined by RuBP regeneration capacity. These results strongly suggest that SBPase does not determine RuBP regeneration capacity in rice. This is likely to be the reason for the unimproved photosynthesis by co-overproduction of SBPase and Rubisco.

Morpho-physiological and proteomic analysis to identify and characterise the traditional rice genotypes for drought tolerance

Rice is the major staple food for one-third of the world’s population. Rice requires high amount of water as compared to other cereal crops. In this context, we screened traditional rice genotypes collected from RARS, Pattambi and Ambalavayal for drought tolerance. Based on the morpho-physiological characters, the plants which showed wilting symptoms were screened according to international standard i.e., IRRI leaf score 7.0. The genotype which showed no wilting symptom even after 90% of the plants were showing wilting was identified as tolerant and the plant which showed early wilting symptom was identified as susceptible genotype. The genotype Chuvanna Modan has taken 25 days and the genotype Parambuvattan has taken only 10 days for wilting. After 90% of plants reached IRRI score 7.0 phenotypic observations i.e., electrical conductivity, measurement of stomatal conductance and transpiration rate using Infra red gas analyser were recorded. Root traits were also measured. The identified tolerant and susceptible genotypes were further subjected to 2D gel electrophoresis to identify and characterize the differentially expressed proteins for drought tolerance. Totally four differentially expressed protein spots were identified in Chuvanna Modan. The differential protein spots were sequenced by Matrix Assisted Laser Desorption/Ionization–Time of Flight Mass Spectrometry (MALDI–TOF/MS). Spot 1 and 2 were identified as ribulose bisphosphate carboxylase small subunit, spot 3 was identified as Protein tyrosine phosphatases and spot 4 was identified as Photosystem II stability/assembly factor HCF136.

Effects of microcompartmentation on flux distribution and metabolic pools in Chlamydomonas reinhardtii chloroplasts.

Cells and organelles are not homogeneous but include microcompartments that alter the spatiotemporal characteristics of cellular processes. The effects of microcompartmentation on metabolic pathways are however difficult to study experimentally. The pyrenoid is a microcompartment that is essential for a carbon concentrating mechanism (CCM) that improves the photosynthetic performance of eukaryotic algae. Using Chlamydomonas reinhardtii, we obtained experimental data on photosynthesis, metabolites, and proteins in CCM-induced and CCM-suppressed cells. We then employed a computational strategy to estimate how fluxes through the Calvin-Benson cycle are compartmented between the pyrenoid and the stroma. Our model predicts that ribulose-1,5-bisphosphate (RuBP), the substrate of Rubisco, and 3-phosphoglycerate (3PGA), its product, diffuse in and out of the pyrenoid, respectively, with higher fluxes in CCM-induced cells. It also indicates that there is no major diffusional barrier to metabolic flux between the pyrenoid and stroma. Our computational approach represents a stepping stone to understanding microcompartmentalized CCM in other organisms.

Revisiting the methionine salvage pathway and its paralogues.

SummaryMethionine is essential for life. Its chemistry makes it fragile in the presence of oxygen. Aerobic living organisms have selected a salvage pathway (the MSP) that uses dioxygen to regenerate methionine, associated to a ratchet‐like step that prevents methionine back degradation. Here, we describe the variation on this theme, developed across the tree of life. Oxygen appeared long after life had developed on Earth. The canonical MSP evolved from ancestors that used both predecessors of ribulose bisphosphate carboxylase oxygenase (RuBisCO) and methanethiol in intermediate steps. We document how these likely promiscuous pathways were also used to metabolize the omnipresent by‐products of S‐adenosylmethionine radical enzymes as well as the aromatic and isoprene skeleton of quinone electron acceptors.

Low pH-responsive proteins revealed by a 2-DE based MS approach and related physiological responses in Citrus leaves

BackgroundRare data are available on the molecular responses of higher plants to low pH. Seedlings of ‘Sour pummelo’ (Citrus grandis) and ‘Xuegan’ (Citrus sinensis) were treated daily with nutrient solution at a pH of 2.5, 3, or 6 (control) for nine months. Thereafter, we first used 2-dimensional electrophoresis (2-DE) to investigate low pH-responsive proteins in Citrus leaves. Meanwhile, we examined low pH-effects on leaf gas exchange, carbohydrates, ascorbate, dehydroascorbate and malondialdehyde. The objectives were to understand the adaptive mechanisms of Citrus to low pH and to identify the possible candidate proteins for low pH-tolerance.ResultsOur results demonstrated that Citrus were tolerant to low pH, with a slightly higher low pH-tolerance in the C. sinensis than in the C. grandis. Using 2-DE, we identified more pH 2.5-responsive proteins than pH 3-responsive proteins in leaves. This paper discussed mainly on the pH 2.5-responsive proteins. pH 2.5 decreased the abundances of proteins involved in ribulose bisphosphate carboxylase/oxygenase activation, Calvin cycle, carbon fixation, chlorophyll biosynthesis and electron transport, hence lowering chlorophyll level, electron transport rate and photosynthesis. The higher oxidative damage in the pH 2.5-treated C. grandis leaves might be due to a combination of factors including higher production of reactive oxygen species, more proteins decreased in abundance involved in antioxidation and detoxification, and lower ascorbate level. Protein and amino acid metabolisms were less affected in the C. sinensis leaves than those in the C. grandis leaves when exposed to pH 2.5. The abundances of proteins related to jasmonic acid biosynthesis and signal transduction were increased and decreased in the pH 2.5-treated C. sinensis and C. grandis leaves, respectively.ConclusionsThis is the first report on low pH-responsive proteins in higher plants. Thus, our results provide some novel information on low pH-toxicity and -tolerance in higher plants.