Accumulation of Polyphenolic Compounds and Osmolytes under Dehydration Stress and Their Implication in Redox Regulation in Four Indigenous Aromatic Rice Cultivars

Present work was undertaken to screen some drought tolerant indigenous aromatic rice cultivars(IARCs),commonly cultivated in West Bengal,India,based on their capacity to produce osmolytes,redox-sensitive phenolic acids and flavonoids,as contrivances for redox-regulation under drought stress.Polyethylene glycol induced post imbibitional dehydration stress mediated changes in redox regulatory properties of the germinating seeds of the four IARCs(Jamainadu,Tulaipanji,Sitabhog,Badshabhog),which were assessed in terms of changes in prooxidant accumulation(in-situ localization of reactive oxygen species(ROS)by confocal microscopy,DCFDA(2′,7′-dichlorofluorescin diacetate)oxidation,O2-and H2O2 accumulation),cumulative antioxidative defense(radical scavenging property and total thiol content),ROS scavenging phenolic acids(gallic acid,protocatechuic acid,gentisic acid,para-hydroxy benzoic acid,chlorogenic acid,caffeic acid,syringic acid,salicylic acid,sinapic acid and p-coumaric acid)and flavonoids(catechin,naringin,rutin,quercetin,kaempferol,myricetin and apigenin).The capability of germinating seeds to accumulate osmolytes(like glycinebetaine,proline,soluble carbohydrates and K+ion)and polyphenolic compounds was also correlated with their corresponding redox status and redox biomarkers(conjugated diene,hydroperoxide,thiobarbituric acid reactive substances and free carbonyl content)produced under the same conditions.The results in general showed that accumulation of osmolytes along with the redox-sensitive phenolics and flavonoids conferred the ability to maintain the redox homeostasis under drought stress for the tolerant IARCs(Badshabhog and Tulaipanji).

Redox Regulation of Arabidopsis Mitochondrial Citrate Synthase

Citrate synthase has a key role in the tricarboxylic （TCA） cycle of mitochondria of all organisms, as it cata- lyzes the first committed step which is the fusion of a carbon-carbon bond between oxaloacetate and acetyl CoA. The regulation of TCA cycle function is especially important in plants, since mitochondrial activities have to be coordinated with photosynthesis. The posttranslational regulation of TCA cycle activity in plants is thus far almost entirely unexplored. Although several TCA cycle enzymes have been identified as thioredoxin targets in vitro, the existence of any thioredoxin-dependent regulation as known for the Calvin cycle, yet remains to be demonstrated. Here we have investigated the redox regulation of the Arabidopsis citrate synthase enzyme by site-directed mutagenesis of its six cysteine residues. Our results indicate that oxidation inhibits the enzyme activity by the formation of mixed disulfides, as the partially oxidized citrate synthase enzyme forms large redox-dependent aggregates. Furthermore, we were able to demonstrate that thioredoxin can cleave diverse intraas well as intermolecular disulfide bridges, which strongly enhances the activity of the enzyme. Activity measurements with the cysteine variants of the enzyme revealed important cysteine residues affecting total enzyme activity as well as the redox sensitivity of the enzyme.

Redox Regulation of Glycogen Biosynthesis in the Cyanobacterium Synechocystis sp. PCC 6803： Analysis of the AGP and Glycogen Synthases

Glycogen constitutes the major carbon storage source in cyanobacteria, as starch in algae and higher plants. Glycogen and starch synthesis is linked to active photosynthesis and both of them are degraded to glucose in the dark to maintain cell metabolism. Control of glycogen biosynthesis in cyanobacteria could be mediated by the regulation of the enzymes involved in this process, ADP-glucose pyrophosphorylase （AGP） and glycogen synthase, which were identified as putative thioredoxin targets. We have analyzed whether both enzymes were subjected to redox modification using purified recombinant enzymes or cell extracts in the model cyanobacterium Synechocystis sp. PCC 6803. Our results indicate that both AGP and glycogen synthases are sensitive to copper oxidation. However, only AGP exhibits a decrease in its enzymatic activity, which is recovered after reduction by DTT or reduced thioredoxin （TrxA）, suggesting a redox control of AGP. In order to elucidate the role in redox control of the cysteine residues present on the AGP sequence （C45, C185, C320, and C337）, they were replaced with serine. All AGP mutant proteins remained active when expressed in Synechocystis, although they showed different electrophoretic mobility profiles after copper oxidation, reflecting a complex pattern of cysteines interaction.

The Deep Thioredoxome in Chlamydomonas reinhardtii： New Insights into Redox Regulation

Thiol-based redox post-translational modifications have emerged as important mechanisms of signaling and regulation in all organisms, and thioredoxin plays a key role by controlling the thiol-disulfide status of target proteins. Recent redox proteomic studies revealed hundreds of proteins regulated by glutathio- nylation and nitrosylation in the unicellular green alga Chlamydomonas reinhardtii, while much less is known about the thioredoxin interactome in this organism. By combining qualitative and quantitative proteomic analyses, we have comprehensively investigated the Chlamydomonas thioredoxome and 1188 targets have been identified. They participate in a wide range of metabolic pathways and cellular pro- cesses. This study broadens not only the redox regulation to new enzymes involved in well-known thiore- doxin-regulated metabolic pathways but also sheds light on cellular processes for which data supporting redox regulation are scarce （aromatic amino acid biosynthesis, nuclear transport, etc）. Moreover, we char- acterized 1052 thioredoxin-dependent regulatory sites and showed that these data constitute a valuable resource for future functional studies in Chlamydomonas. By comparing this thioredoxome with proteomic data for glutathionylation and nitrosylation at the protein and cysteine levels, this work confirms the existence of a complex redox regulation network in Chlamydomonas and provides evidence of a tremendous selectivity of redox post-translational modifications for specific cysteine residues.

The roles of microRNA in redox metabolism and exercise-mediated adaptation

MicroRNAs(miRs)are small regulatory RNA transcripts capable of post-transcriptional silencing of mRNA messages by entering a cellular bimolecular apparatus called RNA-induced silencing complex.miRs are involved in the regulation of cellular processes producing,eliminating or repairing the damage caused by reactive oxygen species,and they are active players in redox homeostasis.Increased mitochondrial biogenesis,function and hypertrophy of skeletal muscle are important adaptive responses to regular exercise.In the present review,we highlight some of the redox-sensitive regulatory roles of miRs.

Proteome-wide identification of S-sulfenylated cysteines response to salt stress in Brassica napus root

Reactive oxygen species(ROS)play a key role in a variety of biological processes,such as the perception of abiotic stress,the integration of different environmental signals,and the activation of stress response networks.Salt stress could induce an increased ROS accumulation in plants,disrupting intracellular redox homeostasis,leading to posttranslational modifications(PTMs)of specific proteins,and eventually causing adaptive changes in metabolism.Here,we performed an iodoTMT-based proteomic approach to identify the sulfenylated proteins in B.napus root responsing to salt stress.Totally,1348 sulfenylated sites in 751 proteins were identified and these proteins were widely existed in different cell compartments and processes.Our study revealed that proteins with changed abundance and sulfenylation level in B.napus root under salt stress were mainly enriched in the biological processes of ion binding,glycolysis,ATP binding,and oxidative stress response.This study displays a landscape of sulfenylated proteins response to salt stress in B.napus root and provides some theoretical support for further understanding of the molecular mechanisms of redox regulation under salt stress in plants.

Redox-Dependent Regulation of the Stress-Induced Zinc-Finger Protein SAP12 in Arabidopsis thaliana

The stress-associated protein SAP12 belongs to the stress-associated protein （SAP） family with 14 members in Arabidopsis thaliana. SAP12 contains two AN1 zinc fingers and was identified in diagonal 2D redox SDS-PAGE as a protein undergoing major redox-dependent conformational changes. Its transcript was strongly induced under cold and salt stress in a time-dependent manner similar to SAP10, with high levels after 6 h and decreasing levels after 24 and 48 h. The tran- script regulation resembled those of the stress marker peroxiredoxin PrxllD at 24 and 48 h. Recombinant SAP12 protein showed redox-dependent changes in quaternary structure as visualized by altered electrophoretic mobility in non-reducing SDS polyacrylamide gel electrophoresis. The oxidized oligomer was reduced by high dithiothreitol concentrations, and also by E. coli thioredoxin TrxA with low dithiothreitol （DTF） concentrations or NADPH plus NADPH-dependent thioredoxin reductase. From Western blots, the SAP12 protein amount was estimated to be in the range of 0.5 ngμg^-1 leaf protein. SAP12 protein decreased under salt and cold stress. These data suggest a redox state-linked function of SAP12 in plant cells particularly under cold and salt stress.

Quantitative proteomics reveals redox-based functional regulation of photosynthesis under fluctuating light in plants

Photosynthesis involves a series of redox reactions and is the major source of reactive oxygen species in plant cells.Fluctuating light(FL) levels,which occur commonly in natural environments,affect photosynthesis;however,little is known about the specific effects of FL on the redox regulation of photosynthesis.Here,we performed global quantitative mapping of the Arabidopsis thaliana cysteine thiol redox proteome under constant light and FL conditions.We identified8857 redox-switched thiols in 4350 proteins,and1501 proteins that are differentially modified depending on light conditions.Notably,proteins related to photosynthesis,especially photosystem I(PSI),are operational thiol-switching hotspots.Exposure of wild-type A.thaliana to FL resulted in decreased PSI abundance,stability,and activity.Interestingly,in response to PSI photodamage,more of the PSI assembly factor PSA3 dynamically switches to the reduced state.Furthermore,the Cys199 and Cys200 sites in PSA3 are necessary for its full function.Moreover,thioredoxin m(Trx m) proteins play roles in redox switching of PSA3,and are required for PSI activity and photosynthesis.This study thus reveals a mechanism for redox-based regulation of PSI under FL,and provides insight into the dynamic acclimation of photosynthesis in a changing environment.

High-Resolution Crystal Structure and Redox Properties of Chloroplastic Triosephosphate Isomerase from Chlamydomonas reinhardtii

Triosephosphate isomerase （TPI） catalyzes the interconversion of glyceraldehyde-3-phosphate to dihydroxyacetone phosphate. Photosynthetic organisms generally contain two isoforms of TPI located in both cytoplasm and chloroplasts. While the cytoplasmic TPI is involved in the glycolysis, the chloroplastic isoform participates in the Calvin-Benson cycle, a key photosynthetic process responsible for carbon fixation. Compared with its cytoplasmic counterpart, the functional features of chloroplastic TPI have been poorly investigated and its three-dimensional structure has not been solved. Recently, several studies proposed TPI as a potential target of different redox modifications including dithiol/disulfide interchanges, glutathionylation, and nitrosylation. However, neither the effects on protein activity nor the molecular mechanisms underlying these redox modifications have been investigated. Here, we have produced recombinantly and purified TPI from the unicellular green alga Chlamydomonas reinhardtii （Cr）. The biochemical properties of the enzyme were delineated and its crystallographic structure was determined at a resolution of 1.1 A. CrTPI is a homodimer with subunits containing the typical （β/α）8-barrel fold. Although no evidence for TRX regulation was obtained, CrTPI was found to undergo glutathionylation by oxidized glutathione and trans-nitrosylation by nitrosoglutathione, confirming its sensitivity to multiple redox modifications.

Thioredoxins Play a Crucial Role in Dynamic Acclimation of Photosynthesis in Fluctuating Light

Sunlight represents the energy source for photosynthesis and plant growth. When growing in the field, plant photosynthesis has to manage strong fluctuations in light intensities. Regulation based on the thio- redoxin （Trx） system is believed to ensure light-responsive control of photosynthetic reactions in the chlo- roplast. However, direct evidence for a role of this system in regulating dynamic acclimation of photosyn- thesis in fluctuating conditions is largely lacking. In this report we show that the ferredoxin-dependent Trxs ml and m2 as well as the NADPH-dependent NTRC are both indispensable for photosynthetic acclimation in fluctuating light intensities. Arabidopsis mutants with combined deficiency in Trxs ml and m2 show wild- type growth and photosynthesis under constant light condition, while photosynthetic parameters are strongly modified in rapidly alternating high and low light. Two independent trxmlm2 mutants show lower photosynthetic efficiency in high light, but surprisingly significantly higher photosynthetic efficiency in low light. Our data suggest that a main target of Trx ml and m2 is the NADP-malate dehydrogenase involved in export of excess reductive power from the chloroplast. The decreased photosynthetic efficiency in the high-light peaks may thus be explained by a reduced capacity of the trxm lm2 mutants in the rapid light acti-vation of this enzyme. In the ntrc mutant, dynamic responses of non-photochemical quenching of excita- tion energy and plastoquinone reduction state both were strongly attenuated in fluctuating light intensities, leading to a massive decrease in PSII quantum efficiency and a specific decrease in plant growth under these conditions. This is likely due to the decreased ability of the ntrc mutant to control the stromal NADP（H） redox poise. Taken together, our results indicate that NTRC is indispensable in ensuring the full range of dynamic responses of photosynthesis to optimize photosynthesis and maintain growth in fluctu- ating light, while Trxs ml and m2 are indispensable for full activation of photosynthesis in the high-light pe- riods but negatively affect photosynthetic efficiency in the low-light periods of fluctuating light.

Comparative Genomic Study of the Thioredoxin Family in Photosynthetic Organisms with Emphasis on Populus trichocarpa

The recent genome sequencing of Populus trichocarpa and Vitis vinifera, two models of woody plants, of Sorghum bicolor, a model of monocot using C4 metabolism, and of the moss Physcomitrella patens, together with the availability of photosynthetic organism genomes allows performance of a comparative genomic study with organisms having different ways of life, reproduction modes, biological traits, and physiologies. Thioredoxins （Trxs） are small ubiq- uitous proteins involved in the reduction of disulfide bridges in a variety of target enzymes present in all sub-cellular compartments and involved in many biochemical reactions. The genes coding for these enzymes have been identified in these newly sequenced genomes and annotated. The gene content, organization and distribution were compared to other photosynthetic organisms, leading to a refined classification. This analysis revealed that higher plants and bryo- phytes have a more complex family compared to algae and cyanobacteria and to non-photosynthetic organisms, since poplar exhibits 49 genes coding for typical and atypical thioredoxins and thioredoxin reductases, namely one-third more than monocots such as Oryza sativa and S. bicolor. The higher number of Trxs in poplar is partially explained by gene duplication in the Trx m, h, and nucleoredoxin classes. Particular attention was paid to poplar genes with emphasis on Trx-like classes called Clot, thioredoxin-like, thioredoxins of the lilium type and nucleoredoxins, which were not described in depth in previous genomic studies.

Ongoing Applicative Studies of Plant Thioredoxins

Studies triggered by the discovery of the function of thioredoxin （Trx） in photosynthesis have revealed its role throughout biology. Parallel biochemical and proteomic analyses have led to the identification of its numerous puta- tive targets. Recently, to verify the biological significance of these targets, in vivo studies using transformants in which Trx is overexpressed or suppressed are in progress, and the transformants themselves that are being used in such studies show their potential applicative values. Moreover, Trx＇s mitigation of allergenicity for some proteins offers promising prospects in the food industry. Practical studies based on redox regulation, once only on the horizon, are now achieving new dimensions. This short review focuses on the industrial applications of Trx studies, the current situation, and future perspectives. The putative targets obtained by the proteomics approach in comparison with in vivo observations of the transformants are also examined. Applicative studies of glutathione, a counterpart of Trx, are also discussed briefly.

Roles of Thioredoxins in the Obligate Anaerobic Green Sulfur Photosynthetic Bacterium Chlorobaculum tepidum

Thioredoxin is a small ubiquitous protein that is involved in the dithiol-disulfide exchange reaction, byway of two cysteine residues located on the molecule surface. In order to elucidate the role of thioredoxin in Chlorobaculum tepidurn, an anaerobic green sulfur bacterium that uses various inorganic sulfur compounds and H2S as electron donors under strict anaerobic conditions for growth, we applied the thioredoxin affinity chromatography method （Motohashi et al., 2001）. In this study, 37 cytoplasmic proteins were captured as thioredoxin target candidates, including proteins involved in sulfur assimilation. Furthermore, six of the candidate proteins were members of the reductive tricarboxylic acid cycle （pyruvate orthophosphate dikinase, pyruvate flavodoxin/ferredoxin oxidoreductase, ^-oxoglutarate synthase, citrate lyase, citrate synthase, malate dehydrogenase）. The redox sensitivity of three enzymes was then examined： citrate lyase, citrate synthase, and malate dehydrogenase, using their recombinant proteins. Based on the information relating to the target proteins, the significance of thioredoxin as a reductant for the metabolic pathway in the anaerobic photosynthetic bacteria is discussed.

Regulation of Calvin-Benson cycle enzymes under high temperature stress

The Calvin Benson cycle(CBC)consists of three critical processes,including fixation of CO_(2) by Rubisco,reduction of 3-phosphoglycerate(3PGA)to triose phosphate(triose-P)with NADPH and ATP generated by the light reactions,and regeneration of ribulose 1,5-bisphosphate(RuBP)from triose-P.The activ-ities of photosynthesis-related proteins,mainly from the CBC,were found more significantly affected and regulated in plants challenged with high temperature stress,incuding Rubisco,Rubisco activase(RCA) and the enzymes involved in RuBP regeneration,such as sedoheptulose-1,7-bisphosphatase(SBPase).Over the past years,the regulatory mechanism of CBC,especially for redox-regulation,has attracted major interest,because balancing flux at the various enzymatic reactions and maintaining metabolite levels in a range are of critical importance for the optimal operation of CBC under high temperature stress,providing insights into the genetic manipulation of photosynthesis.Here,we summarize recent progress regarding the identification of various layers of regulation point to the key enzymes of CBC for acclimation to environmental temperature changes along with open questions are also discussed.

Methionine oxidation and reduction of the ethylene signaling component MaEIL9 are involved in banana fruit ripening

The ethylene insensitive 3/ethylene insensitive3-like(EIN3/EIL)plays an indispensable role in fruit ripening.However,the regulatory mechanism that links post-translational modification of EIN3/EIL to fruit ripening is largely unknown.Here,we studied the expression of 13 MaE IL genes during banana fruit ripening,among which MaE IL9 displayed higher enhancement particularly in the ripening stage.Consistent with its transcript pattern,abundance of MaE IL9 protein gradually increased during the ripening process,with maximal enhancement in the ripening.DNA affinity purification(DAP)-seq analysis revealed that MaE IL9 directly targets a subset of genes related to fruit ripening,such as the starch hydrolytic genes MaA MY3D and MaB AM1.Stably overexpressing MaE IL9 in tomato fruit hastened fruit ripening,whereas transiently silencing this gene in banana fruit retarded the ripening process,supporting a positive role of MaEIL9 in fruit ripening.Moreover,oxidation of methionines(Met-129,Met-130,and Met-282)in MaEIL9 resulted in the loss of its DNA-binding capacity and transcriptional activation activity.Importantly,we identified MaEIL9 as a potential substrate protein of methionine sulfoxide reductase A MaMsrA4,and oxidation of Met-129,Met-130,and Met-282in MaEIL9 could be restored by MaMsrA4.Collectively,our findings reveal a novel regulatory network controlling banana fruit ripening,which involves MaMsrA4-mediated redox regulation of the ethylene signaling component MaEIL9.

Molecular Characterization of the Calvin Cycle Enzyme Phosphoribulokinase in the Stramenopile Alga Vaucheria litorea and the Plastid Hosting Mollusc Elysia chlorotica

Phosphoribulokinase （PRK）, a nuclear-encoded plastid-localized enzyme unique to the photosynthetic carbon reduction （Calvin） cycle, was cloned and characterized from the stramenopile alga Vaucheria litorea. This alga is the source of plastids for the mollusc （sea slug） Elysia chlorotica which enable the animal to survive for months solely by photoautotrophic CO2 fixation. The 1633-bp V. litorea prk gene was cloned and the coding region, found to be interrupted by four introns, encodes a 405-amino acid protein. This protein contains the typical bipartite target sequence expected of nuclearencoded proteins that are directed to complex （i.e. four membrane-bound） algal plastids. De novo synthesis of PRK and enzyme activity were detected in E. chlorotica in spite of having been starved of V. litorea for several months. Unlike the algal enzyme, PRK in the sea slug did not exhibit redox regulation. Two copies of partial PRK-encoding genes were isolated from both sea slug and aposymbiotic sea slug egg DNA using PCR. Each copy contains the nucleotide region spanning exon 1 and part of exon 2 of V litorea prk, including the bipartite targeting peptide. However, the larger prk fragment also includes intron 1. The exon and intron sequences of prk in E. chlorotica and V/itorea are nearly identical. These data suggest that PRK is differentially regulated in V. litorea and E. chlorotica and at least a portion of the V. litorea nuclear PRK gene is present in sea slugs that have been starved for several months.