An efficient protocol for extracting thylakoid membranes and total leaf proteins from Posidonia oceanica and other polyphenol-rich plants.

BACKGROUND: The extraction of thylakoids is an essential step in studying the structure of photosynthetic complexes and several other aspects of the photosynthetic process in plants. Conventional protocols have been developed for selected land plants grown in controlled conditions. Plants accumulate defensive chemical compounds such as polyphenols to cope with environmental stresses. When the polyphenol levels are high, their oxidation and cross-linking properties prevent thylakoid extraction. RESULTS: In this study, we developed a method to counteract the hindering effects of polyphenols by modifying the grinding buffer with the addition of both vitamin C (VitC) and polyethylene glycol (PEG4000). This protocol was first applied to the marine plant Posidonia oceanica and then extended to other plants synthesizing substantial amounts of polyphenols, such as Quercus pubescens (oak) and Vitis vinifera (grapevine). Native gel analysis showed that photosynthetic complexes (PSII, PSI, and LHCII) can be extracted from purified membranes and fractionated comparably to those extracted from the model plant Arabidopsis thaliana. Moreover, total protein extraction from frozen P. oceanica leaves was also efficiently carried out using a denaturing buffer containing PEG and VitC. CONCLUSIONS: Our work shows that the use of PEG and VitC significantly improves the isolation of native thylakoids, native photosynthetic complexes, and total proteins from plants containing high amounts of polyphenols and thus enables studies on photosynthesis in various plant species grown in natural conditions.

Thiol Reductases in Deinococcus Bacteria and Roles in Stress Tolerance.

Deinococcus species possess remarkable tolerance to extreme environmental conditions that generate oxidative damage to macromolecules. Among enzymes fulfilling key functions in metabolism regulation and stress responses, thiol reductases (TRs) harbour catalytic cysteines modulating the redox status of Cys and Met in partner proteins. We present here a detailed description of Deinococcus TRs regarding gene occurrence, sequence features, and physiological functions that remain poorly characterised in this genus. Two NADPH-dependent thiol-based systems are present in Deinococcus. One involves thioredoxins, disulfide reductases providing electrons to protein partners involved notably in peroxide scavenging or in preserving protein redox status. The other is based on bacillithiol, a low-molecular-weight redox molecule, and bacilliredoxin, which together protect Cys residues against overoxidation. Deinococcus species possess various types of thiol peroxidases whose electron supply depends either on NADPH via thioredoxins or on NADH via lipoylated proteins. Recent data gained on deletion mutants confirmed the importance of TRs in Deinococcus tolerance to oxidative treatments, but additional investigations are needed to delineate the redox network in which they operate, and their precise physiological roles. The large palette of Deinococcus TR representatives very likely constitutes an asset for the maintenance of redox homeostasis in harsh stress conditions.

Plastidial and cytosolic thiol reductases participate in the control of stomatal functioning.

Stomatal movements via the control of gas exchanges determine plant growth in relation to environmental stimuli through a complex signalling network involving reactive oxygen species that lead to post-translational modifications of Cys and Met residues, and alter protein activity and/or conformation. Thiol-reductases (TRs), which include thioredoxins, glutaredoxins (GRXs) and peroxiredoxins (PRXs), participate in signalling pathways through the control of Cys redox status in client proteins. Their involvement in stomatal functioning remains poorly characterized. By performing a mass spectrometry-based proteomic analysis, we show that numerous thiol reductases, like PRXs, are highly abundant in guard cells. When investigating various Arabidopsis mutants impaired in the expression of TR genes, no change in stomatal density and index was noticed. In optimal growth conditions, a line deficient in cytosolic NADPH-thioredoxin reductases displayed higher stomatal conductance and lower leaf temperature evaluated by thermal infrared imaging. In contrast, lines deficient in plastidial 2-CysPRXs or type-II GRXs exhibited compared to WT reduced conductance and warmer leaves in optimal conditions, and enhanced stomatal closure in epidermal peels treated with abscisic acid or hydrogen peroxide. Altogether, these data strongly support the contribution of thiol redox switches within the signalling network regulating guard cell movements and stomatal functioning.

Redox signaling through zinc activates the radiation response in Deinococcus bacteria.

Deinococcus bacteria are extremely resistant to radiation and other DNA damage- and oxidative stress-generating conditions. An efficient SOS-independent response mechanism inducing expression of several DNA repair genes is essential for this resistance, and is controlled by metalloprotease IrrE that cleaves and inactivates transcriptional repressor DdrO. Here, we identify the molecular signaling mechanism that triggers DdrO cleavage. We show that reactive oxygen species (ROS) stimulate the zinc-dependent metalloprotease activity of IrrE in Deinococcus. Sudden exposure of Deinococcus to zinc excess also rapidly induces DdrO cleavage, but is not accompanied by ROS production and DNA damage. Further, oxidative treatment leads to an increase of intracellular free zinc, indicating that IrrE activity is very likely stimulated directly by elevated levels of available zinc ions. We conclude that radiation and oxidative stress induce changes in redox homeostasis that result in IrrE activation by zinc in Deinococcus. We propose that a part of the zinc pool coordinated with cysteine thiolates is released due to their oxidation. Predicted regulation systems involving IrrE- and DdrO-like proteins are present in many bacteria, including pathogens, suggesting that such a redox signaling pathway including zinc as a second messenger is widespread and participates in various stress responses.

Involvement of the MetO/Msr System in Two Acer Species That Display Contrasting Characteristics during Germination.

The levels of methionine sulfoxide (MetO) and the abundances of methionine sulfoxide reductases (Msrs) were reported as important for the desiccation tolerance of Acer seeds. To determine whether the MetO/Msrs system is related to reactive oxygen species (ROS) and involved in the regulation of germination in orthodox and recalcitrant seeds, Norway maple and sycamore were investigated. Changes in water content, MetO content, the abundance of MsrB1 and MsrB2 in relation to ROS content and the activity of reductases depending on nicotinamide adenine dinucleotides were monitored. Acer seeds differed in germination speed-substantially higher in sycamore-hydration dynamics, levels of hydrogen peroxide, superoxide anion radicals (O2•-) and hydroxyl radicals (•OH), which exhibited peaks at different stages of germination. The MetO level dynamically changed, particularly in sycamore embryonic axes, where it was positively correlated with the levels of O2•- and the abundance of MsrB1 and negatively with the levels of •OH and the abundance of MsrB2. The MsrB2 abundance increased upon sycamore germination; in contrast, it markedly decreased in Norway maple. We propose that the ROS-MetO-Msr redox system, allowing balanced Met redox homeostasis, participates in the germination process in sycamore, which is characterized by a much higher speed compared to Norway maple.

Integration of MsrB1 and MsrB2 in the Redox Network during the Development of Orthodox and Recalcitrant Acer Seeds.

Two related tree species, Norway maple (Acer platanoides L.) and sycamore (Acer pseudoplatanus L.), produce desiccation-tolerant (orthodox) and desiccation-sensitive (recalcitrant) seeds, respectively. We compared the seeds of these two species to characterize the developmentally driven changes in the levels of peptide-bound methionine sulfoxide (MetO) and the abundance of methionine sulfoxide reductases (Msrs) B1 and B2, with respect to the cellular redox environment. Protein oxidation at the Met level was dynamic only in Norway maple seeds, and the reduced MsrB2 form was detected only in this species. Cell redox status, characterized by the levels of reduced and oxidized ascorbate, glutathione, and nicotinamide adenine dinucleotide (NAD)/phosphate (NADP), was clearly more reduced in the Norway maple seeds than in the sycamore seeds. Clear correlations between MetO levels, changes in water content and redox status were reported in orthodox Acer seeds. The abundance of Msrs was correlated in both species with redox determinants, mainly ascorbate and glutathione. Our data suggest that MsrB2 is associated with the acquisition of desiccation tolerance and that ascorbate might be involved in the redox pathway enabling the regeneration of Msr via intermediates that are not known yet.

Peptide-Bound Methionine Sulfoxide (MetO) Levels and MsrB2 Abundance Are Differentially Regulated during the Desiccation Phase in Contrasted Acer Seeds.

Norway maple and sycamore produce desiccation-tolerant (orthodox) and desiccation-sensitive (recalcitrant) seeds, respectively. Drying affects reduction and oxidation (redox) status in seeds. Oxidation of methionine to methionine sulfoxide (MetO) and reduction via methionine sulfoxide reductases (Msrs) have never been investigated in relation to seed desiccation tolerance. MetO levels and the abundance of Msrs were investigated in relation to levels of reactive oxygen species (ROS) such as hydrogen peroxide, superoxide anion radical and hydroxyl radical (•OH), and the levels of ascorbate and glutathione redox couples in gradually dried seeds. Peptide-bound MetO levels were positively correlated with ROS concentrations in the orthodox seeds. In particular, •OH affected MetO levels as well as the abundance of MsrB2 solely in the embryonic axes of Norway maple seeds. In this species, MsrB2 was present in oxidized and reduced forms, and the latter was favored by reduced glutathione and ascorbic acid. In contrast, sycamore seeds accumulated higher ROS levels. Additionally, MsrB2 was oxidized in sycamore throughout dehydration. In this context, the three elements •OH level, MetO content and MsrB2 abundance, linked together uniquely to Norway maple seeds, might be considered important players of the redox network associated with desiccation tolerance.

Is There a Role for Glutaredoxins and BOLAs in the Perception of the Cellular Iron Status in Plants?

Glutaredoxins (GRXs) have at least three major identified functions. In apoforms, they exhibit oxidoreductase activity controlling notably protein glutathionylation/deglutathionylation. In holoforms, i.e., iron-sulfur (Fe-S) cluster-bridging forms, they act as maturation factors for the biogenesis of Fe-S proteins or as regulators of iron homeostasis contributing directly or indirectly to the sensing of cellular iron status and/or distribution. The latter functions seem intimately connected with the capacity of specific GRXs to form [2Fe-2S] cluster-bridging homodimeric or heterodimeric complexes with BOLA proteins. In yeast species, both proteins modulate the localization and/or activity of transcription factors regulating genes coding for proteins involved in iron uptake and intracellular sequestration in response notably to iron deficiency. Whereas vertebrate GRX and BOLA isoforms may display similar functions, the involved partner proteins are different. We perform here a critical evaluation of the results supporting the implication of both protein families in similar signaling pathways in plants and provide ideas and experimental strategies to delineate further their functions.

Variability in the redox status of plant 2-Cys peroxiredoxins in relation to species and light cycle.

Plant 2-Cys peroxiredoxins (2-CysPRXs) are abundant plastidial thiol-peroxidases involved in key signaling processes such as photosynthesis deactivation at night. Their functions rely on the redox status of their two cysteines and on the enzyme quaternary structure, knowledge of which remains poor in plant cells. Using ex vivo and biochemical approaches, we thoroughly characterized the 2-CysPRX dimer/monomer distribution, hyperoxidation level, and thiol content in Arabidopsis, barley, and potato in relation to the light cycle. Our data reveal that the enzyme hyperoxidization level and its distribution as a dimer and monomer vary through the light cycle in a species-dependent manner. A differential susceptibility to hyperoxidation was observed for the two Arabidopsis 2-CysPRX isoforms and among the proteins of the three species, and was associated to sequence variation in hyperoxidation resistance motifs. Alkylation experiments indicate that only a minor fraction of the 2-CysPRX pool carries one free thiol in the three species, and that this content does not change during the light period. We conclude that most plastidial 2-CysPRX forms are oxidized and propose that there is a species-dependent variability in their functions since dimer and hyperoxidized forms fulfill distinct roles regarding direct oxidation of partners and signal transmission.

Physiological Roles of Plant Methionine Sulfoxide Reductases in Redox Homeostasis and Signaling.

Oxidation of methionine (Met) leads to the formation of two S- and R-diastereoisomers of Met sulfoxide (MetO) that are reduced back to Met by methionine sulfoxide reductases (MSRs), A and B, respectively. Here, we review the current knowledge about the physiological functions of plant MSRs in relation with subcellular and tissue distribution, expression patterns, mutant phenotypes, and possible targets. The data gained from modified lines of plant models and crop species indicate that MSRs play protective roles upon abiotic and biotic environmental constraints. They also participate in the control of the ageing process, as shown in seeds subjected to adverse conditions. Significant advances were achieved towards understanding how MSRs could fulfil these functions via the identification of partners among Met-rich or MetO-containing proteins, notably by using redox proteomic approaches. In addition to a global protective role against oxidative damage in proteins, plant MSRs could specifically preserve the activity of stress responsive effectors such as glutathione-S-transferases and chaperones. Moreover, several lines of evidence indicate that MSRs fulfil key signaling roles via interplays with Ca2+- and phosphorylation-dependent cascades, thus transmitting ROS-related information in transduction pathways.

Involvement of Arabidopsis glutaredoxin S14 in the maintenance of chlorophyll content.

Plant class-II glutaredoxins (GRXs) are oxidoreductases carrying a CGFS active site signature and are able to bind iron-sulfur clusters in vitro. In order to explore the physiological functions of the 2 plastidial class-II isoforms, GRXS14 and GRXS16, we generated knockdown and overexpression Arabidopsis thaliana lines and characterized their phenotypes using physiological and biochemical approaches. Plants deficient in one GRX did not display any growth defect, whereas the growth of plants lacking both was slowed. Plants overexpressing GRXS14 exhibited reduced chlorophyll content in control, high-light, and high-salt conditions. However, when exposed to prolonged darkness, plants lacking GRXS14 showed accelerated chlorophyll loss compared to wild-type and overexpression lines. We observed that the GRXS14 abundance and the proportion of reduced form were modified in wild type upon darkness and high salt. The dark treatment also resulted in decreased abundance of proteins involved in the maturation of iron-sulfur proteins. We propose that the phenotype of GRXS14-modified lines results from its participation in the control of chlorophyll content in relation with light and osmotic conditions, possibly through a dual action in regulating the redox status of biosynthetic enzymes and contributing to the biogenesis of iron-sulfur clusters, which are essential cofactors in chlorophyll metabolism.

Solanum tuberosum ZPR1 encodes a light-regulated nuclear DNA-binding protein adjusting the circadian expression of StBBX24 to light cycle.

ZPR1 proteins belong to the C4-type of zinc finger coordinators known in animal cells to interact with other proteins and participate in cell growth and proliferation. In contrast, the current knowledge regarding plant ZPR1 proteins is very scarce. Here, we identify a novel potato nuclear factor belonging to this family and named StZPR1. StZPR1 is specifically expressed in photosynthetic organs during the light period, and the ZPR1 protein is located in the nuclear chromatin fraction. From modelling and experimental analyses, we reveal the StZPR1 ability to bind the circadian DNA cis motif 'CAACAGCATC', named CIRC and present in the promoter of the clock-controlled double B-box StBBX24 gene, the expression of which peaks in the middle of the day. We found that transgenic lines silenced for StZPR1 expression still display a 24 h period for the oscillation of StBBX24 expression but delayed by 4 h towards the night. Importantly, other BBX genes exhibit altered circadian regulation in these lines. Our data demonstrate that StZPR1 allows fitting of the StBBX24 circadian rhythm to the light period and provide evidence that ZPR1 is a novel clock-associated protein in plants necessary for the accurate rhythmic expression of specific circadian-regulated genes.

Characterization of the Arabidopsis thaliana 2-Cys peroxiredoxin interactome.

Peroxiredoxins are ubiquitous thiol-dependent peroxidases for which chaperone and signaling roles have been reported in various types of organisms in recent years. In plants, the peroxidase function of the two typical plastidial 2-Cys peroxiredoxins (2-Cys PRX A and B) has been highlighted while the other functions, particularly in ROS-dependent signaling pathways, are still elusive notably due to the lack of knowledge of interacting partners. Using an ex vivo approach based on co-immunoprecipitation of leaf extracts from Arabidopsis thaliana wild-type and mutant plants lacking 2-Cys PRX expression followed by mass spectrometry-based proteomics, 158 proteins were found associated with 2-Cys PRXs. Already known partners like thioredoxin-related electron donors (Chloroplastic Drought-induced Stress Protein of 32kDa, Atypical Cysteine Histidine-rich Thioredoxin 2) and enzymes involved in chlorophyll synthesis (Protochlorophyllide OxidoReductase B) or carbon metabolism (Fructose-1,6-BisPhosphatase) were identified, validating the relevance of the approach. Bioinformatic and bibliographic analyses allowed the functional classification of the identified proteins and revealed that more than 40% are localized in plastids. The possible roles of plant 2-Cys PRXs in redox signaling pathways are discussed in relation with the functions of the potential partners notably those involved in redox homeostasis, carbon and amino acid metabolisms as well as chlorophyll biosynthesis.

Expression and characterization of a barley phosphatidylinositol transfer protein structurally homologous to the yeast Sec14p protein.

Phosphatidylinositol transfer proteins (PITPs) include a large group of proteins implicated in the non-vesicular traffic of phosphatidylinositol (PI) between membranes. In yeast, the structure and function of the PITP Sec14-p protein have been well characterized. In contrast, the knowledge on plant PITP proteins is very scarce. In this work, we characterized a novel type of PITP protein in barley named HvSec14p and related to the yeast Sec14-p protein. Our data reveal that HvSec14p consists of only the Sec14p-domain structurally homologous to the yeast phosphoinositide binding domain. We show that HvSec14p expression is up-regulated at both transcript and protein levels at specific stages of development during seed formation and germination, and in leaves of a drought-tolerant barley genotype under osmotic constraints. Modeling analyses of the protein three-dimensional structure revealed its capacity to dock the phosphoinositides, PtdIns(3)P, PtdIns(4)P, PtdIns(5)P and PtdIns(3,5)P2. Consistently, the recombinant HvSec14p protein is able to bind in vitro most PIP types, the highest affinity being observed with PtdIns(3,5)P2. Based on the high gene expression at specific developmental stages and in drought-tolerant barley genotypes, we propose that HvSec14p plays essential roles in the biogenesis of membranes in expanding cells and in their preservation under osmotic stress conditions.

Physiological relevance of plant 2-Cys peroxiredoxin overoxidation level and oligomerization status.

Peroxiredoxins are ubiquitous thioredoxin-dependent peroxidases presumed to display, upon environmental constraints, a chaperone function resulting from a redox-dependent conformational switch. In this work, using biochemical and genetic approaches, we aimed to unravel the factors regulating the redox status and the conformation of the plastidial 2-Cys peroxiredoxin (2-Cys PRX) in plants. In Arabidopsis, we show that in optimal growth conditions, the overoxidation level mainly depends on the availability of thioredoxin-related electron donors, but not on sulfiredoxin, the enzyme reducing the 2-Cys PRX overoxidized form. We also observed that upon various physiological temperature, osmotic and light stress conditions, the overoxidation level and oligomerization status of 2-Cys PRX can moderately vary depending on the constraint type. Further, no major change was noticed regarding protein conformation in water-stressed Arabidopsis, barley and potato plants, whereas species-dependent up- and down-variations in overoxidation were observed. In contrast, both 2-Cys PRX overoxidation and oligomerization were strongly induced during a severe oxidative stress generated by methyl viologen. From these data, revealing that the oligomerization status of plant 2-Cys PRX does not exhibit important variation and is not tightly linked to the protein redox status upon physiologically relevant environmental constraints, the possible in planta functions of 2-Cys PRX are discussed.

Arabidopsis glutaredoxin S17 and its partner, the nuclear factor Y subunit C11/negative cofactor 2α, contribute to maintenance of the shoot apical meristem under long-day photoperiod.

Glutaredoxins (GRXs) catalyze the reduction of protein disulfide bonds using glutathione as a reductant. Certain GRXs are able to transfer iron-sulfur clusters to other proteins. To investigate the function of Arabidopsis (Arabidopsis thaliana) GRXS17, we applied a strategy combining biochemical, genetic, and physiological approaches. GRXS17 was localized in the nucleus and cytosol, and its expression was elevated in the shoot meristems and reproductive tissues. Recombinant GRXS17 bound Fe2S2 clusters, a property likely contributing to its ability to complement the defects of a Baker's yeast (Saccharomyces cerevisiae) strain lacking the mitochondrial GRX5. However, a grxs17 knockout Arabidopsis mutant exhibited only a minor decrease in the activities of iron-sulfur enzymes, suggesting that its primary function is as a disulfide oxidoreductase. The grxS17 plants were sensitive to high temperatures and long-day photoperiods, resulting in elongated leaves, compromised shoot apical meristem, and delayed bolting. Both environmental conditions applied simultaneously led to a growth arrest. Using affinity chromatography and split-Yellow Fluorescent Protein methods, a nuclear transcriptional regulator, the Nuclear Factor Y Subunit C11/Negative Cofactor 2α (NF-YC11/NC2α), was identified as a GRXS17 interacting partner. A mutant deficient in NF-YC11/NC2α exhibited similar phenotypes to grxs17 in response to photoperiod. Therefore, we propose that GRXS17 interacts with NF-YC11/NC2α to relay a redox signal generated by the photoperiod to maintain meristem function.

Involvement of thiol-based mechanisms in plant development.

BACKGROUND: Increasing knowledge has been recently gained regarding the redox regulation of plant developmental stages. SCOPE OF VIEW: The current state of knowledge concerning the involvement of glutathione, glutaredoxins and thioredoxins in plant development is reviewed. MAJOR CONCLUSIONS: The control of the thiol redox status is mainly ensured by glutathione (GSH), a cysteine-containing tripeptide and by reductases sharing redox-active cysteines, glutaredoxins (GRXs) and thioredoxins (TRXs). Indeed, thiol groups present in many regulatory proteins and metabolic enzymes are prone to oxidation, ultimately leading to post-translational modifications such as disulfide bond formation or glutathionylation. This review focuses on the involvement of GSH, GRXs and TRXs in plant development. Recent studies showed that the proper functioning of root and shoot apical meristems depends on glutathione content and redox status, which regulate, among others, cell cycle and hormone-related processes. A critical role of GRXs in the formation of floral organs has been uncovered, likely through the redox regulation of TGA transcription factor activity. TRXs fulfill many functions in plant development via the regulation of embryo formation, the control of cell-to-cell communication, the mobilization of seed reserves, the biogenesis of chloroplastic structures, the metabolism of carbon and the maintenance of cell redox homeostasis. This review also highlights the tight relationships between thiols, hormones and carbon metabolism, allowing a proper development of plants in relation with the varying environment and the energy availability. GENERAL SIGNIFICANCE: GSH, GRXs and TRXs play key roles during the whole plant developmental cycle via their antioxidant functions and the redox-regulation of signaling pathways. This article is part of a Special Issue entitled Redox regulation of differentiation and de-differentiation.

Interplay between circadian rhythm, time of the day and osmotic stress constraints in the regulation of the expression of a Solanum Double B-box gene.

BACKGROUND AND AIMS: Double B-box zinc finger (DBB) proteins are recently identified plant transcription regulators that participate in the response to sodium chloride-induced stress in arabidopsis plants. Little is known regarding their subcellular localization and expression patterns, particularly in relation to other osmotic constraints and the day/night cycle. This study investigated natural variations in the amount of a Solanum DBB protein, SsBBX24, during plant development, and also under various environmental constraints leading to cell dehydration in relation to the circadian clock and the time of day. METHODS: SsBBX24 transcript and protein abundance in various organs of phytotron-grown Solanum tuberosum and S. sogarandinum plants were investigated at different time points of the day and under various osmotic constraints. The intracellular location of SsBBX24 was determined by western blot analysis of subcellular fractions. KEY RESULTS: Western blot analysis of SsBBX24 protein revealed that it was located in the nucleus at the beginning of the light period and in the cytosol at the end, suggesting movement ('trafficking') during the light phase. SsBBX24 gene expression exhibited circadian cycling under control conditions, with the highest and lowest abundances of both transcript and protein occurring 8 and 18 h after dawn, respectively. Exposing Solanum plants to low temperature, salinity and polyethylene glycol (PEG), but not to drought, disturbed the circadian regulation of SsBBX24 gene expression at the protein level. SsBBX24 transcript and protein accumulated in Solanum plants in response to salt and PEG treatments, but not in response to low temperature or water deficit. Most interestingly, the time of the day modulated the magnitude of SsBBX24 expression in response to high salt concentration. CONCLUSIONS: The interplay between circadian rhythm and osmotic constraints in the regulation of the expression of a Solanum DBB transcriptional regulator is demonstrated. It is proposed that stress-dependent, post-transcriptional mechanisms alter the regulation by the circadian clock of the amount of SsBBX24.

Overexpression of plastidial thioredoxins f and m differentially alters photosynthetic activity and response to oxidative stress in tobacco plants.

Plants display a remarkable diversity of thioredoxins (Trxs), reductases controlling the thiol redox status of proteins. The physiological function of many of them remains elusive, particularly for plastidial Trxs f and m, which are presumed based on biochemical data to regulate photosynthetic reactions and carbon metabolism. Recent reports revealed that Trxs f and m participate in vivo in the control of starch metabolism and cyclic photosynthetic electron transfer around photosystem I, respectively. To further delineate their in planta function, we compared the photosynthetic characteristics, the level and/or activity of various Trx targets and the responses to oxidative stress in transplastomic tobacco plants overexpressing either Trx f or Trx m. We found that plants overexpressing Trx m specifically exhibit altered growth, reduced chlorophyll content, impaired photosynthetic linear electron transfer and decreased pools of glutathione and ascorbate. In both transplastomic lines, activities of two enzymes involved in carbon metabolism, NADP-malate dehydrogenase and NADP-glyceraldehyde-3-phosphate dehydrogenase are markedly and similarly altered. In contrast, plants overexpressing Trx m specifically display increased capacity for methionine sulfoxide reductases, enzymes repairing damaged proteins by regenerating methionine from oxidized methionine. Finally, we also observed that transplastomic plants exhibit distinct responses when exposed to oxidative stress conditions generated by methyl viologen or exposure to high light combined with low temperature, the plants overexpressing Trx m being notably more tolerant than Wt and those overexpressing Trx f. Altogether, these data indicate that Trxs f and m fulfill distinct physiological functions. They prompt us to propose that the m type is involved in key processes linking photosynthetic activity, redox homeostasis and antioxidant mechanisms in the chloroplast.

A drought-sensitive barley variety displays oxidative stress and strongly increased contents in low-molecular weight antioxidant compounds during water deficit compared to a tolerant variety.

Barley displays a great genetic diversity, constituting a valuable source to delineate the responses of contrasted genotypes to environmental constraints. Here, we investigated the level of oxidative stress and the participation of antioxidant systems in two barley genotypes: Express, a variety known to be sensitive to drought, and Saïda, an Algerian landrace selected for its tolerance to water deficit. Soil-grown 15-day-old plants were subjected to water deficit for 8 days and then rewatered. We observed that upon water stress Express exhibits compared to Saïda accelerated wilting and a higher level of oxidative stress evaluated by HPLC measurements of lipid peroxidation and by imaging techniques. In parallel, Express plants also display lower levels of catalase and superoxide dismutase activity. No great difference was observed regarding peroxiredoxins and methionine sulfoxide reductases, enzymes detoxifying peroxides and repairing oxidized proteins, respectively. In contrast, upon water stress and recovery, much higher contents and oxidation ratios of glutathione and ascorbate were measured in Express compared to Saïda. Express also shows during water deficit greater increases in the pools of lipophilic antioxidants like xantophyll carotenoids and α-tocopherol. Altogether, these data show that the differential behavior of the two genotypes involves distinct responses regarding antioxidant mechanisms. Indeed, the drought sensitivity of Express compared with Saïda is associated with oxidative damage and a lower enzymatic ROS-scavenging capacity, but in parallel with a much stronger enhancement of most mechanisms involving low-molecular weight antioxidant compounds.