Roland Cazalis

An antioxidant redox system in the nucleus of wheat seed cells suffering oxidative stress

Cereal seed cells contain different mechanisms for protection against the oxidative stress that occurs during maturation and germination. One such mechanism is based on the antioxidant activity of a 1-Cys peroxiredoxin (1-Cys Prx) localized in the nuclei of aleurone and scutellum cells. However, nothing is known about the mechanism of activation of this enzyme. Here, we describe the pattern of localization of NADPH thioredoxin reductase (NTR) in developing and germinating wheat seeds using an immunocytochemical analysis. The presence of NTR in transfer cells, vascular tissue, developing embryo and root meristematic cells, agrees with the localization of thioredoxin h (Trx h), and supports the important function of the NTR/Trx system in cell proliferation and communication. Interestingly, NTR is found in the nuclei of seed cells suffering oxidative stress, thus showing co-localization with Trx h and 1-Cys Prx. To test whether the NTR/Trx system serves as a reductant of the 1-Cys Prx, we cloned a full-length cDNA encoding 1-Cys Prx from wheat, and expressed the recombinant protein in Escherichia coli. Using the purified components, we show NTR-dependent activity of the 1-Cys Prx. Mutants of the 1-Cys Prx allowed us to demonstrate that the peroxidatic residue of the wheat enzyme is Cys46, which is overoxidized in vitro under oxidant conditions. Analysis of extracts from developing and germinating seeds confirmed 1-Cys Prx overoxidation in vivo. Based on these results, we propose that NADPH is the source of the reducing power to regenerate 1-Cys Prx in the nuclei of seed cells suffering oxidative stress, in a process that is catalyzed by NTR.

Sulfhydryl-disulfide changes in storage proteins of developing wheat grain: Influence on the SDS-unextractable glutenin polymer formation

Abstract To understand more precisely the function of free glutenin SH and SS groups in glutenins of developing wheat for UPP formation, a specific sulfhydryl probe, monobromobimane (mBBr), was used for an in vitro protein labeling. By applying this procedure to two varieties of wheat differing in high molecular weight glutenin subunit composition (2*, 7+8, 5+10 and 0, 6+8, 2+12, respectively, for Soissons and Thesee), we showed that the major wheat glutenin subunits residing in the protein body undergo redox change during the development and the maturation of the grain. Indeed, during the cell division and the cell enlargement phase, glutenin subunits and particularly LMW-GS have a large amount of free SH groups and become oxidized during grain dehydration which coincided with the formation of UPP. Moreover, mBBr derivatization of free glutenin SH groups before the artificial grain desiccation inhibits the UPP deposition. As HPSEC-MALLS analysis showed, the alkylation of free glutenin SH groups before the desiccation induces an increase in the SDS solubility of the polymeric proteins by reducing both their molecular weight distribution and their compactness. These results are discussed in connection with an ‘hyperaggregation model’ which has been proposed to explain the formation of glutenin polymer in wheat kernel.

PsTRXh1 and PsTRXh2 Are Both Pea h-Type Thioredoxins with Antagonistic Behavior in Redox Imbalances

Thioredoxins (TRXs) are small ubiquitous oxidoreductases involved in disulfide bond reduction of a large panel of target proteins. The most complex cluster in the family of plant TRXs is formed by h-type TRXs. In Arabidopsis (Arabidopsis thaliana), nine members of this subgroup were described, which are less well known than their plastidial counterparts. The functional study of type-h TRXs is difficult because of the high number of isoforms and their similar biochemical characteristics, thus raising the question whether they have specific or redundant functions. Type-h TRXs are involved in seed germination and self incompatibility in pollen-pistil interaction. Their function as antioxidants has recently been proposed, but further work is needed to clarify this function in plants. In this study, we describe two new h-type TRXs from pea (Pisum sativum; stated PsTRXh1 and PsTRXh2). By functional complementation of a yeast (Saccharomyces cerevisiae) trx1Δ trx2Δ double mutant, we demonstrate that PsTRXh1 is involved in the redox-imbalance control, possibly through its interaction with peroxiredoxins. In contrast, PsTRXh2 provokes a phenotype of hypersensitivity to hydrogen peroxide in the yeast mutant. Furthermore, we show differential gene expression and protein accumulation of the two isoforms, PsTRXh1 protein being abundantly detected in vascular tissue and flowers, whereas PsTRXh2 gene expression was hardly detectable. By comparison with previous data of additional PsTRXh isoforms, our results indicate specific functions for the pea h-type TRXs so far described.

Immunocytochemical localization of Pisum sativum TRXs f and m in non-photosynthetic tissues

Plants are the organisms containing the most complex multigenic family for thioredoxins (TRX). Several types of TRXs are targeted to chloroplasts, which have been classified into four subgroups: m, f, x, and y. Among them, TRXs f and m were the first plastidial TRXs characterized, and their function as redox modulators of enzymes involved in carbon assimilation in the chloroplast has been well-established. Both TRXs, f and m, were named according to their ability to reduce plastidial fructose-1,6-bisphosphatase (FBPase) and malate dehydrogenase (MDH), respectively. Evidence is presented here based on the immunocytochemistry of the localization of f and m-type TRXs from Pisum sativum in non-photosynthetic tissues. Both TRXs showed a different spatial pattern. Whilst PsTRXm was localized to vascular tissues of all the organs analysed (leaves, stems, and roots), PsTRXf was localized to more specific cells next to xylem vessels and vascular cambium. Heterologous complementation analysis of the yeast mutant EMY63, deficient in both yeast TRXs, by the pea plastidial TRXs suggests that PsTRXm, but not PsTRXf, is involved in the mechanism of reactive oxygen species (ROS) detoxification. In agreement with this function, the PsTRXm gene was induced in roots of pea plants in response to hydrogen peroxide.

Circadian regulation of chloroplastic f and m thioredoxins through control of the CCA1 transcription factor

Chloroplastic thioredoxins f and m (TRX f and TRX m) mediate light regulation of carbon metabolism through the activation of Calvin cycle enzymes. The role of TRX f and m in the activation of Calvin cycle enzymes is best known among the TRX family. However, the discoveries of new potential targets extend the functions of chloroplastic TRXs to other processes in non-photosynthetic tissues. As occurs with numerous chloroplast proteins, their expression comes under light regulation. Here, the focus is on the light regulation of TRX f and TRX m in pea and Arabidopsis during the day/night cycle that is maintained during the subjective night. In pea (Pisum sativum), TRX f and TRX m1 expression is shown to be governed by a circadian oscillation exerted at both the transcriptional and protein levels. Binding shift assays indicate that this control probably involves the interaction of the CCA1 transcription factor and an evening element (EE) located in the PsTRX f and PsTRX m1 promoters. In Arabidopsis, among the multigene family of TRX f and TRX m, AtTRX f2 and AtTRX m2 mRNA showed similar circadian oscillatory regulation, suggesting that such regulation is conserved in plants. However, this oscillation was disrupted in plants overexpressing CCA1 (cca1-ox) or repressing CCA1 and LHY (cca1-lhy). The physiological role of the oscillatory regulation of chloroplastic TRX f and TRX m in plants during the day/night cycle is discussed.

Effect of Oxidative Treatment on Corn Seed Germination Kinetics

Corn seeds were treated with high purity oxygen ([O3] = 0 g/m3) and oxygen mixed with ozone ([O3] = 20 g/m3) during 6.8 or 20.5 minutes. Germination tests started immediately or 48 h after treatment. Effects of oxidative treatments on germination were determined by measuring seedlings and roots (>3 and >20 mm) rate at 3, 4 and 5 days of germination test. Results obtained for treated seed samples were higher than for untreated ones. A faster start of germination was observed for treated samples. This early germination start led to a larger number of germinated seeds with longer roots at 4 and 5 days. Nevertheless, too long an ozone treatment seemed to be unfavorable for seed growth, whereas a short one seemed to be most beneficial.

Plastid thioredoxins f and m are related to the developing and salinity response of post-germinating seeds of Pisum sativum.

Plastid thioredoxins (TRXs) f and m have long been considered to regulate almost exclusively photosynthesis-related processes. Nonetheless, some years ago, we found that type-f and m TRXs were also present in non-photosynthetic organs such as roots and flowers of adult pea plants. In the present work, using pea seedlings 2-5 days old, we have determined the mRNA expression profile of the plastid PsTRX f, m1, and m2, together with the ferredoxin NADP reductase (FNR). Our results show that these TRX isoforms are expressed in cotyledons, underlying similar expression levels in roots for PsTRX m2. We have also noted plastid TRX expression in cotyledons of etiolated seedlings of Arabidopsis thaliana lines carrying constructs corresponding to PsTRX f and m1 promoters fused to the reporter gene GUS, pointing to a role in reserve mobilization. Furthermore, the response of plastid TRXs to NaCl and their capacity in restoring the growth of a TRX-deficient yeast under saline conditions suggest a role in the tolerance to salinity. We propose that these redox enzymes take part of the reserve mobilization in seedling cotyledons and we suggest additional physiological functions of PsTRX m2 in roots and PsTRX m1 in the salinity-stress response during germination.

Construction of chimeric cytosolic fructose-1,6-bisphosphatases by insertion of a chloroplastic redox regulatory cluster.

In order to transform cytosolic fructose-1,6-bisphosphatases (FBPase)(EC 3.1.3.11) into potential reductively-modulated chloroplast-type enzymes, we have constructed four chimeric FBPases, which display structural viability as deduced by previous modelling. In the X1-type BV1 and HL1 chimera the N-half of cytosolic sugar beet (Beta vulgaris L.) and human FBPases was fused with the C-half of the pea (Pisum sativum L.) chloroplast enzyme, which carries the cysteine-rich light regulatory sequence. In the X2-type BV2 and HL2 chimera this regulatory fragment was inserted in the corresponding site of the sugar beet cytosolic and human enzymes. Like the plant cytosolic FBPases, the chimeric enzymes show a low rise of activity by dithiothreitol. Both BV1 and BV2, but not HL1 and HL2, display a negligible activation by Trxf, but neither of them by Trxm. Antibodies raised against the pea chloroplast enzyme showed a positive reaction against the four chimeric FBPases and the human enzyme, but not against the sugar beet one. The four chimera display typical kinetics of cytosolic FBPases, with Km values in the 40–140 μM range. We conclude the existence of a structural capacity of cytosolic FBPases for incorporating the redox regulatory cluster of the chloroplast enzyme. However, the ability of these chimeric FBPases for anin vitro redox regulation seems to be scarce, limiting their use from a biotechnology standpoint inin vivo regulation of sugar metabolism.ResumenCon el objetivo de transformar fructosa-1,6-bisfosfatasas (FBPasa)(EC 3.1.3.11) citosólicas en otras de características cloroplastídicas potencialmente modulables por reducción, se han construido cuatro FBPasas quiméricas, cuyo modelaje previo las define como estructuralmente viables. En las quimeras llamadas de tipo X1, BV1 y HL1, las mitades N-terminal de la FBPasa citosólica de remolacha azucarera (Beta vulgaris L.) y humana se fusionaron con la mitad C-terminal de la FBPasa cloroplastídica de guisante (Pisum sativum L.), que lleva la secuencia, rica en cisteina, responsable de la regulación por luz de la enzima. En las denominadas tipo X2, BV2 y HL2, dicho fragmento regulatorio se insertó en el lugar correspondiente de las FBPasas citosólica de remolacha y humana. Como ocurre con las FBPasas citosólicas de plantas, las cuatro enzimas quiméricas mostraron un bajo aumento de actividad por DTT. Al mismo tiempo las quimeras BV1 y BV2, pero no las HL1 y HL2, exhibieron una escasa activación por tiorredoxinaf (Trxf), mientras que ninguna de ellas por Trxm. Anticuerpos frente a la enzima cloroplastídica de guisante mostraron una reacción positiva (Western) frente a las FBPasas quiméricas y humana, pero no frente a la enzima citosólica de remolacha. Las cuatro quimeras exhibieron cinéticas características de FBPasas citosólicas, con valores de Km en el intervalo 40–140 μM. De todo ello se deduce una capacidad estructural de las FBPasas citosólicas para incorporar la secuencia de la enzima cloroplastídica responsable de la regulación por reducción. Sin embargo, la capacidad de estas FBPasas quiméricas para una regulación rédox, al menosin vitro, parece ser escasa, lo que puede limitar su uso biotecnológicopara la regulaciónin vivo del metabolismo de los azúcares en plantas.

Homology modeling and molecular dynamics simulations of the N‐terminal domain of wheat high molecular weight glutenin subunit 10

High molecular weight glutenin subunits (HMW‐GS) are of a particular interest because of their biomechanical properties, which are important in many food systems such as breadmaking. Using fold‐recognition techniques, we identified a fold compatible with the N‐terminal domain of HMW‐GS Dy10. This fold corresponds to the one adopted by proteins belonging to the cereal inhibitor family. Starting from three known protein structures of this family as templates, we built three models for the N‐terminal domain of HMW‐GS Dy10. We analyzed these models, and we propose a number of hypotheses regarding the N‐terminal domain properties that can be tested experimentally. In particular, we discuss two possible ways of interaction between the N‐terminal domains of the y‐type HMW glutenin subunits. The first way consists in the creation of interchain disulfide bridges. According to our models, we propose two plausible scenarios: (1) the existence of an intrachain disulfide bridge between cysteines 22 and 44, leaving the three other cysteines free of engaging in intermolecular bonds; and (2) the creation of two intrachain disulfide bridges (involving cysteines 22–44 and cysteines 10–55), leaving a single cysteine (45) for creating an intermolecular disulfide bridge. We discuss these scenarios in relation to contradictory experimental results. The second way, although less likely, is nevertheless worth considering. There might exist a possibility for the N‐terminal domain of Dy10, Nt‐Dy10, to create oligomers, because homologous cereal inhibitor proteins are known to exist as monomers, homodimers, and heterooligomers. We also discuss, in relation to the function of the cereal inhibitor proteins, the possibility that this N‐terminal domain has retained similar inhibitory functions.

Thioredoxin h System and Wheat Seed Quality

ABSTRACT Thioredoxin is one of the key systems controlling cellular redox balance in all living organisms. Plant thioredoxins are a diverse multigene family divided into two systems, the chloroplastic and the cytoplasmic systems, which are distinguishable by the electron donor and by the enzyme that catalyses thioredoxin reduction. In cereal seed, the thioredoxin (Trx) h system acts in the developing phase, controlling the delivery of compounds during seed filling. Early in the development of the imbibed seed, it promotes the mobilization of storage nitrogen and carbon in the endosperm by inhibiting the inactivators of amylolytic enzyme and by activating a specific serine protease, thiocalsin. During seed maturation and germination, the Trx h system controls oxidative stress in the living tissues, specifically in the scutellum and the aleurone layer, where it accumulates in the nucleus. The overexpression and the suppression of Trx confirm these features and constitute a powerful tool to manage seed quali...