Christophe Laloi

Transcriptomic Footprints Disclose Specificity of Reactive Oxygen Species Signaling in Arabidopsis

Reactive oxygen species (ROS) are key players in the regulation of plant development, stress responses, and programmed cell death. Previous studies indicated that depending on the type of ROS (hydrogen peroxide, superoxide, or singlet oxygen) or its subcellular production site (plastidic, cytosolic, peroxisomal, or apoplastic), a different physiological, biochemical, and molecular response is provoked. We used transcriptome data generated from ROS-related microarray experiments to assess the specificity of ROS-driven transcript expression. Data sets obtained by exogenous application of oxidative stress-causing agents (methyl viologen, Alternaria alternata toxin, 3-aminotriazole, and ozone) and from a mutant (fluorescent) and transgenic plants, in which the activity of an individual antioxidant enzyme was perturbed (catalase, cytosolic ascorbate peroxidase, and copper/zinc superoxide dismutase), were compared. In total, the abundance of nearly 26,000 transcripts of Arabidopsis (Arabidopsis thaliana) was monitored in response to different ROS. Overall, 8,056, 5,312, and 3,925 transcripts showed at least a 3-, 4-, or 5-fold change in expression, respectively. In addition to marker transcripts that were specifically regulated by hydrogen peroxide, superoxide, or singlet oxygen, several transcripts were identified as general oxidative stress response markers because their steady-state levels were at least 5-fold elevated in most experiments. We also assessed the expression characteristics of all annotated transcription factors and inferred new candidate regulatory transcripts that could be responsible for orchestrating the specific transcriptomic signatures triggered by different ROS. Our analysis provides a framework that will assist future efforts to address the impact of ROS signals within environmental stress conditions and elucidate the molecular mechanisms of the oxidative stress response in plants.

Rapid Induction of Distinct Stress Responses after the Release of Singlet Oxygen in Arabidopsis

The conditional fluorescent (flu) mutant of Arabidopsis accumulates the photosensitizer protochlorophyllide in the dark. After a dark-to-light shift, the generation of singlet oxygen, a nonradical reactive oxygen species, starts within the first minute of illumination and was shown to be confined to plastids. Immediately after the shift, plants stopped growing and developed necrotic lesions. These early stress responses of the flu mutant do not seem to result merely from physicochemical damage. Peroxidation of chloroplast membrane lipids in these plants started rapidly and led to the transient and selective accumulation of a stereospecific and regiospecific isomer of hydroxyoctadecatrieonic acid, free (13S)-HOTE, that could be attributed almost exclusively to the enzymatic oxidation of linolenic acid. Within the first 15 min of reillumination, distinct sets of genes were activated that were different from those induced by superoxide/hydrogen peroxide. Collectively, these results demonstrate that singlet oxygen does not act primarily as a toxin but rather as a signal that activates several stress-response pathways. Its biological activity in Arabidopsis exhibits a high degree of specificity that seems to be derived from the chemical identity of this reactive oxygen species and/or the intracellular location at which it is generated.

Cross-talk between singlet oxygen- and hydrogen peroxide-dependent signaling of stress responses in Arabidopsis thaliana

Upon a dark-to-light shift, the conditional fluorescent (flu) mutant of Arabidopsis releases singlet oxygen (1O2) within the plastid compartment. Distinct sets of nuclear genes are activated that are different from those induced by superoxide (O2•−) and/or hydrogen peroxide (H2O2), suggesting that different types of reactive oxygen species activate distinct signaling pathways. It is not known whether the pathways operate separately or interact with each other. We have addressed this problem by modulating noninvasively the level of H2O2 in plastids by means of a transgenic line that overexpresses the thylakoid-bound ascorbate peroxidase (tAPX). The overexpression of the H2O2-specific scavenger reduced strongly the activation of nuclear genes in plants treated with the herbicide paraquat that in the light leads to the enhanced generation of O2•− and H2O2. In the flu mutant overexpressing tAPX, the intensity of 1O2-mediated cell death and growth inhibition was increased when compared with the flu parental line. Also, the expression of most of the nuclear genes that were rapidly activated after the release of 1O2 was significantly higher in flu plants overexpressing tAPX, whereas in wild-type plants, overexpression of tAPX did not lead to visible stress responses and had only a very minor impact on nuclear gene expression. The results suggest that H2O2 antagonizes the 1O2-mediated signaling of stress responses as seen in the flu mutant. This cross-talk between H2O2- and 1O2-dependent signaling pathways might contribute to the overall stability and robustness of wild-type plants exposed to adverse environmental stress conditions.

Identification and characterization of a mitochondrial thioredoxin system in plants.

Plants possess two well described thioredoxin systems: a cytoplasmic system including several thioredoxins and an NADPH-dependent thioredoxin reductase and a specific chloroplastic system characterized by a ferredoxin-dependent thioredoxin reductase. On the basis of biochemical activities, plants also are supposed to have a mitochondrial thioredoxin system as described in yeast and mammals, but no gene encoding plant mitochondrial thioredoxin or thioredoxin reductase has been identified yet. We report the characterization of a plant thioredoxin system located in mitochondria. Arabidopsis thaliana genome sequencing has revealed numerous thioredoxin genes among which we have identified AtTRX-o1, a gene encoding a thioredoxin with a potential mitochondrial transit peptide. AtTRX-o1 and a second gene, AtTRX-o2, define, on the basis of the sequence and intron positions, a new thioredoxin type up to now specific to plants. We also have characterized AtNTRA, a gene encoding a protein highly similar to the previously described cytosolic NADPH-dependent thioredoxin reductase AtNTRB but with a putative presequence for import into mitochondria. Western blot analysis of A. thaliana subcellular and submitochondrial fractions and in vitro import experiments show that AtTRX-o1 and AtNTRA are targeted to the mitochondrial matrix through their cleavable N-terminal signal. The two proteins truncated to the estimated mature forms were produced in Escherichia coli; AtTRX-o1 efficiently reduces insulin in the presence of DTT and is reduced efficiently by AtNTRA and NADPH. Therefore, the thioredoxin and the NADPH-dependent thioredoxin reductase described here are proposed to constitute a functional plant mitochondrial thioredoxin system.

The Arabidopsis Cytosolic Thioredoxin h5 Gene Induction by Oxidative Stress and Its W-Box-Mediated Response to Pathogen Elicitor

The AtTRXh5 protein belongs to the cytosolic thioredoxins h family that, in Arabidopsis, contains eight members showing very distinct patterns and levels of expression. Here, we show that the AtTRXh5 gene is up-regulated during wounding, abscission, and senescence, as well as during incompatible interactions with the bacterial pathogen Pseudomonas syringae. By electrophoretic mobility shift assays, a binding activity on a W-box in the AtTRXh5 promoter region was found induced by treatments with the P. syringae-derived elicitor peptide flg22, suggesting that a WRKY transcription factor controls AtTRXh5 induction upon elicitor treatment. Remarkably, AtTRXh5 was up-regulated in plants overexpressing WRKY6. More generally, AtTRXh5 is induced in response to oxidative stress conditions. Collectively, our data indicate a possible implication of the cytosolic thioredoxin AtTRXh5 in response to pathogens and to oxidative stresses. In addition, this regulation is unique to AtTRXh5 among the thioredoxin h family, arguing in favor of a speciation rather than to a redundancy of the members of this multigenic family.

No single way to understand singlet oxygen signalling in plants.

When plant cells are under environmental stress, several chemically distinct reactive oxygen species (ROS) are generated simultaneously in various intracellular compartments and these can cause oxidative damage or act as signals. The conditional flu mutant of Arabidopsis, which generates singlet oxygen in plastids during a dark‐to‐light transition, has allowed the biological activity of singlet oxygen to be determined, and the criteria to distinguish between cytotoxicity and signalling of this particular ROS to be defined. The genetic basis of singlet‐oxygen‐mediated signalling has been revealed by the mutation of two nuclear genes encoding the plastid proteins EXECUTER (EX)1 and EX2, which are sufficient to abrogate singlet‐oxygen‐dependent stress responses. Conversely, responses due to higher cytotoxic levels of singlet oxygen are not suppressed in the ex1/ex2 background. Whether singlet oxygen levels lower than those that trigger genetically controlled cell death activate acclimation is now under investigation.

Covariations in the nuclear chloroplast transcriptome reveal a regulatory master-switch

The evolution of the endosymbiotic progenitor into the chloroplast organelle was associated with the transfer of numerous chloroplast genes into the nucleus. Hence, inter‐organellar signalling, and the co‐ordinated expression of sets of nuclear genes, was set up to control the metabolic and developmental status of the chloroplast. Here, we show by the differential‐expression analysis of 3,292 genes, that most of the 35 environmental and genetic conditions tested, including plastid signalling mutations, elicit only three main classes of response from the nuclear chloroplast transcriptome. Two classes, probably involving GUN (genomes uncoupled)‐type plastid signalling, are characterized by alterations, in opposite directions, in the expression of largely overlapping sets of genes.

iTRAQ-based analysis of changes in the cassava root proteome reveals pathways associated with post-harvest physiological deterioration.

The short storage life of harvested cassava roots is an important constraint that limits the full potential of cassava as a commercial food crop in developing countries. We investigated the molecular changes during physiological deterioration of cassava root after harvesting using isobaric tags for relative and absolute quantification (iTRAQ) of proteins in soluble and non-soluble fractions prepared during a 96 h post-harvest time course. Combining bioinformatic approaches to reduce information redundancy for unsequenced or partially sequenced plant species, we established a comprehensive proteome map of the cassava root and identified quantitatively regulated proteins. Up-regulation of several key proteins confirmed that physiological deterioration of cassava root after harvesting is an active process, with 67 and 170 proteins, respectively, being up-regulated early and later after harvesting. This included regulated proteins that had not previously been associated with physiological deterioration after harvesting, such as linamarase, glutamic acid-rich protein, hydroxycinnamoyl transferase, glycine-rich RNA binding protein, β-1,3-glucanase, pectin methylesterase, maturase K, dehydroascorbate reductase, allene oxide cyclase, and proteins involved in signal pathways. To confirm the regulation of these proteins, activity assays were performed for selected enzymes. Together, our results show that physiological deterioration after harvesting is a highly regulated complex process involving proteins that are potential candidates for biotechnology approaches to reduce such deterioration.

The multigenic family of thioredoxin h in Arabidopsis thaliana: specific expression and stress response

Abstract In the model plant Arabidopsis thaliana, cytosolic thioredoxins h (TRXh) are encoded by a multigenic family of eight genes. Genomic studies have revealed that a number of these genes are duplicated genes originating from a common ancestor. This multiplicity of thioredoxin h genes raises questions of the specificity of plant thioredoxins and the function of such a large multigenic family in plant. The results from studies using northern blots, semi-quantitative RT-PCR and transgenic promoter–GUS fusions provide strong evidence that the members of the AtTRXh gene family show expression levels that vary among different plant organs. Moreover, distinct AtTRXh genes are induced in response to pathogenic elicitors. Together, our data suggest that the members of the multigenic family of AtTRXh may not have redundant functions.

Arabidopsis mutants reveal multiple singlet oxygen signaling pathways involved in stress response and development.

Shortly after the release of singlet oxygen (1O2) in chloroplasts drastic changes in nuclear gene expression occur in the conditional flu mutant of Arabidopsis that reveal a rapid transfer of signals from the plastid to the nucleus. Factors involved in this retrograde signaling were identified by mutagenizing a transgenic flu line expressing a 1O2-responsive reporter gene. The reporter gene consisted of the luciferase open reading frame and the promoter of an AAA-ATPase gene (At3g28580) that was selectively activated by 1O2 but not by superoxide or hydrogen peroxide. A total of eight second-site mutants were identified that either constitutively activate the reporter gene and the endogenous AAA-ATPase irrespectively of whether 1O2 was generated or not (constitutive activators of AAA-ATPase, caa) or abrogated the 1O2-dependent up-regulation of these genes as seen in the transgenic parental flu line (non-activators of AAA-ATPase, naa). The characterization of the mutants strongly suggests that 1O2-signaling does not operate as an isolated linear pathway but rather forms an integral part of a signaling network that is modified by other signaling routes and impacts not only stress responses of plants but also their development.