A dual role for glutathione transferase U7 in plant growth and protection from methyl viologen-induced oxidative stress.

Plant glutathione S-transferases (GSTs) are glutathione-dependent enzymes with versatile functions, mainly related to detoxification of electrophilic xenobiotics and peroxides. The Arabidopsis (Arabidopsis thaliana) genome codes for 53 GSTs, divided into seven subclasses; however, understanding of their precise functions is limited. A recent study showed that class II TGA transcription factors TGA2, TGA5, and TGA6 are essential for tolerance of UV-B-induced oxidative stress and that this tolerance is associated with an antioxidative function of cytosolic tau-GSTs (GSTUs). Specifically, TGA2 controls the expression of several GSTUs under UV-B light, and constitutive expression of GSTU7 in the tga256 triple mutant is sufficient to revert the UV-B-susceptible phenotype of tga256. To further study the function of GSTU7, we characterized its role in mitigation of oxidative damage caused by the herbicide methyl viologen (MV). Under non-stress conditions, gstu7 null mutants were smaller than wild-type (WT) plants and delayed in the onset of the MV-induced antioxidative response, which led to accumulation of hydrogen peroxide and diminished seedling survival. Complementation of gstu7 by constitutive expression of GSTU7 rescued these phenotypes. Furthermore, live monitoring of the glutathione redox potential in intact cells with the fluorescent probe Grx1-roGFP2 revealed that GSTU7 overexpression completely abolished the MV-induced oxidation of the cytosolic glutathione buffer compared with WT plants. GSTU7 acted as a glutathione peroxidase able to complement the lack of peroxidase-type GSTs in yeast. Together, these findings show that GSTU7 is crucial in the antioxidative response by limiting oxidative damage and thus contributes to oxidative stress resistance in the cell.

Oligogalacturonides Enhance Resistance against Aphids through Pattern-Triggered Immunity and Activation of Salicylic Acid Signaling.

The remarkable capacity of the generalist aphid Myzus persicae to resist most classes of pesticides, along with the environmental and human health risks associated with these agrochemicals, has necessitated the development of safer and greener solutions to control this agricultural pest. Oligogalacturonides (OGs) are pectin-derived molecules that can be isolated from fruit industry waste. OGs have been shown to efficiently stimulate plant defenses against pathogens such as Pseudomonas syringae and Botrytis cinerea. However, whether OGs confer resistance against phytophagous insects such as aphids remains unknown. Here, we treated Arabidopsis plants with OGs and recorded their effects on the feeding performance and population of M. persicae aphids. We also identified the defense mechanism triggered by OGs in plants through the analysis of gene expression and histological approaches. We found that OG treatments increased their resistance to M. persicae infestation by reducing the offspring number and feeding performance. Furthermore, this enhanced resistance was related to a substantial accumulation of callose and reactive oxygen species and activation of the salicylic acid signaling pathway.

The TGA Transcription Factors from Clade II Negatively Regulate the Salicylic Acid Accumulation in Arabidopsis.

Salicylic acid (SA) is a hormone that modulates plant defenses by inducing changes in gene expression. The mechanisms that control SA accumulation are essential for understanding the defensive process. TGA transcription factors from clade II in Arabidopsis, which include the proteins TGA2, TGA5, and TGA6, are known to be key positive mediators for the transcription of genes such as PR-1 that are induced by SA application. However, unexpectedly, stress conditions that induce SA accumulation, such as infection with the avirulent pathogen P. syringae DC3000/AvrRPM1 and UV-C irradiation, result in enhanced PR-1 induction in plants lacking the clade II TGAs (tga256 plants). Increased PR-1 induction was accompanied by enhanced isochorismate synthase-dependent SA production as well as the upregulation of several genes involved in the hormone's accumulation. In response to avirulent P. syringae, PR-1 was previously shown to be controlled by both SA-dependent and -independent pathways. Therefore, the enhanced induction of PR-1 (and other defense genes) and accumulation of SA in the tga256 mutant plants is consistent with the clade II TGA factors providing negative feedback regulation of the SA-dependent and/or -independent pathways. Together, our results indicate that the TGA transcription factors from clade II negatively control SA accumulation under stress conditions that induce the hormone production. Our study describes a mechanism involving old actors playing new roles in regulating SA homeostasis under stress.

TGA class II transcription factors are essential to restrict oxidative stress in response to UV-B stress in Arabidopsis.

Plants possess a robust metabolic network for sensing and controlling reactive oxygen species (ROS) levels upon stress conditions. Evidence shown here supports a role for TGA class II transcription factors as critical regulators of genes controlling ROS levels in the tolerance response to UV-B stress in Arabidopsis. First, tga256 mutant plants showed reduced capacity to scavenge H2O2 and restrict oxidative damage in response to UV-B, and also to methylviologen-induced photooxidative stress. The TGA2 transgene (tga256/TGA2 plants) complemented these phenotypes. Second, RNAseq followed by clustering and Gene Ontology term analyses indicate that TGA2/5/6 positively control the UV-B-induced expression of a group of genes with oxidoreductase, glutathione transferase, and glucosyltransferase activities, such as members of the glutathione S-transferase Tau subfamily (GSTU), which encodes peroxide-scavenging enzymes. Accordingly, increased glutathione peroxidase activity triggered by UV-B was impaired in tga256 mutants. Third, the function of TGA2/5/6 as transcriptional activators of GSTU genes in the UV-B response was confirmed for GSTU7, GSTU8, and GSTU25, using quantitative reverse transcription-PCR and ChIP analyses. Fourth, expression of the GSTU7 transgene complemented the UV-B-susceptible phenotype of tga256 mutant plants. Together, this evidence indicates that TGA2/5/6 factors are key regulators of the antioxidant/detoxifying response to an abiotic stress such as UV-B light overexposure.

PROHIBITIN3 Forms Complexes with ISOCHORISMATE SYNTHASE1 to Regulate Stress-Induced Salicylic Acid Biosynthesis in Arabidopsis.

Salicylic acid (SA) is a major defense signal in plants. In Arabidopsis (Arabidopsis thaliana), the chloroplast-localized isochorismate pathway is the main source of SA biosynthesis during abiotic stress or pathogen infections. In the first step of the pathway, the enzyme ISOCHORISMATE SYNTHASE1 (ICS1) converts chorismate to isochorismate. An unknown enzyme subsequently converts isochorismate to SA. Here, we show that ICS1 protein levels increase during UV-C stress. To identify proteins that may play roles in SA production by regulating ICS1, we analyzed proteins that coimmunoprecipitated with ICS1 via mass spectrometry. The ICS1 complexes contained a large number of peptides from the PROHIBITIN (PHB) protein family, with PHB3 the most abundant. PHB proteins have diverse biological functions that include acting as scaffolds for protein complex formation and stabilization. PHB3 was reported previously to localize to mitochondria. Using fractionation, protease protection, and live imaging, we show that PHB3 also localizes to chloroplasts, where ICS1 resides. Notably, loss of PHB3 function led to decreased ICS1 protein levels in response to UV-C stress. However, ICS1 transcript levels remain unchanged, indicating that ICS1 is regulated posttranscriptionally. The phb3 mutant displayed reduced levels of SA, the SA-regulated protein PR1, and hypersensitive cell death in response to UV-C and avirulent strains of Pseudomonas syringae and, correspondingly, supported increased growth of P. syringae The expression of a PHB3 transgene in the phb3 mutant complemented all of these phenotypes. We suggest a model in which the formation of PHB3-ICS1 complexes stabilizes ICS1 to promote SA production in response to stress.

Modulation of Auxin Levels in Pollen Grains Affects Stamen Development and Anther Dehiscence in Arabidopsis.

Auxin regulates diverse aspects of flower development in plants, such as differentiation of the apical meristem, elongation of the stamen, and maturation of anthers and pollen. It is known that auxin accumulates in pollen, but little information regarding the biological relevance of auxin in this tissue at different times of development is available. In this work, we manipulated the amount of free auxin specifically in developing pollen, using transgenic Arabidopsis lines that express the bacterial indole-3-acetic acid-lysine synthetase (iaaL) gene driven by a collection of pollen-specific promoters. The iaaL gene codes for an indole-3-acetic acid-lysine synthetase that catalyzes the conversion of free auxin into inactive indole-3-acetyl-l-lysine. The transgenic lines showed several abnormalities, including the absence of short stamina, a diminished seed set, aberrant pollen tubes, and perturbations in the synchronization of anther dehiscence and stamina development. This article describes the importance of auxin accumulation in pollen and its role in stamina and anther development.

Chemical inhibition of the histone acetyltransferase activity in Arabidopsis thaliana.

Chemical inhibition of chromatin regulators provides an effective approach to investigate the roles of chromatin modifications in plant and animals. In this work, chemical inhibition of the Arabidopsis histone acetyltransferase activity by γ-butyrolactone (MB-3), the inhibitor of the catalytic activity of mammalian GENERAL CONTROL NON-REPRESSIBLE 5 (GCN5) is evaluated. Arabidopsis seedlings were germinated in LS medium supplemented with different concentrations of MB-3, and inhibition in the root length and yellowed leaves were observed. The yellowed leaves phenotype of the plants grown in 100 µM of MB-3 was reverted when plants were additionally treated with 1 µM of TSA, a histone deacetylase inhibitor. Using an immunoblot assay with specific antibodies revealed a reduction of H3K14 acetylation levels at 3 and 24 h post-treatment. At 24 h post-treatment a reduction of H3K9 acetylation levels was observed. Targets of GCN5 related to stress were downregulated at 3 h post-treatment but no change was observed in target genes related to developmental transition. Our results indicate that MB-3 is a chemical inhibitor of the histone acetyltransferase in Arabidopsis and suggest that this inhibitor could function in other plants species.

Transcriptional Control of Glutaredoxin GRXC9 Expression by a Salicylic Acid-Dependent and NPR1-Independent Pathway in Arabidopsis.

Salicylic acid (SA) is a key hormone that mediates gene transcriptional reprogramming in the context of the defense response to stress. GRXC9, coding for a CC-type glutaredoxin from Arabidopsis, is an SA-responsive gene induced early and transiently by an NPR1-independent pathway. Here, we address the mechanism involved in this SA-dependent pathway, using GRXC9 as a model gene. We first established that GRXC9 expression is induced by UVB exposure through this pathway, validating its activation in a physiological stress condition. GRXC9 promoter analyses indicate that SA controls gene transcription through two activating sequence-1 (as-1)-like elements located in its proximal region. TGA2 and TGA3, but not TGA1, are constitutively bound to this promoter region. Accordingly, the transient recruitment of RNA polymerase II to the GRXC9 promoter, as well as the transient accumulation of gene transcripts detected in SA-treated WT plants, was abolished in a knockout mutant for the TGA class II factors. We conclude that constitutive binding of TGA2 is essential for controlling GRXC9 expression, while binding of TGA3 in a lesser extent contributes to this regulation. Finally, overexpression of GRXC9 indicates that the GRXC9 protein negatively controls its own gene expression, forming part of the complex bound to the as-1-containing promoter region. These findings are integrated in a model that explains how SA controls transcription of GRXC9 in the context of the defense response to stress.

Molecular and Genomic Characterization of the Pseudomonas syringae Phylogroup 4: An Emerging Pathogen of Arabidopsis thaliana and Nicotiana benthamiana.

Environmental fluctuations such as increased temperature, water availability, and air CO2 concentration triggered by climate change influence plant disease dynamics by affecting hosts, pathogens, and their interactions. Here, we describe a newly discovered Pseudomonas syringae strain found in a natural population of Arabidopsis thaliana collected from the southwest of France. This strain, called Psy RAYR-BL, is highly virulent on natural Arabidopsis accessions, Arabidopsis model accession Columbia 0, and tobacco plants. Despite the severe disease phenotype caused by the Psy RAYR-BL strain, we identified a reduced repertoire of putative Type III virulence effectors by genomic sequencing compared to P. syringae pv tomato (Pst) DC3000. Furthermore, hopBJ1Psy is found exclusively on the Psy RAYR-BL genome but not in the Pst DC3000 genome. The plant expression of HopBJ1Psy induces ROS accumulation and cell death. In addition, HopBJ1Psy participates as a virulence factor in this plant-pathogen interaction, likely explaining the severity of the disease symptoms. This research describes the characterization of a newly discovered plant pathogen strain and possible virulence mechanisms underlying the infection process shaped by natural and changing environmental conditions.

Draft Genome Sequence of Pseudomonas syringae RAYR-BL, a Strain Isolated from Natural Accessions of Arabidopsis thaliana Plants.

Here, we report the genome sequence of the P. syringae strain RAYR-BL, isolated from natural accessions of Arabidopsis plants. The draft genome sequence consists of 5.85 Mbp assembled in 110 contigs. The study of P. syringae RAYR-BL is a valuable tool to investigate molecular features of plant-pathogen interaction under environmental conditions.

WRKY7, -11 and -17 transcription factors are modulators of the bZIP28 branch of the unfolded protein response during PAMP-triggered immunity in Arabidopsis thaliana.

Plants must defend themselves against pathogens. The defense response requires greater protein synthesis, which generates endoplasmic reticulum (ER) stress, yet failure to attenuate this stress has detrimental effects. WRKY7/11/17 transcription factors (TFs) are negative regulators of immunity since mutants are more resistant to Pseudomonas syringae pv tomato (Pst) infection. Here, we reveal a connection between ER-stress and the molecular mechanisms underlying the wrky mutant phenotype. The bZIP28 TF upregulates ER-chaperone expression (BiP1/2, ERdj3B, and SDF2) upon exposure of Arabidopsis to a bacterial defense elicitor, flagellin 22 (Flg22). Also, the activation of ER-chaperones is more sustained in double and triple wrky mutants treated with Flg22, suggesting that WRKY7/11/17 TFs downregulate these genes. Moreover, wrky mutants accumulate more bZIP28 transcripts in response to Flg22, indicating that WRKY7/11/17 transcriptionally repress this TF. Using Arabidopsis protoplasts, we also demonstrate that WRKYs bind to the bZIP28 promoter via W-box elements. Additionally, triple wrky mutants are more resistant, whilst bzip28 mutants are more susceptible, to Pst infection. Finally, we postulate a model of PAMP-Triggered Immunity regulation, where Flg22 activates bZIP28-signaling inducing the expression of ER-stress genes, as well as WRKY7/11/17 expression, which in turn inhibits PTI by downregulating bZIP28, controlling physiological responses in the Arabidopsis-Pst interaction.

Corrigendum: Salicylic acid and reactive oxygen species interplay in the transcriptional control of defense genes expression.

[This corrects the article on p. 171 in vol. 6, PMID: 25852720.].

Salicylic acid and reactive oxygen species interplay in the transcriptional control of defense genes expression.

It is well established that salicylic acid (SA) plays a critical role in the transcriptional reprograming that occurs during the plant defense response against biotic and abiotic stress. In the course of the defense response, the transcription of different sets of defense genes is controlled in a spatio-temporal manner via SA-mediated mechanisms. Interestingly, different lines of evidence indicate that SA interplays with reactive oxygen species (ROS) and glutathione (GSH) in stressed plants. In this review we focus on the evidence that links SA, ROS, and GSH signals to the transcriptional control of defense genes. We discuss how redox modifications of regulators and co-regulators involved in SA-mediated transcriptional responses control the temporal patterns of gene expression in response to stress. Finally, we examine how these redox sensors are coordinated with the dynamics of cellular redox changes occurring in the defense response to biotic and abiotic stress.