Reactive Oxygen Species Production In Arabidopsis Research Articles

The calcium-dependent protein kinase CPK16 regulates hypoxia-induced ROS production by phosphorylating the NADPH oxidase RBOHD in Arabidopsis.

Reactive oxygen species (ROS) production is a key event in modulating plant responses to hypoxia and post-hypoxia reoxygenation. However, the molecular mechanism by which hypoxia-associated ROS homeostasis is controlled remains largely unknown. Here, we showed that the calcium-dependent protein kinase CPK16 regulates plant hypoxia tolerance by phosphorylating the plasma membrane-anchored NADPH oxidase respiratory burst oxidase homolog D (RBOHD) to regulate ROS production in Arabidopsis (Arabidopsis thaliana). In response to hypoxia or reoxygenation, CPK16 was activated through phosphorylation of its Ser274 residue. The cpk16 knockout mutant displayed enhanced hypoxia tolerance, whereas CPK16-overexpressing (CPK16-OE) lines showed increased sensitivity to hypoxic stress. In agreement with these observations, hypoxia and reoxygenation both induced ROS accumulation in the rosettes of CPK16-OEs more strongly than in the rosettes of the cpk16-1 mutant or the wild type. Moreover, CPK16 interacted with and phosphorylated the N-terminus of RBOHD at 4 serine residues (Ser133, Ser148, Ser163, and Ser347) that were necessary for hypoxia- and reoxygenation-induced ROS accumulation. Furthermore, the hypoxia-tolerant phenotype of cpk16-1 was fully abolished in the cpk16 rbohd double mutant. Thus, we have uncovered a regulatory mechanism by which the CPK16-RBOHD module shapes the ROS production during hypoxia and reoxygenation in Arabidopsis.

CRK2 and C-terminal Phosphorylation of NADPH Oxidase RBOHD Regulate Reactive Oxygen Species Production in Arabidopsis.

Reactive oxygen species (ROS) are important messengers in eukaryotic organisms, and their production is tightly controlled. Active extracellular ROS production by NADPH oxidases in plants is triggered by receptor-like protein kinase-dependent signaling networks. Here, we show that CYSTEINE-RICH RLK2 (CRK2) kinase activity is required for plant growth and CRK2 exists in a preformed complex with the NADPH oxidase RESPIRATORY BURST OXIDASE HOMOLOG D (RBOHD) in Arabidopsis (Arabidopsis thaliana). Functional CRK2 is required for the full elicitor-induced ROS burst, and consequently the crk2 mutant is impaired in defense against the bacterial pathogen Pseudomonas syringae pv tomato DC3000. Our work demonstrates that CRK2 regulates plant innate immunity. We identified in vitro CRK2-dependent phosphorylation sites in the C-terminal region of RBOHD. Phosphorylation of S703 RBOHD is enhanced upon flg22 treatment, and substitution of S703 with Ala reduced ROS production in Arabidopsis. Phylogenetic analysis suggests that phospho-sites in the C-terminal region of RBOHD are conserved throughout the plant lineage and between animals and plants. We propose that regulation of NADPH oxidase activity by phosphorylation of the C-terminal region might be an ancient mechanism and that CRK2 is an important element in regulating microbe-associated molecular pattern-triggered ROS production.

Comparative Genomics Screen Identifies Microbe-Associated Molecular Patterns from 'Candidatus Liberibacter' spp. That Elicit Immune Responses in Plants.

Citrus huanglongbing (HLB), caused by phloem-limited 'Candidatus Liberibacter' bacteria, is a destructive disease threatening the worldwide citrus industry. The mechanisms of pathogenesis are poorly understood and no efficient strategy is available to control HLB. Here, we used a comparative genomics screen to identify candidate microbe-associated molecular patterns (MAMPs) from 'Ca. Liberibacter' spp. We identified the core genome from multiple 'Ca. Liberibacter' pathogens, and searched for core genes with signatures of positive selection. We hypothesized that genes encoding putative MAMPs would evolve to reduce recognition by the plant immune system, while retaining their essential functions. To efficiently screen candidate MAMP peptides, we established a high-throughput microtiter plate-based screening assay, particularly for citrus, that measured reactive oxygen species (ROS) production, which is a common immune response in plants. We found that two peptides could elicit ROS production in Arabidopsis and Nicotiana benthamiana. One of these peptides elicited ROS production and defense gene expression in HLB-tolerant citrus genotypes, and induced MAMP-triggered immunity against the bacterial pathogen Pseudomonas syringae. Our findings identify MAMPs that boost immunity in citrus and could help prevent or reduce HLB infection.

Regulation of mitochondrial NAD pool via NAD+ transporter 2 is essential for matrix NADH homeostasis and ROS production in Arabidopsis

Reactive oxygen species (ROS) play a crucial role in numerous biological processes in plants, including development, responses to environmental stimuli, and programmed cell death (PCD). Deficiency in MOSAIC DEATH 1 (MOD1), a plastid-localized enoyl-ACP reductase essential for de novo fatty acid biosynthesis in Arabidopsis thaliana, leads to the increased malate export from chloroplasts to mitochondria, and the subsequent accumulation of mitochondria-generated ROS and PCD. In this study, we report the identification and characterization of a mod1 suppressor, som592. SOM592 encodes mitochondrion-localized NAD+ transporter 2 (NDT2). We show that the mitochondrial NAD pool is elevated in the mod1 mutant. The som592 mutation fully suppressed mitochondrial NADH hyper-accumulation, ROS production, and PCD in the mod1 mutant, indicating a causal relationship between mitochondrial NAD accumulation and ROS/PCD phenotypes. We also show that in wild-type plants, the mitochondrial NAD+ uptake is involved in the regulation of ROS production in response to continuous photoperiod. Elevation of the alternative respiration pathway can suppress ROS accumulation and PCD in mod1, but leads to growth restriction. These findings uncover a regulatory mechanism for mitochondrial ROS production via NADH homeostasis in Arabidopsis thaliana that is likely important for growth regulation in response to altered photoperiod.

Autophagy regulates glucose-mediated root meristem activity by modulating ROS production in Arabidopsis

ABSTRACTGlucose produced from photosynthesis is a key nutrient signal regulating root meristem activity in plants; however, the underlying mechanisms remain poorly understood. Here, we show that, by modulating reactive oxygen species (ROS) levels, the conserved macroautophagy/autophagy degradation pathway contributes to glucose-regulated root meristem maintenance. In Arabidopsis thaliana roots, a short exposure to elevated glucose temporarily suppresses constitutive autophagosome formation. The autophagy-defective autophagy-related gene (atg) mutants have enhanced tolerance to glucose, established downstream of the glucose sensors, and accumulate less glucose-induced ROS in the root tips. Moreover, the enhanced root meristem activities in the atg mutants are associated with improved auxin gradients and auxin responses. By acting with AT4G39850/ABCD1 (ATP-binding cassette D1; Formerly PXA1/peroxisomal ABC transporter 1), autophagy plays an indispensable role in the glucose-promoted degradation of root peroxisomes, and the atg mutant phenotype is partially rescued by the overexpression of ABCD1. Together, our findings suggest that autophagy is an essential mechanism for glucose-mediated maintenance of the root meristem.Abbreviation: ABA: abscisic acid; ABCD1: ATP-binding cassette D1; ABO: ABA overly sensitive; AsA: ascorbic acid; ATG: autophagy related; CFP: cyan fluorescent protein; Co-IP: co-immunoprecipitation; DAB: 3’,3’-diaininobenzidine; DCFH-DA: 2’,7’-dichlorodihydrofluorescin diacetate; DR5: a synthetic auxin response element consists of tandem direct repeats of 11 bp that included the auxin-responsive TGTCTC element; DZ: differentiation zone; EZ, elongation zone; GFP, green fluorescent protein; GSH, glutathione; GUS: β-glucuronidase; HXK1: hexokinase 1; H2O2: hydrogen peroxide; IAA: indole-3-acetic acid; IBA: indole-3-butyric acid; KIN10/11: SNF1 kinase homolog 10/11; MDC: monodansylcadaverine; MS: Murashige and Skoog; MZ: meristem zone; NBT: nitroblue tetrazolium; NPA: 1-N-naphtylphthalamic acid; OxIAA: 2-oxindole-3-acetic acid; PIN: PIN-FORMED; PLT: PLETHORA; QC: quiescent center; RGS1: Regulator of G-protein signaling 1; ROS: reactive oxygen species; SCR: SCARECROW; SHR, SHORT-ROOT; SKL: Ser-Lys-Leu; SnRK1: SNF1-related kinase 1; TOR: target of rapamycin; UPB1: UPBEAT1; WOX5: WUSCHEL related homeobox 5; Y2H: yeast two-hybrid; YFP: yellow fluorescent protein

A Lectin Receptor-Like Kinase Mediates Pattern-Triggered Salicylic Acid Signaling.

Plant surface-localized pathogen recognition receptors (PRRs) perceive conserved microbial features, termed pathogen-associated molecular patterns (PAMPs), resulting in disease resistance. PAMP perception leads to calcium influx, MAPK activation, a burst of reactive oxygen species (ROS) mediated by RbohD, accumulation of the defense hormone salicylic acid (SA), and callose deposition. Lectin receptor-like kinases (LecRKs) belong to a specific PRR family and are important players in plant innate immunity. Here, we report that LecRK-IX.2 is a positive regulator of PRR-triggered immunity. Pathogen infection activated the transcription of Arabidopsis (Arabidopsis thaliana) LecRK-IX.2, and the LecRK-IX.2 knockout lines exhibited enhanced susceptibility to virulent Pseudomonas syringae pv tomato DC3000. In addition, LecRK-IX.2 is capable of inducing RbohD phosphorylation, likely by recruiting calcium-dependent protein kinases to trigger ROS production in Arabidopsis. Overexpression of LecRK-IX.2 resulted in elevated ROS and SA and enhanced systemic acquired resistance to P. syringae pv tomato DC3000. Our data highlight the importance of LecRKs in plant immune signaling and SA accumulation.

Manipulation of ABA Content in Arabidopsis thaliana Modifies Sensitivity and Oxidative Stress Response to Dickeya dadantii and Influences Peroxidase Activity.

The production of reactive oxygen species (ROS) is one of the first defense reactions induced in Arabidopsis in response to infection by the pectinolytic enterobacterium Dickeya dadantii. Previous results also suggest that abscisic acid (ABA) favors D. dadantii multiplication and spread into its hosts. Here, we confirm this hypothesis using ABA-deficient and ABA-overproducer Arabidopsis plants. We investigated the relationships between ABA status and ROS production in Arabidopsis after D. dadantii infection and showed that ABA status modulates the capacity of the plant to produce ROS in response to infection by decreasing the production of class III peroxidases. This mechanism takes place independently of the well-described oxidative stress related to the RBOHD NADPH oxidase. In addition to this weakening of plant defense, ABA content in the plant correlates positively with the production of some bacterial virulence factors during the first stages of infection. Both processes should enhance disease progression in presence of high ABA content. Given that infection increases transcript abundance for the ABA biosynthesis genes AAO3 and ABA3 and triggers ABA accumulation in leaves, we propose that D. dadantii manipulates ABA homeostasis as part of its virulence strategy.

Stomatal behaviors reflect enantioselective phytotoxicity of chiral herbicide dichlorprop in Arabidopsis thaliana

Stomata in plants play vital roles in water transpiration and gas exchange necessary for photosynthesis, which are critical for the plants growth. Until now, however, the effect of chiral herbicides on the response of stomata was poorly understood. To unveil this puzzle, the enantioselective effect of chiral herbicide dichloroprop (DCPP) on stomata in Arabidopsis thaliana was investigated. It was found that (R)-DCPP preferentially promoted the extent of stomatal opening in Arabidopsis leaves, resulting in 59.84% enhancement at 0.3μmol·L−1 comparing to the control, where (S)- and (Rac)-DCPP exhibited no significant differences. Enantioselectivity was also observed in the response of stomata to DCPP. To better understand the mechanism involved, the reactive oxygen species (ROS) production and antioxidant system defense were measured. Interestingly, the ROS production in Arabidopsis leaves was also enantioselective. The (R)-DCPP treatments resulted in 6.08-fold enhancement compared with the control, whereas 1.35- and 2.51-fold increases occurred in (S)-DCPP and (Rac)-DCPP treatments, respectively. The promoting of stomatal opening was positively correlated with ROS production. In addition, the antioxidant system response provided evidence of oxidative stress and damage caused by DCPP. This study confirmed that the ROS produced by DCPP promoted stomatal opening and suggested a potential sight to elucidate the phytotoxicity of chiral herbicides.

Universal stress protein HRU1 mediates ROS homeostasis under anoxia

Plant survival is greatly impaired when oxygen levels are limiting, such as during flooding or when anatomical constraints limit oxygen diffusion. Oxygen sensing in Arabidopsis thaliana is mediated by Ethylene Responsive Factor (ERF)-VII transcription factors, which control a core set of hypoxia- and anoxia-responsive genes responsible for metabolic acclimation to low-oxygen conditions. Anoxic conditions also induce genes related to reactive oxygen species (ROS). Whether the oxygen-sensing machinery coordinates ROS production under anoxia has remained unclear. Here we show that a low-oxygen-responsive universal stress protein (USP), Hypoxia Responsive Universal Stress Protein 1 (HRU1), is induced by RAP2.12 (Related to Apetala 2.12), an ERF-VII protein, and modulates ROS production in Arabidopsis. We found that HRU1 is strongly induced by submergence, but that this induction is abolished in plants lacking RAP2.12. Mutation of HRU1 through transfer DNA (T-DNA) insertion alters hydrogen peroxide production, and reduces tolerance to submergence and anoxia. Yeast two-hybrid and bimolecular fluorescence complementation (BiFC) analyses reveal that HRU1 interacts with proteins that induce ROS production, the GTPase ROP2 and the NADPH oxidase RbohD, pointing to the existence of a low-oxygen-specific mechanism for the modulation of ROS levels. We propose that HRU1 coordinates oxygen sensing with ROS signalling under anoxic conditions.

Methylglyoxal-induced stomatal closure accompanied by peroxidase-mediated ROS production in Arabidopsis

Methylglyoxal (MG) is an oxygenated short aldehyde and a glycolytic intermediate that accumulates in plants under environmental stresses. Being a reactive α-oxoaldehyde, MG may act as a signaling molecule in plants during stresses. We investigated whether MG induces stomatal closure, reactive oxygen species (ROS) production, and cytosolic free calcium concentration ([Ca2+]cyt) to clarify roles of MG in Arabidopsis guard cells. MG induced production of ROS and [Ca2+]cyt oscillations, leading to stomatal closure. The MG-induced stomatal closure and ROS production were completely inhibited by a peroxidase inhibitor, salicylhydroxamic acid (SHAM), but were not affected by an NAD(P)H oxidase mutation, atrbohD atrbohF. Furthermore, the MG-elicited [Ca2+]cyt oscillations were significantly suppressed by SHAM but not by the atrbohD atrbohF mutation. Neither endogenous abscisic acid nor endogenous methyl jasmonate was involved in MG-induced stomatal closure. These results suggest that intrinsic metabolite MG can induce stomatal closure in Arabidopsis accompanied by extracellular ROS production mediated by SHAM-sensitive peroxidases, intracellular ROS accumulation, and [Ca2+]cyt oscillations.

Yeast Elicitor-Induced Stomatal Closure and Peroxidase-Mediated ROS Production in Arabidopsis

Yeast elicitor (YEL) induces stomatal closure. We investigated reactive oxygen species (ROS) production, nitric oxide (NO) production and [Ca(2+)](cyt) oscillations to clarify YEL signaling in Arabidopsis guard cells. YEL induced ROS accumulation in guard cells. A peroxidase inhibitor [salicylhydroxamic acid (SHAM)] inhibited the stomatal closure and the ROS accumulation, but neither the atrbohD atrbohF mutation nor an NADPH oxidase inhibitor [diphenylene iodonium chloride (DPI)] had any effect. An NO scavenger [2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO)] inhibited the YEL-induced stomatal closure and SHAM abolished NO production. YEL-elicited [Ca(2+)](cyt) oscillations were inhibited by SHAM but not by the atrbohD atrbohF mutation. These results indicate that YEL induces stomatal closure accompanied by ROS production mediated by peroxidases and NO production.