Reactive Oxygen Species Production In Plants Research Articles

Plant PR1 rescues condensation of the plastid iron-sulfur protein by a fungal effector.

Plant pathogens secrete numerous effectors to promote host infection, but whether any of these toxic proteins undergoes phase separation to manipulate plant defence and how the host copes with this event remain elusive. Here we show that the effector FolSvp2, which is secreted from the fungal pathogen Fusarium oxysporum f. sp. lycopersici (Fol), translocates a tomato iron-sulfur protein (SlISP) from plastids into effector condensates in planta via phase separation. Relocation of SlISP attenuates plant reactive oxygen species production and thus facilitates Fol invasion. The action of FolSvp2 also requires K205 acetylation that prevents ubiquitination-dependent degradation of this protein in both Fol and plant cells. However, tomato has evolved a defence protein, SlPR1. Apoplastic SlPR1 physically interacts with and inhibits FolSvp2 entry into host cells and, consequently, abolishes its deleterious effect. These findings reveal a previously unknown function of PR1 in countering a new mode of effector action.

RsRbohD1 Plays a Significant Role in ROS Production during Radish Pithiness Development.

Pithiness is one of the physiological diseases of radishes, which is accompanied by the accumulation of reactive oxygen species (ROS) during the sponging of parenchyma tissue in the fleshy roots. A respiratory burst oxidase homolog (Rboh, also known as NADPH oxidase) is a key enzyme that catalyzes the production of ROS in plants. To understand the role of Rboh genes in radish pithiness, herein, 10 RsRboh gene families were identified in the genome of Raphanus sativus using Blastp and Hmmer searching methods and were subjected to basic functional analyses such as phylogenetic tree construction, chromosomal localization, conserved structural domain analysis, and promoter element prediction. The expression profiles of RsRbohs in five stages (Pithiness grade = 0, 1, 2, 3, 4, respectively) of radish pithiness were analyzed. The results showed that 10 RsRbohs expressed different levels during the development of radish pithiness. Except for RsRbohB and RsRbohE, the expression of other members increased and reached the peak at the P2 (Pithiness grade = 2) stage, among which RsRbohD1 showed the highest transcripts. Then, the expression of 40 genes related to RsRbohD1 and pithiness were analyzed. These results can provide a theoretical basis for improving pithiness tolerance in radishes.

Effect of riboflavin on redox balance, osmolyte accumulation, methylglyoxal generation and nutrient acquisition in indian squash (Praecitrullus fistulosus L.) under chromium toxicity.

The presence of high chromium (Cr) levels induces the buildup of reactive oxygen species (ROS), resulting in hindered plant development. Riboflavin (vitamin B2) is produced by plants, fungi, and microbes. It serves as a precursor to the coenzymes flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN), which play a crucial role in cellular metabolism. The objective of this work was to clarify the underlying mechanisms by which riboflavin alleviates Cr stress in Praecitrullus fistulosus L. Further, the role of riboflavin in growth, ions homeostasis, methylglyoxal detoxification, and antioxidant defense mechanism are not well documented in plants under Cr toxicity. We found greater biomass and minimal production of ROS in plants pretreated with riboflavin under Cr stress. Results manifested a clear abridge in growth, chlorophyll content, and nutrient uptake in Indian squash plants exposed to Cr stress. Findings displayed that Cr stress visibly enhanced oxidative injury reflected as higher malondialdehyde (MDA), hydrogen peroxide (H2O2), superoxide radical (O2•‒), methylglyoxal (MG) levels alongside vivid lipoxygenase activity. Riboflavin strengthened antioxidant system, enhanced osmolyte production and improved membrane integrity. Riboflavin diminished Cr accumulation in aerial parts that led to improved nutrient acquisition. Taken together, riboflavin abridged Cr phytotoxic effects by improving redox balance because plants treated with riboflavin had strong antioxidant system that carried out effective ROS detoxification. Riboflavin protected membrane integrity that, in turn, improved nutrient uptake in plants.

Reactive oxygen species in plants: an invincible fulcrum for biotic stress mitigation.

Climate change-associated environmental vagaries have amplified the incidence of pests and pathogens on plants, thus imparting the increased quest for management strategies. Plants respond to stresses through intricate signaling networks that regulate diverse cellular mechanisms. Reactive oxygen species (ROS) are cardinal towards the maintenance of normal plant activities as well as improving stress management. Plants that exhibit a fine balance between ROS levels and its management apparently mitigate stresses better. There have been very many compendiums on signaling and management of ROS during several abiotic stresses. However, expansion of knowledge related to ROS induction and homeostasis during biotic stresses is pertinent. Hence, considering its importance, we provide insights in this review on how plants signal and manage ROS upon an oxidative burst during their interaction with pathogens and herbivores. Substantial degree of molecular changes and pivotal roles of ROS have been detected during phyto-pathogen/herbivore interactions, opening novel platforms to understand signaling/management of events under varied biotic stresses. It is interesting to know that, though plants react to biotic stresses through oxidative burst, receptors and elicitors involved in the signal transduction differ across stresses. The review provides explicit details about the specific signaling of ROS production in plants under pathogen and herbivore attack. Furthermore, we also provide an update about tackling the accumulated ROS under biotic stresses as another pivotal step. ROS signaling and homeostasis can be exploited as critical players and a fulcrum to tackle biotic stresses, thus paving the way for futuristic combinatorial stress management strategies. KEY POINTS: •The review is a comprehension of redox signaling and management in plants during herbivory and pathogen infection •Reactive oxygen species (ROS) is an important factor during normal plant activities as well as in their response to stresses. Diverse modes of ROS signaling and management have been observed during both biotic stresses independently •Exploration of plant biology in multi-stress resistant plants like the crop wild relatives could pave the way for combinatorial management of stress for a better tomorrow.

2-Hydroxymelatonin, Rather Than Melatonin, Is Responsible for RBOH-Dependent Reactive Oxygen Species Production Leading to Premature Senescence in Plants.

Unlike animals, plants amply convert melatonin into 2-hydroxymelatonin (2-OHM) and cyclic 3-hydroxymelatonin (3-OHM) through the action of melatonin 2-hydroxylase (M2H) and melatonin 3-hydroxylase (M3H), respectively. Thus, the effects of exogenous melatonin treatment in plants may be caused by melatonin, 2-OHM, or 3-OHM, or some combination of these compounds. Indeed, studies of melatonin’s effects on reactive oxygen species (ROS) production have reported conflicting results. In this study, we demonstrated that 2-OHM treatment induced ROS production, whereas melatonin did not. ROS production from 2-OHM treatment occurred in old arabidopsis leaves in darkness, consistent with an ethylene-mediated senescence mechanism. Transgenic tobacco plants containing overexpressed rice M2H exhibited dwarfism and leaf necrosis of the upper leaves and early senescence of the lower leaves. We also demonstrated that 2-OHM-mediated ROS production is respiratory burst NADPH oxidase (RBOH)-dependent and that 2-OHM-induced senescence genes require ethylene and the abscisic acid (ABA) signaling pathway in arabidopsis. In contrast to melatonin, 2-OHM treatment induced senescence symptoms such as leaf chlorosis and increased ion leakage in arabidopsis. Senescence induction is known to begin with decreased levels of proteins involved in chloroplast maintenance, including Lhcb1 and ClpR1. Together, these results show that 2-OHM acts as a senescence-inducing factor by inducing ROS production in plants.

Interaction between TaNOX7 and TaCDPK13 Contributes to Plant Fertility and Drought Tolerance by Regulating ROS Production.

Reactive oxygen species (ROS) homeostasis is critical for both physiological processes and stress responses of plants. NADPH oxidases (NOXs) are the key producers of ROS in plants. However, their functions in ROS homeostasis and plant growth regulation in wheat (Triticum aestivum) are little investigated. Here, we cloned and characterized a NOX isoform TaNOX7 in wheat. Overexpression of TaNOX7 in rice led to enhanced root length, ROS production, drought tolerance as well as bigger panicles and higher yield but shorter growth period duration. Further results indicate that TaCDPK13, a member of calcium-dependent protein kinases (CDPKs), can directly interact with TaNOX7 and enhance ROS production in plants. These results demonstrate that TaNOX7 plays crucial roles in wheat development, fertility, and drought tolerance via interaction with TaCDPK13, which may act as an upstream regulator of TaNOX7 to regulate ROS production in wheat.

Unravelling efficient applications of agriculturally important microorganisms for alleviation of induced inter-cellular oxidative stress in crops

Abiotic stresses like high temperature, cold, freezing, drought, salinity, flooding or oxidizing agents cause significant loss in the crop yield and quality. Abiotic stresses cause reactive oxygen species (ROS) production such as singlet oxygen (1O2), hydrogen peroxide (H2O2), superoxide radical (O2•−), hydroxyl radical (OH-), etc., that leads to a significant reduction of crop yield. A major source of ROS production in plants through aerobic metabolism is chloroplast, mitochondria, and peroxisome. The tripartite interactions involving Trichoderma- Phytopathogen-Host have received less attention in contrast to the plant–antagonist, plant–pathogen or pathogen–antagonist interactions. This article explores the possibilities of employing thermotolerant strains of agriculturally important microorganisms (AIMs) for alleviating the oxidative stress induced due heat stress in crops by modulating oxidative and defense network of the host.

Cadmium activates ZmMPK3-1 and ZmMPK6-1 via induction of reactive oxygen species in maize roots

Both mitogen-activated protein kinase (MAPK) cascades and reactive oxygen species (ROS) are critical to signaling in eukaryotes. Cadmium induces MAPK activation and ROS production in plants. This study aims to identify specific MAPKs activated by CdCl2 in maize roots, and examine the relationship between MAPK activation and ROS production under CdCl2 treatment. Using in-gel kinase assays, immunoprecipitation, and immunoblot analysis, we identified 43 and 45 kDa ERK-like MAPKs in response to CdCl2. Further analysis revealed that ZmMPK3-1 and ZmMPK6-1 correspond to the 43 and 45 kDa MAPKs, respectively. In addition, CdCl2 induced ROS production prior to the activation of ZmMPK3-1 and ZmMPK6-1. Inhibition of ROS attenuated Cd-activation of ZmMPK3-1 and ZmMPK6-1, whereas inhibition of MAPK signaling did not disturb Cd-induced ROS production. Collectively, these results indicate that, in maize roots, cadmium stress activates ZmMPK3-1 and ZmMPK6-1 via ROS induction.

Survival factor 1 contributes to the oxidative stress response and is required for full virulence of Sclerotinia sclerotiorum.

Summary Sclerotinia sclerotiorum is a devastating necrotrophic fungal pathogen that infects over 400 species of plants worldwide. Reactive oxygen species (ROS) modulations are critical for the pathogenic development of S. sclerotiorum. The fungus applies enzymatic and non‐enzymatic antioxidants to cope with the oxidative stress during the infection processes. Survival factor 1 was identified and characterized to promote survival under conditions of oxidative stress in Saccharomyes cerevisiae. In this research, a gene named SsSvf1 was predicted to encode a survival factor 1 homologue in S. sclerotiorum. SsSvf1 transcripts showed high expression levels in hyphae under oxidative stress. Silencing of SsSvf1 resulted in increased sensitivity to oxidative stress in culture and increased levels of intracellular ROS. Transcripts of SsSvf1 showed a dramatic increase during the initial stage of infection and the gene‐silenced strains displayed reduced virulence on oilseed rape and Arabidopsis thaliana. Inhibition of plant ROS production partially restores virulence of SsSvf1 gene‐silenced strains. SsSvf1 gene‐silenced strains exhibited normal oxalate production, but were impaired in compound appressorium formation and cell wall integrity. The results suggest that SsSvf1 is involved in coping with ROS during fungal‐host interactions and plays a crucial role in the pathogenicity of S. sclerotiorum.

The Chromatin Factor HNI9 and ELONGATED HYPOCOTYL5 Maintain ROS Homeostasis under High Nitrogen Provision

Reactive oxygen species (ROS) can accumulate in cells at excessive levels, leading to unbalanced redox states and to potential oxidative stress, which can have damaging effects on the molecular components of plant cells. Several environmental conditions have been described as causing an elevation of ROS production in plants. Consequently, activation of detoxification responses is necessary to maintain ROS homeostasis at physiological levels. Misregulation of detoxification systems during oxidative stress can ultimately cause growth retardation and developmental defects. Here, we demonstrate that Arabidopsis (Arabidopsis thaliana) plants grown in a high nitrogen (N) environment express a set of genes involved in detoxification of ROS that maintain ROS at physiological levels. We show that the chromatin factor HIGH NITROGEN INSENSITIVE9 (HNI9) is an important mediator of this response and is required for the expression of detoxification genes. Mutation in HNI9 leads to elevated ROS levels and ROS-dependent phenotypic defects under high but not low N provision. In addition, we identify ELONGATED HYPOCOTYL5 as a major transcription factor required for activation of the detoxification program under high N. Our results demonstrate the requirement of a balance between N metabolism and ROS production, and our work establishes major regulators required to control ROS homeostasis under conditions of excess N.

Reduction in Trace Element Mediated Oxidative Stress towards Cropped Plants via Beneficial Microbes in Irrigated Cropping Systems: A Review

Summer crops grown in Australia, including rice, cotton, and sugar cane, require high volumes of water, which is applied using irrigation systems. Yields from these crops are influenced by abiotic stressors. Fluctuations in the abiotic stressors, including soil pH and trace element availability, can increase levels of reactive oxygen species (ROS) in plants leading to increased oxidative stress and subsequent reduced crop growth and yield. One potential way of reducing plant ROS production and levels in these systems is through inoculation of these crops with beneficial microbes. The ability of beneficial microbes to enhance plant growth is well characterized, and it is also clear that many of them produce antioxidant enzymes. Presented in this review are the potential modes of action for microbes to reduce abiotic stress in cropped systems.

Pre-treatment of soybean plants with calcium stimulates ROS responses and mitigates infection by Sclerotinia sclerotiorum

Considering the high incidence of white mold caused by Sclerotinia sclerotiorum in a variety of field crops and vegetables, different control strategies are needed to keep the disease under economical threshold. This study assessed the effect of foliar application of a calcium formulation on disease symptoms, oxalic acid production, and on the oxidative stress metabolism in soybean plants inoculated with each of two isolates of the pathogen that have contrasting aggressiveness (HA, highly-aggressive versus WA, weakly-aggressive). Changes in reactive oxygen species (ROS) levels in soybean plants inoculated with S. sclerotiorum isolates were assessed at 6, 24, 48 and 72 h post inoculation (hpi). Generation of ROS including hydrogen peroxide (H2O2), anion superoxide (O2−) and hydroxyl radical (OH) was evaluated. Inoculation with the WA isolate resulted in more ROS accumulation compared to the HA isolate. Pre-treatment with the calcium formulation restored ROS production in plants inoculated with the HA isolate. We also noted a marked decrease in oxalic acid content in the leaves inoculated with the HA isolate in presence of calcium, which coincided with an increase in plant ROS production. The expression patterns of genes involved in ROS detoxification in response to the calcium treatments and/or inoculation with S. Sclerotiorum isolates were monitored by RT-qPCR. All of the tested genes showed a higher expression in response to inoculation with the WA isolate. The expression of most genes tested peaked at 6 hpi, which preceded ROS accumulation in the soybean leaves. Overall, these data suggest that foliar application of calcium contributes to a decrease in oxalic acid production and disease, arguably via modulation of the ROS metabolism.

Abiotic stress tolerance and ABA responses of transgenic Glycine max plants with modulated RACK1 expression

The receptor for activated C kinase 1 (RACK1) belongs to a protein subfamily that contains a tryptophan-aspartic acid-domain (WD) repeat structure. In this study, a soybean RACK1 gene (GmRACK1) was cloned. The amino acid sequence of GmRACK1 had seven WD repeats, which contained typical glycine-histidine (GH) and WD dipeptides. Tissue-specific expression showed that GmRACK1 is expressed at differential levels in all examined tissues and strongly down-regulatd by abscisic acid (ABA) and abiotic stress. Subcellular localization suggested that GmRACK1 is located in the plasma membrane, cytoplasm, and nucleus. In response to drought and salt stress, the GmRACK1-RNAi lines showed significantly higher dry weight of whole plants, chlorophyll content, and survival rate of soybean seedlings than wild-type and homozygous (OE) lines. GmRACK1-RNAi plants were observed to be more sensitive to ABA-mediated seed germination and root growth. Furthermore, we found that the level of ABA and transcript levels of stress-responsive genes were clearly up-regulated under drought and salt stress conditions in GmRACK1-RNAi plants. Consistent with the accumulation of reactive oxygen species (ROS), elevated electrolyte leakage and malondialdehyde, and lower expression of ROS-scavenging genes were found in the GmRACK1-OE lines. The GmRACK RNAi lines exhibited increased tolerance to drought and salt stress compared with the wild-type and OE lines. In addition, yeast two-hybrid assays showed that GmRACK1 could interact with WNK8, eIF6, and RbohC-N. These results therefore indicate that GmRACK1 responds to drought and salt stress through ABA signaling and the regulation of cellular ROS production in plants.

Regulation of plant reactive oxygen species (ROS) in stress responses: learning from AtRBOHD.

Reactive oxygen species (ROS) are constantly produced in plants, as the metabolic by-products or as the signaling components in stress responses. High levels of ROS are harmful to plants. In contrast, ROS play important roles in plant physiology, including abiotic and biotic tolerance, development, and cellular signaling. Therefore, ROS production needs to be tightly regulated to balance their function. Respiratory burst oxidase homologue (RBOH) proteins, also known as plant nicotinamide adenine dinucleotide phosphate oxidases, are well studied enzymatic ROS-generating systems in plants. The regulatory mechanisms of RBOH-dependent ROS production in stress responses have been intensively studied. This has greatly advanced our knowledge of the mechanisms that regulate plant ROS production. This review attempts to integrate the regulatory mechanisms of RBOHD-dependent ROS production by discussing the recent advance. AtRBOHD-dependent ROS production could provide a valuable reference for studying ROS production in plant stress responses.

Reactive oxygen species (ROS) and response of antioxidants as ROS-scavengers during environmental stress in plants

Reactive oxygen species (ROS) were initially recognized as toxic by-products of aerobic metabolism. In recent years, it has become apparent that ROS plays an important signaling role in plants, controlling processes such as growth, development and especially response to biotic and abiotic environmental stimuli. The major members of the ROS family include free radicals like O2● −, OH● and non-radicals like H2O2 and 1O2. The ROS production in plants is mainly localized in the chloroplast, mitochondria and peroxisomes. There are secondary sites as well like the endoplasmic reticulum, cell membrane, cell wall and the apoplast. The role of the ROS family is that of a double edged sword; while they act as secondary messengers in various key physiological phenomena, they also induce oxidative damages under several environmental stress conditions like salinity, drought, cold, heavy metals, UV irradiation etc., when the delicate balance between ROS production and elimination, necessary for normal cellular homeostasis, is disturbed. The cellular damages are manifested in the form of degradation of biomolecules like pigments, proteins, lipids, carbohydrates and DNA, which ultimately amalgamate in plant cellular death. To ensure survival, plants have developed efficient antioxidant machinery having two arms, (i) enzymatic components like superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX), guaiacol peroxidase (GPX), glutathione reductase (GR), monodehydroascorbate reductase (MDHAR) and dehydroascorbate reductase (DHAR); (ii) non-enzymatic antioxidants like ascorbic acid (AA), reduced glutathione (GSH), α-tocopherol, carotenoids, flavonoids and the osmolyte proline. These two components work hand in hand to scavenge ROS. In this review, we emphasize on the different types of ROS, their cellular production sites, their targets, and their scavenging mechanism mediated by both the branches of the antioxidant systems, highlighting the potential role of antioxidant

The cotton WRKY transcription factor GhWRKY17 functions in drought and salt stress in transgenic Nicotiana benthamiana through ABA signaling and the modulation of reactive oxygen species production.

Drought and high salinity are two major environmental factors that significantly limit the productivity of agricultural crops worldwide. WRKY transcription factors play essential roles in the adaptation of plants to abiotic stresses. However, WRKY genes involved in drought and salt tolerance in cotton (Gossypium hirsutum) are largely unknown. Here, a group IId WRKY gene, GhWRKY17, was isolated and characterized. GhWRKY17 was found to be induced after exposure to drought, salt, H2O2 and ABA. The constitutive expression of GhWRKY17 in Nicotiana benthamiana remarkably reduced plant tolerance to drought and salt stress, as determined through physiological analyses of the germination rate, root growth, survival rate, leaf water loss and Chl content. GhWRKY17 transgenic plants were observed to be more sensitive to ABA-mediated seed germination and root growth. However, overexpressing GhWRKY17 in N. benthamiana impaired ABA-induced stomatal closure. Furthermore, we found that GhWRKY17 modulated the increased sensitivity of plants to drought by reducing the level of ABA, and transcript levels of ABA-inducible genes, including AREB, DREB, NCED, ERD and LEA, were clearly repressed under drought and salt stress conditions. Consistent with the accumulation of reactive oxygen species (ROS), reduced proline contents and enzyme activities, elevated electrolyte leakage and malondialdehyde, and lower expression of ROS-scavenging genes, including APX, CAT and SOD, the GhWRKY17 transgenic plants exhibited reduced tolerance to oxidative stress compared with wild-type plants. These results therefore indicate that GhWRKY17 responds to drought and salt stress through ABA signaling and the regulation of cellular ROS production in plants.

Glutathione and glutathione reductase: A boon in disguise for plant abiotic stress defense operations

Abiotic stresses such as salinity, drought, clilling, heavy metal are the major limiting factors for crop productivity. These stresses induce the overproduction of reactive oxygen species (ROS) which are highly reactive and toxic, which must be minimized to protect the cell from oxidative damage. The cell organelles, particularly chloroplast and mitochondria are the major sites of ROS production in plants where excessive rate of electron flow takes place. Plant cells are well equipped to efficiently scavenge ROS and its reaction products by the coordinated and concerted action of antioxidant machinery constituted by vital enzymatic and non-enzymatic antioxidant components. Glutathione reductase (GR, EC 1.6.4.2) and tripeptide glutathione (GSH, γ-Glutamyl-Cysteinyl-Glycine) are two major components of ascorbate-glutathione (AsA-GSH) pathway which play significant role in protecting cells against ROS and its reaction products-accrued potential anomalies. Both GR and GSH are physiologically linked together where, GR is a NAD(P)H-dependent enzymatic antioxidant and efficiently maintains the reduced pool of GSH - a cellular thiol. The differential modulation of both GR and GSH in plants has been widely implicated for the significance of these two enigmaticantioxidants as major components of plant defense operations. Considering recent informations gained through molecular-genetic studies, the current paper presents an overview of the structure, localization, biosynthesis (for GSH only), discusses GSH and GR significance in abiotic stress (such as salinity, drought, clilling, heavy metal)-exposed crop plants and also points out unexplored aspects in the current context for future studies.

Involvement of coronatine-inducible reactive oxygen species in bacterial speck disease of tomato

Pseudomonas syringae pv. tomato DC3000 (Pst DC3000) produces a chlorosis-inducing phytotoxin coronatine (COR), which has multiple virulence functions in planta. One of the hallmarks of bacterial speck disease on tomato leaves is the formation of necrotic lesions surrounded by chlorosis. The physiological significance of COR-induced chlorosis in disease development is still unknown. In our recent publication in New Phytologist, we demonstrated that COR-induced effects on photosynthetic machinery resulted in the accumulation of reactive oxygen species (ROS). Tomato seedlings inoculated with Pst DC3000 and incubated in light showed more disease-associated necrotic cell death than inoculated seedlings incubated in either dark or low light conditions. Furthermore, COR suppressed the expression of thylakoid-localized Cu/Zn superoxide dismutase (Cu/Zn SOD), but not the cytosolic-localized Cu/Zn SOD. In this addendum, we propose a model for the function of COR as a regulator of plant ROS production in different cellular sites leading to disease-associated necrotic cell death during bacterial speck of tomato.

NADPH oxidase-dependent reactive oxygen species formation required for root hair growth depends on ROP GTPase

Reactive oxygen species (ROS) production by an NADPH oxidase (NOX) encoded by AtrbohC/RHD2 is required for root hair growth in Arabidopsis thaliana. ROP (RHO of plants) GTPases are also required for normal root hair growth and have been proposed to regulate ROS production in plants. Therefore, the role of ROP GTPase in NOX-dependent ROS formation by root hairs was investigated. Plants overexpressing wild-type ROP2 (ROP2 OX), constitutively active (CA-rop2), or dominant negative (DN-rop2) rop2 mutant proteins were used. Superoxide formation by root hairs was detected by superoxide dismutase-sensitive nitroblue tetrazolium reduction, and ROS production in the root hair differentiation zone was detected by dihydrofluorescein diacetate oxidation. Both probes showed that ROS production was increased in ROP2 OX and CA-rop2 plants, and decreased in DN-rop2 plants, relative to wild-type plants. When CA-rop2 was expressed in the NOX loss-of-function rhd2-1 mutant, ROS formation and root hair growth were impaired, suggesting that RHD2 is required for this ROP2-dependent ROS formation.

The plant Mo‐hydroxylases aldehyde oxidase and xanthine dehydrogenase have distinct reactive oxygen species signatures and are induced by drought and abscisic acid

The plant molybdenum-cofactor (Moco) and flavin-containing enzymes, xanthine dehydrogenase (XDH; EC 1.2.1.37) and aldehyde oxidase (AO; EC 1.2.3.1) are thought to play important metabolic roles in purine metabolism and hormone biosynthesis, respectively. Their animal counterparts contribute to reactive oxygen species (ROS) production in numerous pathologies and here we examined these enzymes as potential sources of ROS in plants. Novel in-gel assay techniques and Moco sulfurase mutants, lacking a sulfur ligand in their Moco active center, were employed to demonstrate that the native tomato and Arabidopsis XDHs are capable of producing O, but not H2O2, while the animal counterpart was shown to produce both, O and H2O2. Superoxide production was dependent on Moco sulfuration when using hypoxanthine/xanthine but not NADH as substrates. The activity was inhibited by diphenylene iodonium (DPI), a suicide inhibitor of FAD containing enzymes. Analysis of XDH in an Arabidopsis Atxdh1 T-DNA insertion mutant and RNA interference lines revealed loss of O activity, providing direct molecular evidence that plant XDH generates superoxides. Contrary to XDH, AO activity produced only H2O2 dissimilar to native animal AO, that can produce O as well. Surprisingly, H2O2 accumulation was not sensitive to DPI. Plant ROS production and transcript levels of AO and XDH were rapidly upregulated by application of abscisic acid and in water-stressed leaves and roots. These results, supported by in vivo measurement of ROS accumulation, indicate that plant AO and XDH are possible novel sources for ROS increase during water stress.