Aquaporin as a membrane transporter of hydrogen peroxide in plant response to stresses

Hydrogen peroxide (H₂O₂) is a reactive oxygen species that signals between cells, and H₂O₂ signaling is essential for diverse cellular processes, including stress response, defense against pathogens, and the regulation of programmed cell death in plants. Although plasma membrane intrinsic proteins (PIPs) have been known to transport H₂O₂ across cell membranes, the permeability of each family member of PIPs toward H₂O₂ has not yet been determined in most plant species. In a recent study, we showed that certain isoforms of Arabidopsis thaliana AtPIPs, including AtPIP2;2, AtPIP2;4, AtPIP2;5, and AtPIP2;7, are permeable for H₂O₂ in yeast cells. Since the expression of PIPs is differently modulated in Arabidopsis by abiotic stress or H₂O₂ treatment, it is important to investigate the integrated regulation of aquaporin expression and their physiological significance in H₂O₂ transport and plant response to diverse abiotic stresses.     

Revealing on hydrogen sulfide and nitric oxide signals co-ordination for plant growth under stress conditions

In the recent times, plants are facing certain types of environmental stresses, which give rise to formation of reactive oxygen species (ROS) such as hydroxyl radicals, hydrogen peroxides, superoxide anions and so on. These are required by the plants at low concentrations for signal transduction and at high concentrations, they repress plant root growth. Apart from the ROS activities, hydrogen sulfide (H₂S) and nitric oxide (NO) have major contributions in regulating growth and developmental processes in plants, as they also play key roles as signaling molecules and act as chief plant immune defense mechanisms against various biotic as well as abiotic stresses. H₂S and NO are the two pivotal gaseous messengers involved in growth, germination and improved tolerance in plants under stressed and non-stress conditions. H₂S and NO mediate cell signaling in plants as a response to several abiotic stresses like temperature, heavy metal exposure, water and salinity. They alter gene expression levels to induce the synthesis of antioxidant enzymes, osmolytes and also trigger their interactions with each other. However, research has been limited to only cross adaptations and signal transductions. Understanding the change and mechanism of H₂S and NO mediated cell signaling will broaden our knowledge on the various biochemical changes that occur in plant cells related to different stresses. A clear understanding of these molecules in various environmental stresses would help to confer biotechnological applications to protect plants against abiotic stresses and to improve crop productivity.     

Reactive oxygen species in leaf abscission signaling

Reactive oxygen species (ROS) are produced in response to many environmental stresses, such as UV, chilling, salt and pathogen attack. These stresses also accompany leaf abscission in some plants, however, the relationship between these stresses and abscission is poorly understood. In our recent report, we developed an in vitro abscission system that reproduces stress-induced pepper leaf abscission in planta. Using this system, we demonstrated that continuous production of hydrogen peroxide (H₂O₂) is involved in leaf abscission signaling. Continuous H₂O₂ production is required to induce expression of the cell wall-degrading enzyme, cellulase and functions downstream of ethylene in abscission signaling. Furthermore, enhanced production of H₂O₂ occurs at the execution phase of abscission, suggesting that H₂O₂ also plays a role in the cell-wall degradation process. These data suggest that H₂O₂ has several roles in leaf abscission signaling. Here, we propose a model for these roles.Key words: leaf abscission, reactive oxygen species, H₂O₂, in vitro, ethylene, auxin, pepper, NADPH oxidase     

Hydrogen peroxide-mediated cell-wall stiffening in vitro in maize coleoptiles

It has recently been proposed that H₂O₂-dependent peroxidative formation of phenolic cross-links between cell-wall polymers serves as a mechanism for fixing the viscoelastically extended wall structure and thus confers irreversibility to wall extension during cell growth (M. Hohl et al. 1995, Physiol. Plant. 94: 491–498). In the present paper the isolated cell wall (operationally, frozen/thawed maize coleoptile segments) was used as an experimental system to investigate H₂O₂-dependent cell-wall stiffening in vitro. Hydrogen peroxide inhibited elongation growth (in vivo) and decreased cell-wall extensibility (in vitro) in the concentration range of 10–10000 mol·1^–1. In rheological measurements with a constant-load extensiometer the stiffening effect of H₂O₂ could be observed with both relaxed and stressed cell walls. In-vitro cell-wall stiffening was a time-dependent reaction that lasted about 60 min in the presence of saturating concentrations of H₂O₂. The presence of peroxidase in the growth-limiting outer epidermal wall of the coleoptile was shown by histochemical assays. Peroxidase inhibitors (azide, ascorbate) suppressed the wall-stiffening reaction by H₂O₂ in vitro. Hydrogen peroxide induced the accumulation of a fluorescent, insoluble material in the cell walls of living coleoptile segments. These results demonstrate that primary cell walls of a growing plant organ contain all ingredients for the mechanical fortification of the wall structure by H₂O₂-inducible phenolic cross-linking.Supported by Deutsche Forschungsgemeinschaft. I thank Ms. Bärbel Huvermann for expert technical assistance.     

Defence-related biochemical changes by elicitor of malformation pathogen,Fusarium mangiferae,in mango

Malformation of mango (Mangifera indica L.) induced by Fusarium moniliforme var. subglutinans is a plant disease of international importance. The paper reports the downstream defence responses at the initial stage in a susceptible host (cultivar Amrapali) after treatment with biotic (isolated from the pathogen cell wall) (BEL) and abiotic (salicylic acid, SA) elicitors, and inoculation of vegetative buds with the pathogen (IVB). The SA was further tested to induce resistance in field trials. The inoculation and application of elicitors increased β-1, 3 glucanase that causes lysis of fungal hyphae by many folds. Hydrogen peroxide (H₂O₂) (active oxygen species) that induces hypersensitive cell death was reduced to the minimum level after treatment with BEL. The reduction of H₂O₂ in the inoculated vegetative buds was also substantial; however, comparatively less with SA treatment. Consequently, there was no hypersensitive cell death in the malformed mango. Salicylic acid that enhances H₂O₂ content by suppressing H₂O₂-degradation by catalase, increased marginally with the SA treatment and in the IVB, but reduced with the BEL. The reduction of SA in BEL-treated buds concomitantly reduced its H₂O₂ content. The activity of catalase, suppressor of resistance mechanism, was reduced in all the treatments, but the reduction was not enough to arrest H₂O₂-degradation. Magiferin (1, 3, 6, 7-tetrahdroxyxanthone C₂-β-D glucoside), a defence metabolite of mango, increased substantially in all the treatments; maximum with the BEL. A pathogenesis-related (PR) protein of 20 KDa that resists symptom development appeared in all the treatments except the control. But light colour of the spots for the PR-protein indicated low protein accumulation. The maximum accumulation was with the IVB followed by SA and BEL treatments. The amount of total protein reduced considerably in all the treatments. The SA treatment on healthy plants failed to induce defence against malformation. Contrarily, the treatment on malformed seedlings restored normal growth within two months. Hence, SA acted better over the infected plants in presence of the pathogen. Thus, a signal transduction system involving SA and H₂O₂ remained nonfunctional and enough defence chemicals could not be synthesised. Defence genes that produce phenolic and β-1, 3 glucanase, however, became activated and saved the plants from death although could not prevent symptom manifestations.     

Overexpression of sweetpotato swpa4 peroxidase results in increased hydrogen peroxide production and enhances stress tolerance in tobacco

Plant peroxidases (POD) reduce hydrogen peroxide (H₂O₂) in the presence of an electron donor. Extracellular POD can also induce H₂O₂ production and may perform a significant function in responses to environmental stresses via the regulation of H₂O₂ in plants. We previously described the isolation of 10 POD cDNA clones from cell cultures of sweetpotato (Ipomoea batatas). Among them, the expression of the swpa4 gene was profoundly induced by a variety of abiotic stresses and pathogenic infections (Park et al. in Mol Gen Genome 269:542–552 2003; Jang et al. in Plant Physiol Biochem 42:451–455 2004). In the present study, transgenic tobacco (Nicotiana tabacum) plants overexpressing the swpa4 gene under the control of the CaMV 35S promoter were generated in order to assess the function of swpa4 in planta. The transgenic plants exhibited an approximately 50-fold higher POD specific activity than was observed in control plants. Both transient expression analysis with the swpa4-GFP fusion protein and POD activity assays in the apoplastic washing fluid revealed that the swpa4 protein is secreted into the apoplastic space. In addition, a significantly enhanced tolerance to a variety of abiotic and biotic stresses occurred in the transgenic plants. These plants harbored increased lignin and phenolic content, and H₂O₂ was also generated under normal conditions. Furthermore, they showed an increased expression level of a variety of apoplastic acidic pathogenesis-related (PR) genes following enhanced H₂O₂ production. These results suggest that the expression of swpa4 in the apoplastic space may function as a positive defense signal in the H₂O₂-regulated stress response signaling pathway.     

Role of salicylic acid in induction of plant defense system in chickpea (Cicer arietinum L.)

Salicylic acid (SA), a plant hormone plays an important role in induction of plant defense against a variety of biotic and abiotic stresses through morphological, physiological and biochemical mechanisms. A series of experiments were carried out to evaluate the biochemical response of the chickpea (Cicer arietinum L.) plants to a range of SA concentrations (1, 1.5, and 2 mM). Water treated plants were maintained as control. Activities of peroxidase (POD) and polyphenol oxidase (PPO) were evaluated and amounts of total phenols, hydrogen peroxide (H₂O₂), and proteins were calculated after 96 h of treatment. Plants responded very quickly to SA at 1.5 mM and showed higher induction of POD and PPO activities, besides the higher accumulation of phenols, H₂O₂ and proteins. Plants treated with SA at 2 mM showed phytotoxic symptoms. These results suggest that SA at 1.5 mM is safe to these plants and could be utilized for the induction of plant defense.     

Hydrogen peroxide produced by NADPH oxidase: a novel downstream signaling pathway in melatonin‐induced stress tolerance in Solanum lycopersicum

The beneficial effects of melatonin on abiotic stress have been demonstrated in several plants. However, little is known about the signal transduction pathway of melatonin involved in the plant stress response. Here, we manipulated the melatonin levels in tomato plants through a chemical approach. The roles of melatonin in stress tolerance were studied by assessing the symptoms, chlorophyll fluorescence and stress‐responsive gene expression. Moreover, both chemical and genetic approaches were used to study the roles of hydrogen peroxide (H₂O₂) in melatonin‐induced signal transduction in tomato plants. We found that melatonin activates NADPH oxidase (RBOH) to enhance H₂O₂ levels by reducing its S‐nitrosylation activity. Furthermore, melatonin‐induced H₂O₂ accumulation was accompanied by obtainable stress tolerance. Inhibition of RBOH or chemical scavenging of H₂O₂ significantly reduced the melatonin‐induced defense response, including reduced expression of several stress‐related genes (CDPK1, MAPK1, TSPMS, ERF4, HSP80 and ERD15) and reduced antioxidative enzyme activity (SOD, CAT and APX), which were responsible for the stress tolerance. Collectively, these results revealed a novel mechanism in which RBOH activity and H₂O₂ signaling are important components of the melatonin‐induced stress tolerance in tomato plants.     

Multifaceted roles of aquaporins as molecular conduits in plant responses to abiotic stresses

Abiotic stress has become a challenge to food security due to occurrences of climate change and environmental degradation. Plants initiate molecular, cellular and physiological changes to respond and adapt to various types of abiotic stress. Understanding of plant response mechanisms will aid in strategies aimed at improving stress tolerance in crop plants. One of the most common and early symptoms associated with these stresses is the disturbance in plant–water homeostasis, which is regulated by a group of proteins called “aquaporins”. Aquaporins constitute a small family of proteins which are classified further on the basis of their localization, such as plasma membrane intrinsic proteins, tonoplast intrinsic proteins, nodulin26-like intrinsic proteins (initially identified in symbiosomes of legumes but also found in the plasma membrane and endoplasmic reticulum), small basic intrinsic proteins localized in ER (endoplasmic reticulum) and X intrinsic proteins present in plasma membrane. Apart from water, aquaporins are also known to transport CO₂, H₂O₂, urea, ammonia, silicic acid, arsenite and wide range of small uncharged solutes. Besides, aquaporins also function to modulate abiotic stress-induced signaling. Such kind of versatile functions has made aquaporins a suitable candidate for development of transgenic plants with increased tolerance toward different abiotic stress. Toward this endeavor, the present review describes the versatile functions of aquaporins in water uptake, nutrient balancing, long-distance signal transfer, nutrient/heavy metal acquisition and seed development. Various functional genomic studies showing the potential of specific aquaporin isoforms for enhancing plant abiotic stress tolerance are summarized and future research directions are given to design stress-tolerant crops.     

Role of reactive oxygen species-generating enzymes and hydrogen peroxide during cadmium, mercury and osmotic stresses in barley root tip

The effect of cadmium (Cd) on the expression and activity of NADPH oxidase, peroxidase and oxalate oxidase as well as on the expression of aquaporins and dehydrins was studied in barley root tip. The root tip represented intact apical part of the barley root containing the root cap, meristems and elongation zone. Except stress induced by Cd, barley root tips were analysed after their exposure to phytotoxic concentration of mercury (Hg)-, hydrogen peroxide (H₂O₂)- or polyethylene glycol (PEG)-induced water stress in order to compare the Cd-induced changes with changes induced by these other stress factors. Cd, Hg, H₂O₂ and with some exceptions also PEG treatments caused similar alterations in the gene expression of reactive oxygen species (ROS)-generating and water deficiency-related genes, and in the activity of ROS-generating enzymes. These evidences support our opinion that ROS accumulation and water imbalance are the common symptoms of these stress factors and that the elevated production of H₂O₂ plays, probably as a signal molecule, a key role in the induction of plant responses to abiotic stresses in barley root tip. On the other hand, H₂O₂ at permanent high concentration is probably the main toxic factor during stress conditions.     

Polyamines, polyamine oxidases and nitric oxide in development, abiotic and biotic stresses

Nitric oxide (NO), polyamines (PAs), diamine oxidases (DAO) and polyamine oxidases (PAO) play important roles in wide spectrum of physiological processes such as germination, root development, flowering and senescence and in defence responses against abiotic and biotic stress conditions. This functional overlapping suggests interaction of NO and PA in signalling cascades. Exogenous application of PAs putrescine, spermidine and spermine to Arabidopsis seedlings induced NO production as observed by fluorimetry and fluorescence microscopy using the NO-binding fluorophores DAF-2 and DAR-4M. The observed NO release induced by 1 mM spermine treatment in the Arabidopsis seedlings was very rapid without apparent lag phase. These observations pave a new insight into PA-mediated signalling and NO as a potential mediator of PA actions. When comparing the functions of NO and PA in plant development and abiotic and biotic stresses common to both signalling components it can be speculated that NO may be a link between PA-mediated stress responses filing a gap between many known physiological effects of PAs and amelioration of stresses. NO production indicated by PAs could be mediated either by H₂O₂, one reaction product of oxidation of PAs by DAO and PAO, or by unknown mechanisms involving PAs, DAO and PAO.     

LSD1‐, EDS1‐ and PAD4‐dependent conditional correlation among salicylic acid,hydrogen peroxide,water use efficiency and seed yield in Arabidopsis thaliana

In Arabidopsis thaliana, LESION SIMULATING DISEASE 1 (LSD1), ENHANCED DISEASE SUSCEPTIBILITY 1 (EDS1) and PHYTOALEXIN DEFICIENT 4 (PAD4) proteins are regulators of cell death (CD) in response to abiotic and biotic stresses. Hormones, such as salicylic acid (SA), and reactive oxygen species, such as hydrogen peroxide (H₂O₂), are key signaling molecules involved in plant CD. The proposed mathematical models presented in this study suggest that LSD1, EDS1 and PAD4 together with SA and H₂O₂ are involved in the control of plant water use efficiency (WUE), vegetative growth and generative development. The analysis of Arabidopsis wild‐type and single mutants lsd1, eds1, and pad4, as well as double mutants eds1/lsd1 and pad4/lsd1, demonstrated the strong conditional correlation between SA/H₂O₂ and WUE that is dependent on LSD1, EDS1 and PAD4 proteins. Moreover, we found a strong correlation between the SA/H₂O₂ homeostasis of 4‐week‐old Arabidopsis leaves and a total seed yield of 9‐week‐old plants. Altogether, our results prove that SA and H₂O₂ are conditionally regulated by LSD1/EDS/PAD4 to govern WUE, biomass accumulation and seed yield. Conditional correlation and the proposed models presented in this study can be used as the starting points in the creation of a plant breeding algorithm that would allow to estimate the seed yield at the initial stage of plant growth, based on WUE, SA and H₂O₂ content.     

Pretreatment with H(2) O(2) alleviates aluminum-induced oxidative stress in wheat seedlings

Hydrogen peroxide (H₂O₂) is a key reactive oxygen species (ROS) in signal transduction pathways leading to activation of plant defenses against biotic and abiotic stresses. In this study, we investigated the effects of H₂O₂ pretreatment on aluminum (Al) induced antioxidant responses in root tips of two wheat (Triticum aestivum L.) genotypes, Yangmai‐5 (Al‐sensitive) and Jian‐864 (Al‐tolerant). Al increased accumulation of H₂O₂ and O₂^?? leading to more predominant lipid peroxidation, programmed cell death and root elongation inhibition in Yangmai‐5 than in Jian‐864. However, H₂O₂ pretreatment alleviated Al‐induced deleterious effects in both genotypes. Under Al stress, H₂O₂ pretreatment increased the activities of superoxide dismutase, catalase, peroxidase, ascorbate peroxidase and monodehydroascorbate reductase, glutathione reductase and glutathione peroxidase as well as the levels of ascorbate and glutathione more significantly in Yangmai‐5 than in Jian‐864. Furthermore, H₂O₂ pretreatment also increased the total antioxidant capacity evaluated as the 2, 2‐diphenyl‐1‐picrylhydrazyl‐radical scavenging activity and the ferric reducing/antioxidant power more significantly in Yangmai‐5 than in Jian‐864. Therefore, we conclude that H₂O₂ pretreatment improves wheat Al acclimation during subsequent Al exposure by enhancing the antioxidant defense capacity, which prevents ROS accumulation, and that the enhancement is greater in the Al‐sensitive genotype than in the Al‐tolerant genotype.     

Comparative proteomics analysis of the root apoplasts of rice seedlings in response to hydrogen peroxide

Background

Plant apoplast is the prime site for signal perception and defense response, and of great importance in responding to environmental stresses. Hydrogen peroxide (H₂O₂) plays a pivotal role in determining the responsiveness of cells to stress. However, how the apoplast proteome changes under oxidative condition is largely unknown. In this study, we initiated a comparative proteomic analysis to explore H₂O₂-responsive proteins in the apoplast of rice seedling roots.

Methodology/Principal Findings

14-day-old rice seedlings were treated with low concentrations (300 and 600 µM) of H₂O₂ for 6 h and the levels of relative electrolyte leakage, malondialdehyde and H₂O₂ were assayed in roots. The modified vacuum infiltration method was used to extract apoplast proteins of rice seedling roots, and then two-dimensional electrophoresis gel analysis revealed 58 differentially expressed protein spots under low H₂O₂ conditions. Of these, 54 were successfully identified by PMF or MS/MS as matches to 35 different proteins including known and novel H₂O₂-responsive proteins. Almost all of these identities (98%) were indeed apoplast proteins confirmed either by previous experiments or through publicly available prediction programs. These proteins identified are involved in a variety of processes, including redox homeostasis, cell wall modification, signal transduction, cell defense and carbohydrate metabolism, indicating a complex regulative network in the apoplast of seedling roots under H₂O₂ stress.

Conclusions/Significance

The present study is the first apoplast proteome investigation of plant seedlings in response to H₂O₂ and may be of paramount importance for the understanding of the plant network to environmental stresses. Based on the abundant changes in these proteins, together with their putative functions, we proposed a possible protein network that provides new insights into oxidative stress response in the rice root apoplast and clues for the further functional research of target proteins associated with H₂O₂ response.     

Nitric oxide and hydrogen sulfide crosstalk during heavy metal stress in plants

Gases such as ethylene, hydrogen peroxide (H₂O₂), nitric oxide (NO), carbon monoxide (CO) and hydrogen sulfide (H₂S) have been recognized as vital signaling molecules in plants and animals. Of these gasotransmitters, NO and H₂S have recently gained momentum mainly because of their involvement in numerous cellular processes. It is therefore important to study their various attributes including their biosynthetic and signaling pathways. The present review provides an insight into various routes for the biosynthesis of NO and H₂S as well as their signaling role in plant cells under different conditions, more particularly under heavy metal stress. Their beneficial roles in the plant's protection against abiotic and biotic stresses as well as their adverse effects have been addressed. This review describes how H₂S and NO, being very small-sized molecules, can quickly pass through the cell membranes and trigger a multitude of responses to various factors, notably to various stress conditions such as drought, heat, osmotic, heavy metal and multiple biotic stresses. The versatile interactions between H₂S and NO involved in the different molecular pathways have been discussed. In addition to the signaling role of H₂S and NO, their direct role in posttranslational modifications is also considered. The information provided here will be helpful to better understand the multifaceted roles of H₂S and NO in plants, particularly under stress conditions.     

Cloning and characterization of three genes encoding Qb-SNARE proteins in rice

Qb-SNARE proteins belong to the superfamily of SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) and function as important components of the vesicle trafficking machinery in eukaryotic cells. Here, we report three novel plant SNARE (NPSN) genes isolated from rice and named OsNPSN11, OsNPSN12 and OsNPSN13. They have about 70% nucleotide identity over their entire coding regions and similar genomic organization with ten exons and nine introns in each gene. Multiple alignment of deduced amino acid sequences indicate that the OsNPSNs proteins are homologous to AtNPSNs from Arabidopsis, containing a Qb-SNARE domain and a membrane-spanning domain in the C-terminal region. Semi-quantitative RT-PCR assays showed that the OsNPSNs were ubiquitously and differentially expressed in roots, culms, leaves, immature spikes and flowering spikes. The expression of OsNPSNs was significantly activated in rice seedlings treated with H₂O₂, but down-regulated under NaCl and PEG6000 stresses. Transient expression method in onion epidermal cells revealed that OsNPSNs were located in the plasma membrane. Transformed yeast cells with OsNPSNs had better growth rates than empty-vector transformants when cultured on either solid or liquid selective media containing various concentrations of H₂O₂, but more sensitive to NaCl and mannitol stresses. The 35S:OsNPSN11 transgenic tobacco also showed more tolerance to H₂O₂ and sensitivity to NaCl and mannitol than non-transgenic tobacco. These results indicate that OsNPSNs may be involved in different aspects of the signal transduction in plant and yeast responses to abiotic stresses. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.