Rup Kumar Kar

Plant responses to water stress: Role of reactive oxygen species

Terrestrial plants most often encounter drought stress because of erratic rainfall which has become compounded due to present climatic changes.Responses of plants to water stress may be assigned as either injurious change or tolerance index. One of the primary and cardinal changes in response to drought stress is the generation of reactive oxygen species (ROS), which is being considered as the cause of cellular damage. However, recently a signaling role of such ROS in triggering the ROS scavenging system that may confer protection or tolerance against stress is emerging. Such scavenging system consists of antioxidant enzymes like SOD, catalase and peroxidases, and antioxidant compounds like ascorbate, reduced glutathione; a balance between ROS generation and scavenging ultimately determines the oxidative load. As revealed in case of defence against pathogen, signaling via ROS is initiated by NADPH oxidase-catalyzed superoxide generation in the apoplastic space (cell wall) followed by conversion to hydrogen peroxide by the activity of cell wall-localized SOD. Wall peroxidase may also play role in ROS generation for signaling. Hydrogen peroxide may use Ca2+ and MAPK pathway as downstream signaling cascade. Plant hormones associated with stress responses like ABA and ethylene play their role possibly via a cross talk with ROS towards stress tolerance, thus projecting a dual role of ROS under drought stress.

Profiling of selected indigenous rice (Oryza sativa L.) landraces of Rarh Bengal in relation to osmotic stress tolerance.

A total of ten rare indigenous rice landraces of West Bengal were screened for germination potential and seedling growth under varying concentrations of sodium chloride (NaCl) and polyethylene glycol (PEG) solutions as osmotic stress inducing agents. Among the studied rice landraces Kelas and Bhut Moori showed highest degree of tolerance to induced osmotic stresses. Proline content of the studied lines was also determined. Genetic relationship among the studied rice landraces was assessed with 22 previously reported osmotic stress tolerance linked Simple Sequence Repeat (SSR) markers. The identified allelic variants in form of amplified products size (molecular weight) for each SSR marker were documented to find out allele mining set for the linked markers of the studied genotypes in relation to osmotic stress tolerance. A Microsatellite Panel was constructed for the different allelic forms (size of amplified products) of each used marker. Among 22 SSR markers, ten showed unique alleles in form of single specific amplified product for the studied four genotypes which can be used for varietal identification. Genetic relationship among the studied rice lines was determined and a dendrogram was constructed to reveal their genetic inter-relationship. Polymorphism Information Content (PIC) for each used marker was also calculated for the studied rice lines.

Comparative responses of three fuel wood yielding plants to PEG-induced water stress at seedling stage

Three species of fast growing fuel wood yielding plants locally available (Acacia holosericea, Bauhinia variegata and Cassia siamea) were characterized in respect of their responses to water stress. Seedlings (25 days) of these species, exposed to two levels of water stress (−0.5 and −1.0 MPa) induced by PEG-6000 for 24 h, were analysed for relative water content (RWC) and the contents of chlorophyll, protein, soluble sugars and proline in leaves along with activities of catalase, peroxidase and superoxide dismutase (SOD). RWC was lower in stressed compared to the unstressed seedlings. However, stress-induced decline in RWC was lowest in B. variegata. Chlorophyll and protein contents declined with increasing levels of water stress, decline being least in B. variegata. Soluble sugar and proline contents increased under water stress particularly in B. variegata. The enzyme activity of catalase (EC-1.11.1.6), peroxidase (EC-1.11.1.7) and SOD (EC-1.15.1.1) decreased with increased levels of water stress. Such decline in the activity of these enzymes was least in B. variegata. Apparently, B. variegata is potentially the species most tolerant to water stress among these three fuel wood-yielding plants.

Role of peroxidase activity and Ca(2+) in axis growth during seed germination.

AbstractMain conclusionAxis growth during seed germination is mediated by reactive oxygen species and apoplastic peroxidase plays a role by producing OH·from H2O2. Ca2+activates both apoplastic peroxidase and NADPH oxidase. Role of reactive oxygen species (ROS) in seed germination and axis growth has been demonstrated in our earlier works with Vigna radiata seeds by studying superoxide generation and its metabolism in axes (Singh et al. in Plant Signal Behav doi:10.4161/psb.29278, 2014). In the present study, the participation of apoplastic peroxidase along with the involvement of Ca2+ in axis growth during germination and post-germination stage has been investigated. Pharmacological studies using peroxidase (POX) inhibitors (salicylhydroxamic acid, SHAM; sodium azide, NaN3) and OH· scavenger (sodium benzoate, NaBz) indicated that seed germination and early axis growth (phase I) depend much on POX activity. Subapical region of axes corresponding to radicle that elongated much particularly in phase II suggested high POX activity as well as high NADPH oxidase (Respiratory burst oxidase homologue, Rboh, in plants) activity as indicated from localization by staining with TMB (3,3′,5,5′-tetramethyl benzidine dihydrochloride hydrate) and NBT (nitroblue tetrazolium chloride), respectively. Apoplastic class III peroxidase (Prx) and also cellular POX activity reached maximum at the time of radicle emergence as revealed by TMB staining, spectrophotometric and in-gel assay for POX activity. Treatment with Ca2+ antagonists (La3+, plasma membrane-located Ca2+ channel blocker and EGTA, Ca2+ chelator in apoplast) retarded seed germination and strongly inhibited axis growth, while Li+ (blocks endosomal Ca2+ release) was effective only in retarding phase II axis growth suggesting an involvement of Ca2+ influx during early axis growth. From the effect of Ca2+ antagonists on the localization of activities of POX and Rboh using stains, it appears that Ca2+ plays a dual role by activating Prx activity in apoplast while activating Rboh by entering into cytosol.

Superoxide and its metabolism during germination and axis growth of Vigna radiata (L.) Wilczek seeds

Involvement of reactive oxygen species in regulation of plant growth and development is recently being demonstrated with various results depending on the experimental system and plant species. Role of superoxide and its metabolism in germination and axis growth was investigated in case of Vigna radiata seeds, a non-endospermous leguminous species having epigeal germination, by studying the effect of different reactive oxygen species (ROS) inhibitors, distribution of O2•ˉ and H2O2 and ROS enzyme profile in axes. Germination percentage and axis growth were determined under treatment with ROS inhibitors and scavengers. Localization of O2•ˉ and H2O2 was done using nitroblue tetrazolium (NBT) and 3,3′,5,5′-tetramethyl benzidine dihydrochloride hydrate (TMB), respectively. Apoplastic level of O2•ˉ was monitored by spectrophotometric analysis of bathing medium of axes. Profiles of NADPH oxidase and superoxide dismutase (SOD) were studied by in-gel assay. Germination was retarded by treatments affecting ROS level except H2O2 scavengers, while axis growth was retarded by all. Superoxide synthesis inhibitor and scavenger prevented H2O2 accumulation in axes in later phase as revealed from TMB staining. Activity of Cu/Zn SOD1 was initially high and declined thereafter. Superoxide being produced in apoplast possibly by NADPH oxidase activity is further metabolized to •OH via H2O2. Germination process depends possibly on •OH production in the axes. Post-germinative axis growth requires O2•ˉ while the differentiating zone of axis (radicle) requires H2O2 for cell wall stiffening.

Reactive Oxygen Species-Mediated Promotion of Root Growth Under Mild Water Stress During Early Seedling Stage of Vigna radiata (L.) Wilczek

Plants often show promotion of root growth under water stress while inhibiting shoot growth. Regulation of such differential growth response is not very clearly understood. Our earlier observation on the role of reactive oxygen species (ROS) in embryonic axis growth during seed germination prompted us to explore a possible role for ROS in water stress-induced root growth promotion during early seedling development of Vigna radiata. Normal and water stress-induced root growth were dependent on generation of ROS, for example, superoxide (O2·−), hydrogen peroxide (H2O2) and hydroxyl radical (OH·), as evident from pharmacological experiments using ROS scavengers and inhibitors. However, in the case of the hypocotyl, neither normal growth nor growth inhibition under water stress was associated with ROS. Apoplastic O2·− and H2O2 production monitored by spectrophotometric analysis of the bathing medium using XTT and xylenol orange, respectively and localization of O2·− and H2O2 by NBT and TMB, respectively confirmed the involvement of ROS in root growth. Increased NADPH oxidase (NOX) activity (putative plasma membrane located apoplastic O2·− producer) in the membrane fraction, as determined by in-gel assay and concomitant increase in apoplastic O2·− and H2O2 generation, as evident by NBT and TMB stain in water stressed roots corroborated the view that promotion of root growth in the early seedling stage is effected by ROS being initiated with O2·− generation by NOX in the apoplastic space.

Congruence between PM H+-ATPase and NADPH oxidase during root growth: a necessary probability

Plasma membrane (PM) H+-ATPase and NADPH oxidase (NOX) are two key enzymes responsible for cell wall relaxation during elongation growth through apoplastic acidification and production of ˙OH radical via O2˙−, respectively. Our experiments revealed a putative feed-forward loop between these enzymes in growing roots of Vigna radiata (L.) Wilczek seedlings. Thus, NOX activity was found to be dependent on proton gradient generated across PM by H+-ATPase as evident from pharmacological experiments using carbonyl cyanide m-chlorophenylhydrazone (CCCP; protonophore) and sodium ortho-vanadate (PM H+-ATPase inhibitor). Conversely, H+-ATPase activity retarded in response to different ROS scavengers [CuCl2, N, N’ –dimethylthiourea (DMTU) and catalase] and NOX inhibitors [ZnCl2 and diphenyleneiodonium (DPI)], while H2O2 promoted PM H+-ATPase activity at lower concentrations. Repressing effects of Ca+2 antagonists (La+3 and EGTA) on the activity of both the enzymes indicate its possible mediation. Since, unlike animal NOX, the plant versions do not possess proton channel activity, harmonized functioning of PM H+-ATPase and NOX appears to be justified. Plasma membrane NADPH oxidase and H+-ATPase are functionally synchronized and they work cooperatively to maintain the membrane electrical balance while mediating plant cell growth through wall relaxation.

Integrated role of ROS and Ca+2 in blue light-induced chloroplast avoidance movement in leaves of Hydrilla verticillata (L.f.) Royle

Directional chloroplast photorelocation is a major physio-biochemical mechanism that allows these organelles to realign themselves intracellularly in response to the intensity of the incident light as an adaptive response. Signaling processes involved in blue light (BL)-dependent chloroplast movements were investigated in Hydrilla verticillata (L.f.) Royle leaves. Treatments with antagonists of actin filaments [2,3,5-triiodobenzoic acid (TIBA)] and microtubules (oryzalin) revealed that actin filaments, but not microtubules, play a pivotal role in chloroplast movement. Involvement of reactive oxygen species (ROS) in controlling chloroplast avoidance movement has been demonstrated, as exogenous H2O2 not only accelerated chloroplast avoidance but also could induce chloroplast avoidance even in weak blue light (WBL). Further support came from experiments with different ROS scavengers, i.e., dimethylthiourea (DMTU), KI, and CuCl2, which inhibited chloroplast avoidance, and from ROS localization using specific stains. Such avoidance was also partially inhibited by ZnCl2, an inhibitor of NADPH oxidase (NOX) as well as 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), a photosynthetic electron transport chain (ETC) inhibitor at PS II. However, methyl viologen (MV), a PS I ETC inhibitor, rather accelerated avoidance response. Exogenous calcium (Ca+2) induced avoidance even in WBL while inhibited chloroplast accumulation partially. On the other hand, chloroplast movements (both accumulation and avoidance) were blocked by Ca+2 antagonists, La3+ (inhibitor of plasma membrane Ca+2 channel) and ethylene glycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid (EGTA, Ca+2 chelator) while LiCl that affects Ca+2 release from endosomal compartments did not show any effect. A model on integrated role of ROS and Ca+2 (influx from apolastic space) in actin-mediated chloroplast avoidance has been proposed.

ROS Signaling: Relevance with Site of Production and Metabolism of ROS

One of the inevitable consequences of aerobic metabolism is the production of ROS in biological organisms including plants. Accordingly, plants have evolved antioxidant system (antioxidant enzymes and antioxidant molecules) to protect the cell components from oxidative threat. Usually cells accumulate ROS to a fatal level due to imbalance between generation and scavenging under stress or pathogenic attack. Recent observations led to an idea of involvement of ROS in signaling for plant growth and development. Although common sites of ROS generation are chloroplasts, mitochondria, and other organelles, ROS produced by plasma membrane localized NADPH oxidase (Rboh) in extracellular space has been implicated to participate in signaling process. Calcium, being most important signal molecule, has a cross talk with Rboh through a positive feedback loop that forms the basis of ROS-driven signaling network. Such signaling most often works behind plant growth and developmental processes like seed germination, root growth, stomatal regulation, and stress tolerance. Recent researches establish a ROS wave with an integration of Ca2+ signal that may operate for long distance signaling in plants. This may help to explain the event of systemic signaling in case of systemic acquired resistance (SAR) during pathogen attack and systemic acquired acclimation (SAA), which is achieved by gradual exposure to stress. Establishment of the possibilities of intracellular ROS signaling through vesicular trafficking and involvement in regulation of nuclear activities are on the way.

Possible interaction of ROS, antioxidants and ABA to survive osmotic stress upon acclimation in Vigna radiata L. Wilczek seedlings

Acclimation is a process of adjustment to gradual environmental change that enables plants to survive further stress by triggering some tolerance mechanism possibly involving ABA, ROS and oxidative metabolism. Here we have studied acclimation responses in terms of the performances with regard to physiological (growth and relative water content) and biochemical (chlorophyll, carotenoids, protein, malondialdehyde, sugar content) attributes, ABA production and stomatal sensitivity to exogenous ABA, extracellular ROS production and activation of antioxidant system. Our study reveals that repeated exposure to short-term mild water stress simulated by polyethylene glycol (PEG-6000) induces acclimation in mung bean (Vigna radiata L. Wilczek) seedlings. Acclimation induced tolerance was associated with reduced leaf size and enhanced root growth, accumulation of soluble sugar as osmoprotectant, maintenance of water potential, lessening of membrane damage as indicated by lower MDA content. Acclimated mung bean seedlings have shown greater degree of tolerance through increased production of and enhanced sensitivity to ABA (as reflected by faster stomatal closure), enhanced production of extracellular O2.- and H2O2 and the elevated activities of antioxidative enzymes to control the oxidative burst. Taken together, the results convey that acclimated seedlings minimize osmotic stress-induced damage through a possible network of ABA, ROS and antioxidants.