Concurrent effect of drought and heat stress in rice (Oryza sativa L.): physio-biochemical and molecular approach.

The present study was carried out to investigate the physio-biochemical and molecular responses of two rice genotypes (Noichi and N22) under drought, heat and combined drought/heat stress conditions. The antagonistic stomatal activity was found under the combined stress conditions; stomata were open under control and heat stress, conversely, stomata remained closed under drought and combined stress levels. Photosynthetic activity and chlorophyll content are decreased by the overproduction of reactive oxygen species and increased lipid peroxidation in both rice genotypes. To prevent oxidative damage, many antioxidant enzymes like catalase (CAT), ascorbate peroxidase (APX) and superoxide dismutase (SOD) are produced in both genotypes under these conditions. Under the single stress conditions, CAT activity were increased in N22, whereas combined stress levels, SOD and APX activity were higher for both genotypes. Proline accumulation was also increased under single as well as combined stress conditions for both genotypes to combat stress injuries. Pollen viability was lost under all stress levels but severe loss was found under combined stress levels, which causes spikelet sterility leading to yield losses for both genotypes. As evident from transcript levels, HSP71.18 and HSP71.10 expressions were higher under single and combined conditions, butHSP72.57 gene expression increased only by individual stress levels. WRKY11, WRKY 55, DREB 2A, LEA3 and DHN1 were positively expressed under all stress levels. Conversely, expression of DREB2B genes was higher only under single stress levels. In summary, these results suggest that the effect of combined stress is different from the single stress and it is more severe than the individual stress. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-024-03980-1.

Chloroplast avoidance movement: a novel paradigm of ROS signalling.

The damaging effects of supra-optimal irradiance on plants, often turning to be lethal, may be circumvented by chloroplast avoidance movement which realigns chloroplasts to the anticlinal surfaces of cells (parallel to the incident light), essentially minimizing photon absorption. In angiosperms and many other groups of plants, chloroplast avoidance movement has been identified to be a strong blue light (BL)-dependent process being mediated by actin filaments wherein phototropins are identified as the photoreceptor involved. Studies through the last few decades have identified key molecular mechanisms involving Chloroplast Unusual Positioning 1 (CHUP1) protein and specific chloroplast-actin (cp-actin) filaments. However, the signal transduction pathway from strong BL absorption down to directional re-localization of chloroplasts by actin filaments is complex and ambiguous. Being the immediate cellular products of high irradiance absorption and having properties of remodelling actin as well as phototropin, reactive oxygen species (ROS) deemed to be more able and prompt than any other signalling agent in mediating chloroplast avoidance movement. Although ROS are presently being identified as fundamental component for regulating different plant processes ranging from growth, development and immunity, its role in avoidance movement have hardly been explored in depth. However, few recent reports have demonstrated the direct stimulatory involvement of ROS, especially H2O2, in chloroplast avoidance movement with Ca2+ playing a pivotal role. With this perspective, the present review discusses the mechanisms of ROS-mediated chloroplast avoidance movement involving ROS-Ca2+-actin communication system and NADPH oxidase (NOX)-plasma membrane (PM) H+-ATPase positive feed-forward loop. A possible working model is proposed.

Orchestration of Cu-Zn SOD and class III peroxidase with upstream interplay between NADPH oxidase and PM H+-ATPase mediates root growth in Vigna radiata (L.) Wilczek.

Post-germination plant growth depends on the regulation of reactive oxygen species (ROS) metabolism, spatiotemporal pH changes and Ca+2 homeostasis, whose potential integration has been studied during Vigna radiata (L.) Wilczek root growth. The dissipation of proton (H+) gradients across plasma membrane (PM) by CCCP (protonophore) and the inhibition of PM H+-ATPase by sodium orthovanadate repressed SOD (superoxide dismutase; EC 1.15.1.1) activity as revealed by spectrophotometric and native PAGE assay results. Similar results derived from treatment with DPI (NADPH oxidase inhibitor) and Tiron (O2- scavenger) denote a functional synchronization of SOD, PM H+-ATPase and NOX, as the latter two enzymes are substrate sources for SOD (H+ and O2-, respectively) and are involved in a feed-forward loop. After SOD inactivation, a decline in apoplastic H2O2 content was observed in each treatment group, emerging as a possible cause of the diminution of class III peroxidase (Prx; EC 1.11.1.7), which utilizes H2O2 as a substrate. In agreement with the pivotal role of Ca+2 in PM H+-ATPase and NOX activation, Ca+2 homeostasis antagonists, i.e., LaCl3 (Ca+2 channel inhibitor), EGTA (Ca+2 chelator) and LiCl (endosomal Ca+2 release blocker), inhibited both SOD and Prx. Finally, a drastic reduction in apoplastic OH (hydroxyl radical) concentrations (induced by each treatment, leading to Prx inhibition) was observed via fluorometric analysis. A consequential inhibition of root growth observed under each treatment denotes the importance of the orchestrated functioning of PM H+-ATPase, NOX, Cu-Zn SOD and Prx during root growth. A working model demonstrating postulated enzymatic synchronization with an intervening role of Ca+2 is proposed.

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.

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.

Abscisic acid mediated differential growth responses of root and shoot of Vigna radiata (L.) Wilczek seedlings under water stress.

Water stress is a common problem in Indian agriculture and recent global climate change has aggravated this problem further. Plants have an adaptive response to water scarcity as reflected in differential root and shoot growth. In case of Vigna radiata seedlings we have observed earlier promotion of root growth while inhibition of shoot (hypocotyl) growth under mild water stress, which is probably mediated by ABA. However, the exact mechanism of ABA action is far from clear. The present study attempts to elucidate the action of ABA through apoplastic reactive oxygen species (ROS) and its impact upon antioxidant defence system during the growth of root and shoot under water stress. Thus promotion of root growth by application of exogenous ABA (10 µM) and mild water stress (ψ -0.5 MPa) may be correlated with enhanced apoplastic ROS production possibly by activating plasma membrane located NADPH oxidase (NOX) enzyme. On the contrary, in hypocotyls where growth was rather inhibited by application of water stress or ABA, neither NOX activity nor ROS accumulation was significantly detected upon these treatments. Overall activity of antioxidant enzymes [superoxide dismutase (SOD, EC 1.15.1.1), peroxidase (POX, EC 1.11.1.7), ascorbate peroxidase (APX, EC 1.11.1.1) and catalase (CAT, EC 1.11.1.6)] was high and somewhat promoted by ABA and water stress treatment in roots compared to hypocotyls. A possible ROS-mediated role of ABA in promoting growth and antioxidant activity in roots under water stress has been proposed.

Early axis growth during seed germination is gravitropic and mediated by ROS and calcium.

In plant establishment, seed germination is characterized by the emergence of a radicle for secured anchorage to the soil and nutrient and water uptake. Early growth of germinating axes appears to be gravisensitive, and the regulation of this process is largely uncharacterized, particularly in case of epigeally germinating species. Our previous work on the germination of Vigna radiata seeds demonstrated the role of apoplastic reactive oxygen species (ROS) in germination-associated axis growth. This study attempts to explore a possibly similar role of ROS in the gravitropic bending of germinating axes. Pharmacological and histological studies correlated the curvature growth of the axis (due to cell elongation in the cortical region of the upper side) with apoplastic superoxide accumulation. The superoxide was produced by diphenylene iodonium chloride (DPI)-insensitive NADH oxidase, which was different from the DPI-sensitive NADPH oxidase active in the apical elongation zone of the radicle. This NADH oxidase was differentially controlled by IAA, and its activation required influx of apoplastic Ca2+. This study shows that the early axis growth in germinating seeds is gravisensitive, which is distinct spatially as well as temporally from the elongation growth of the axis (radicle) and controlled by auxin and cytosolic Ca2+ through NADH oxidase-dependent ROS production.

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.

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(•)- 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(•)- and H2O2 was done using nitroblue tetrazolium (NBT) and 3,3',5,5'-tetramethyl benzidine dihydrochloride hydrate (TMB), respectively. Apoplastic level of O2(•)- 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(•)- while the differentiating zone of axis (radicle) requires H2O2 for cell wall stiffening.

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.

Plant responses to water stress: role of reactive oxygen species.

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.