| Reactive oxygen species (ROS) are highly toxic to plants. However, they can act as second messagers and play key roles in signaling transduction in plants. Plasma-membrane NADPH oxidase transfers electrons from cytoplasmic NADPH to O2 to form superoxide radical (O2· ) in plants. It has been documented that NADPH oxidase is involved in plant defense reactions to pathogen or elicitor attack and in plant response to abiotic stresses.The plant hormone abscisic acid (ABA) plays important roles in responses to many abiotic stresses including drought, salinity and cold in plants. Recently, NADPH oxidase has been addressed to function in the production of ROS in ABA-induced stomatal closing in Arabidopsis. However, little information is available on ABA-stimulated immediate production of ROS (oxidative burst) and the origin of the ROS in plant cells except in guard cells. In this study, we investigated the rapid production of hydrogen peroxide (H2O2) induced by ABA in suspension culture cells of tobacco (Nicotiana tabacum L.) BY-2 cells. The results showed that the immediate generation of H2O2. which was derived from superoxide dismutase-catalyzed dismutation of superoxide radical, was significantly induced by ABA. Furthermore, treatment of the cultured tobacco cells with ABA also resulted in obvious increase in plasma membrane (PM) NADPH oxidase activity, and these enhanced effects of H2O2 production and NADPH oxidase activity induced by ABA were pronouncedly inhibited by two widely used mammalian neutrophil NADPH oxidase inhibitors, diphenylene iodonium (DPI) and imidazole. These results indicated that PM NADPH oxidase plays important roles in ABA-induced rapid accumulation of H2O2 in cultured tobacco cells. In addition, two transgenic cultured tobacco cell lines overexpressing NtrbohD, a PM NADPH oxidase gene in tobacco, were generated and the expression levels of the gene were analyzed by RT-PCR and western blot The results revealed that ABA obviously enhanced the expression levels of NtrbohD in cultured tobacco cells, suggesting that NtrbohD participates in ABA-stimulated generation of H2O2.Nickel, a kind of heavy metal, is one of essential micronutrients for plant growth and development. However, it becomes toxic at high concentration. It has been addressed that excessive nickel can lead to significant increase in the levels of membrane lipid peroxidation in plants. However, the mechanism for the generation of ROS and the factors that affect the syntheses of ROS in Ni-treated plants are largely unknown. Here, we investigated the roles of the PM NADPH oxidase in nickel-induced enhancement in production of ROS and the activity of antioxidant enzymes such as SOD, CAT and GR in roots of wheat (Triticum durum D.) seedlings. Treatment with nickel resulted in significant increase not only in levels of membrane lipid peroxidation and content of H2O2, but also in the production rate of O2'- and the activity of the PM NADPH oxidase in wheat roots. The increased effects caused by nickel were pronouncedly inhibited by pretreatments with three NADPH oxidase inhibitors (DPI, imidazole and pyridine). Moreover, the change patterns of the Ni-induced increase and inhibitor-caused decrease in these parameters were quite similar. These data suggest that theNi-induced enhancements in levels of H2O2 and O2·-, which appear to cause membrane lipid peroxidation, are mainly originated from PM NADPH oxidase. In addition, data suggest that Ca2+ may be involved in the oxidative stress and PM NADPH oxidase also participates in the rise of the activity of SOD, CAT and GR induced by nickel in wheat roots.The regulation of NADPH oxidase activity is the main subject for plant research. Previous investigations revealed that calcium-dependent protein kinase 1 of Arabidopsis (Ak1) is involed in the modulation of NADPH oxidase activity. However, the detailed mechanism is unclear. We checked the interaction of AK1 with the C-terminal and N-terminal cytosolic regions of AtrbohD and AtrbohF using yeast two-hybrid technology, and there are no interactions were...
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