Investigation On Functions Of NADPH Oxidase AtrbohD In Arabidopsis Thaliana Under Waterlogging Stress

Flooding is one of the major disasters in agriculture. It often causes waterlogging stress which results in oxygen deprivation in plants. Evidence indicated that reactive oxygen species (ROS) as signal molecules play important roles in plant response to hypoxia stress. However, the questions whether ROS are involved in waterlogging stress response and what are the molecularmechanisms remain largely unknown to date. In this paper, we demonstrated that AtrbohD is involved in the regulation of waterlogging stress via producing H₂O₂ in Arabidopsis by using NADPH oxidase AtrbohD mutant atrbohD, transgenic plants and other mutants related to oxygen deprivation signal transduction, and methods of bioinformatics, molecular biology and plant physiology. Firstly, T-DNA insertion homozygous null mutant atrbohD and transgenic plants overexpressing AtrbohD and GUS were obtained. RT-PCR results showed that AtrbohD transcripts were completely eliminated in atrbohD. Phenotype analysis suggests that atrbohD seedlings were very sensitive to waterlogging stress than the wild-type (WT) and overexpression transgenic plants in soil as the survival rate of atrbohD was apparently smaller than those of WT and transgenic plants. Moreover, by using RT-PCR and real-time PCR technologies, we found that the expression level of AtrbohD increased obviously in WT plants and the expression level of ADH in atrbohD was significantly lowerand that in transgenic plants was remarkable higer than WT under waterlogging stress. Then, the transgenic plants of AtrbohD::GUS were stained and GUS activity was determined. The results revealed that waterlogging treatment markedly elevated the gene expression of AtrbohD. In addition, H₂O₂ contents in atrbohD plants were significantly lower than those in WT plants under waterlogging stress. By contrast, H₂O₂ contents in transgenic plants overexpressing AtrbohD were pronouncedly higher than those in WT. These results suggest that AtrbohD is involved in waterlogging stress signal transduction in Arabidopsis.In order to further determine the action mechanism of AtrbohD, we obtained the GUS transgenic plants driven by promoters of AtrbohD, ROP2 and ADH respectively. After treatments with waterlogging, the WT seedlings transformed with ADH::GUS stained dark whereas the atrbohD1 and DN-rop2 plants transformed with the same vector had the unchanged staining results. WT and CA-rop2 plants transformed with AtrbohD::GUS stained dark and no significant difference in GUS staining results of DN-rop2 plants transformed with the same vector before and after waterlogging treatments. Furthermore, GUS activity data were consistent with the GUS staining results of these plants. We also measured the expression patterns of ROP2 and ADH1 by RT-PCR and real time PCR. Waterlogging treatments evidently induced the expression of ROP2 in atrbohD1 and WT plants, and the numbers of the transcripts increased with prolonged time.The ADH specific activities of WT, the mutants and transgenic plants were similar under normal growth conditiosns. However, under waterlogging stress, ADH activities in both WT and atrbohD increased. Compared with WT, atrbohD had relatively lower ADH activity. Additionally, ADH activities induced by waterlogging treatments were obviously inhibited in rop2 and DN-rop2 mutants. In DN-rop2, the ADH activity did not changed before and after waterlogging stress. We also observed that the H₂O₂ accumulation triggered by waterlogging in atrbohD was less than that in WT and such effects were enhanced with the prolonged treatment time. Furthermore, the increased generation rates of H₂O₂ evoked by waterlogging in rop2,DN-rop2 apparently were lower than WT while the parameters in CA-rop2 and transgenic plant overexpression of AtrbohD were higher than WT. Taken together, these results suggest that H₂O₂ produced by AtrbohD plays important roles in waterlogging stress signaling and AtrbohD probably places downstream of ROP2 in Arabidopsis.