Functional Analysis Of NADPH Oxidase Genes AtRbohA And AtRbohI In Response To Salt And Drought Stress In Arabidopsis

In the natural environment,plants are frequently exposed to multi-abiotic stresses,such as drought and salt stress.Plants have evolved various strategies to adapt to the saline environments.ROS are the important signaling molecules playing a vital role in response to various abiotic stresses.A number of enzymes could generate or degrade ROS.Among these enzymes,the NADPH oxidases are known as respiratory burst oxidase homologs(RBOHs)functions in transporting electron and synthesize the ROS.Previous studies have shown that AtRbohD and AtRbohF are involved in response to salt and drought stress.However,whether the other AtRbohs participate in this process remains to be determined.In this study,we investigated the roles of AtRbohA and AtRbohl in response to salt and drought stresses.Also,we preliminarily analyzed the mechanisms of salt and drought tolerance using their deletion and overexpression mutants.The results are as follows:The expression levels of AtRbohA were significantly reduced during NaCl and exogenous ABA treatments.The AtRbohl transcripts were strongly detected in siliques and roots.Loss of function in AtRbohA did not affect the sensitivity to NaCl and ABA treatments during germination compared to wild type.However,a gain of function in AtRbohA(g-rbohA)is more sensitivity to NaCl and ABA treatments during germination.To further confirm this result,we obtained two AtRbohA overexpressors(OE-3 and OE-16)by qRT-PCR assay.The results showed that overexpression of AtRbohA in wild type significantly enhanced the sensitivity to NaCl and ABA treatments during germination.Moreover,the H2O2 contents in dry seeds of AtRbohA overexpressors(OE-3 and OE-16)and g-rbohA are more higher compared to wild type.By predicting and searching the potential cis-acting element in AtRbohA promoter,we found that there exist a CACCG box which could recognized by transcription factor ABI4.The AtRbohA expression level in abi4-1 mutant is more lower compared to wild type.However,ABI4 could not directly bind to the AtRbohA promoter,which was verified by Yeast one-hybrid system in vitro.There results showed that AtRbohA participates in the response to ABA and NaCl treatments during germination and can regulated by ABI4.Exogenous ABA application can decrease AtRbohI expression at transcript level,while mannitol increases AtRbohl expression level significantly.The AtRbohI transcripts were strongly detected in dry seeds and roots.The loss-of-function mutant rbohl exhibited sensitivity to ABA and mannitol treatments during germination.Furthermore,the lateral root growth of Atrbohl was severely inhibited after treatment with mannitol compared to wild type.Reversely,the lateral root growth of AtRbohl overexpressors(OE-3,OE-4 and OE-5)was less inhibited exposed to mannitol treatment.Overexpression of AtRbohI in Arabidopsis significantly improved drought tolerance.Moreover,more H2O2 accumulated in AtRbohI overexpressors than in wild type plants in response to mannitol treatment.Our conclusion is that AtRbohI functions in drought-stress response in Arabidopsis thaliana.