The Ethylene Biosynthesis Factors Mediate Arabidopsis Tolerance To Nacl Stress In Dependence On ROS Metabolism

Ethylene (C2H4) has long been known to be a signaling molecule that regulates a variety of developmental processes and stress responses in plants. Ethylene is synthesized from methionine by its conversion to S-adenosylmethionine (AdoMet), which is converted by the enzyme 1-aminocyclopropane- 1-carboxylate synthase (ACS) into methylthioadenosine (MTA) and 1-aminocyclopropane-1-carboxylic acid (ACC), the precursor of ethylene. ACS is a multigene family. The Arabidopsis thaliana genome contains 12 genes annotated as ACS (ACS1–12), dispersed among the five chromosomes. We choose two active genes in the Arabidopsis thaliana ACS gene family, and name them as AtREBA and AtREBB. A series of experiments have been performed:The homozygous T-DNA insertion mutants atreba,atrebb was identify and atreba/b double mutant(the three mutant lines were got from ABRC)were used for the following experiments:In the control experiments, the three lines mutant have no significant differences with WT. By the treatment of 150mmol / L NaCl, the germination rates of the three mutant strains is significantly lower than WT, which atreba,atrebb,atreba/b germination rate has dropped 52%,92%,80%, respectively. It can be seen, inactivation of AtREBA and AtREBB significantly inhibit plant tolerence to salt stress.RT-PCR results shows that the inactivation of AtREBA and AtREBB has not impact the expression of the another, suggesting that the interaction betweent AtREBA and AtREBB does not occur in the transcription level. GUS expressions were detected, the treatment of 150mmol/L NaCl increase the AtREBA and AtREBB expression levels, indicating that the salt stress will significantly influence the expression of these two genes.The yeast two-hybrid experiment show that AtREBA and AtREBB interaction in vitro. Bimolecular fluorescence complementation (BiFC) experiments show that two genes interact at the plasma membrane. Additionally, the interaction between two genes increase 10 times in the 60s by the treatment of 150mmol/L NaCl. So AtREBA and AtREBB affect the stress response of plants through the interaction, and the location in the plasma membrane. Compared with the result of the RT-PCR, we infer that the interaction between genes occur on the protein level.Salt stress cause the reactive oxygen species (ROS) production. Detecting H₂O₂ accumulation in atreba,atrebb and atreba/b plants show: 150mmol/L NaCl treatment encreased the H₂O₂ accumulationin in wild-type plants by DAB staining, while H₂O₂ accumulation was buffered in different degrees in the three mutants. Using H₂O₂ probe marking and 150mmol/L NaCl treatment, the fluorescence intensity increased in seedling roots of WT (18.37%) , atreba (64.34%) , atrebb(58.09%) , and atreba/b(51.35%). The lack of AtREBA obviously affect the expression of AtRBOHD/F, ROS producing enzyme NADPH oxidase.Catalase (CAT) exclusively remove ROS H₂O₂. 150mmol/L NaCl decreased CAT activity of WT,atreba,atrebb,atreba/b by 19.01%,58.33%,25.26%,44.76%. These data indicate that AtREBA and AtREBB regulate CAT activity in response to salt stress.In conclusion, AtREBA and AtREBB affect the seed germination and growth by regulating ROS metabolism in responding to salt stress. The two genes interact at the plasma membrane, and changing with salt stress.