Functional Analysis Of AtRabE1c In Respones To ABA

Among endogenous plant hormones,abscisic acid(ABA)plays an indispensable role in plant tolerance to drought and salt.In plant cells,there is an ABA signaling pathway in which ABA receptors PYL/PYR/RCAR,PP2 Cs and Sn RK2 s work as the main proteins.Currently,it is found that some proteins can interact with ABA receptor proteins,including those related to vesicle trafficking.For example,ESCRT system protein FREE1,VPS23 A and ALIX,can interact with ABA receptors such as PYL1,PYL4 and so on.These proteins are playing a role in the endocytic vacuolar degradation pathway for ABA receptors.However,the molecular mechanism of this pathway is still not clear.At present,the existing literatures shown that a small G protein RabE1 c,which can play an important role in vesicles trafficking,maybe interact with ABA receptors PYL1,PYL4 and so on.Therefore,the purpose of this study is to investigate whether RabE1 c has an influence on ABA signaling pathway,and its possible mechanism in mediating ABA receptor vacuolar degradation.In this study,we use active or inactive transgenic mutants of the GFP-RabE1c-Q74 L,GFP-RabE1c-S29 N and GFPRabE1c-N128 I.GFP-RabE1c-Q74 L is the active form that could only bind to GTP,GFP-RabE1c-S29 N is the inactive form that could only bind to GDP,and GFPRabE1c-N128 I is the inactive form that could not bind to GTP or GDP.We studied the responses of these RabE1 c mutant lines and wild type line Col-0 under ABA treatment in germination rate,primary root growth,stomatal morphology and water loss rate.It was found that all mutants of RabE1 c showed hypersensitive phenotypes to ABA and GFP-RabE1c-N128 I line has deformed stomatal.In addition,under the stimulation of ABA,the transcription level of RabE1 c gene decreased.At the same time,the transcription levels of downstream response genes RD29 A,COR15A and ABI5 in the ABA signaling channel were significantly increased in RabE1 c mutant GFP-RabE1c-Q74 L and GFP-RabE1c-N128 I than in Col-0 under ABA stimulation,while the transcription levels of these genes were slightly increased in GFP-RabE1cS29 N.The results suggest that RabE1 c may take part in the regulation of ABA signaling pathway.In order to analyze the subcellular localization differences among RabE1 c mutants,we treated RabE1 c mutant seedlings with FM4-64 dyes and vesicles transport inhibitor BFA.It was found that GFP-RabE1c-Q74 L and GFP-RabE1cS29 N could be located in the endomembrane system,mainly in TGN,while GFPRabE1c-N128 I could only exist in the cytosol.Since it was reported that there is an interaction between RabE1 c and ABA receptors,we attempted to analyze whether the RabE1 c mutation could affect the subcellular distribution of ABA receptors in RabE1 c mutant background was observed.To conduct this study,the RabE1 c mutant plants were hybridized with ABA receptor overexpressed plants to obtain ABA receptor-overexpressed plants under RabE1 c mutant background.By observing the subcellular distribution of ABA receptors in the root cells of the seedlings,it was found that in the RabE1 c mutant background,both the ABA receptors PYL1 and PYR1 can be showed the punctate structure aggregation which was not present in the wild type background.Also,it was found that the clustered punctate structures were distributed in the cytosol and vacuoles.Since ABA receptors associated with plasma membrane can be transported to vacuoles for degradation through the endocytic pathway,we analyzed the endocytic rates of mutants using endocytosis pathway marker FM4-64 and vesicle trafficking inhibitor BFA to understand whether ABA receptor aggregation is related to possible endocytic pathway abnormalities caused by RabE1 c mutations.found that compared with wild type,the BFA body formation rate became more slowly in RabE1 c mutants,suggesting that endocytosis rate is slower in RabE1 c mutant.According to the above results,we speculate that the function of ABA receptor vacuolar degradation pathway is negatively affected by RabE1 c mutations.Furthermore,our laboratory colleagues used ABA receptor antibody to do western blot to show elevated protein levels of ABA receptors in RabE1 c mutants,which may be caused by inhibition of their endocytic vacuolar degradation pathway.The accumulation of ABA receptor proteins in mutants may interpret,the cellular and phenotypic results described above.The above studies lay a foundation for analysis of ABA receptor degradation pathways.Also,it is helpful for subsequent studies on plant drought resistance,which is of great significance to the development of crop drought tolerance.