Gene Expression Pattern Of Guanine Nucleotide Exchange Factors RopGEF4/8in Arabidopsis And The Response To ABA Signaling

Rho-related GTPase of plants (ROP) plays an important role in plant growth and development as a signaling protein. The function of ROP as a binary switch is performed by cycling between an inactive GDP-bound state and an active GTP-bound state in cells. Guanine nucleotide exchange factor (GEF) activates ROP by stimulating the exchange from GDP bound form to GTP bound form of GTPases. Arabidopsis thaliana RopGEF family of14members contains a conserved central domain, the domain of unknown function315(DUF315), and variable N-and C-terminal regions. However, the functions and roles of RopGEFs in the plant development or stress response are still unclear. ROPs act as regulators of various physiological processes of plant growth and development and also involved in the ABA signaling by suppressing the ABA-induced responses. In view of the above-mentioned facts, we want to know the role of RopGEFs involved in ABA signaling pathway.In this study, we characterized gene expression patterns of Arabidopsis RopGEF4and RopGEF8; Functions of RopGEF4and RopGEF8in ABA-mediated physiological processes were also discussed.We investigated the tissue-specific expression of RopGEF4and RopGEF8by performing the qRT-PCR analysis from different tissues:roots, stem, rosette leaves, capsules, seedlings and flowers, including early bud and open flower. Our data show that RopGEF4was expressed in all tissues. Whereas, RopGEF8was distinct tissue-specific expression pattern with predominant expression at flowers. To investigate further, we also constructed GUS expression vector drived by RopGEF4and RopGEF8promoter and transformed to Arabidopsis by floral dipping. The results of histochemical staining for GUS were consistent with the qRT-PCR analysis. Interestingly, RopGEF4was expressed at specific parts of seedlings such like the tip of the main vein of cotyledon. RopGEF8was mainly expressed in pollen. In order to study the subcelluar localization of RopGEF4, we contrasted the35S: RopGEF4:GFP and RopGEF4pro:RopGEF4:YFP transiently expression vector and transformed to Arabidopsis protoplasts by PEG. Both GFP and YFP fluorescences suggested that RopGEF4localized in plasma menmbrane, cytoplasm and nucleus.Next, we performed that RopGEF4and RopGEF8were involved in ABA signaling. The expression levels of RopGEF4and RopGEF8were changed after100μM ABA treatment. QRT-PCR data and GUS staining results indicate that RopGEF4transcription was up-regulated at3h after ABA treatment and then down-regulated at6h in leaf. Meanwhile, the expression was up-regulated at0.5h after ABA treatment and then down-regulated in root.RopGEF8transcription was up-regulated until1h after ABA treatment both in leaf and root. Then expression was down-regulated.T-DNA insert ropgef4mutant decreased insensitive to ABA-induced stomatal closure. Then, the phenotypes such as seed germination, cotyledon formation and root length between the mutants (ropgef4and ropgef8) and wild-type (WT) under ABA treatment were observed.ropfgef8decreased insensitive to ABA-induced seed germination.Whereas, there was no difference between ropgef4and WT in ABA-induced seed germination. Both ropgef4and ropgef8show the same phenotypes as WT in ABA-induced root length.Taken together, we performed the gene expression pattern of Arabidopsis RopGEF4and RopGEF8.And we preliminarily note that RopGEF4and RopGEF8are participated in ABA signaling pathway. However, more researches are needed to prove the definite function of RopGEFs in ABA signaling pathway.