Functional Characterization Of Swap70-A Guanine Nucleotide Exchange Factor In Arabidopsis Thaliana

Small GTPases function as key molecular signaling switches to regulate many important cellular processes in plants, for example, growth and development of plant, biotic and abiotic stress responses and plant immunity. And the guanine nucleotide exchange factor(GEF) promotes the activation of small GTPases.SWAP-70( Switch-associated protein 70), is a phosphatidylinositol-3,4,5-trisphosphate(Ptd Ins(3,4,5)P3)-dependent guanine nucleotide exchange factor(GEF),the molecular weight of the protein is 70-k Da.In recent years, it was widely researched in animal kingdom, and it was reported that has involved in animal innate immune, membrane ruffling, virus invasion, and played a key role in regulating F-actin rearrangements. However, the study of SWAP70 in plant kingdom is tiny, let alone in Arabidopsis thaliana.Here, we analysised the function of At SWAP70 in Arabidopsis using the evidence of phylogenetic tree of SWAP70 homologs, RT-PCR and Quantitative Real-Time PCR, laser scanning confocal microscopy and physiological experiment of mutant. In this study, we try to identify the genetic and molecular mechanisms of At SWAP70 actives the ROP GTPases as a GEF, which regulate various cellular processes, such as membrane trafficking and abiotic stresses.And we intend to provide evidence that At SWAP70 involved in the process of plant growth and development and response stresses from environment.The main results of this paper were summarized as following:1. SWAP70 had originated from the early differentiation between monocotyledon and dicotyledon.The analysis of the evolution of SWAP70 homologs shows that the amino acid sequence analysis of SWAP70 in monocotyledon and dicotyledon are divided into two classes, indicate that SWAP70 had originated from the early differentiation between above both.2. The mutant of gene At SWAP70 called 232, which is a loss-of-function mutant.RT-PCR and q RT-PCR results indicat that the T-DNA insert site is located in the fourth intron, and the mutant is a knock-out mutant.3. AtSWAP70 maybe involved in the process of vesicle trafficking, and mainly function in PVC/MVB and TGN.The analysis of subcellular localization indicates that a high degree of colocalization was observed in PVC/MVB between At SWAP70-YFP and markers-RFP, and shows a modest degree of colocalization in TGN and little degree in vacuole.So the results showed At SWAP70 has a closely connection with vesicle trafficking, and At SWAP70 mainly localized in PVC/MVB and TGN.4. At SWAP70 widely function in cellular process of various tissues. QRT-PCR results suggested that At SWAP70 is a constitutive expressional protein, because it could be detected in root, stem, leaves, flower, bud, pollen and so on.5. Gene At SWAP70 could response fastly under many abiotic stress(Na Cl、ABA and H2O2) and biotic stress(flg22、elf18). q RT-PCR results showed that in the treatment of Na Cl, ABA, H2O2, flg22 and elf18, the expression of gene At SWAP70 all could be regulate remarkably up or down.6. At SWAP70 had tended to response to abiotic stress rather than to biotic one, especially response the stress of Na Cl and ABA. In the mutant seeds greening experiment, the mutant 232 is much sensitive than WT under the treatment of Na Cl and ABA, but nearly have no difference under KCl. On the other hand, in the biotic stress experiment(Pst DC3000 and Pst DC3000 Avr Rpt2), WT and 232 also have no visible difference.Taken together, we speculate that the At SWAP70 maybe involved in the process of vesicle trafficking, biotic and abiotic stress responses and ABA responses in Arabidopsis thaliana as a guanine nucleotide exchange factor.Our findings provide insights into the molecular mechanisms by which At SWAP70 function as a GEF to regulate the vesicle trafficking and to adapt stress from environment.