Functional Analysis Of Arabidopsis GCIA2 In Regulating Carbohydrate Synthesis And ABA Response

The ubiquitin proteasome system(UPS)is a rapid regulatory mechanism for selectively protein degradation in eukaryotes and plays an important role in regulating plant growth and development and the responses to abiotic stress.RING type ubiquitin ligase E3 is one of the key components of UPS.Fructose-1,6-bisphosphatase(FBPase)is one of the key enzymes in glucose metabolism.Previous research showed that FBPase can be degraded via UPS in yeast,and the E3 in this process is the Gid complex.The subunits Gid2 and Gid9 containing RING domains carry the E3 activity of the Gid complex.Homology analysis showed that yeast Gid2 / Gid9 had four homologous genes in Arabidopsis(GCIA1/2/3/4).In this thesis,the biological function of Arabidopsis GCIA2 was analyzed by both in vitro and in vivo methods.Firstly,the GCIA2 protein was expressed and purified in prokaryotic system and then E3 activity was checked by in vitro ubiquitination assay.But no positive E3 activity was detected.This result may due to the lack of protein post-translational modification which is important for protein activities.Subsequently,GCIA2 protein was expressed and purified via tobacco transient system,but still showed no E3 activity in the following in vitro ubiqutination assay.Since it is reported that the in vitro E3 activities of both yeast and human Gid2 / Gid9 were all detected in the form of Gid complex,the in vitro E3 activity of Arabidopsis GCIA2 may also need the binding with other subunits of the Gid complex.We will continue to perform the experimental analysis in this regard.Subcellular localization analysis showed that GCIA2 was mainly located in the nucleus and cytoplasm.Expression pattern analysis showed that the expression level of GCIA2 in cauline leaves and mature siliques was significantly higher than that in other tissues,and its expression was induced by ABA and salt stress treatment.To analyze the function of GCIA2 in plants,we first identified three T-DNA insertion mutants: gcia2,gcia2-2 and gcia2-3.To avoid the influence of gene redundancy on gene function analysis,we created and identified related double mutants based on the gcia2 single mutant by conventional plant cross.The homozygous double mutants of gcia1 gcia2,gcia2 gcia3,gcia2 gcia4 have been successfully obtained so far.At the same time,the homozygous transgenic plants of the overexpression lines and functional complementary lines of GCIA2 were successfully constructed for further gene function analysis.The yeast Gid2 is known to regulate the ubiquitination and degradation of FBPase as the key E3 subunit of the Gid complex.There are two functional FBPase in Arabidopsis:the cytosolic FBPase(cyFBPase)and chloroplast FBPase(cp FBPase).They are all the key enzymes in carbohydrate synthesis and play an important role in plant growth and development.To explore whether GCIA2 regulates the ubiquitin degradation of FBPase in Arabidopsis,we first checked the sugar-related phenotype of gcia2 mutant.The results showed under no sugar condition gcia2 seeds could germinate normally,but partial seedlings exhibited growth arrest after germination.This phenotype suggests that the gene function of GCIA2 may indeed relate to sugar metabolism or signal transduction in plants.Pull-down and Co-IP analysis also showed that GCIA2 protein interacted with both cyFBPase and cp FBPase.However,there was no significant difference in the protein level and the degradation rate of the two FBPase between wild-type and gcia2 seedlings.These results suggest that the gene function of GCIA2 in Arabidopsis is different from its yeast homolog Gid2.The main function of GCIA2 may not be involved in the UPS degradation of FBPase in Arabidopsis.However,the results of no difference between the two FBPase protein level and stability in gcia2 and the wild-type seedlings could be due to the genetic redundancy in Arabidopsis.GCIA2 related double mutant and multiple mutant materials will be used for analysis in the future work.The expression of GCIA2 gene was induced by ABA and salt stress.There is an obvious crosstalk between the sugar and ABA signal transduction pathway.The information from ABRC showed that GCIA1 which is the homologous gene of GCIA2 was co-expressed with SnRK2.3,one of the key components in ABA signaling pathway.Based on the above information,it is speculated that GCIA2 might affect both ABA signaling pathway and drought response.Phenotype analysis showed that gcia2 is highly sensitive to ABA,and GCIA2 mutation mostly affected post-germination growth of roots under ABA condition.And drought stress tolerance of gica2 was significantly reduced compared to wild type plants.GST pull-down assay proved that GCIA2 interacted with SnRK2.3.In addition,SnRK1 is reported to be the central integrator of stress and energy transduction in Arabidopsis,and its overexpression plants is sensitive to ABA which is similar to gcia2.Therefore,maybe the function of GCIA2 also be related to SnRK1.1.The protein-protein interaction between GCIA2 and SnRK1.1 was also confirmed by GST pull-down assay.All the results indicated that GCIA2 may affect plant ABA response and energy metabolism via regulating the function of SnRK2.3 and SnRK1.1.