Functional Analysis Of Ubiquitin Ligase GCIA1 In Arabidopsis

Ubiquitin modification plays an important role in regulating the plant growth and development and response to abiotic stress.E3 ubiquitin ligase is the key component of protein ubiquitination.Fructose-1,6-bisphosphatase(FBPase)is one of the key enzymes in glucose metabolism,so the regulation of FBPase is crucial for all the organisms.Previous research showed that FBPase can be degraded through the ubiquitin-proteasome system in yeast,and the E3 in this process is the GID complex composed of seven Gid proteins.Among them the two subunits Gid2/Gid9 have the characteristic RING domain of E3 ligase.GCIA1 is one of the Arabidopsis homologous genes of yeast Gid2/Gid9.In this thesis,the biological function of Arabidopsis GCIA1 was analyzed both in vitro and in vivo.Firstly,we detected the ubiquitin ligase activity of GCIA1 protein via prokaryotic expression system and tobacco transient expression system respectively.The results implied that GCIA1 can be autoubiquitinated and then degraded by 26 S proteasome.In order to analyze the function of GCIA1 in plants,we first identified two T-DNA insertion mutant gcia1 and gcia1-2 ordered from ABRC.We also created gene-editing mutant using CRISPR/Cas9 technology.Unexpectedly,we couldn't obtain homozygous offspring from the heterozygote of the editing plants.According to the comparation of the specific editing site and the T-DNA insertion site of gcia1 mutant,Lis H domain of GCIA1 protein might be important for its fertility.Besides the gene mutants,we also constructed the homozygous transgenic plants of GCIA1 overexpression and complementary lines for further gene function analysis.There are two functional FBPase in Arabidopsis: the cytosolic FBPase(cy FBPase)is the key enzyme of gluconeogenesis,while the chloroplast FBPase(cp FBPase)is involved in Calvin cycle and starch synthesis in chloroplast.Therefore,both two FBPase regulate carbon assimilation and distribution which are crucial for autotrophy in green plants.To verify whether GCIA1 regulates the ubiquitin degradation of FBPase in plants,we first analyzed the growth phenotype of gcia1 mutant at different sucrose concentration medium.It was found that gcia1 had a higher rate of seedling growth arrest compared to wild-type under no sugar condition.The Western Blot results showed that protein levels of both of the two FBPase were increased in gcia1 mutant.The protein interaction assay between GCIA1 and two kinds of the FBPase indicated that GCIA1 can interact with cp FBPase;and the result of co-expression in tobacco leaves showed that Myc-cp FBPase protein level was obviously decreased at the present of Flag-GCIA1,indicating that GCIA1 may affect seedlings development by regulating the ubiquitin degradation of cp FBPase in plants.It is known that FBPase affects the content of sugar which can be used as signaling molecule and important components of intracellular energy metabolism.There is obvious crosstalk between the sugar and ABA signal transduction pathway,and the information from ABRC showed that GCIA1 was co-expressed with Sn RK2.3,one of the key components in ABA signaling pathway,so we also explored the ABA phenotype of gcia1 mutant.The results showed that gcia1 is highly sensitive to ABA,GST pull-down assay showed GCIA1 interacted with Sn RK2.3.These results indicated that GCIA1 might negatively regulate ABA signaling pathway by influencing the function of Sn RK2.3 in Arabidopsis.In addition,Sn RK1.1 is an important regulator of energy metabolism in cells.The protein-protein interaction between GCIA1 and Sn RK1.1 was also confirmed by GST pull-down assay.All the results indicated that GCIA1 may affect plant ABA response and energy metabolism via regulating the function of Sn RK2.3 or Sn RK1.1.At present,the double mutants of gcia1 snrk2.3 and gcia1 snrk1.1 have been successfully constructed.These plants are important genetic materials which can help us to analyse the molecular mechanism of GCIA1 in plants,especially its function in influencing plant development and response to ABA stress via regulating the functions of SnRK2.3 and SnRK1.1.