Function And Expression Regulation Of Maize Galactinol Synthase2 Gene(ZmGOLS2)

Maize(Zea mays L.)is an importment food crop in the world.Its growth is impacted by abiotic stress(such as drought,heat and salt)throughout its life cycle.Raffinose,a tri-oligosacchrides,is accumulated in plant leaves when plants are encountered abiotic stress or accumulated in seeds during late stage development.Maize GALACTINOL SYNTHASE(GOLS)is the first committed enzyme in raffinose bio-synthesis pathway.Previous studies shows that there are two members(ZmGOLS1 and ZmGolS2)in maize GOLS gene family.Both ZmGOLS1 and ZmGOLS2 are induced by dehydration or heat stress in germinating maize seeds and cultured cells.A heat shock response element(HSE)in the promoter of ZmGOLS2 has been identified.In spite of these known information,the function and the expression regulation remain largely unknown.In this thesis,we have successfully characterized two transcription factors regulating ZmGOLS2 expression under drought and heat stress.We have also investigated the regulation of galactinol bio-synthesis using genome-wide association analysis(GWAS)of the galactinol content,ZmGOLS2 mRNA and ZmGOLS2 protein in 35 DAP(days after pollination)embryos and the SNP database from 368 different maize inbred lines.The main results are shown as follows:There are two galactinol synthase genes(ZmGOLS1 and ZmGOLS2)in maize genome.Characterization of the mutator interrupted ZmGOLS1 mutnt(zmgols1)showed that the function of this two GOLS is not redundant,and both of these two GOLS genes function in maize galactinol biosynthesis.Over-expression of ZmGOLS2 in Arabidopsis increased both galactinol and raffinose content in mature leaves and enhanced the abiotic stress tolerance of the transgentic lines.Over-expression of ZmDREB2 A in Arabidopsis also enhanced the plant abiotic stress tolerance,however,it did not increase the galactinol and raffinose accumulation in leaf blade.Compared with ZmGOLS2 over expressed plants,ZmDREB2 A over-expression lines showed growth retardation phenotype under normal growth conditions.ZmGOLS2-overexpressing Arabidopsis plants are more sensitive to ABA during seed germination or seedlings growth.The expression of some genes in the ABA signaling pathway were up-regulated and the stomatal aperture was reduced after ABA treatment.ZmGOLS2 protein expression in 35 DAP embryos from 201 maize inbred lines were characterized using western blot analysis.Genome association analysis(GWAS)was performed using the protein expression data and the transcriptome SNP database of 368 maize inbred lines.We found a candidate gene ZmRLP5(GRMZM2G163081)was significantly associated with ZmGOLS2 protein expression.This gene encodes a ribosome protein,which is a component of eukaryotic 60 S ribosome subunit.There are two types of this ribosome protein according to the SNP of the 118 th amino acid(glutamine and lysine)in 368 inbred lines.The glutamine type was associated with the higher protein expression level of ZmGOLS2 and thelysine type was associated with the lower expression level of ZmGOLS2 protein in 368 maize inbred lines.Two dehydration response elements(DRE)in the ZmGOLS2 promoter have been identified.Further more,ZmDREB2 A has been characterized directly binding to the DRE element near the TATA frame and up-regulates Zm GOLS2 expression.By homologous comparison,a maize heat shock transcription factor(HSF)gene(ZmHSFA2,GRMZM2G010871)was identified.ZmHSFA2 is responsive to heat and salt stress and its expressed protein is localized in nuclear.ZmHSFA2 directly binds to the HSE element of ZmGOLS2 promoter and up-regulates ZmGOLS2 expression.The transcriptional activity of ZmHSFA2 is regulated by phosphorylation.Over-expression of ZmHSFA2 in Arabidopsis increased raffinose content in mature leaves and enhanced the heat stress tolerance of the plant.However,ZmHSFA2 over-expression caused growth retardation under normal growth conditions.ZmHSFA2 physically interacts with maize HEAT SHOCK FACTOR BINDING PROTEIN 2(ZmHSBP2)and ZmHSBP2 negatively regulates the transcriptional activity of ZmHSFA2.Over-expression of ZmHSBP2 in Arabidopsis significantly reduced raffinose content in mature leaves after heat shock,and reduced the heat stress tolerance of seeds and seedlings of the Arabidopsis transgenic lines.ZmHSBP2 expression is regulated by both ZmHSFA2 and ZmHSBP2.We postulate that the regulation of HSBPs by HSFA and HSBP might be conservative in plant kingdom.In summary,this study found that galactinol may act as a signal substance to induce stomatal closure.ZmRLP5 may regulate the translation of ZmGOLS2 in maize seeds.Over-experssion of ZmGOLS2 enhanced the plant abotic stress tolerance.We also have identified and characterized two transcription factors,ZmDREB2 A and ZmHSFA2 that directly bind to the promoter of ZmGOLS2 and up-regulate its expression under abiotic stress.Over-expression of the ZmDREB2 A or ZmHSFA2 also enhanced the plant abiotic stress tolerance,but the transgenic plants showed growth retardation.These results provide insightful information of the function and expression regulation of ZmGOLS2 gene and indicate that it is practical to manuipulate GOLS gene,rather than to regulate transcription factor genes,to enhance the crop abiotic stress tolerance.