The Function Of Raffinose Family Oligosaccharides In Plant Drought Stress Tolerance And Seed Vigor Of Maize And Arabidopsis

Plant drought stress tolerance and seed vigor are crucial for crop production.Raffinose family oligosaccharides（RFOs）are thought to play important roles in plant abiotic stress tolerance and seed vigor.GALACTINOL SYNTHASE（GOLS）,RAFFINOSE SYNTHASE（RS）and STACHYOSE SYNTHASE（STS）are three key enzymes in plant RFOs metabolism.Extensive studies about the protein function and regulation of expression of GOLS gene,in response to plant abiotic stress tolerance,have been conducted.However,the RS gene has been poorly characterized.The role of each individual RFOs sugar member in abiotic stress tolerance and seed vigor remain poorly understood.In this thesis,the function of the RS gene product and the role of individual members of RFOs,have been investigated under the hypothesis that they are involved in plant drought stress tolerance and seed vigor of maize（Zea mays）and Arabidopsis（Arabidopsis thaliana）plants using maize zmrs mutants and Arabidopsis plants ectopically expressing ZmGOLS2,ZmRS,ZmGOLS2/ZmRS or AtSTS.Using homology cloning methods,a unique maize RS gene（ZmRS,GRMZM2G150906）was identified.Bacterially-or Arabidopsis-expressed ZmRS was able to hydrolyze galactinol-or synthesize raffinose depending on the availability of sucrose.Kinetic analysis showed that ZmRS has a lower Km for the galactosyl-donor（galactinol）than the receptor（sucrose）when catalyzing the synthesis of raffinose.The molar ratio of the two substrates（sucrose/galactinol）determines whether ZmRS is to hydrolyze galactinol or to synthesize raffinose.The 264^nd aspartic acid of the ZmRS protein was essential for its raffinose synthetic activity but not for galactinol hydrolytic activity.Two ZmRS maize mutant lines from different genetic backgrounds were identified.The zmrs lines failed to express ZmRS protein,completely abolished raffinose,and accumulated more galactinol than its NS（Counterpart null segregant）plants in embryos,endosperms and leaves.zmrs mutant plants were more sensitive to drought stress than NS plants.The percentage water loss of the leaves from both zmrs alleles was significantly greater than that from respective NS leaves.The greater water loss of zmrs leaves could be partially rescued by exogenous raffinose supplementation.These results indicated that raffinose may protect plants from drought stress via its water-holding capacity.Over-expression of ZmRS enhanced drought tolerance of Arabidopsis plants.ZmRS expressing lines showed lower raffinose content and higher myo-inositol content than control plants under drought conditions due to the galactinol hydrolytic activity of Zm RS and free sucrose deficiency in leaves.The RFOs content and the accelerated aging（AA）tolerance was compared between zmrs mutants and NS control lines,the results clearly showed that zmrs seeds were more sensitive to accelerated aging（AA）treatment than NS seeds.Manipulation of Arabidopsis RFOs content by overexpressing ZmGOLS2,ZmRS or AtSTS demonstrated that co-overexpression of ZmGOLS2 and ZmRS,or overexpression of ZmGOLS2 alone,significantly increased total RFOs and enhanced seed vigor.Surprisingly,while over-expression of ZmRS increased raffinose it dramatically decreased seed vigor,galactinol,stachyose and verbascose amounts,while the mutant（atrs5）was similar to WT in all respects except for stachyose,which it accumulated.Total RFOs,RFOs:Sucrose ratio,but not absolute individual RFOs amounts,positively correlated with Arabidopsis seed vigor,with stachyose and verbascose contributing more than raffinose.The RFOs biosynthetic pathway evolution might be split into two parts,the synthesis of raffinose and the synthesis of higher degrees of polymerization（DP）RFOs.Evolutionary studies showed that Plant RFOs SYNTHASE gene are evolved from ALKALINE-α-GALACTOSIDASE genes.RFOs SYNTHASE genes appeared in seed plants and was positively selected by evolutionary pressures.The copy numbers and structures of RFOs SYNTHASE genes were obviously different between monocotyledons and dicotyledons.Compared with dicotyledons,the copy number and the exon number of RFOs SYNTHASE genes of monocotyledons were dramatically decreased.In some of the gramineae,like maize and rice,the HDP-RFOs（STACHYOSE）SYNTHASE genes were lost due to domestication selection.In summary,the function-,the enzyme activity-and the evolution-of RAFFINOSE SYNTHASE have been studied in this thesis.The findings of this study provide new insights of the roles of raffinose in plant drought stress tolerance and seed vigor.This study proves that it is practical to improve plant drought stress tolerance and seed vigor through manipulation of the RFOs metabolic pathway.