The Expression Pattern Analyses Of Guard Cell Highly Expressed GDSL Lipase Genes In Arabidopsis And Functional Identification Of The Gdsl3-1 Mutant

During the process of plant growth and development,they require CO₂ for photosynthesis;simultaneously water is exchanged for transpiration.This process is strictly regulated by stomatal switch on plant epidermal surface.The surface of the plant has a layer of cuticle,preventing plants from excessive water loss through transpiration,which has an important role in maintaining the necessary water in the plant.GDSL-esterase/lipase is a big subfamily of lipolytic enzyme which widely exists in microbes and plants.In the process of lipid metabolism,it will produce a variety of metabolites;some are involved in the synthesis of plant cuticle,wax and content of cell membrane.Thus,the GDSL-lipases may be involved in the plant cuticle and wax synthesis and may play an important role in the guard cell signal transduction pathway.However,little have been known about their functions in plant development and stress responses.In this research,we found that there are 19 GDSL-lipase genes highly expressed in guard cells through the microarray data among the 112 GDSL-lipase genes in Arabidopsis thaliana.In order to explore the functions of these 19 genes,we performed fine analyses of the expression patterns and the corresponding protein subcellular localizations;moreover,we screened the mutants of these genes in response to CO₂ concentration changes,and found gdsl3-1 was insensitive to the changes of CO₂ concentration.The main results of this research are as follows:The promoters of 11 from 19 GDSL-lipase genes were cloned into the vectors to control GUS reporter expression when expressed in Col-0 plants.GUS staining analyses of the transgentic plants showed that 7 of them do highly express in guard cells,while 4of them are lower or not detected.We also performed the analyses of the subcellular localizations of 19 GDSL-lipases by GFP or YFP fusion.The fusion expression vectors of 19 GDSL-lipases-GFP or-YFP were generated and transiently transformated into the tobacco epidermal cells,and observed by confocal fluorescence microscopy.The results show that 11 of the 19proteins were located at endoplasmic reticulum（ER）,the subcellular localization of one of them was not clearly determined;one of the 11 ER proteins was also localized at the cell membrane,and one of them was also localized at the cell membrane,in nucleus and chloroplast.To further determine the functions of these GDSL-lipase genes,we screened T-DNA mutants of these genes and found one mutant gdsl3-1 was insensitive to the changes of CO₂ concentrations.However,the expression of genomic GDSL3 could not complement the phenotype.Finally we found that a 5Kb region containing MPK12 and BPS2 genes were deleted in gdsl3-1,confirmed by southern blotting,sequencing and q RT-PCR.When genomic DNA of MPK12 was transformed into gdsl3-1 mutants,the CO₂ insensitive phenotype was rescued.This demonstrated that MPK12 is responsible for the CO₂sensitivity,and is a key regulator of CO₂-induced stomatal closing pathway.In order to get a better understanding of the function of MPK12 in the guard cells CO₂ signaling pathway,EMS mutagenesis was performed in the background of mpk12-d（an MPK12deletion mutant isolated from gdsl3-1）,and the genetic enhancer and suppressor of mpk12-d are under screening and their function will be identified.