Study On ECS1 Gene Regulating Pavement Cell Expansion Of Leaf Epidermis And Ethylene Biosynthesis In Arabidopsis

Pavement cell is a main type of leaf epidermal cells in dicotyledonous plants.It is an ideal model system for study of cell differentiation,cell polarity,cell fate determination,organogenesis,and morphogenesis in plants.Therefore,it is important for understanding the mechanisms of plant development.Through screening an EMS-mutagenized Arabidopsis pool,we isolated a mutant with abnormal epidermal cells.In this mutant,pavement cells and mesophyll cells become large,and the epidermal cells of hypocotyls and roots are "swollen".So it was named as ecsl(epidermal cell swelling).Most importantly,ecsl displays typical ethylene triple responses.Genetic analysis revealed that ecs1 is a dominant and gain-of-function mutant controlled by a single gene.By map-based cloning,we identified that ECS1 encodes a previously reported disease resistant protein.Our phenotypic and physiological analyses suggested that ECSl probably controls cell expansion by enhancing endoreduplication,and participate in ethylene biosynthesis by negatively regulating ACSs stability.1.Through dental resin impression method,we observed that leaf pavement cells of ecsl become larger and swollen.Leaf tissue section revealed that cells of palisade parenchyma and spongy parenchyma of ecsl mutant also dramatic become larger.The number of membrane lamellosa in chloroplast thylakoids is increased in this mutant.In dark,the etiolated ecsl seedlings display ethylene triple responses,which consist of radial swelling of the hypocotyl,exaggeration of the apical hook,inhibition of hypocotyl and root elongation,and excessive proliferation of root hairs.In light,root hairs of ecsl are excessively proliferated.The primary roots are shortened due to short and wide epidermal cells in ecsl compared to wild-type.In addition,ecsl mutant displays dark green rosette leaves,increased chlorophyll content and trichome numbers,shortened siliques,symmetrically growing flower bolting.2.Flow cytometry analysis showed that leaf tissue cells of ecsl have higher nuclei ploidy level compared to wild type.Fluorescence in situ hybridization(FISH)using specific 45s rDNA as a probe revealed that the endoreduplication is enhanced in ecsl,and high ploidy nuclei(above 8C)ratio is elevated in the cells of ecsl leaf tissues.These results indicate that the enlarged pavement cells and mesophyll cells in ecsl are correlated with its enhanced endoreduplication in the nulei.At the same time,we observed a number of large nulei in ecsl pavement cells by staining with DAPI.We introduced the CYCB1;1:GUS construct into the ecsl background and examined the accumulation of CYCB1;1 transcripts.As a result,GUS activity was decreasd in ecs1,indicating that mitosis activity is reduced,in turn,endoreduplication is enhanced in ecsl.3.ecsl mutant seedlings produce about two times of ethylene more than that of wild type by Gas Chromatograph analysis.Moremove,ethylene inhibitor AVG or AgNO3 can partially restore the defects of hypocotyls and roots in ecs1.These data suggest that ecsl is an ethylene constitutive mutant.In addition,hypocotyls and roots of ecsl show less sensitive response to ACC.4.A map-based cloning approach was used to identify mutated gene in ecs1.We design many SSLP and Indel markers for first and fine mapping.The mutation gene was finally mapped to a 110 kilo-bases region on bottom of Chromosome 5.Approximately fifty-six candidate genes in this region were sequenced,and a C to T substitution at 278 bp in the first coding region of ECS1 was found,which leads to a conversion of a serine to phenylalanine within the highly conserved TIR domain of ECS1.Therefore,we suppose that ECS1 is possibly responsible for the mutant defects in epidermal cell shape and abnormal ethylene triple responses.5.We constructed two expression vectors in which ECS1 was fused to GFP reporter under the control of 35S promoter or ECS1 promoter was fused to GUS reporter,and they were transformed into wild type plants.ECS1 is generally expressed almost in all organs of Arabidopsis,especially strongly in the roots and flowers.ECS1 protein is mainly localized at plasma membrane of epidermal cells and in chloroplasts.Given that the heterozygous mutant has partial homozygote phenotype,we constructed a vector in which mutation gene ecsl was fused to GFP reporter under the control of 35S promoter to verify whether ecs1 is a gain-of-function mutant.Both ECS1 OX and ecsl OX transgenic plants nearly phenocopy partial phenotypes of ecsl.They show enlarged pavement cells and mesophyll cells,but lack of ethylene triple responses.Endogenous ethylene is dramaticly decreased in ECS1 OX transgenic plants.6.Real-time quantitative RT-PCR was used to analyze the relative transcript levels of several key genes controlling cell cycle.The result showed the expression levels of several genes promoting cycle progression are downregulated,for example,CYCB1,RBR,CDC2B and CYCD3.But some genes promoting endoreduplication are upregulated in ecsly for instance,KRP1,KRP2 and E2FC.These results further indicate that endoreduplication is enhanced in ecsl.ACS2 and ACS6 transcripts are significantly upregulated in ecs1,resulting in ethylene overproduction.Ethylene-induced genes,for example,CHIB,ERF1,PDF1.2 and EBP are also upregulated in ecsl.These results further confirm ethylene overproduction in ecsl mutant.