The Primary Analysis Of Arabidopsis Es2-D Mutant

Senescence,the last stage of leaf development in higher plants,is phenotypically defined by leaf yellowing,which results from the quantitative loss of chlorophyll.Leaf senescence is critical for plants' fitness as nutrient relocation from leaves to reproducing seeds is achieved through this process.We isolated a mutant that enhances sicl-1 phenotype from screening of an Arabidopsis activation-tagged mutant pool,which was generated by randomly integrating the CaMV 35S enhancer element into the genome of sic1-1 mutant.The mutant,designated es2-D(enhancer of sic1-1),exhibited pleiotropic phenotypes during the growth and development process,including reduced plant height,increased branching,shortened internodes,accelerated flowering time,decreased leaves' areas,reduced fertility and accelerated senescence.We identified a single T-DNA insertion which is located on chromosome 5 by thermal asymmetric interlaced PCR(TAIL-PCR).The expression levels of six genes in the vicinity of the insertion site were analyzed by reverse transcriptase PCR(RT-PCR).Only one gene,ES2 gene,was up-regulated.Transgenic plants overexpressing ES2 gene under the control of the CaMV 35S promoter could recapitulate the phenotypes of the es2-D mutant.These data indicate that the pleiotropic phenotypes of es2-D are indeed caused by over-expression of ES2 gene.ES2 gene has no intron and encodes a protein of 508 amino acid residues,sharing sequence similarity with the Arabidopsis MATE proteins.HMMTOP transmembrane topology prediction suggested that ES2 possesses 12 transmembrane domains.We analyzed the expression patterns of ES2 gene and subcellular localization of ES2.ES2 gene expresses in various tissues and organs.ES2 protein is localized to particles with different sizes and shapes,which is distributed ubiquitously in the cells,suggesting that ES2 protein is retained in the endo-membrane organelles.Notably,the mutant leaves are pale green and accelerated senescence.We measured chlorophyll content of rosette leaves at different growth stages,the results showed that chlorophyll content of es2-D mutant rosette leaf was decreased compared with the wild type.Under dark conditions,es2-D mutant seedlings exhibited accelerated senescence,which was proved by reduced chlorophyll content and the expression of senescence-related marker gene SAG12.In summary,Arabidopsis ES2 protein may play a role in the chlorophyll degradation of leaf senescence.Studying leaf senescence of es2-D mutant not only contributes to our knowledge about this fundamental developmental process but also may improve ways of manipulating senescence for agricultural applications.