Functional Analysis Of Nine Cotton Genes Related To Leaf Senescence In Gossypium Hirsutum L.

Cotton?Gossypium spp.?is one of the leading natural fiber crops worldwide and is a vital source of seed oil and protein meal?J.Li et al.2013?.Upland cotton accounts for more than 95% of world production.Cotton production is constrained by many biotic and abiotic factors such as drought,nutrient limitation,extreme temperature,and salinity.These abiotic stresses adversely influence plant growth and production,leading to large economic losses.Leaf senescence is defined as a deterioration process that continues to the final developmental stage of the leaf.This process is usually regulated by both external and internal factors.There are about 5,356 Senescence associated genes?SAGs?belonging to 44 plant species.A great number of these genes were identified in Arabidopsis.Leaf senescence can be regulated by many transcription factors.In this study,nine cotton genes were selected according to their expression levels during leaf senescence from our laboratory database.The phylogenetic tree was constructed by MEGA6.Cultivated cotton CCRI-10 seeds were sown in the experimental field of Institute of Cotton Research of CAAS?Anyang,Henan?for profiling and leaf development stages analysis.For abiotic?drought and salt?stress and phytohormone?ABA,SA,ET,and JA?treatments,CCRI-10 seeds were sown in potting soil at 25 °C in a chamber room.Arabidopsis?ecotype Columbia-0?was selected for transgenic studies.Total RNA was isolated from various samples and the c DNA prepared for q RT-PCR.The comparative CT method was applied to calculate the relative expression levels of genes.For the phylogenetic tree,nine cotton genes were divided into two groups,most of the homologus genes in previous studies showed roles in phytohormones and abiotic stress.Expression profiling of the nine genes showed different patterns of tissue specific expression.In leaf development stages,the majority of cotton genes showed high expression in early and complete senescence stage.Furthermore,most of the cotton genes have a positive or negative response to phytohormones and abiotic stress.Based on the results of this study,we found four cotton genes Cot AD₀7559,Cot AD₃7422,Cot AD₂1204 and Cot AD₅4353 as candidate genes for leaves senescence and abiotic stress.The thioredoxin family was characterized and studied in details.Furthermore,two genes Cot AD₂1204?RPL2?and Cot AD₅4353?CDSP32,Gh TRX134?were transformed into Arabidopsis to study their biological function.Thioredoxins?TRX?are small molecules of proteins that are present in all organisms.TRXs play an important role in diverse functions of plant growth and development.In this study,we performed genome-wide,characterization and expression levels of TRX gene family in cotton.A total of 150 Gh TRX proteins were identified in upland cotton and classified into five subfamilies based on their domain compositions.Phylogenetic tree analysis divided TRX genes into seven subgroups.Gh TRX genes covered all upland cotton chromosomes,with duplicated gene events.Ka/Ks ratio of three gene pairs was less than 1,suggesting purifying selection.The functions of Gh TRX genes were studied using gene ontology,protein localization,and promoter analysis.Furthermore,six Gh TRX genes were randomly selected to examine their expression level in cotton development and under various exogenous treatments.The genes showed high expressions in various tissues and at different stages of leaf senescence,also showed high expression under abscisic acid,ethylene,drought,and salinity.This study reveals the first report of TRX family genes in upland cotton.However further studies are needed to elucidate their specific functions in the cotton plant.Two genes Gh TRX134 and Gh RPL2 were transformed into Arabidopsis?Col-0?produced transgenic Arabidopsis plants.Gene expression and phenotypic analysis in T3 lines for each gene were analyzed under drought,salt,methyl viologen?MV?,and cold stress.VIGS system was used to silencing the two genes on cotton plants.Furthermore,responsive genes expression,proline content and antioxidant enzymes activity levels were studied in the Gh TRX134 and Gh RPL2 overexpressing and Wt plants under abiotic stress.The results showed that overexpression of Gh TRX134 and Gh RPL2 enhanced plant tolerance to drought,salt and MV stress.While silencing these genes in cotton plants inhibited their tolerance to drought.The responsive genes showed higher expression level in the transgenic plants more than Wt under abiotic stress.In addition,the Gh TRX134 overexpression plants showed higher enzymes activities level and proline content under drought and salt respectively.Whereas the Gh RPL2 overexpression plants exhibited higher proline content and CAT activity level under drought and salt.Our results strongly suggest that Gh TRX134 and Gh RPL2 regard as key factors for abiotic stress in the cotton plant.Therefore,the Gh TRX134 gene can be transformed into cotton plants to generate transgenic lines that can be used in cotton breeding programs for drought and salt stress tolerance.