Genetic Analyses Of NAC Gene Family For Leaf Senescence And Stress-Responses In Cotton (Gossypium Hirsutum L.)

NAC (NAM, ATAF, and CUC) is a plant-specific transcription factor family with diverse roles in plant development and stress regulation. NAC family proteins also contribute to a variety of developmental processes in plants, such as leaf senescence, floral morphogenesis, stress induced flowering, development of shoot apical meristem, shoot branching determination, lateral root development, and hormone signaling. The NAC domain proteins has been associated with plant responses to biotic and abiotic stresses including salinity, cold shock, drought, mechanical wounding, and viral infections. Some other areas where their involvement has been reported are fiber development, xylogenesis and wood formation. Despite a few NAC TFs in Arabidopsis and Oryza model plants have been characterized for their functions, no reports are available for their functional characterization in cotton specifically for leaf senescence.In this study, we identified the GhNAC gene family, characterized for leaf senescence and diverse stresses in cotton(Gossypium hirsutum L.). The qRT-PCR analysis revealed that these genes have high expression during leaf senescence and response to stresses. GhNAC8and GhNAC7overexpression lines in Arabidopsis depict essential role during leaf senescence. GhNAC7being a member of cotton NAC family has important functions in leaf senescence as elucidated by qRT-PCR, immunoblot analysis, overexpression in Arabidopsis and tobacco. GhNAC genes promoter regions possess several cis-elements inducible by physiological and environmental factors.The main results are summarized as follows:1. Characterization of GhNAC8-GhNACl7genes for leaf senescence and responses to stressesStress-responsive NAC genes (GhNAC8-GhNAC17) isolated from cotton (Gossypium hirsutum L.) were characterised in the context of leaf senescence and stress tolerance. Based on the sequence characterisation, these GhNACs could be classified into three groups belonging to three known NAC sub-families. Their predicted amino acid sequences exhibited similarities to NAC genes from other plant species. All GhNAC genes were highly expressed in senescent leaves, except GhNAC10and GhNAC13, which had higher expression levels in fibres collected from25days post anthesis (DPA) plants. Ectopic expression of GhNAC8and GhNAC9show early leaf senescence compared to wild-types plant, revealed biological function for leaf senescence in Arabidopsis. The ten GhNAC genes displayed differential expression patterns and levels during natural and induced leaf senescence. Quantitative RT-PCR and promoter analyses suggest that these genes are induced by ABA, ethylene, drought, salinity, cold, heat and other hormonal treatments.2. Expression analysis of NAC family gene (GhNAC18-77) for multiple abiotic stresses in cottonA total of60full-length GhNAC genes were isolated from upland cotton, and they were phylogeneticly clustered into seven distinct subfamilies. We also constructed the phylogenetic tree for GhNAC genes and their motif, which show similarities among the subfamilies. The60GhNAC genes were located in the chromosome of D sub-genome and were found to be distributed on13chromosomes. Majority of the NACs showed specific temporal and spatial expression patterns for tissue-specific studies based on qRT-PCR analyses. Furthermore, the roles of GhNAC genes were monitored by qRT-PCR for leaf senescence, ethylene, ABA, GA3, drought and salinity.3. GhNAC7, a novel GhNAC family transcription factor gene functions in leaf senescenceGhNAC7, a gene encoding a NAC family transcription factor was studied for functional analysis. Expression of this gene is closely associated with the leaf senescence process of cotton as revealed by tissue-specific and natural leaf senescence studies through qRT-PCR and immunoblot assays. The GhNAC7expression also induced by ethylene, ABA, drought, salinity, cold and high temperature treatments in cotton. In two model plant species, Arabidopsis and tobacco overexpression of GhNAC7showed visible symptoms for leaf senescence. Protein expression in BL21E. Coli and immunoblot assay in senescent leaf confirmed that molecular weight of GhNAC7protein was approximately29kDa. Our data strongly suggest that GhNAC7play an important role in cotton leaf senescence.