Functional Analysis Of GhNAC18 And GhNAC20 During Leaf Senescence And Stress Responses In Cotton (Gossypium Hirsutum L.)

Leaf senescence is a complex developmental phase that involves both degenerative and nutrient recycling processes. This process marks the beginning of the end of plant life span. It is characterized by loss of chlorophyll and the degradation of proteins, nucleic acids, lipids, and nutrient remobilization. Its onset and progression of leaf senescence are controlled by an array of environmental cues (such as drought, darkness, extreme temperatures, and pathogen attack) and endogenous factors (including age, ethylene, jasmonic acid, salicylic acid, abscisic acid, and cytokinin).While this process is precautious in nature, premature leaf senescence has a negative impact on quantity and quality of crop yield. In short season cotton that is characterized with premature leaf senescence, mature faster howerve its yield performance is affected by premature leaf senescence. This study aimed at understanding the molecular regulation of leaf senescence in cotton with the objective of identify and manipulating candidate genes to delay senescence or synchronize it with normal maturity. Several genes have been found to regulate leaf senescence and their regulation is under transcription factors control. However, many underlying transcription factors (TFs) are yet to be identified. NAC (NAM, ATAF1,2 and CUC2) is one of the largest plant specific transcription factors with diverse roles in plant development and stress responses. NAC TFs contribute to a variety of developmental processes in plants which include leaf senescence, floral morphogenesis, lateral root development, development of apical meristems, shoot branching and hormone signaling. This family of genes constitutes a domain that has been associated with plant abiotic and biotic stresses such as salinity, cold shock, drought, wounding, and viral infection. In plants NAC family genes have been characterized especially in Arabidopsis and rice. In cotton a few have been reported but still many of these genes remain uncharacterized despite being implicated with many functions in plant development and stress responses.In this study, we cloned and characterized two GhNAC genes for leaf senescence and stress responses in cotton. The full-length of cDNAs of these two genes were 1511bp and 1090bp for GhNAC 18 and GhANAC20 respectively.GhNAC18 encode a protein of 419aa while GhNAC20 encoded a protein of 281aa. Each of these genes contained a single open reading frame. Using WoLF PSORT program both genes were located in the nucleus, confirming their role as nuclear transcription factors. Like many other NAC family TFs, both genes had three exons and two introns. The first two exons encoded the N-terminal domain while the last exon encoded the highly divergent C-terminal region which is the likely cause of their distinguishable functional differences. The transcriptional activation activities were located at the C-terminal region.The result from tissues-specific expression analysis indicated that that the genes were constitutively expressed in all tissues with significant variations in specific organs implying that they play a crucial role in cotton. For example, although both genes were highly expressed in the cotyledon and young leaves, there was apparent deferential expression in senescing leaves. GhNAC20 was strongly expressed in the senescing leaves while GhNAC18 was highly expressed in young and cotyledon leaves. This implied that their role in leaf senescence was antagonistic such that, while GhNAC20 was promoting senescence, GhNAC18 was playing inhibitory role.During hormone treatment, four signaling molecules; ABA (Abscisic acid), MeJA (Methyl Jasmonate), SA (Salicylic acid) or ET (Ethylene) induced these two genes suggesting that both genes are responsive. However the induction time and strength varied between these genes. For instance GhNAC20 was upregulated by ABA while GhNAC18 was down regulated. Ethylene increased the transcript levels of both gene, but GhNAC18 reached its maximum at the 6h while GhNAC20 peaked at 36h after treatment. GhNAC20 was strongly induced by MeJA peaking at 36h while GhNAC18 showed no significant expression under the same treatment. Expression level for GhNAC20 was significantly higher under SA treatment than GhNAC18. GhNAC20 was induced by salinity while GhNAC18 was downregulated. Both genes were sensitive to H2O2 treatment at 2h while wounding upregulated GhNAC20 and peaked after two hours whereas GhNAC18 reached maximum at the 8h.To confirm their role in leaf senescence, the transcript levels were measured during natural and dark-induced senescence. Strikingly, GhNAC20 was strongly upregulated during these types of senescence while GhNAC18 was downregulated. When GhNAC20 was knocked down by VIGS technique, GhNAC20-silenced plants showed delayed leaf senescence compared to control cotton plants. GhNAC18 was overexpressed in Arabidopsis and the analysis of T2 transgenic lines showed delayed senescence compared to wild type plants. These result take together indicate that GhNAC20 is a positive promoter of leaf senescence while GhNAC18 play an inhibitory role. However both genes participate in regulation of stress responses in cotton.