The Function Of NAC TFS In Leaf Senescence Of Upland Cotton

Senescence is the last stage of plant development,and it is the result of plants evolution.Leaf is the main organ for photosynthesis in plant,so leaf senescence plays an important role in plant survival.As one of the economic crops,cotton is the main source of fiber.Short-season cotton resolves the contradiction between wheat and cotton,but early-maturing comes along with premature senility,which results in a great loss of fiber yield and quality.NAC?NAM,ATAF1/ATAF2,CUC1?transcription factor?TFs?is one of the largest transcription factor families in plants,and it involves in plant development and responses to different stimuli,including various biotic and abiotic stresses.In recent years,lots of studies prove that NAC transcription factors regulate leaf senescence in different varieties,such as Arabidopsis,rice and wheat.With cotton genomes?A,D and AD?databases,it becomes possible for analysis of NAC transcription factors in cotton at genomes level.Those studies prove that NAC transcription factors play important roles in cotton development and stresses responses,but those studies are mainly focus on family analysis,functional analysis is rare,so our studies describe functions of two NAC factors and its related GT genes in detail,which are list below:1.Function identify of GhNAC63 in upland cottonWith the sequence of JUNGBRUNNENI in Arabidopsis,combined with the mRNA database of upland cotton,we got the mRNA sequence of GhNAC63.According to the information in genome,we isolated this gene and its promoter;With qRT-PCR,we detected its expression patterns in different cotton tissues,in developmental stages of cotyledons,in responses to different stresses with different cultivation conditions;We constructed35S-GhNAC63 vector with XbaI and SacI cutting sites,transferred it into Arabidopsis through floral dip transformation with Agrobacterium LBA4404,phenotype observed in T₄generation;Based on the principle of virus induced gene silence,we suppressed the expression level of GhNAC63 in cotton with pYL156-pYL192 virus system,phenotype observed later.Sequence analysis showed that GhNAC63 was located on scaffold246.1,its promoter containing a lot of stresses related cis-elements;The expression pattern of GhNAC63 showed that GhNAC63 expressed in most tissues,and its highest expression level was detected in early developmental stage of fiber,but it was relative low in roots,in the same time,GhNAC63 was sensitive to ethylene treatment;Overexpression of GhNAC63 led to a high sensitivity phenotype of ethylene and drought in Arabidopsis,but repression of GhNAC63 in cotton made cotton seedlings growing better compared with empty control.This study proved that GhNAC63 negatively regulated plants development,and might involved in ethylene and drought signaling pathway,so this study enriched functional studies of NACs in cotton.2.GhNAC79 promoted flowering and improved drought resistance in plantsAccording to the senescence-related RNA-seq database and mRNA database of upland cotton,we isolated a NAC transcription factor from senescent cotyledon,which was named with GhNAC79 in NCBI;Based on the conserved domain of GhNAC79,we divided GhNAC79 into 4 parts,and inserted the four parts into pGBKT7 vector separately,then transferred into yeast stain Y₂H to detect the transcriptional activation activity of GhNAC79;With Sca I and Xba I cutting sites,promoter of GhNAC79 was inserted into pBI121,structured pGhNAC79-GUS expression vector was then transferred into Arabidopsis,after GUS staining,the expression pattern of promoter was detected under a stereomicroscope;With qRT-PCR,we explored the expression pattern of GhNAC79 during cotyledon development,in responses to different stresses;The constructed vector 35S-GhNAC79 was transferred into Arabidopsis and cotton,phenotype observed later;With two virus induced gene silence system,pYL156-pYL192 and pCLCrVA-pCLCrVB,the expression level of GhNAC79 was repressed in cotton,phenotype observed later.Results showed that GhNAC79 was located on scaffold42.1,containing 3 exons and 2 introns,and its transcriptional activating domain was in its C-terminal.Except for basic cis-elements,its promoter contained lots of stresses-related cis-elements;GhNAC79 was highly expressed in later stage of cotyledon development in CCRI10,and GhNAC79 was sensitive to drought stress.Overexpression of GhNAC79improved drought resistance in Arabidopsis and cotton,on the contrary,repression of GhNAC79 led to a more drought sensitivity phenotype in cotton.After drought treatment,the aperture of stoma was smaller in GhNAC79 overexpression plants than in control,but there was no difference in the number of stoma,which proved that GhNAC79 regulated drought stress through stomatal aperture.The result of yeast one-hybrid proved that GhNAC79 was regulated by drought-related gene,which further indicated the function of GhNAC79 in drought signaling pathway.This study proved that GhNAC79 positively regulated drought stress,and it involved in cotyledon development of cotton.It not only enriched functional studies of GhNACs in cotton,but also provided an important cotton material for drought-resistance breeding.3.Analysis of GT transcription factors in upland cottonBased on promoter analysis of GhNAC63 and GhNAC79,we found that their promoter all contained GT cis-element,which related with light response,played important roles in photosynthesis.This study analyzed the feature of GT factors in three cotton genomes,and cloned 8 senescence-related GhGTs in upland cotton,then their functions was verified in detail.According to the GT homologous genes in Arabidopsis and rice,based on the three mRNA databases of cotton,we got 67 GT factors from cotton through bioinformatics,39 in Gossypium hirsutum,14 in Gossypium arboreum and 14 in Gossypium raimondii,then analyzed their chromosomal localization,motifs features,and clustering feature;In the same time,8 senescence-related gene were isolated from upland cotton.With qRT-PCR,the expression level of the 8 GhGTs were detected in different cotton tissues,in responses to various stresses,and during leaf development;Finally,GhGT31 was isolated and transferred into Arabidopsis.Results showed that the variation of GT factors was small during cotton evolution,which was a purifying selection process.The distribution of 39 GhGTs were evenly in different clusters,which proved the function of GhGTs were complex.The expression pattern of 8 senescence associated genes proved that 8 GhGTs were highly expressed in later developmental stage of fiber,and except GhGT20,others 7 GhGTs had a relative high expression level in roots.The expression pattern also proved that 8 senescence associated genes had different expression patterns in cotyledon and true leaf development,in response to different treatments.GhGT12 was sensitive to ABA,ethylene and MeJA treatments,in the same time,the expression level of GhGT12 was high in later developmental stage of cotyledon,which indicated that GhGT12 might be an important factor in hormone mediated leaf senescence pathway.GhGT31 was highly expressed during CCRI10 cotyledon development,and showed high sensitivity to drought treatment.35S-GhGT31 plants showed an early flowering phenotype,which indicated the function of GhGT31 in drought mediated leaf senescence pathway.This work proved that GhGTs were involved in upland cotton development and stresses responses,which laid the foundation of GT researches in cotton.