Study On The Roles Of Drought Stress-responsive Genes GhBLH5-A05 And GhHDT4D In Cotton Response To Drought Stress

Since 1950s,the global temperature has increased by 0.13℃ per 10 years and rainfall has also decreased year by year.The water shortage has caused crops to often suffer drought stress Drought stress affects the plant growth and result in crops yield reduction.Cotton is an important cash crop,which provides major natural fiber and oil supply for textile industry and food industry.Previous report shows 57%of cotton worldwide is distributed in dry areas such as Xinjiang Province in China,Central Asia,and South Africa.Drought has become a huge threat to cotton yield and quality.Explore the genetic basis of cotton response to drought stress and using genetic engineering to cultivate new cotton cultivar with high tolerance to drought stress will help us to better confront this threat.In this study,we identify two drought response genes,GhBLH5-A05 from BEL 1-like gene family and GhHDT4D from HDAC gene family using drought related transcriptome data of cotton.Genetic analysis and biochemical experiments were performed to reveal their functions and regulatory mechanisms in cotton response to drought stress.The main findings are as follows:1.Functional study of GhBLH5-A05 gene in cotton response to drought stress.BEL 1-like family transcription factor is involved in a variety of plant development processes,but the biological functions of these proteins in the regulation of plant stress responses is not clear.Here,we isolated a BEL 1-like transcription factor from cotton genome,which play an important role in cotton(Gossypium hirsutum)response to drought stress.We firstly identified 50 non-redundant BEL-like genes in cotton(Gossypium hirsutum).Phylogenetic analysis revealed GhBEL1-likes could be classified into six clades.Furthermore,members within the same subfamily share a similar gene structure and motif composition.Tissue expression profiles showed that GhBELl-like genes have different expression patterns in different tissues,and most of them were highly expressed in leave,stem and torus of cotton.The transcription levels of most GhBELl-like genes were markedly altered in cotton under heat,cold and PEG treatments.Drought related transcriptome data of cotton showed that expressions of some GhBELl-like genes were induced in cotton under drought stress.The expression levels of GhBLH5-A05 and GhBLH3-A06 genes were significantly up-regulated on the second and fourth day of drought treatment,and down-regulated on sixth day of drought treatment,and up-regulated again on the eighth day of drought treatment.PEG treatment and drought stress can significantly induce the expression of GhBLH5-A05.We conduct a functional analysis of GhBLH5-A05 in cotton response to drought stress.Overexpression of GhBLH5-A05 in Arabidopsis and cotton enhanced tolerance to drought stress,whereas down-regulation of GhBLH5-A05 in cotton resulted in elevated sensitivity to drought stress.Luciferase reporter analysis,yeast one-hybrid assay and electrophoretic mobility shift assay illustrate that GhBLH5-A05 directly interacts with promoter of GhRD20-A09 and GhDREB2C-D05 to activate their expression.Moreover,GhBLH5-A05 can interact with GhKNAT6-A03,a KNOX transcription factor.Co-expression of GhBLH5-A05 and GhKNAT6-A03 significantly increased transcriptional activity of GhRD20-A09 and GhDREB2C-D05.Taken together,our data indicate that GhBLH5-A05 function as an important regulatory factor in cotton response to drought stress.2.Functional study of GhHDT4D gene in cotton response to drought stress.Acetylation and deacetylation of histones are important for regulating a series of biological processes in plants.Histone deacetylases(HDACs)control regulate the histone deacetylation that plays an important role in plant response to abiotic stress.In our study,we show the evidence that GhHDT4D(a member of the HD2 subfamily of HDACs)is involved in cotton(Gossypium hirsutum)response to drought stress.Overexpression of GhHDT4D in Arabidopsis increased plant tolerance to drought,whereas silencing GhHDT4D in cotton resulted in elevated sensitivity to drought.Simultaneously,the H3K9 acetylation level was altered in the GhHDT4D silenced cotton,compared with the controls.GhHDT4D can suppress the transcription of GhWRKY33,which plays a negative role in cotton defense drought,by reducing its H3K9 acetylation level.Furthermore,the expression of some stress-related genes,such as GhDREB2A,GhDREB2C,GhSOS2,GhRD20-1,GhRD20-2 and GhRD29A,were significantly decreased in the GhHDT4D silenced cotton,but increased in the GhWRKY33 silenced cotton.Given these data together,our findings suggest that GhHDT4D may enhance drought tolerance by suppressing the expression of GhWRKY33,thereby releasing the inhibition of the downstream drought response genes by GhWRKY33 in cotton.