Analysis Of Chromatin Accessibility Response To Drought Stress And Functional Study Of StNF-Y Gene In Potato

Potato is one of the most important food crops in the world where it ranks only behind rice and wheat in terms of production.Potato growth is susceptible to drought stress due to shallow root systems.During the plant evolution in the long term,plants evolved a variety of mechanisms of the drought stress response.Chromatin structure plays an important role in the regulation of gene expression,the transcription factors binding cis-acting elements that are usually associated with open chromatin regions.The genomic regions were bound by transcription factors(TFs)and other regulatory proteins that are thought to be depleted of nucleosomes or have undergone dynamic nucleosome modifications.The ATAC-seq technology has been successfully applied to the detection of chromatin dynamic changes in the genome-wide level and key stress response regulatory elements in plants.In the present study,the ATAC-seq and RNA-seq technology were employed to analyze chromatin open regions and TF binding,a transcriptional regulatory network was constructed for potato plants response to drought stress.Among the identified key transcription factors,St NF-Y transcription factor was selected for further study,the St NF-Y transcription factors were identified in the genome wide level,and further functional study of the crucial transcription factors by molecular biology and physiological and biochemical methods.The main results are as follows:1.RNA-seq and ATAC-seq were used to analyze the potato cultivar “Desiree” under normal and drought conditions.A total of 4180 DEGs were identified by RNA-seq in potato,of which 1951 were up-regulated and 2229 were down-regulated.A total of 96,540 THSs were identified in potato leaves by ATAC-seq.The distribution pattern of genome element THSs in potato CK and DT samples was similar,and the order of numerical values from large to small is intergenic region,CDS,intron,promoter region,3’ UTR and 5’ UTR.The significantly enriched motif-bind transcription factors identified in the promoter region,CDS region,intron and intergenic region of potato are TBP,b HLH,CBF and CDF,respectively.2.The genes obtained from RNA-seq and ATAC-seq were analyzed for integrated analysis.These genes were divided into the genes with expression and accessible chromatin no differently changed,the genes with expression no differently changed and increased chromatin accessibility,the genes with no different chromatin accessibility changed and expression increased,and the genes with chromatin accessibility and expression increased.A total of 533 genes with increased chromatin accessibility and increased expression were identified.These genes significantly participate in the pathways of plant hormone signal transduction,cutin,suberine and wax biosynthesis and Linoleic acid metabolism.The transcriptional regulation network of potato responding to drought stress was constructed by using the drought-specific motif“TTTCCACD”、“ACTATAAGTGA”、“AAAGKCAG”、“GATGTTGACA”and“AACRTGTTTGGT”.3.A total of 41 St NF-Y genes(10 St NF-YA,22 St NF-YB and 9 St NF-YC)were identified in potato genome.In addition,the phylogenetic,gene structure,motif,and chromosomal location of St NF-Y genes were analyzed.The results suggested that,St NF-Ys in the same class shared similar gene structures and conserved motifs.The chromosomal location analysis showed that 41 St NF-Y genes were located in 12 chromosomes.The tissue expression profiles based on RNA-seq data showed that 24 St NF-Y genes had different expression patterns,while the remaining 12 had low expression in all tissues.In addition,St NF-Y genes showed to respond to stress factors such as heat,mannitol and ABA.The results of q RT-PCR to further suggested that,St NF-Y genes are involved in salt,drought and dehydration stress responses and showed different expression patterns.4.The St NF-YC9 gene was cloned from the cultivar “Desiree”.The ORF of St NF-YC9 gene was 759 bp,encoding 252 amino acids,which was induced by dehydration and drought stress.The subcellular localization results showed that St NF-YC9 protein was mainly located in the cell membrane and nucleus and might locate in the cell wall.By constructed the overexpression vector that under the control of Ca MV 35 S promoter to generate transgenic potato plants by Agrobacterium-mediated transformation,thereby overexpressing St NF-YC9 gene.The results showed that,Overexpressing St NF-YC9 potato lines(Ox St NF-YC9)had significantly increased in root length and exhibited stronger stomatal closure in potato treated by polyethylene-glycol and abscisic acid.In addition,Ox St NF-YC9 lines had higher photosynthetic rates and decreased water loss under short-term drought stress compared to wild-type plants.During long-term drought stress,Ox St NF-YC9 lines had higher proline levels,lower malondialdehyde content,and increased activity of several antioxidant enzymes,including superoxide dismutase,catalase,and peroxidase The expression of St NF-YC9 gene was silenced by VIGS technology,and the drought tolerance of TRV:C9 was reduced.The above results indicate that St NF-YC9 positively regulates drought tolerance in potato.5.The results of yeast two-hybrid showed that the proteins interacted with St NF-YC9 were involved in potato response to drought stress.Y2 H point-to-point results showed that St NF-YC9 interacts with St NF-YB19 and St NF-YA9 respectively,and Bimolecular fluorescence complementation(Bi FC)assay was used for verifies the interaction between St NF-YC9 and St NF-YB19.The results of yeast three-hybrid showed that there existed a St NF-YC9-B19-A9 complex.Silencing the St NF-YA9 gene in potato by VIGS reduced the RWC of TRV: A9 and increased the water loss rate of isolated leaves.Under drought stress,the proline content in TRV: A9 was decreased,that led to TRV: A9 became more sensitive to drought stress.In addition,the results of yeast one-hybrid showed that St NF-YA9 bind to the promoter region of the rate-limiting enzyme gene St P5 CS and St OSML gene.In summary,RNA-seq and ATAC-seq were used to analyze the molecular mechanism of potato in response to drought stress at the genome-wide level,and the transcription regulation network in response to drought stress was constructed.The molecular mechanism of St NF-Y gene participating in drought stress was further studied.This study aims to provide theoretical basis for improving drought tolerance in potato.