Cell Elongation Mechanism Of Gossypium Barbadense Regulated By DNA Methylation Under Alkaline Stress

The growth of cotton was threatened by unfavorable stresses such as alkaline or salinity in the soil.This abiotic stress leaded to a significant decrease in cotton yield and fiber quality.G.barbadense is a cultivated cotton known not only for producing high-quality fiber,but also for its excellent resistance to salinity.Although high intracellular p H was known to affect the cellular elongation of plant root hairs,little was known about the molecular mechanisms of stress tolerance in G.barbadense.In this study,the hypocotyl and fibers of G.barbadense were used as the research materials,and the WGBS sequencing technology was used to compare and analyze 6 samples of multicellular hypocotyls and single-cell fibers under Na₂CO₃,Na Cl stress and normal growth conditions.A comparative analysis was performed to map single-base cytosine methylation.Then,by combining RNA-seq data,the metabolic pathways of epicotyl and fiber elongation in response to alkaline stress methylation epigenetic changes and transcriptional changes were further mined.The underlying epigenetic and transcriptional mechanisms of alkaline stress-induced cell elongation were analyzed.The main findings of this study were as follows:1.Taking the hypocotyl as an example,the elongation of the hypocotyl was inhibited by alkaline stress.The methylation levels in the cytosine site,CG,CHG and CHH sequences sequenced in this tissue were19.74%,71.03%,53.87%and 6.26%,respectively.The methylation levels of CHH sequences increased the most in the three sequence environments under alkaline stress,and high and low levels of m C occurred mostly in CT-and CA-sequences,respectively.Different types of TEs exhibit low methylation levels in the context of CHH sequences.In the three sequence environments,the methylation level of the promoter region was higher,and the differentially expressed and methylated differential genes were enriched in the phytohormone signaling pathway.2.The study on the enrichment genes of carboxylesterase in the plant hormone signal transduction pathway found that 72 GBCXE genes were identified in the G.barbadense,and the CXE gene family in the G.barbadense was relatively conservative.The analysis of promoter action elements indicated that carboxylesterase also played a role in plant signal transduction pathway and participates in the regulation mechanism of cotton alkaline stress.The GBCXE49 gene contains typical conserved domains characteristic of HGG and GXSGX carboxylesterases.GBPIF1 transcription factor represses the transcription of GBCXE49 gene,silencing of GBXE49 gene affects the contents of IAA and GA in cotton leaves,and overexpression of GBCXE49 gene in transgenic Arabidopsis promotes main stem meristems and elongation,indicating that GBCXE49 under alkaline stress Involved in cell elongation mediated by phytohormones.3.Alkaline stress decreased the TFU of fibers in elongation.DNA methylation during fiber elongation accounted for 23%,22%and 55%of the total methylation sites in the three sequence contexts of CG,CHG and CHH,respectively.Transcription start site（TSS）and transcription DNA methylation around the termination site（TES）was drastically reduced.Saline-alkali stress resulted in decreased methylation levels in the background promoter regions of elongation fiber CG and CHG sequences.Likewise,TE showed higher methylation levels in the CG and CHG contexts and lowest in the CHH context.The environmental methylation of CHG and CHH sequences was negatively correlated with the expression levels of most gene regions.Under alkaline stress,3468 high-level DMR-anchored genes and 7407 low-level DMR-anchored genes were identified in the exon region.The region identified 2142 hypermethylated region-anchored genes and 5880 low-level DMR-anchored genes.DNA methylation levels affected the transcriptional expression of some genes in the fatty acid biosynthesis and metabolism pathway（ko00062）.4.The study on the rate-limiting enzyme gene KCS of fatty acid metabolism pathway showed that:38GBKCS genes were identified in G.barbadense,and the KCS family in cotton underwent expansion during evolution.GBKCS was relatively conserved in specific subgroups,and most promoters of GBKCS gene family members contain response elements such as auxin,ABA,GA,Me JA,drought,light,and MYB.The transcription factor GBMYB88 positively regulates the transcription of the GBKCS24 gene.It indicated that GBKCS24 gene promoted the growth of epidermal hair and plant height of Arabidopsis,and enhanced the tolerance of alkaline stress.In cotton,the GBKCS24 gene was silenced,and dark brown patches appeared,and the expression levels of the VLCFAs-related genes GBKCR1,GBHCD2,and GBECR were significantly reduced.5.The integrated analysis of hypocotyl and elongation fibers under alkaline stress found that the differentially methylated regions were positively correlated with the number of genes in the three sequence backgrounds,but negatively correlated with the density of TEs.Hypocotyls had significantly more hypermethylated DMRs than hypomethylated DMRs under alkaline stress,while elongation fibers had significantly more hypomethylated DMRs than hypermethylated DMRs under alkaline stress.27methylation-related genes were widely expressed in different tissues of cotton.The enrichment of hypocotyl phytohormone signal transduction pathways and fibrolipid and fatty acid metabolism pathways reflected the differences between unicellular and multicellular elongation life processes.