Studies On Transcriptional Regulation Of HvNramp5 And Epigenetic Regulatory Network Of Cadmium Stress In Barley Roots

Cadmium（Cd）is one of the most prevalent and toxic heavy metal pollutants in agricultural soils.Cd²⁺is rapidly uptaken by plant roots from the soil andtranslocated to edible sections and seeds of plants,which has adverse effects on crop growth,production and food safety.Root absorption is the first stage in the entry of cadmium into plant cells in various physiological processes.Barley（Hordeum vulgare）is the world’s fourth largest cereal crop,and its seeds are used for food,fodder,and as a raw material in the production of beer.Due to its ecological flexibility,strong tolerance for infertile environments and capacity to grow in a variety of soil conditions,it poses a significant risk of soil Cd contamination.Therefore,it is important to clarify the molecular mechanism of cadmium uptake in barley roots in order to reduce the cadmium content in the above ground,especially in seeds.According to previous research,HvNramp5 is a major transporter for Cd and Mn absorption in barley roots,and its coding gene expression is induced by iron deficiency condition,but the mechanism of its transcriptional regulation is still unknown.To reveal the regulatory mechanisms of HvNramp5 gene expression in barley roots and epigenetic regulatory networks in response to cadmium stress,we used multi-omics approaches such as expression association analysis（e GWAS）of HvNramp5 gene,yeast-one-hybrid assay,transcriptome and m6A methylation（N⁶-methyladenosine）sequencing analysis in this study.The main findings were as following:1.The regulatory mechanism of HvNramp5 expressionin barley roots.The expression of HvNramp5 was determined in barley roots of 100 core accessions under cadmium treatment（0.5μM Cd Cl₂）for 7 days.The results demonstrated that the expression of HvNramp5 varied among genotypes,which was positively linked with cadmium concentration in barley roots.A total of 73 assocaited SNPs with 105annotated genes,including 11 kinase genes and 6 transcription factors,were found in the e GWAS study using high-quality SNPs derived from transcriptome sequencing.The yeast-one-hybrid system was then used to screen genes that interacted with HvNramp5,and 42 positive clones were identided.Point-to-point validation was used to screen four transcription factors or genes with transcriptional activation activity for interactions with HvNramp5.Finally,using CRISPR/Cas9 technology,gene editing mutants of barley for the above genes were created,and the expression of the HvNramp5 in the roots of barley T₁ generationmutants was examined under control and iron deficiency conditions,to determine the effect of the HORVU5Hr1G095410（HMG-Y-related protein A）on HvNramp5 expression under iron deficiency.The HORVU5Hr1G095410positively regulates the expression of HvNramp5 gene,and knocking out this gene significantly reduced the HvNramp5 expression.2.Epigenetic regulatory networks of barley roots in response to cadmium stress.The m6A methylation（N⁶-methyladenosine）and transcriptome in the root of Golden Promise cultivar under control and Cd treated（5μM Cd Cl₂,7 days）condition were performed.The results revealed that the level of m6A methylation was higher in the Cd treated group than in the control.In both groups,the m6A methylation alteration was abundant in the stop codon,3’UTR region,and near the chromosomal telomeres.A total of 8151 differential m6A peak were identified（|log₂（fold change）|≥1,P<0.05）,which contained 4,277 genes.Compared the transcriptome data of the Cd-treated and the control groups,3,920 differentially expressed genes（|log₂（fold change）|≥1,P<0.05）were discovered,while 2,015 genes were up-regulated and 1,905 genes were down-regulated.According to KEGG,these genes were mostly found in the pathways of"phenylalanine biosynthesis","MAPK plant signaling pathway","phytohormone signaling".The degree of m6A methylation modification was positively linked with gene expression,and methylation occurred at the start and stop codons to enhance gene expression,according to the combined analysis of the m6A methylation and transcriptome data.Further analysis revealed MAPK,WRKY and MYB members with significant differences in both m6A methylation modification and expression,and we proposed transcriptional regulatory networks in barley roots in response to Cd stress,which may provide new insights into barley breeding with low Cd content.In summary,the results of this study not only enrich transcriptional regulatory mechanisms of HvNramp5 expression and Cd stress response in barley roots,but also provide theoretical references for genetic improvement of barley.