Genome-Wide Identification Of RNA Methylation Regulation Related Genes And Their Roles In Response To Salt Stress In Wild Emmer Wheat

Wild emmer wheat is the wild ancestor of cultivated tetraploid and hexaploid wheat,and has long been an important secondary gene pool for wheat genetic improvement due to its superior characteristics such as large grain size,high protein content,and strong stress tolerance.N~6-adenosine methylation of RNA,abbreviated as m~6A,is the most conserved and widely distributed RNA modification in organisms and plays an important regulatory role in plant organogenesis,growth and development,and stress response.However,there have been relatively few studies on m~6A-related genes in wild emmer wheat,especially those involved in regulating salt stress response.In this study,using bioinformatics analysis,we identified and analyzed the m~6A regulatory gene family members in wild emmer wheat at the whole-genome level and constructed a co-expression network based on their expression characteristics under salt stress.We identified and explored 7 salt-tolerant co-expression modules and 9 core m~6A regulatory genes involved in salt stress response.We also identified the salt tolerance of a key candidate gene TdFIP37 functional loss mutant and preliminarily clarified its biological function based on RNA-seq analysis.In addition,we analyzed the expression profile and transcription characteristics of wild emmer wheat under salt stress using direct RNA sequencing technology and preliminarily analyzed the m~6A methylation characteristics of wild emmer wheat under salt stress,laying a foundation for further revealing the molecular mechanism of m~6A regulation of salt tolerance in wild emmer wheat.The main results of this study are as follows:1)The whole-genome identification of the m~6A regulatory gene family in wild emmer wheat.We systematically identified the writer,the eraser,and reader families of m~6A regulatory machinery in wild emmer wheat at the whole-genome level using HMM search and BLASTP tools.Through analysis,we identified 64 candidate genes,including 21 writers,17 erasers,and 26 readers.Phylogenetic analysis divided them into three subgroups,each with similar protein structural domains and conserved motifs.Collinearity analysis showed that segmental duplication and polyploidization significantly expanded the m~6A genes in the wild emmer wheat genome.Predictions of cis-acting elements suggested that cis-acting elements involved in stress response and hormone regulation,such as MBS,LTR,and ABRE,were highly present in their promoters,implying their involvement in regulating stress response and growth and development in wild emmer wheat.Finally,we analyzed the genetic variation and differentiation of m~6A genes in tetraploid wheat populations based on resequencing data,with Pi values of 4.18E-06 and 3.77E-06 in wild and cultivated emmer wheat populations,respectively,and a genetic differentiation index of 0.262,indicating that m~6A genes underwent significant genetic bottleneck effects during emmer wheat domestication.2)Discovery of salt stress-related m~6A regulatory genes and their network analysis.Based on 54 RNA-seq datasets from different time points of salt-tolerant variety A5 and salt-sensitive variety C2 under normal and salt stress conditions,the expression profiles of the identified m~6A genes under salt stress were analyzed.Thirteen differentially expressed m~6A genes were identified under salt stress by spatiotemporal sequence analysis,including 9 upregulated and 4 downregulated genes,providing preliminary evidence of salt stress-responsive m~6A genes.Furthermore,a co-expression regulatory network was constructed based on RNA-seq data,and combined with functional enrichment analysis,7key co-expression modules significantly associated with salt stress were identified,and 9 key candidate m~6A regulatory genes responsive to salt stress were uncovered,revealing an m~6A-mediated regulatory network involved in salt stress response.3)The identification and transcriptome analysis of Tdfip37 in salt tolerance were conducted.A functional loss-of-function mutant of the key candidate gene FIP37 was screened from a tetraploid EMS mutant library,and its salt tolerance was evaluated.It was found that compared with the wild-type,the mutant was more sensitive to salt stress and showed significantly reduced salt tolerance.RNA-seq was employed to analyze the expression profiles of Tdfip37 under normal and salt stress conditions,and it was found that the gene expression level of Tdfip37 mutant changed more dramatically than that of WT after salt stress treatment,with a total of 5910 differentially expressed genes(DEGs),among which 3691 were up-regulated DEGs and 2219 were down-regulated DEGs.These DEGs were enriched in numerous functions related to enzyme activity,purine metabolism,and sugar degradation.4)Analysis of m~6A and transcription features in wild emmer wheat under salt stress.Using Nanopore’s RNA Direct Sequencing technology,we analyzed the m~6A sites and expression profiles in response to salt stress in the B5 strain.A total of 46,009 m~6A sites were identified,distributed on 14 chromosomes of wild emmer wheat,with the most on chromosome 5A(3,918)and the second most on chromosome 5B(3,842),with the characteristic motif being ATCTC.Transcripts undergoing m~6A methylation were significantly enriched in pathways and functional entries responsive to salt stress,such as plant hormone signal transduction,hyperosmotic salt response,and potassium ion transmembrane transport.In addition,184,958 m5C modification sites were identified.3,356differentially expressed transcripts with methylation after salt stress treatment were identified by correlating transcript expression levels and m~6A methylation status,and 68differentially expressed transcripts were selected for response to salt stress based on homologous gene functional annotation and a protein interaction network was constructed.Furthermore,2,475 new genes,26,779 new transcripts,and 693 transcription factors were predicted,and 1,318 common lncRNAs were predicted using three methods:CNCI analysis,CPC2,and pfam protein structure domain analysis.After salt stress treatment,the overall level of polyA tail length increased,and the number of alternative splicing events increased.In summary,this study identified key gene modules and core genes involved in m~6A regulation and salt stress response in wild diploid wheat,shedding light on the molecular mechanisms underlying m~6A-mediated salt tolerance in this species.The target genes identified here may also serve as potential targets for genetic improvement of salt tolerance in tetraploid and common wheat.These findings were published in Plant Science and similar academic journals.