The Molecular Regulation Mechanism Of M~6A Modification On Anther Fertility Of YS-type Thermo-sensitive Male Sterile Line In Wheat (Triticum Aestivum L.)

Heterosis has made a significant contribution to ensuring food security.Plant male sterility has become the most widely and efficient way for crop heterosis utilization.Exploring the molecular mechanism of plant male sterility can help breeders to establish stable sterile lines,produce high-combination hybrids,and improve crop yield and quality.Methylation modification plays potential regulatory roles in plant male sterility.As one of the most prevalent methylation modifications in RNA,N~6 methyladenosine(m~6A)has been shown to be involved in the regulation of male sterility in rice.However,there is not adequate research undertaken on the mechanism of wheat male sterility from the perspective of m~6A methylation modification.Therefore,we used the transcriptome and epi-transcriptomics resources to investigate the regulatory roles of m~6A methylation modification on the wheat thermo-sensitive cytoplasmic male sterile line(TCMS)YS3038.We identified the m~6A methyltransferase genes that are related to male sterility and verified the regulatory roles of two genes in the anther fertility of YS3038.The main results are as follows:1.The m~6A level in YS3038 anthers changed significantly under sterile and fertile conditions.Compared to YS3038B,the total RNA m~6A level,the m RNA m~6A enrichment,the methylation modification sites,and the number of m~6A modified genes were significantly reduced in YS3038A.2.A total of 8,127 differential m~6A methylation modification sites were identified by Me RIP-Seq,including 164 up-regulated m~6A sites and 7,963 down-regulated m~6A sites.The differential m~6A methylation sites were mainly deposited in the 3’UTR region of the genes and modified 7,997 genes.Differentially expressed m~6A modified genes were highly enriched in biological processes related to pollen development,pollen tube germination,and pollen mother cell differentiation.3.A total of 9,511 differentially expressed genes were identified by RNA-Seq.There were7,386 up-regulated genes and 2,215 down-regulated genes in YS3038A compared to YS3038B.GO annotation showed that the up-regulated DEGs were mainly involved in redox,carbohydrate metabolism,and translation processes.The down-regulated DEGS were mainly enriched in protein phosphorylation,transmembrane transport,carbohydrate metabolism,positive regulation of gene expression,and epigenetics processes.KEGG annotation showed that the DEGs were mainly enriched in carbohydrate metabolism and lipid metabolism pathways.These pathways are related to anther development.4.There were 1,595 genes with significant changes in m~6A and m RNA levels.More than80%of the differentially expressed genes showed an expression pattern of m~6A down and m RNA up.The m~6A sites of 90%of DEMGs distributed in the 3’UTR region.GO and KEGG annotation revealed that 1,595 differentially expressed genes were mainly enriched in sugar metabolism and lipid metabolism pathways.These pathways were closely related to nutrient accumulation and pollen wall development in anthers.The m~6A methylation modification deposited in the 3’UTR region negatively regulated the expression of genes in the above pathways,participating in the regulation of YS3038 anther fertility.5.Eighteen putative m~6A methyltransferase genes were identified in the whole genome of wheat.They were divided into five categories,namely Ta MTA,Ta MTB and Ta MTC,Ta FIP37,Ta HAKAI,and Ta VIR.Multiple sequence alignments and evolutionary analysis showed that the protein sequences and gene structures of the same type of wheat methyltransferase genes were highly conserved.Colinearity analysis showed that segmental duplication was the only way to expand m~6A methyltransferase genes in wheat.The expression analysis of 18 genes in YS3038 anthers under sterile and fertile conditions showed that these genes were involved in the regulation of YS3038 male fertility.6.Two genes,Ta MTA-2 and Ta FIP37-3,were successfully cloned from the c DNA of YS3038 anthers.These two genes were located in the nucleus.Overexpressed Ta MTA-2 and Ta FIP37-3 significantly increased RNA m~6A levels in S.cerevisia.Ta MTA and Ta FIP37 were silenced by using VIGS technology,which reduced the seed setting rate of YS3038 under fertile conditions.These results indicated that Ta MTA-2 and Ta FIP37-3 were related to the m~6A methylation levels and Ta MTA and Ta FIP37 were closely related to the anther fertility of YS3038.Taken together,we suggested that the RNA m~6A methylation modification and m~6A methyltransferase genes Ta MTA and Ta FIP37 were involved in the regulation of anther fertility in the wheat thermo-sensitive cytoplasmic male sterile line YS3038.The m~6A peaks,which were deposited in the 3’UTR region,negatively regulated the expression of genes in the anther of YS3038.Abnormal gene expression disrupted the normal sugar metabolism and lipid metabolism pathways and may affect the accumulation of nutrients,resulting in the pollen abortion of YS3038A.This study preliminarily revealed the regulatory roles of m~6A in anther fertility of the wheat thermo-sensitive cytoplasmic male sterile line YS3038 and provided a theoretical basis for the study of epigenetic regulation of m~6A in wheat and the utilization of wheat heterosis.