Dissecting the roles of increased mRNA m6A methylation in autotetraploidization in Stevia rebaudiana

Abstract

N6-methyladenosine (m⁶A) is the most abundant modification in eukaryotic mRNA. m⁶A functions in embryo development, flowering time regulation, and fruit ripening. Although polyploidization, a significant factor in plant evolution, leads to phenotypic changes, the roles of m⁶A in plant polyploidization remain unclear. Here, we observed increased leaf area, fresh weight, and thickness upon autotetraploidization in stevia (Stevia rebaudiana). To examine phenotypic and molecular changes following polyploidization, we quantified m⁶A abundance in RNA and conducted m⁶A immunoprecipitation sequencing (m⁶A-seq) and transcriptome analysis of autotetraploid and diploid stevia. Polyploidization led to increased m⁶A levels in RNA, especially in mRNA. m⁶A-seq methylome profiling revealed ~ 20,000 transcripts containing m⁶A, primarily in 3′ untranslated regions. Moreover, 2642 differentially modified m⁶A peaks (DMPs) were hypermethylated (hyper-DMPs) post polyploidization, and transcripts with hyper-DMPs were mainly associated with zeatin and flavonoid biosynthesis. Comparative analysis unveiled a possible correlation between m⁶A methylation and mRNA abundance, as confirmed by in vitro mRNA stability assays. The transcripts of many candidate genes involved in auxin, cytokinin, wax, and DNA biosynthesis, the cell cycle, and the cell wall exhibited hypermethylation and higher abundance in autotetraploid stevia. The contents of wax and auxin compounds significantly increased in autotetraploid stevia, suggesting that m⁶A modification helps maintain higher expression of these target genes. Our findings point to an m⁶A-orchestrated regulatory circuit where m⁶A hypermethylates and upregulates DMP-marked transcripts of auxin and wax biosynthesis genes, thereby determining the accumulation of auxin and wax compounds following stevia polyploidization, which contributes to the phenotypic changes following its autotetraploidization.