Integrated Methylome and Transcriptome Analyses Reveal the Molecular Mechanism by Which DNA Methylation Regulates Kenaf Flowering

Abstract

DNA methylation regulates key biological processes in plants. In this study, kenaf seedlings were pretreated with the DNA methylation inhibitor 5-azacytidine (5-azaC) (at concentrations of 0, 100, 200, 400, and 600 μM), and the results showed that pretreatment with 200 μM 5-azaC promoted flowering most effectively. To elucidate the underlying mechanism, phytohormone, adenosine triphosphate (ATP), and starch contents were determined, and genome-wide DNA methylation and transcriptome analyses were performed on anthers pretreated with 200 μM 5-azaC (5-azaC200) or with no 5-azaC (control conditions; 5-azaC0). Biochemical analysis revealed that 5-azaC pretreatment significantly reduced indoleacetic acid (IAA) and gibberellic acid (GA) contents and significantly increased abscisic acid (ABA) and ATP contents. The starch contents significantly increased in response to 200 and 600 μM 5-azaC. Further genome-wide DNA methylation analysis revealed 451 differentially methylated genes (DMGs) with 209 up- and 242 downregulated genes. Transcriptome analysis showed 3,986 differentially expressed genes (DEGs), with 2,171 up- and 1,815 downregulated genes. Integrated genome-wide DNA methylation and transcriptome analyses revealed 72 genes that were both differentially methylated and differentially expressed. These genes, which included ARFs, PP2C, starch synthase, FLC, PIF1, AGL80, and WRKY32, are involved mainly in plant hormone signal transduction, starch and sucrose metabolism, and flowering regulation and may be involved in early flowering. This study serves as a reference and theoretical basis for kenaf production and provides insights into the effects of DNA methylation on plant growth and development.

Keywords: 5-azacytidine (5-azaC); DNA methylation; flowering; kenaf (Hibiscus cannabinus L.); transcriptome.