Genome-wide identification and expression pattern analysis of the YTH domain in Rosa chinensis 'Old Blush' and validation of the m6A binding capability of RcDF1A

Abstract

N⁶-methyladenosine (m⁶A), a vital emerging epigenetic modification in eukaryotic RNA, exerts critical roles in diverse biological processes including hormone signaling, stress response regulation, RNA stability maintenance, translation control and cell differentiation through recognition by its readers, notably the YTH domain-containing proteins in plants. Rosa chinensis ‘Old Blush’, an ancient Chinese rose variety, holds a prominent position in rose breeding due to its long history and excellent genetic characteristics. This study comprehensively identified YTH family members in ‘Old Blush’, systematically naming them based on their chromosomal locations and subfamily affiliations. Physical and chemical properties analysis, along with subcellular localization studies, revealed that most of these family members are located in the nucleus, hinting at their potential roles in nuclear processes. Furthermore, our findings suggest that these proteins may engage in liquid-liquid phase separation, a pivotal aspect that sheds light on their functional localization and dynamics within the cell. Our analysis of cis-acting elements in the promoter region of RcYTH genes uncovered a close association with light stress and abscisic acid (ABA) signaling pathway, implying a pivotal role in the response to environmental stress. Expression pattern analyses further reinforced this notion, revealing distinct expression profiles across various tissues and significant alterations in response to dark treatment, intense light exposure, and ABA stress. To directly assess the m⁶A binding ability of RcYTH proteins, we performed electrophoretic mobility shift assays (EMSA) using RCDF1A as a representative. Our results conclusively demonstrated that RcDF1A possesses the ability to bind m⁶A in vitro, thereby providing direct evidence for its involvement in the metabolism of m⁶A-modified mRNAs. This comprehensive study not only establishes a solid foundation for exploring the multifaceted functions of YTH family members in R.chinensis ‘Old Blush’, but also enriches the understanding of epigenetic marker m⁶A of rose, offering novel insights for genetic improvement and breeding of rose and other plants via epitranscriptomic engineering.