Identification of Rice m6A Methyltransferase Complex Subunits MTA/MTB and Their Effects on Heading Date and Grain Weight

Abstract

N6-methyladenosine (m6A) is a vital epitranscriptomic modification that regulates plant development and stress responses. In rice (Oryza sativa L.), the identity of the catalytic subunit (MTA) of the core m6A writer complex has remained controversial because of inconsistent annotations based primarily on sequence homology. Here, phylogenetic analysis identified LOC_Os02g45110 as the putative catalytic subunit OsMTA, whereas LOC_Os01g16180 and LOC_Os03g05420 were classified as the structural subunits OsMTB1 and OsMTB2, respectively. All three proteins showed nuclear enrichment. Yeast two-hybrid, luciferase complementation imaging, and bimolecular fluorescence complementation assays demonstrated that OsMTA interacts independently with OsMTB1 and OsMTB2. Moreover, transient co-expression of OsMTA with either OsMTB1 or OsMTB2 increased global m6A levels in a heterologous system. No homozygous plants carrying frameshift mutations in OsMTA were obtained, suggesting that complete loss of OsMTA function is lethal. Knockdown of OsMTA or CRISPR/Cas9-mediated knockout of OsMTB1 and OsMTB2 significantly delayed heading date and reduced 1000-grain weight. Conversely, overexpressing OsMTA accelerated heading, whereas overexpressing OsMTB1 or OsMTB2 generally accelerated heading but significantly reduced 1000-grain weight. Together, these findings define the core composition of the rice m6A writer complex and reveal its effects on heading date and grain weight.