Evolutionary Divergence, Predicted Interaction Interface, and Regulatory Specialization of MTB as a Non-Catalytic...

N6-methyladenosine (m6A) is the most prevalent internal modification of eukaryotic mRNA and a central regulator of plant development and stress adaptation. The plant m6A writer complex requires two MT-A70 family proteins, the catalytic subunit MTA70 and its non-catalytic partner MTB, yet the evolutionary basis and structural logic underlying this functional division remain unresolved across land plant lineages. Here, we present an integrative computational analysis of MTA70 and MTB across 15 phylogenetically representative species spanning bryophytes, lycophytes, charophyte algae, monocots, and dicots. Phylogenomic reconstruction resolved three strongly supported clades, namely MTA70, MTB, and an intermediate MTA70-like group, demonstrating that catalytic-to-regulatory divergence predates the separation of major land plant lineages. MTA70 proteins exhibited strict conservation of gene structure, catalytic motifs, and domain architecture, reflecting selective constraint at functionally critical residues, whereas MTB showed extensive divergence in exon-intron organization and surface-exposed residues, consistent with relaxed structural constraints. AlphaFold2-based structural modeling and data-driven protein-protein docking predicted a stable MTA70-MTB heterodimer with a buried surface area of 1435 Ų and a binding free energy of -8.1 kcal/mol, with Lys746 and Lys637 of MTB identified as primary interface hotspots by computational alanine scanning. Expression profiling across six species revealed preferential MTB accumulation in reproductive tissues, while promoter analysis identified statistically significant enrichment of jasmonate-responsive elements (TGACG-motif) in MTB promoters (Mann-Whitney U, p = 0.025) and a 3.4-fold higher abundance of ABA-responsive elements (ABRE) in MTB relative to MTA70, suggesting potential responsiveness to multiple phytohormone signals. Together, these findings establish an evolutionary and regulatory framework for MTB as a conserved scaffold coupling m6A deposition to developmental and environmental signaling in land plants.