Complex origin, evolution, and diversification of non-canonically organized OVATE-OFP and OVATE-Like OFP gene pair across Embryophyta

Ovate Family Proteins (OFP) is a plant-specific gene family of negative transcriptional regulators. Till-date, a handful of in-silico studies have provided glimpses into family size, expansion patterns, and genic features across all major plant lineages. A major lacuna exists in understanding origin of organisation complexity of members such as those arranged in a head-to-head manner which may lead to transcriptional co-regulation via a common bi-directional promoter. To address this gap, we investigated the origin, organization and evolution of two head-to-head arranged gene pairs of homologs of AtOFP2-AtOFP17, and, AtOFP4-AtOFP20 across Archaeplastida. The ancestral forms of AtOFP2, AtOFP4, AtOFP17, and AtOFP20 are likely to have evolved in last common ancestors of Embryophyta (land plants) given their complete absence in Rhodophyta and Chlorophyta. The OFP gene family originated and expanded in Bryophyta, including protein variants with complete (OVATE-OFP) or partial (OVATE-Like OFP) OVATE domain; with head-to-head organization present only in Spermatophyta (gymnosperms and angiosperms). Ancestral State Reconstruction revealed the origin of head-to-head organized gene pair in gymnosperms, with both genes being OVATE-OFP (homologs of AtOFP2/4). Phylogenetic reconstruction and copy number analysis suggests the presence of a single copy of the head-to-head arranged pair of OFP2/4 (OVATE)-OFP17/20 (OVATE-Like) in all angiosperms except Brassicaceae, and a duplication event in last common ancestor of core Brassicaceae approximately 32-54 MYA leading to origin of AtOFP2-AtOFP17 and AtOFP4-AtOFP20 as paralogs. Synteny analysis of genomic regions harbouring homologs of AtOFP2-AtOFP17, AtOFP4-AtOFP20 and AtOFP2/4-AtOFP17/20 across angiosperms suggested ancestral nature of AtOFP2-AtOFP17 gene pair. The present study thus establishes the orthology and evolutionary history of two non-canonically organised gene pairs with variation in their OVATE domain. The non-canonical organisation, atleast in Brassicaceae, has the potential of generating complex transcriptional regulation mediated via a common bi-directional promoter. The study thus lays down a framework to understand evolution of gene and protein structure, transcriptional regulation and function across a phylogenetic lineage through comparative analyses.