Splicing Variant and In Silico Functional Analysis of OsPLR3 in a Thai Landrace Rice Associated with Lignan Biosynthesis

Background: Lignans are plant-derived polyphenols with antioxidant and phytoes-tro-genic activities. Although pinoresinol–lariciresinol reductase (PLR) enzymes are well characterized in dicots, their roles in monocots remain poorly understood. In this study, we characterize OsPLR3, a putative PLR gene in the monocot Oryza sativa cv. Mae Phaya Thong Dam. Methods: OsPLR3 was cloned and shown to produce three isoforms (OsPLR3-X1, OsPLR3-X2, OsPLR3-X3) via alternative splicing. We performed domain prediction, 3D modeling (I-TASSER), docking (CB-Dock2), RT-/qRT-PCR, HPLC, PlantCARE cis-element mapping, and phylogenetic reconstruction (MEGA11). Results: OsPLR3-X1 and OsPLR3-X2 retained complete NADPH binding domains with the cat-alytic lysine (K133), whereas X3 lacked essential motifs and its expression was un-de-tected. Only the OsPLR3-X2 isoform was transcriptionally active in stems, leaves, flow-ers, and developing seeds, and its expression rose from the milky to mature seed stages, paralleling secoisolariciresinol accumulation. Docking suggested the strong binding of OsPLR3-X2 to pinoresinol (−8.4 kcal/mol) and lariciresinol (−9.2 kcal/mol). Phylogenetic analysis further positioned the three OsPLR3 isoforms within a mono-cot-specific PLR3 lineage distinct from the classical PLR1/2 group of dicots. Conclusions: Evidence from the structure, docking, expression, metabolite profiling, and phylogeny experiments indicates that OsPLR3-X2 is a catalytically competent isoform contributing to lignan biosynthesis in rice seeds. These findings advance our understanding of monocot secondary metabolism and highlight OsPLR3-X2 as a target for metabolic en-gineering and nutraceutical enhancement.