OsPAL2;1 and OsPAL2;3 are Key Regulators of Phenolic Acid to Modulate Allelopathy and Rhizosphere Microbiome in Rice

Phenylalanine ammonia-lyase (PAL; EC 4.3.1.5) is encoded by a multigene family in rice (Oryza sativa L.), and its transcriptional abundance is tightly coupled with allelopathic potential. Rice chromosome 2 harbors a tandemly duplicated cluster of four OsPAL paralogs: OsPAL2;1, OsPAL2;2, OsPAL2;3, and OsPAL2;4. To dissect their precise roles in regulating allelopathy, this study generated independent overexpression lines for each OsPAL gene in both the allelopathic rice genotype 'PI312777' and the non-allelopathic cultivar 'Lemont'. Overexpression of individual OsPAL genes significantly enhanced the inhibitory effects of root exudates on barnyardgrass growth, with OsPAL2;1 and OsPAL2;3 exhibiting the most pronounced weed-suppressive phenotypes. Mechanistically, OsPAL overexpression drove distinct tissue-specific metabolic alterations: in transgenic 'PI312777', concentrations of protocatechuic acid, p-coumaric acid, ferulic acid, salicylic acid, and cinnamic acid significantly accumulated in both roots and leaves; conversely, 'Lemont' overexpression lines displayed selective increases in protocatechuic acid, p-hydroxybenzoic acid, and cinnamic acid. Beyond direct allelochemical mediation, OsPAL overexpression reshaped the rhizosphere microbiome. Transgenic 'PI312777' lines displayed reduced alpha diversity and species richness within the root-associated bacterial community. Most strikingly, OsPAL2;1 and OsPAL2;3 overexpression lines showed a marked enrichment of Flavisolibacter, Ohtaekwangia, Lysobacter, and Myxococcota. Collectively, our findings demonstrate that OsPAL2;1 and OsPAL2;3 emerge as prime candidates for engineering next-generation rice varieties with enhanced natural weed-suppressive capacity through integrated metabolic and microbiome engineering.