Essential Yet Dispensable: The Role of CINNAMATE 4-HYDROXYLASE in Rice Cell Wall Lignification

Abstract A comprehensive understanding of the intricate lignin biosynthesis in grasses could contribute to enhancing our ability to utilize grass biomass. CINNAMATE 4-HYDROXYLASE (C4H), in conjunction with PHENYLALANINE AMMONIA-LYASE (PAL), initiates the entry of phenylalanine into the cinnamate/monolignol pathway, leading to the production of diverse phenylpropanoids, including lignin monomers. Despite extensive research on C4H in eudicots, genetic studies of C4H in grasses remain considerably limited. Notably, the role of C4H in the presence of PHENYLALANINE/TYROSINE AMMONIA-LYASE (PTAL), a grass-specific ammonia-lyase that can bypass the conserved PAL-C4H pathway by recruiting tyrosine into the cinnamate/monolignol pathway, remains unclear. To address this gap, a set of genome-edited rice mutants harboring knockout mutations in rice C4H genes were generated and subjected to the analysis of growth phenotype and cell wall chemotype, alongside isotopic feeding and chemical inhibitor assays to test the contributions of the PAL-C4H and PTAL pathways. The phenotype and chemotype characterizations of C4H -knockout rice mutants demonstrated that class I (OsC4H1/CYP73A38) and class II (OsC4H2a/CYP73A39 and OsC4H2b/CYP73A40) C4Hs cooperatively contribute to lignin biosynthesis in rice. Nevertheless, the impacts of C4H -deficiency on plant development and lignin formation in rice appeared to be less prominent compared to those reported in eudicots. The 13 C-labeled phenylalanine and tyrosine feeding experiments demonstrated that even with the phenylalanine-derived PAL-C4H pathway completely blocked, the C4H -knockout rice can still produce significant levels of lignin and maintain sound cell walls by utilizing the tyrosine-derived PTAL pathway. Overall, this study demonstrates the essential but dispensable role of C4H in grass cell wall lignification.