Altered development and lignin deposition in rice p‐ <i>COUMAROYL ESTER 3‐HYDROXYLASE</i> loss‐of‐function mutants

The aromatic composition of lignin greatly influences the potential utility of lignocellulosic biomass. Previously, we generated transgenic rice plants with altered lignin aromatic composition and enhanced biomass utilization properties by suppressing the expression of p-COUMAROYL ESTER 3-HYDROXYLASE (C3'H). While RNAi-derived C3'H-knockdown lines displayed relatively normal growth with substantially augmented levels of p-hydroxyphenyl-type lignin units, genome-edited C3'H-knockout lines exhibited severely impaired growth phenotype, leading to arrested seedling development. In this study, we further characterized the genome-edited C3'H-knockout rice by analyzing gene expression and phenolic metabolite profiles alongside phenotypic traits and cell wall lignin structure. The seedlings of the C3'H-knockout rice displayed irregular vasculature and ectopic lignification. RNA-sequencing analysis detected widespread changes in the expression of genes associated with plant growth, hormone biosynthesis and signaling, and stress responses in the C3'H-knockout rice. Overall, our data suggested that C3'H disruption activates metabolic sensor-mediated signaling pathways, which in turn regulate phenylpropanoid metabolism. In line with this, phenolic metabolite profiling of the C3'H-knockout rice revealed not only shifts in monolignol-associated phenylpropanoids but also reductions in flavonoids and salicylic acid derivatives. Moreover, changes in the aromatic composition of the mutant lignin and phenolic metabolites indicated the presence of parallel monolignol pathways enabling rice to produce guaiacyl- and syringyl-type monolignol derivatives in the absence of C3'H activity. Our findings contribute to a deeper understanding of the mechanisms underlying the growth defects of lignin-modified mutants, with implications for optimizing the utility of grass lignocellulose.