Simultaneous Down-Regulation of Dominant Cinnamoyl CoA Reductase and Cinnamyl Alcohol Dehydrogenase Dramatically Altered Lignin Content in Mulberry.

Mulberry (Morus alba L.) is a significant economic tree species in China. The lignin component serves as a critical limiting factor that impacts both the forage quality and the conversion efficiency of mulberry biomass into biofuel. Cinnamoyl CoA reductase (CCR; EC 1.21.1.44) and cinnamyl alcohol dehydrogenase (CAD; EC 1.1.1.95) are the key enzymes that catalyze the final two reductive steps in the biosynthesis of monolignols. In this study, we conducted a comprehensive functional analysis to validate the predominant CCR genes involved in monolignol biosynthesis. In this study, we initially validated the predominant CCR genes implicated in monolignol biosynthesis through an extensive functional analysis. Phylogenetic analysis, tissue-specific expression profiling and enzymatic assays indicated that MaCCR1 is the authentic CCR involved in lignin biosynthesis. Furthermore, the expression level of MaCCR1 exhibited a significant positive correlation with lignin content, and the down-regulation of MaCCR1 via virus-induced gene silencing resulted in altered lignin content in mulberry. The down-regulation of MaCCR1 and MaCAD3/4, both individually and concurrently, exhibited markedly different effects on lignin content and mulberry growth. Specifically, the simultaneous down-regulation of MaCCR1 and MaCAD3/4 significantly altered lignin content in mulberry, resulting in dwarfism of the plants. Conversely, the down-regulation of MaCAD3/4 alone not only decreased lignin content but also led to an increase in biomass. These findings offer compelling evidence elucidating the roles of MaCCRs in mulberry and identify specific target genes, thereby providing a crucial foundation for the genetic modification of lignin biosynthesis.