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

Mulberry (<i>Morus alba</i> 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 <i>CCR</i> genes involved in monolignol biosynthesis. In this study, we initially validated the predominant <i>CCR</i> genes implicated in monolignol biosynthesis through an extensive functional analysis. Phylogenetic analysis, tissue-specific expression profiling and enzymatic assays indicated that <i>MaCCR1</i> is the authentic CCR involved in lignin biosynthesis. Furthermore, the expression level of <i>MaCCR1</i> exhibited a significant positive correlation with lignin content, and the down-regulation of <i>MaCCR1</i> via virus-induced gene silencing resulted in altered lignin content in mulberry. The down-regulation of <i>MaCCR1</i> and <i>MaCAD3/4,</i> both individually and concurrently, exhibited markedly different effects on lignin content and mulberry growth. Specifically, the simultaneous down-regulation of <i>MaCCR1</i> and <i>MaCAD3/4</i> significantly altered lignin content in mulberry, resulting in dwarfism of the plants. Conversely, the down-regulation of <i>MaCAD3/4</i> alone not only decreased lignin content but also led to an increase in biomass. These findings offer compelling evidence elucidating the roles of <i>MaCCRs</i> in mulberry and identify specific target genes, thereby providing a crucial foundation for the genetic modification of lignin biosynthesis.