Characterization of two reductases MaLAR and MaANR revealed their roles in proanthocyanidin biosynthesis in mulberry

Proanthocyanidins (PAs), polymers of flavan-3-ols, are crucial for the sensory quality and stress defense of mulberry (<i>Morus</i> spp.). The biosynthesis of their monomers, catechin and epicatechin, are catalyzed by leucoanthocyanidin reductase (<i>LAR</i>) and anthocyanidin reductase (<i>ANR</i>), respectively, representing key rate-limiting steps that determine PA composition and abundance. In this study, systematic functional analyses including phylogenetic analysis, quantified spatio-temporal expression profiles during fruit development (S1-S4 stages), <i>in vitro</i> enzymatic assay, knock-down using Virus-Induced Gene Silencing (VIGS) in mulberry leaves and heterologous overexpression in <i>Arabidopsis thaliana</i> were conducted to reveal their roles in proanthocyanidin biosynthesis in mulberry. Results showed that <i>MaLAR</i> (969 bp) and <i>MaANR</i> (1014 bp) were successfully cloned and phylogenetically conserved. Spatio-temporal expression analysis revealed distinct patterns: <i>MaLAR</i> expression continuously increased, reaching highest expression level at the fully ripe stage (S4), whereas <i>MaANR</i> showed highest expression level at the color-turning stage (S2). <i>In vitro</i> enzymatic assays confirmed that <i>MaLAR</i> catalyzed the formation of catechin from leucoanthocyanidin, and <i>MaANR</i> catalyzed the formation of epicatechin from anthocyanidin. VIGS-mediated silencing of either gene in mulberry leaves led to significant reduction in total PA content. Conversely, heterologous overexpression of <i>MaLAR</i> or <i>MaANR</i> in <i>Arabidopsis</i> resulted in significant increase of PA in the seed coat. Our findings confirm that <i>MaLAR</i> and <i>MaANR</i> are conserved, key positive regulators of PA biosynthesis in mulberry. The differential expression patterns during fruit ripening suggest they play distinct, temporally regulated roles in determining the final PA composition and content. These results provide an important theoretical basis and represent important targets for the metabolic engineering and molecular breeding of mulberry for improved fruit quality.