Dihydrochalcones (DHCs) are highly accumulated in tender leaves of Lithocarpus litseifolius but their biosynthetic pathway and accumulation mechanism remain unclear. In this study, candidate genes including one cinnamoyl-CoA reductase (LlCCR), two double bond reductases (LlDBR1 ~ 2), three aldehyde hydrogenases (LlALDH1 ~ 3), two 4-coumaroyl:CoA ligases (Ll4CL1 ~ 2) and four phloretin glycosyltransferases (LlP4′GT, LlP2′GT1 ~ 3) were comprehensively investigated. The substrate specificities and catalytic kinetics of these gene-encoded enzymes were achieved. Through successive catalysis of LlALDH1, Ll4CL2, and chalcone synthase 1 (LlCHS1) or combined action of LlCCR and LlCHS1, phloretin was biosynthesized from direct precursor dihydro-p-coumaraldehyde, which had been converted from initial precursor p-coumaroyl-CoA by LlCCR-mediated carboxylic acid reduction and LlDBR1-catalyzed α,β-double bond saturation. High accumulation of the DHCs in tender leaves of L. litseifolius was mainly driven by efficient catalysis of LlCCR toward p-coumaroyl-CoA and highly expressed genes in the pathway, especially the LlP4′GT and LlP2′GT1 which contributed to biosynthesis of trilobatin and phlorizin, respectively. Antisense oligodeoxyribonucleotide treatments against the LlCCR, LlDBR1, LlALDH1, Ll4CL2, LlP4′GT, and LlP2′GT1 significantly reduced transcripts of the target genes and content of DHCs, confirming these genes might be involved in the pathway. This finding provides insight into the biosynthesis and accumulation mechanism of DHCs in planta.

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