Dihydrochalcones (DHCs) constitute a specific class of flavonoids widely known for their various health-related advantages. Melatonin (MLT) has received attention worldwide as a master regulator in plants, but its roles in DHC accumulation remain unclear. Herein, the elicitation impacts of MLT on DHC biosynthesis were examined in Lithocarpus litseifolius, a valuable medicinal plant famous for its sweet flavor and anti-diabetes effect. Compared to the control, the foliar application of MLT significantly increased total flavonoid and DHC (phlorizin, trilobatin, and phloretin) levels in L. litseifolius leaves, especially when 100 μM MLT was utilized for 14 days. Moreover, antioxidant enzyme activities were boosted after MLT treatments, resulting in a decrease in the levels of intracellular reactive oxygen species. Remarkably, MLT triggered the biosynthesis of numerous phytohormones linked to secondary metabolism (salicylic acid, methyl jasmonic acid (MeJA), and ethylene), while reducing free JA contents in L. litseifolius. Additionally, the flavonoid biosynthetic enzyme activities were enhanced by the MLT in leaves. Multiple differentially expressed genes (DEGs) in RNA-seq might play a crucial role in MLT-elicited pathways, particularly those associated with the antioxidant system (SOD, CAT, and POD), transcription factor regulation (MYBs and bHLHs), and DHC metabolism (4CL, C4H, UGT71K1, and UGT88A1). As a result, MLT enhanced DHC accumulation in L. litseifolius leaves, primarily by modulating the antioxidant activity and co-regulating the physiological, hormonal, and transcriptional pathways of DHC metabolism.

Lithocarpus litseifolius is a medicinal tea plant recognized for its sweet flavor and anti-diabetic properties, but its limited leaf yield under cultivation restricts its economic sustainability. Melatonin (MLT) is a multifunctional plant growth regulator, but its roles in leaf growth under normal conditions remain not fully understood. Herein, we investigated the effects and mechanisms of foliar-applied MLT on L. litseifolius seedlings, including growth indices, phytohormone profiles, photosynthetic characteristics, and transcriptome alterations. The 100 μM MLT treatment significantly enhanced leaf dry weight by 33.8% and leaf dry matter content by 22.2% compared to the control group. MLT decreased both free and bound abscisic acid (ABA), while increasing gibberellins (GAs), 5-deoxystrigol, auxins (e.g., IAM), and cytokinins (e.g., cZ9G). Additionally, exogenous MLT improved photosynthetic rate, stomatal conductance, chlorophyll content, and soluble sugars in leaves. RNA-seq revealed that MLT up-regulated DEGs involved in hormone biosynthesis and signaling (CYP707A, BAK1, D14, CCD1, and IAA6), photosynthesis (PsbC/B, PetH, PsaB, and ATPase β), and sugar metabolism (WAXY, glgC, and otsB). Our results demonstrate that MLT promotes leaf dry matter accumulation through coordinated phytohormone homeostasis, photosynthetic enhancement, and transcriptional regulation, offering a cost-effective strategy to improve leaf yield in L. litseifolius.