For decades, scientists have known that DHCs—particularly phlorizin and trilobatin—accumulate to remarkably high levels in sweet tea leaves, sometimes exceeding 30% of dry weight. These compounds have attracted growing interest as natural sugar substitutes that support healthy, low-sugar diets. However, the biosynthetic pathway remained unclear, with conflicting reports about key steps, especially how the critical double bond gets reduced. Compounding the confusion, some enzymes proposed to be involved in apples—the other major DHC-producing plant—showed inconsistent activity in laboratory tests. Based on these challenges, a systematic investigation of the entire pathway in L. litseifolius was urgently needed.
A team led by Jian-Liang Lu at Zhejiang University's Tea Research Institute, in collaboration with Shaoxing Jianming Tea Industry Co., Ltd, reports (DOI: 10.1093/hr/uhag061) these findings on July 6, 2026, in Horticulture Research. By cloning and characterizing more than 20 candidate genes from sweet tea, the researchers identified the complete set of enzymes responsible for DHC biosynthesis and uncovered two parallel routes for producing the core molecule phloretin—a discovery that rewrites the textbook understanding of how these valuable compounds are made.
The research team systematically screened the sweet tea transcriptome and cloned 24 candidate genes, including cinnamoyl-CoA reductases (CCRs), double bond reductases (DBRs), aldehyde dehydrogenases (ALDHs), 4-coumaroyl-CoA ligases (4CLs), and phloretin glycosyltransferases (PGTs). Through in vitro enzyme assays, they demonstrated that LlCCR—an enzyme traditionally known for its role in lignin biosynthesis—is remarkably multifunctional. It not only reduces *p*-coumaroyl-CoA to *p*-coumaraldehyde but also catalyzes the reverse oxidation of dihydro-*p*-coumaraldehyde to dihydro-*p*-coumaroyl-CoA, effectively serving as a metabolic switch between lignin and DHC pathways. Critically, the team discovered that the double bond reduction occurs not on the CoA-activated form (as previously assumed) but on the aldehyde intermediate, catalyzed by LlDBR1. This finding resolves years of conflicting data about DBR activity. Using four in vitro reaction systems, the researchers showed that phloretin can be generated through two routes from dihydro-*p*-coumaraldehyde: either via sequential catalysis by LlALDH1, Ll4CL2, and chalcone synthase (CHS), or directly through LlCCR and CHS. Gene silencing experiments using antisense oligodeoxyribonucleotides (asODNs) confirmed that suppressing any of these key genes significantly reduced DHC content, validating their in vivo roles.
“We were surprised to find that the traditional lignin enzyme LlCCR plays a dual role in sweet tea—it not only participates in lignin formation but also actively channels metabolites into the DHC pathway,” the authors said. “What's even more exciting is that the double bond reduction happens at the aldehyde stage, not the CoA-ester stage as previously believed. This fundamentally changes how we think about DHC biosynthesis.” They added that the exceptionally high DHC accumulation in tender leaves is driven by the combination of LlCCR's efficient catalysis and the strong expression of downstream glycosyltransferases, particularly LlP4'GT and LlP2'GT1, which respectively produce trilobatin and phlorizin.
The findings have immediate practical implications for the food industry and natural sweetener production. With global demand for low-calorie, plant-derived sweeteners rising sharply, understanding the complete DHC biosynthetic pathway provides a blueprint for metabolic engineering. The two parallel routes to phloretin offer flexibility for optimizing production in microbial systems, potentially enabling more efficient and cost-effective manufacturing of these healthy sweeteners. Moreover, the discovery that LlCCR acts as a metabolic branch-point enzyme—competing with LlHCT (hydroxycinnamoyl-CoA:shikimate/quinate hydroxycinnamoyl transferase) for the same substrate—sheds light on how plants balance lignin production (for structural support) against DHC accumulation (for defense or other functions). This knowledge could guide breeding strategies to enhance DHC content in sweet tea and other crops, delivering healthier, naturally sweet products to consumers.
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References
DOI
10.1093/hr/uhag061
Original Source URL
https://doi.org/10.1093/hr/uhag061
Funding information
This work was supported by the National Natural Science Foundation of China (grant 32272763), the China Agriculture Research System of MOF and MARA, and the Zhejiang Science and Technology Major Program on Agricultural New Variety Breeding – Tea Plant (grant 2021C02067-6).
About Horticulture Research
Horticulture Research is an open access journal of Nanjing Agricultural University and ranked number one in the Horticulture category of the Journal Citation Reports ™ from Clarivate, 2023. The journal is committed to publishing original research articles, reviews, perspectives, comments, correspondence articles and letters to the editor related to all major horticultural plants and disciplines, including biotechnology, breeding, cellular and molecular biology, evolution, genetics, inter-species interactions, physiology, and the origination and domestication of crops.