Flavan-3-ols and oligomeric proanthocyanidins (PAs) are the main nutritional polyphenols in green tea (Camellia sinensis), which provide numerous benefits to human health. To date, the regulatory mechanism of flavan-3-ol biosynthesis in green tea remains open to study. Herein, we report the characterization of a C. sinensis tryptophan-aspartic acid repeat protein (CsWD40) that interacts with myeloblastosis (MYB) and basic helix-loop-helix (bHLH) transcription factors (TFs) to regulate the biosynthesis of flavan-3-ols. Full length CsWD40 cDNA was cloned from leaves and was deduced to encode 342 amino acids. An in vitro yeast two-hybrid assay demonstrated that CsWD40 interacted with two bHLH TFs (CsGL3 and CsTT8) and two MYB TFs (CsAN2 and CsMYB5e). The overexpression of CsWD40 in Arabidopsis thaliana transparent testa glabra 1 (ttg1) restored normal trichome and seed coat development. Ectopic expression of CsWD40 alone in tobacco resulted in a significant increase in the anthocyanins of transgenic petals. CsWD40 was then coexpressed with CsMYB5e in tobacco plants to increase levels of both anthocyanins and PAs. Furthermore, gene expression analysis revealed that CsWD40 expression in tea plants could be induced by several abiotic stresses. Taken together, these data provide solid evidence that CsWD40 partners with bHLH and MYB TFs to form ternary WBM complexes to regulate anthocyanin, PA biosynthesis, and trichome development.

The tea plant (Camellia sinensis) is rich in polyphenolic compounds. Particularly, flavan-3-ols and proanthocyanidins (PAs) are essential for the flavor and disease-resistance property of tea leaves. The fifth subgroup of R2R3-MYB transcription factors comprises the primary activators of PA biosynthesis. This study showed that subgroup 5 R2R3-MYBs in tea plants contained at least nine genes belonging to the TT2, MYB5, and MYBPA types. Tannin-rich plants showed an expansion in the number of subgroup 5 R2R3-MYB genes compared with other dicotyledonous and monocot plants. The MYBPA-type genes of tea plant were slightly expanded. qRT–PCR analysis and GUS staining analysis of promoter activity under a series of treatments revealed the differential responses of CsMYB5s to biotic and abiotic stresses. In particular, CsMYB5a, CsMYB5b, and CsMYB5e responded to high-intensity light, high temperature, MeJA, and mechanical wounding, whereas CsMYB5f and CsMYB5g were only induced by wounding. Three genetic transformation systems (C. sinensis, Nicotiana tabacum, and Arabidopsis thaliana) were used to verify the biological function of CsMYB5s. The results show that CsMYB5a, CsMYB5b, and CsMYB5e could promote the gene expression of CsLAR and CsANR. However, CsMYB5f and CsMYB5g could only upregulate the gene expression of CsLAR but not CsANR. A series of site-directed mutation and domain-swapping experiments were used to verify functional domains and key amino acids of CsMYB5s responsible for the regulation of PA biosynthesis. This study aimed to provide insight into the induced expression and functional diversity model of PA biosynthesis regulation in tea plants.

Strawberry fruits, known for their excellent taste and potential health benefits, are particularly valued for their rich content of hydrolyzable tannins (HTs). These compounds play key roles in regulating growth and development. However, the molecular mechanisms underlying HT synthesis in plants remains poorly elucidated. In this study, based on a correlation analysis between the transcriptome and metabolome of HTs, galloyl glucosyltransferase (UGT84A22), serine carboxypeptidase-like acyltransferases (SCPL-ATs), and carboxylesterases (CXEs) were screened. Furthermore, in vitro enzymatic assays confirmed that FaSCPL3-1 acted as a hydrolyzable tannins synthase (HTS), catalyzing the continuous galloylation of glucose to form simple gallotannins (GTs). Additionally, FaCXE1/FaCXE3/FaCXE7 catalyzed the degalloylation of simple GTs and ellagitannins (ETs), and FaUGT84A22 catalyzed the glycosylation of gallic acid (GA) to produce 1-O-β-glucogallin (βG), a galloyl donor. Moreover, in FvSCPL3-1-RNAi transgenic strawberry plants, the contents of simple GT and some ET compounds were reduced, whereas, in FaCXE7 overexpressing strawberry plants, these compounds were increased. These enzymes constituted a biosynthetic pathway of galloyl derivatives, termed the “galloylation-degalloylation cycle” (G-DG cycle). Notably, the overexpression of FaCXE7 in strawberry plants not only promoted HT synthesis but also interfered with plant growth and development by reducing lignin biosynthesis. These findings offer new insights into the mechanisms of HT accumulation in plants, contributing to improving the quality of berry fruits quality and enhancing plant resistance.

Long non-coding RNAs (lncRNAs) have gathered significant attention due to their pivotal role in plant growth, development, and biotic and abiotic stress resistance. Despite this, there remains little understanding regarding the functions of lncRNA in these domains in the tea plant (Camellia sinensis, C. sinensis), mainly attributable to the insufficiencies in gene manipulation techniques for tea plants. In this study, we designed a novel strategy to identify evolutionarily conserved trans-lncRNA (ECT-lncRNA) pairs in plants. We used highly consistent base sequences in the exon overlapping region between trans-lncRNAs and their target gene transcripts. Based on this method, we successfully screened 24 ECT-lncRNA pairs from at least two or more plant species. In tea, as observed in model plants such as Arabidopsis, alfalfa, potatoes, and rice, there exists a trans-lncRNA capable of forming an ECT-lncRNA pair with transcripts of 12-oxophytodienoate reductase (OPR) family, denoted as the OPRL/OPR pair. Considering evolutionary perspectives, the OPRL gene cluster in each species likely originates from a replication event of the OPR gene cluster. Gene manipulation and gene expression analysis revealed that CsOPRL influences disease resistance by regulating CsOPRs expression in tea plants. Furthermore, the knockout of StOPRL1 in Solanum tuberosum led to aberrant growth characteristics and strong resistance to fungal infection. This study provides insights into a strategy for the screening and functional verification of ECT-lncRNA pairs.

Tea plants are rich in phenolic compounds, the content of which varies with plant organs. However, the accumulation pattern of flavonoids during the development of tea seedlings has yet to be systematically clarified. This study explored the accumulation of flavonoids of Mengku Dayecha' (MKDY) and 'Huangkui' (HK) seedlings during multiple developmental stages. The results demonstrate that the amounts of accumulated flavonoids, including flavan-3-ols, proanthocyanidins, and flavonols, have mild differences among stages of the same organ. However, there are significant differences among organs. The results of TOF-MS and UPLC-QQQ-MS/MS indicate that the types of flavonoids accumulated in the aboveground part and the underground part are distinct. The aboveground part mainly accumulates B-ring di/tri-hydroxyl catechins, gallate catechins monomers, and polymers, while the underground part amasses B-ring di-hydroxyl catechins and special flavonol glycosides. The qRT-PCR analysis exhibits that CsF3'5'Ha, CsSCPL4, and CsSCPL5, which control B-ring tri-hydroxyl and gallate catechins biosynthesis, respectively, are highly expressed in the aboveground part, especially the shoots, but barely in the underground part. In contrast, CsANRa is expressed in all organs, particularly the root tips. This study identified the accumulation patterns of flavonoids in the seedling stage of tea plants, with the prospect of providing a theoretical reference for further biological research of tea plants.