Caffeine, a primary flavor component in tea, has been the subject of intense research. With the goal of shedding light on the complex regulatory processes governing caffeine biosynthesis in tea plants, the liquid chromatography coupled with mass spectrometry (LC-MS), transcriptomics, and small RNA analyses were employed on diverse tea cultivars such as ‘Jianghua Kucha’ (including ‘Xianghong 3’ (XH3H) and ‘Kucha 3’ (KC3H)), ‘Fuding Dabaicha’ (FDDB), ‘Yaoshan Xiulv’ (YSXL), and ‘Bixiangzao’ (BXZ). The results showed that the caffeine level in ‘Jianghua Kucha’ was significantly higher than that in other tea plant cultivars. In addition, weighted gene co-expression network analysis (WGCNA) indicated that that the CsbHLH1 gene might play a pivotal role as a potential hub gene related to the regulation of caffeine biosynthesis. Subcellular localization analysis showed that the CsbHLH1 protein was localized in the nucleus of the cells. Moreover, CsbHLH1 suppresses the transcription of TCS1 through binding to the TCS1 promoter, as evidenced by a yeast one-hybrid assay, and an electrophoretic mobility shift assay (EMSA) assay and dual luciferase analysis. In addition, a microRNA, miR1446a, was identified that directly cleaves the downstream transcription factor CsbHLH1 at position 88 bp, leading to an increase in caffeine levels. Therefore, our findings imply that CsbHLH1 binds to the TCS1 promoter (-971 bp to -1019 bp) to reduce its expression, thereby negatively regulating caffeine biosynthesis. On the other hand, miR1446a enhances the biosynthesis of caffeine by suppressing the expression of CsbHLH1. These works enhanced our understanding of the molecular mechanisms of caffeine biosynthesis in tea plants and offered potential directions for manipulating caffeine levels in future tea cultivation.

Drought stress significantly alters the metabolic homeostasis of tea plants; however, few studies have examined the role of specific metabolites, particularly tea polyphenols, in drought resistance. This study reveals that the tea polyphenol content in drought-tolerant tea cultivars tends to increase under drought conditions. Notably, in environments characterized by staged and repeated drought, changes in tea polyphenol are significantly positively correlated with drought resistance. To investigate this further, we irrigated the roots with exogenous tea polyphenols before subjecting the plants to drought. Our findings indicated that the absorptive roots of the experimental group exhibited enhanced development, improved cellular integrity, and a significant increase in peroxidase activity. A comprehensive analysis of the transcriptome and metabolome revealed that tea polyphenols are closely associated with the phenylpropanoid metabolism pathway. Notably, CsMYB77 and CsPOD44 genes were identified as highly correlated with this pathway. Overexpression experiments in Arabidopsis thaliana demonstrated that CsMYB77 promotes the expression of phenylpropanoid pathway genes, thereby enhancing drought resistance. Conversely, antisense oligonucleotide silencing of CsMYB77 decreased drought resistance in tea plants. Additional experiments, including yeast one-hybrid assays, luciferase complementation imaging, dual-luciferase assays, and electrophoretic mobility shift assays, confirmed that CsMYB77 positively regulates the expression of CsPOD44. In summary, our findings indicate that the differences in drought tolerance among tea cultivars are closely linked to phenylpropanoid metabolism. Specifically, tea polyphenols may mediate the regulatory network involving CsMYB77 and CsPOD44, thereby enhancing stress resistance by promoting root development. This study offers new insights into the breeding of drought-resistant tea cultivars.

Tea (Camellia sinensis L.), an important economic crop, is recalcitrant to Agrobacterium-mediated transformation (AMT), which has seriously hindered the progress of molecular research on this species. The mechanisms leading to low efficiency of AMT in tea plants, related to the morphology, growth, and gene expression of Agrobacterium tumefaciens during tea-leaf explant infection, were compared to AMT of Nicotiana benthamiana leaves in the present work. Scanning electron microscopy (SEM) images showed that tea leaves induced significant morphological aberrations on bacterial cells and affected pathogen–plant attachment, the initial step of a successful AMT. RNA sequencing and transcriptomic analysis on Agrobacterium at 0, 3 and 4 days after leaf post-inoculation resulted in 762, 1923 and 1656 differentially expressed genes (DEGs) between the tea group and the tobacco group, respectively. The expressions of genes involved in bacterial fundamental metabolic processes, ATP-binding cassette (ABC) transporters, two-component systems (TCSs), secretion systems, and quorum sensing (QS) systems were severely affected in response to the tea-leaf phylloplane. Collectively, these results suggest that compounds in tea leaves, especially gamma-aminobutyrate (GABA) and catechins, interfered with plant–pathogen attachment, essential minerals (iron and potassium) acquisition, and quorum quenching (QQ) induction, which may have been major contributing factors to hinder AMT efficiency of the tea plant.

Trichomes, which develop from epidermal cells, are considered one of the important characteristics of the tea plant [Camellia sinensis (L.) O. Kuntze]. Many nutritional and metabolomic studies have indicated the important contributions of trichomes to tea products quality. However, understanding the regulation of trichome formation at the molecular level remains elusive in tea plants. Herein, we present a genome-wide comparative transcriptome analysis between the hairless Chuyeqi (CYQ) with fewer trichomes and the hairy Budiaomao (BDM) with more trichomes tea plant genotypes, toward the identification of biological processes and functional gene activities that occur during trichome development. In the present study, trichomes in both cultivars CYQ and BDM were unicellular, unbranched, straight, and soft-structured. The density of trichomes was the highest in the bud and tender leaf periods. Further, using the high-throughput sequencing method, we identified 48,856 unigenes, of which 31,574 were differentially expressed. In an analysis of 208 differentially expressed genes (DEGs) encoding transcription factors (TFs), five may involve in trichome development. In addition, on the basis of the Gene Ontology (GO) annotation and the weighted gene co-expression network analysis (WGCNA) results, we screened several DEGs that may contribute to trichome growth, including 66 DEGs related to plant resistance genes (PRGs), 172 DEGs related to cell wall biosynthesis pathway, 29 DEGs related to cell cycle pathway, and 45 DEGs related to cytoskeleton biosynthesis. Collectively, this study provided high-quality RNA-seq information to improve our understanding of the molecular regulatory mechanism of trichome development and lay a foundation for additional trichome studies in tea plants.