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.

Calcium-dependent protein kinases (CDPKs) are important Ca²⁺ sensors that play crucial roles in plant responses to environmental stresses. However, the functions of CDPK genes in the tea plant (Camellia sinensis), especially in disease resistance, remain poorly understood. In this study, 32 CsCDPK genes were identified from the tea plant genome and classified into four phylogenetic groups. These genes were unevenly distributed across 14 chromosomes. Analyses of gene structure, conserved motifs, physicochemical properties, promoter cis-elements, and syntenic relationships indicated that the CsCDPK family is highly conserved but also shows some evolutionary divergence. Expression analyses based on transcriptomic data and RT-qPCR revealed that several CsCDPK genes were strongly induced by infection with the gray blight pathogen Pseudopestalotiopsis spp. and by exogenous salicylic acid (SA) and methyl jasmonate (MeJA) treatments. Endogenous SA, jasmonic acid (JA), and MeJA levels also increased rapidly after pathogen infection. Six genes, CsCDPK3, CsCDPK8, CsCDPK11, CsCDPK12, CsCDPK21, and CsCDPK26, were further functionally analyzed using antisense oligodeoxynucleotide-mediated silencing. Silencing of these genes significantly increased lesion size after pathogen inoculation, indicating reduced resistance. These findings suggest that the selected CsCDPK genes are likely involved in tea plant defense against gray blight disease, providing candidate genes for further functional characterization and potential resistance improvement.