MRNA-miRNA analyses reveal the involvement of CsbHLH1 and miR1446 in the regulation of caffeine biosynthesis in Camellia sinensis

Abstract

Abstract 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.