mRNA-miRNA analyses reveal the involvement of CsbHLH1 and miR1446a in the regulation of caffeine biosynthesis in Camellia sinensis

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, 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 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 by binding to the TCS1 promoter, as evidenced by a yeast one-hybrid assay, an electrophoretic mobility shift assay, and dual luciferase analysis. In addition, a microRNA, miR1446a, was identified that directly cleaves CsbHLH1, leading to an increase in caffeine levels. Therefore, our findings imply that CsbHLH1 binds to the TCS1 promoter ( − 971 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. This work enhances our understanding of the molecular mechanisms of caffeine biosynthesis in tea plants and offers potential directions for manipulating caffeine levels in future tea cultivation.

Acknowledgements

This work was supported by the Natural Science Foundation of Hunan Province (2021JC0007), the National Natural Science Foundation of China (U22A20500, U19A2030, 32172629), and the key Science and Technology Research Projects of Hunan Province (2021NK0008, 2021NK1020).

Author contributions

Q.J. designed and performed the experiments and drafted the paper. Z.W. analyzed the data and prepared the figures and tables. DS performed data analysis and reviewed and revised the manuscript. L.C., C.S., F.S., S.X., F.H., Z.C., X.Z., J.H., G.L., Q.S., and M.H. participated in the experiments and data analysis. Z.L., J.H., N.T., and S.L. conceived the study, participated in the coordination, data analysis, and interpretation, and drafted and reviewed the manuscript. All authors read and approved the final manuscript.

Data availability statement

All the data supporting our findings are contained within the manuscript. All raw transcriptome data reported in this article have been deposited in the Sequence Read Archive (SRA) under accession number PRJNA986690 and PRJNA1056246.

Conflict of interests

The authors declare they have no competing interests.

Supplementary information

Supplementary data is available at Horticulture Research online.

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