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

Authors: JIN, QIFANG - Hunan Agricultural University; ZHONG, WANG - Hunan Agricultural University; Sandhu, Devinder; CHEN, LAN - Hunan Agricultural University; SHAO, CHENYU - Hunan Agricultural University; SHANG, FANGHUIZI - Hunan Agricultural University; XIE, SIYI - Hunan Agricultural University; HUANG, FEIYI - Hunan Academy Of Agricultural Sciences; CHEN, ZHENYAN - Hunan Agricultural University; ZHANG, XIANGQIN - Hunan Agricultural University; HU, JINYU - Hunan Agricultural University; LIU, GUIZHI - Hunan Agricultural University; SU, QIN - Hunan Agricultural University; HUANG, MENGDI - Hunan Agricultural University; LIU, ZHONGHUA - Hunan Agricultural University; HUANG, JIANAN - Hunan Agricultural University; TIAN, NA - Hunan Agricultural University; LIU, SHUOQIAN - Hunan Agricultural University

Submitted to: Horticulture Research
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 12/17/2023
Publication Date: 2/20/2024
Citation: Jin, Q., Zhong, W., Sandhu, D., Chen, L., Shao, C., Shang, F., Xie, S., Huang, F., Chen, Z., Zhang, X., Hu, J., Liu, G., Su, Q., Huang, M., Liu, Z., Huang, J., Tian, N., Liu, S. 2024. mRNA-miRNA analyses reveal the involvement of CsbHLH1 and miR1446a in the regulation of caffeine biosynthesis in Camellia sinensis. Horticulture Research. 11 (2):uhad282. https://doi.org/10.1093/hr/uhad282.
DOI: https://doi.org/10.1093/hr/uhad282

Interpretive Summary: Our research investigates the interesting process of how caffeine, which is a key ingredient contributing to the flavor of tea, is produced within tea plants. We used a number of scientific techniques and looked at a range of tea plant varieties to unravel this mystery. We found that one particular variety, called Jianghua kucha, contained more caffeine than the others. We discovered that an important gene, named CsbHLH1, could be like a control center for producing caffeine, and the protein made from this was found in the nucleus of the cell. Our experiments indicated that the CsbHLH1 protein could bind to another gene, called TCS1, to slow down its function. This, in turn, reduces the amount of caffeine that the plant makes. Adding another layer to this complex process, we found a small molecule called miR1446a that can cut the CsbHLH1 gene at a specific location, making it less effective and leading to more caffeine being produced. So, it appears that the interplay between the CsbHLH1 gene and miR1446a controls how much caffeine a tea plant produces. Our research may help to fine-tune the caffeine levels in tea plants, potentially leading to teas with different caffeine content to suit everyone's preference. The information generated in this study will be beneficial to geneticists and molecular biologists in fine-tuning the caffeine levels in tea plants, potentially leading to teas with different caffeine content to suit everyone's preference.

Technical 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.