miR828a-CsMYB114 Module Negatively Regulates the Biosynthesis of Theobromine 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; XIE, SIYI - Hunan Agricultural University; SHANG, FANGHUIZI - Hunan Agricultural University; WU, TING - Hunan Agricultural University; JIN, HUIYING - Hunan Agricultural University; HUANG, FEIYI - Hunan Academy Of Agricultural Sciences; LIU, GUIZHI - Hunan Agricultural University; HU, JINYU - Hunan Agricultural University; SU, QIN - Hunan Agricultural University; HUANG, MENGDI - Hunan Agricultural University; ZHU, QIAN - Hunan Agricultural University; ZHOU, BIAO - Hunan Agricultural University; ZHU, LIHUA - Hunan Agricultural University; PENG, LVWEN - 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: Journal of Agricultural and Food Chemistry
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 2/2/2024
Publication Date: 2/20/2024
Citation: Jin, Q., Zhong, W., Sandhu, D., Chen, L., Shao, C., Xie, S., Shang, F., Wu, T., Jin, H., Huang, F., Liu, G., Hu, J., Su, Q., Huang, M., Zhu, Q., Zhou, B., Zhu, L., Peng, L., Liu, Z., Huang, J., Tian, N., Liu, S. 2024. miR828a-CsMYB114 Module Negatively Regulates the Biosynthesis of Theobromine in Camellia sinensis. Journal of Agricultural and Food Chemistry. 72 (8): 4464-4475. https://doi.org/10.1021/acs.jafc.3c07736.
DOI: https://doi.org/10.1021/acs.jafc.3c07736

Interpretive Summary: Theobromine is a compound found in tea that's closely related to caffeine, and it's made by a specific enzyme known as theobromine synthase. In our study, we wanted to understand how theobromine is produced in tea plants and what factors influence its levels. We discovered that a gene called CsMYB114 plays a critical role in regulating the production of theobromine. Our results showed that when CsMYB114 is active, it binds to the DNA of the tea plant and increases the activity of the theobromine synthase enzyme, leading to more theobromine production. Interestingly, we also found that a microRNA named miR828a can "silence" CsMYB114, leading to reduced theobromine production. A microRNA is a tiny piece of genetic material that can bind to specific genes and reduce their activity. We showed that when miR828a was active, it directly targeted CsMYB114, resulting in less theobromine. Finally, to confirm our findings, we used a technique called antisense oligonucleotide (AsODN) to suppress the activity of CsMYB114. Antisense oligonucleotides are short DNA strands that can bind to specific genes and block their activity. When we used AsODN to silence CsMYB114, we saw a significant reduction in theobromine production, further proving that CsMYB114 is indeed a key player in theobromine biosynthesis. This understanding could potentially be used by tea geneticists and breeders in the future to create tea plants with customized levels of theobromine to meet the preferences and needs of tea drinkers around the world.

Technical Abstract: Theobromine is an important quality component in tea plants (Camellia sinensis), which is produced from 7- methylxanthine by theobromine synthase (CsTbS), the key rate-limiting enzyme in theobromine biosynthetic pathway. Our transcriptomics and widely targeted metabolomics analyses suggested that CsMYB114 acted as a potential hub gene involved in the regulation of theobromine biosynthesis. The inhibition of CsMYB114 expression using antisense oligonucleotides (ASO) led to a 70.21% reduction of theobromine level in leaves of the tea plant, which verified the involvement of CsMYB114 in theobromine biosynthesis. Furthermore, we found that CsMYB114 was located in the nucleus of the cells and showed the characteristic of a transcription factor. The dual luciferase analysis, a yeast one-hybrid assay, and an electrophoretic mobility shift assay (EMSA) showed that CsMYB114 activated the transcription of CsTbS, through binding to CsTbS promoter. In addition, a microRNA, miR828a, was identified that directly cleaved the mRNA of CsMYB114. Therefore, we conclude that CsMYB114, as a transcription factor of CsTbS, promotes the production of theobromine, which is inhibited by miR828a through cleaving the mRNA of CsMYB114.