Abstract
Key message
Three novel transcription factors were successfully identified and shown to interact with the trichome-specific THCAS promoter regulatory region.
Abstract
Cannabinoids are important secondary metabolites present in Cannabis sativa L. (cannabis). One cannabinoid that has received considerable attention, 9-tetrahydrocannabinol (THC), is derived from Delta-9-Tetrahydrocannabinolic acid (THCA) and responsible for the mood-altering and pain-relieving effects of cannabis. A detailed understanding of transcriptional control of THCA synthase (THCAS) is currently lacking. The primary site of cannabinoid biosynthesis is the glandular trichomes that form on female flowers. Transcription factors (TFs) have been shown to play an important role in secondary-metabolite biosynthesis and glandular trichome formation in Artemisia annua, Solanum lycopersicum and Humulus lupulus. However, analogous information is not available for cannabis. Here, we characterize a 548 bp fragment of the THCAS promoter and regulatory region that drives trichome-specific expression. Using this promoter fragment in a yeast-one-hybrid screen, we identified 3 novel TFs (CsAP2L1, CsWRKY1 and CsMYB1) and provided evidence that these 3 TFs regulate the THCAS promoter in planta. The O-Box element within the proximal region of the THCAS promoter is necessary for CsAP2L1-induced transcriptional activation of THCAS promoter. Similar to THCAS, the genes for all three TFs have trichome-specific expression, and subcellular localization of the TFs indicates that all three proteins are in the nucleus. CsAP2L1 and THCAS exhibit a similar temporal, spatial and strain-specific gene expression profiles, while those expression patterns of CsWRKY1 and CsMYB1 are opposite from THCAS. Our results identify CsAP2L1 playing a positive role in the regulation of THCAS expression, while CsWRKY1 and CsMYB1 may serve as negative regulators of THCAS expression.
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Acknowledgements
This work was supported by a Mitacs Elevate Post-doctoral Fellowship, in partnership with Anandia Laboratories, to YL. This work was also supported by the State Key Laboratory of Ecological Pest Control for Fujian and Taiwan Crops (SKL2018004), the General Program of the Natural Science Foundation of Fujian Province (2019J01419), the Project of Forestry Peak Discipline of Fujian Agriculture and Forestry University (118/71201800745), the Fujian Agriculture and Forestry University Science Fund for Distinguished Young Scholars (XJQ201902).The authors thank John Coleman for UPLC, and Anandia employees involved in plant cultivation and maintenance throughout the duration of the project. We also thank Dr. Taku Demura (Nara Institute of Science and Technology, Japan) and Dr. Pu Yan (Institute of Tropical Bioscience and Biotechnology, Chinese Academy of Tropical Agricultural Sciences, China) for providing plasmids and technical advice for the dual luciferase transient transfection assay.
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YL, GH and JEP conceived this research. YL, PZ and SC designed and performed experiments, YL, PZ, SC, and GH analyzed data. YL, GH and JEP prepared the article.
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JEP is the Chief Science Officer of Aurora Cannabis Inc., a for-profit cannabis license producer and biotechnology company. The nucleotide sequences and protein sequences encoding the three transcription factors have been filed for the patents 201911286950, 201911289699 and 201911286948.
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Sequence data from this article can be found in the GenBank/EMBL databases under the following accession numbers: CsAP2L1, XM_030640449; CsWRKY1, XM_030654298; CsMYB1, XM_030639466.
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Liu, Y., Zhu, P., Cai, S. et al. Three novel transcription factors involved in cannabinoid biosynthesis in Cannabis sativa L.. Plant Mol Biol 106, 49–65 (2021). https://doi.org/10.1007/s11103-021-01129-9
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DOI: https://doi.org/10.1007/s11103-021-01129-9