Source: UNIV OF CONNECTICUT submitted to
DECIPHERING THE MOLECULAR REGULATORY MECHANISMS CONTROLLING TRICHOME DEVELOPMENT AND CANNABINOID BIOSYNTHESIS IN HEMP PLANTS
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
ACTIVE
Funding Source
AFRI COMPETITIVE GRANT
Reporting Frequency
Annual
Accession No.
1028172
Grant No.
2022-67014-37049
Cumulative Award Amt.
$600,000.00
Proposal No.
2021-07862
Multistate No.
(N/A)
Project Start Date
Apr 15, 2022
Project End Date
Jan 7, 2026
Grant Year
2022
Program Code
[A1103]- Foundational Knowledge of Plant Products
Recipient Organization
UNIV OF CONNECTICUT
438 WHITNEY RD EXTENSION UNIT 1133
STORRS,CT 06269
Performing Department
Plant Science & Landscape Arch
Non Technical Summary
Cannabinoids are a group of terpenophenolic compounds, such as cannabidiolic acid (CBD), cannbigerolic acid (CBG) et al, made primarily in cannabis female flower trichomes. Cannabinoids have significant pharmaceutical values and potential as dietary supplements. However, little is known about the regulation of cannabinoid biosynthesis. Cannabis sativa also provides an excellent model to study glandular trichome development and secondary metabolism. There is no published molecular biology study to date on cannabis trichome development, and no genes responsible for glandular trichome initiation identified in cannabis. There is also not much knowledge about the control of cannabinoid biosynthetic gene expression during flower development. We are interested in the molecular mechanisms mediating glandular trichome development and cannabinoid biosynthesis. We have recently identified transcription factors (TFs) that are responsible for glandular trichome initiation and cannabinoid biosynthesis respectively. The proposed research aims to understand hormone-mediated transcriptional control of glandular trichome development and cannabinoid biosynthesis in hemp. The regulatory network of TFs involved in trichome initiation, and the transcriptional regulation of cannabinoid biosynthetic genes by TFs will be determined. Trichome phenotypes of tobacco plants overexpressing the TFs, and hemp overexpressing of suppressing the TFs will be investigated; cannabinoid production in transgenic hemp will be determined. We envision that the fundamental knowledge obtained can be translated into novel genetic tools and strategies to improve cannabinoid profile, aid hemp farmers with the common problem of overproducing THC, and benefit human health. The findings can also be expanded to other species developing glandular trichomes for secondary metabolite production and storage.
Animal Health Component
(N/A)
Research Effort Categories
Basic
100%
Applied
(N/A)
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
2061730104050%
2061730103020%
2061730105030%
Knowledge Area
206 - Basic Plant Biology;

Subject Of Investigation
1730 - Hemp;

Field Of Science
1040 - Molecular biology; 1030 - Cellular biology; 1050 - Developmental biology;
Goals / Objectives
Cannabinoids are terpenophenolic compounds primarily produced in the glandular trichomes on unfertilized female flowers of Cannabis sativa L. (C. sativa, cannabis). There is no published report on how the development of glandular trichomes on cannabis female flowers are regulated;there is still a lack of knowledge at the molecular level on how the genes are transcriptionally regulated during female flower/glandular trichome development.Our long-term goal of this proposed research is to discover and delineate transcriptional regulatory networks that control glandular trichome initiation and development and cannabinoid biosynthesis in hemp for the improvement and desired modulation of cannabinoid contents; the knowledge generated from this study can be applied to research of other agriculturally important plant species that develop glandular trichomes for secondary metabolite manufacturing and storage. The proposed research is aimed to make achievements in the following objectives:Objective 1: Discover and characterize transcription factors that are critical for glandular trichome initiation and development.Objective 2: Discover and characterize transcription factors that control cannabinoid biosynthetic genes.Objective 3: Investigate hormone mediated transcriptional modulation of glandular trichome development and cannabinoid biosynthesis.Objective 4: Evaluate THC production by CBDAS and/or CBCAS through CRISPR-mediated knockout of cannabinoid synthase(s).
Project Methods
There is no study on the in planta expression patternof genes in the cannabinoid biosynthetic pathway. We will clone the promoters into a vector with a GUS reporter to learn in which tissues the gene is expressed, esp. if it is highly expressed in the glandular trichomes. We will conduct yeast one hybrid (Y1H)to identify putative TF that regulate genes in the cannabinoid biosynthetic pathway.We will use dual luciferase assay to examine the regulation of promoter regions of genes of interest by transcription factor (TF) identified. The binding of the TF to the promoter will be further validated using Y1H.For genes involved in glandular trichome development, we will ectopically express the gene in tobacco to examine glandular trichome phenotype. We will use Y2H to search for the interaction partners of the identified TF, MIXTA. We aim to identify the transcriptional network that controls glandular trichome development in female flowers. These genes can be marker genes for breeding.Plant hormones play vital roles in secondary metabolite production. We will further examine the regulation of the genes by hormones. We will focus on the change of promoter activities after hormone treatment. GUS or LUC activities driven by the promoters will be examined in protoplasts or Nicotiana benthamiana transformed with the gene constructs. Because there is no cannabis plant transformation protocol available, we are developing a cannabis protoplast isolation protocol for transient gene expression, which is useful for the promoter assays.We will recruit undergraduate students research assistant to conduct some of the experiments. Our lab has been offering research opportunities for undergraduate students and will keep offering independent study or work study positions for them. We will also add cannabis research topics in some of our courses. We are preparing students with robust research abilities and will make them competitive in the job market. Students graduated from or studiedin our lab have found satisfactory jobs after they completed their study.We will also present our research results in meetings for cannabis growers, horticulturists and breeders to deliver our knowledge to a broader community. We will publish our findings inrefereedjournals. We will also generate intellectual properties if the identifiedTF genes would show phenomenal phenotypes of enhanced/optimally improvedcannabinoid production.

Progress 04/15/23 to 04/14/24

Outputs
Target Audience:UConn students can learn our research through the Cannabis Horticulture course offered by our department. People outside of UConn who are taking the summer online course 'Horticulture of Cannabis: Seed to Harvest' can also get a chance to learn our cannabis biology research. Research opportunities are continuously provided for students who are interested in cannabis horticulture and biology research. Presentation opportunities at conferences are provided to students as well. Samuel Haiden, a PhD student who is working on this project, gave an oral presentation on the CannMed 23 meeting and our work reached a broader range of audience beyond plant biologists, horticulturists or cannabis growers. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?A previous master student was trained in molecular cloning, gene expression analysis and dual luciferase reporter assay techniques. A PhD student was trained with techniques in molecular cloning, gene expression analysis and yeast cDNA library construction. Two new master students were trained in cannabis growth and cloning, tissue culture propagation, molecular cloning, cannabis genomic DNA and RNA extraction, plasmid extraction as well as cannabinoid measurement using HPLC. The PhD student gave an oral presentation at the CannMed23 conference. How have the results been disseminated to communities of interest?Results obtained from the supported project have led to an article in a top-tier peer-reviewed journal. Results were also presented as a talk at CannMed23 that host scientists, scholars, stakeholders, industries and growers who work with or are interested in any aspects of cannabis study. What do you plan to do during the next reporting period to accomplish the goals?We generated a female flower cDNA library in yeast, which is ready for yeast one or two hybrid. Because full length MIXTA has transactivation capability, we have cloned different portions of MIXTA to identify the section that does not contain transactivation function. Then we will use the truncated MIXTA genes to screen the cDNA library to identify proteins that may interact with MIXTA. We will also clone the promoters of PT1 and PT4 into a Y1H vector and then screen the cDNA library to identify putative TFs that may regulate PT1 or PT4. We will also try the published stable transformation protocol using the autoflowering hemp varieties.

Impacts
What was accomplished under these goals? We expressed PT1 or PT4 promoter driven GUS in cannabis seedlings or sugar leaves and showed that both promoter activities were detected in the leaves of seedlings. Strongest PT4 promoter activity is associated with where capitate-stalked glandular trichomes develop, indicating the enzymatic activity of PT4 in these cells. In silico analysis of the promoters identified putative hormone responsive elements. Based on the analysis, luciferase assays were conducted and further showed that hormones ABA and SA can activate both PT1 and PT4 promoters. While auxin and MeJA responsive elements were identified, we didn't detect promoter activation, which could be due to the promote length we cloned. We also showed that ACC (ethylene), ZR (cytokinin) and GA are all able to activate PT4 promoter. While NAA (auxin) can activate the PT1 promoter, it cannot activate the PT4 promoter and there is no auxin responsive element in the PT4 promoter. Treatment of SA to cannabis plants upregulated expression of cannabinoid biosynthetic genes in the later steps of the pathway.

Publications

  • Type: Journal Articles Status: Published Year Published: 2023 Citation: Sands LB, Haiden SR, Ma Y, Berkowitz GA (2023) Hormonal control of promoter activities of Cannabis sativa prenyltransferase 1 and 4 and salicylic acid mediated regulation of cannabinoid biosynthesis. Scientific Reports, 13(1):112. https://doi.org/10.1038/s41598-023-35303-4
  • Type: Journal Articles Status: Published Year Published: 2022 Citation: Sipahi H, Whyte TD, Ma G, Berkowitz G (2022) Genome-Wide Identification and Expression Analysis of Wall-Associated Kinase (WAK) Gene Family in Cannabis sativa L. Plants, 11(20):2703. https://doi.org/10.3390/plants11202703
  • Type: Conference Papers and Presentations Status: Other Year Published: 2023 Citation: Spectral Diet and the Transcriptional Control of Flowering, Trichome & Cannabinoid Production.


Progress 04/15/22 to 04/14/23

Outputs
Target Audience:Our department offers a Cannabis Horticulture course and students in this course were introduced to our current cannabis research. Research opportunities were also provided for students who are interested in working in the lab; several of the students were exposed to undergraduate research through our SPSS 3999 Independent study course One of these undergraduates, Arsalan Zaki presented his research accomplishment on this project at the Plant Biology 2022 international meeting. A master student and a PhD student working on this project, Ms. Lauren Sands and Dr. Gang Ma respectively, also presented the results of their work on the project at this meeting. Our department and University held a Cannabis Research Symposium in March 2023. A current PhD student and a previous master student gave oral presentations of the results of their work on this project at this international symposium. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?A M.S. student was trained and learned molecular cloning, gene expression analysis and dual luciferase reporter assay techniques. A Ph.D. student was trained with techniques for molecular cloning, gene expression analysis, plant transformation and tissue culture, and yeast transactivation assay. Two undergraduate students did independent study projects and learned basic molecular biology techniques, such as DNA and RNA extraction, plasmid extraction, PCR. Students also learned how to clone cannabis plants using cuttings and how to grow cannabis in the greenhouse. A Ph.D. student, a M.S. student, and an undergraduate student were supported to give poster presentations at the Plant Biology 2022 international meeting. A cannabis symposium was held at UConn in March 2022. Scientists working on cannabis and cannabinoids in various fields, including horticulture, molecular biology, biotechnology, medicinal research and agricultural economics. A previous graduate student and a current PhD student gave oral presentations at the symposium. How have the results been disseminated to communities of interest?Results obtained from the supported projects have led to four publications in peer-reviewed journals. Results were also presented as posters and talks at national and international conferences. What do you plan to do during the next reporting period to accomplish the goals?We plan to perform yeast two hybrid to identify proteins that interact with CsMIXTA. We will also examine CsMIXTA promoter activity in cannabis tissue and the regulation by different hormones. We will also examine the effects of hormones on glandular development and cannabinoid biosynthesis. We will optimize a transient transfection approach in sugar leaves and perform RNAi or CRISPR to knockdown or knockout THCAS and CsMIXTA.

Impacts
What was accomplished under these goals? We examined the expression of cannabinoid biosynthetic genes during seven weeks of female flower/glandular trichome development. The results showed that cannabinoid biosynthetic geneswere upregulated during flower maturation except for THCAS. We identified an R2R3 transcription factor, CsMIXTA. Overexpression of CsMIXTA in tobacco showed the involvement of CsMIXTA in glandular trichome initiation and morphogenesis. Members in the plant-species homeodomain zipper family (HD-ZIP) have been shown to be involved in epidermal cell differentiation. We focused on the HD-ZIP IV subfamily and performed a bioinformatic analysis of the 9 genes in the family. Gene expression analysis showed that 4 are highly expressed in the female flowers, which could be putative candidates for future research.

Publications

  • Type: Conference Papers and Presentations Status: Other Year Published: 2022 Citation: Yi Ma. Genetic regulation of cannabinoid biosynthesis in Cannabis sativa. Oral presentation at Sussex Symposium, November 4, 2022, Yale University, New Haven, CT
  • Type: Conference Papers and Presentations Status: Other Year Published: 2022 Citation: Lauren Sands, Yi Ma, Gerald Berkowitz. Analysis of hormone regulation of a key step in the cannabinoid biosynthetic pathway in Cannabis sativa. Plant biology 2022. July 9 - July 13, 2022. Portland, OR
  • Type: Conference Papers and Presentations Status: Other Year Published: 2022 Citation: Arsalan Zaki, Lauren Sands, Monica Palencia-Prieto1, Yi Ma and Gerald A. Berkowitz. Evaluation of a Commercial Biostimulant for Enhancement of Cannabis Growth. Plant biology 2022. July 9 - July 13, 2022. Portland, OR
  • Type: Conference Papers and Presentations Status: Other Year Published: 2022 Citation: Gang Ma, Alice Kira Zelman, Peter V. Apicella and Gerald Berkowitz. Genome-Wide Identification and Expression Analysis of Homeodomain Leucine Zipper Subfamily IV (HD-ZIP IV) Gene Family in Cannabis sativa L. Plant biology 2022. July 9 - July 13, 2022. Portland, OR
  • Type: Theses/Dissertations Status: Other Year Published: 2022 Citation: Gang Ma. 2022. Delineating cell and tissue signaling cascades involved in plant perception of developmental cues and pathogen infection into altered plant and cell function
  • Type: Conference Papers and Presentations Status: Other Year Published: 2022 Citation: Samuel Haiden, Monica Palencia-Prieto, and Gerald Berkowitz. Identifying the ideal Spectral Diet for cannabis production: how to control cannabinoid biosynthesis through the manipulation of horticultural lighting. Plant biology 2022. July 9 - July 13, 2022. Portland, OR
  • Type: Journal Articles Status: Published Year Published: 2022 Citation: Sands, L.B.; Cheek, T.; Reynolds, J.; Ma, Y.; Berkowitz, G.A. Effects of Harpin and Flg22 on Growth Enhancement and Pathogen Defense in Cannabis sativa Seedlings. Plants 2022, 11, 1178. https://doi.org/10.3390/plants11091178
  • Type: Journal Articles Status: Published Year Published: 2022 Citation: Ma, G.; Zelman, A.K.; Apicella, P.V.; Berkowitz, G. Genome-Wide Identi?cation and Expression Analysis of Homeodomain Leucine Zipper Subfamily IV (HD-ZIP IV) Gene Family in Cannabis sativa L. Plants 2022, 11, 1307. https://doi.org/10.3390/plants11101307
  • Type: Journal Articles Status: Published Year Published: 2023 Citation: Haiden, S.R.; Apicella, P.V.; Ma, Y.; Berkowitz, G.A. Overexpression of CsMIXTA, a Transcription Factor from Cannabis sativa, Increases Glandular Trichome Density in Tobacco Leaves. Plants. 2022, 11, 1519. https://doi.org/10.3390/plants11111519
  • Type: Journal Articles Status: Published Year Published: 2022 Citation: Apicella, P. V., Sands, L. B., Ma, Y., & Berkowitz, G. A. (2022). Delineating genetic regulation of cannabinoid biosynthesis during female flower development in Cannabis sativa. Plant Direct, 6(6), e412. https://doi.org/10.1002/pld3.412
  • Type: Theses/Dissertations Status: Other Year Published: 2022 Citation: Lauren Sands. 2022. Studies of Cannabis Genes Impacting Growth, Pathogen Defense, and Hormone Regulation of Cannabinoid Synthesis.
  • Type: Conference Papers and Presentations Status: Other Year Published: 2023 Citation: Samuel Haiden. Cloning a cannabis transcription factor that facilitate trichome morphogenesis in developing flowers. Oral presentation at UConn Cannabis Symposium, March 16, 2023  March 17, 2023.
  • Type: Conference Papers and Presentations Status: Other Year Published: 2023 Citation: Lauren Sands. Identification of hormone responsive elements in the promoter of a cannabis gene encoding the rate-limiting step in cannabinoid synthesis: hormone regulation during flower development. Oral presentation at UConn Cannabis Symposium, March 16, 2023  March 17, 2023.