Progress 04/15/24 to 04/14/25
Outputs
Target Audience:A number of invited presentations were made in the past year (not listed in prior annual reports) at national/international conferences targeted to a) commercial agricultural producers, b) researchers, and c) teachers who provide instruction relevant to the focus of the USDA funded work we are doing. These presentations included: 1. Haiden, S, Berkowitz GA 2025MJUnpacked Cannabis Resesarch Conference, Atlantic City, NJ 2. Haiden S, Berkowitz GA 2025 SUNY Niagra Cannabis Convention, Niagra Falls, NY 3. Berkowitz GA 2025 Cannabis cholarship; integration of research and teaching. 4th Natioanl Cannabis Curriculum Convening, 4/25-4-26. 4. Ma, Y, Berkowitz GA 2024 Transcriptional regulation of cannabinoid biosynthesis. USDA-NIFA Foundational Knowledge of Plant Products Principal Ingestigators' Conference. 5. Haiden S, Berkowitz GA 2024 CannMed, Marcp Os;amd, FL Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?A Research Asistant Professor (Dr. Yi Ma) umdertook a research leave while funded on this award and developed new research skills at the Carnegie Inst. (Stanford CA) in the lab of Dr. Zhiyong Wang, a leader in the area of studying the effects of thehormone brassinosteroid on cannabis. Dr. Ma was able to obtain new training in this area of plant biology. Two graduate students working on projects related to the award's objectives were able to receive training, attend conferences, and meet other scientists working in this area. Two undergraduate students also received training in plant molecular biology research. How have the results been disseminated to communities of interest?Through the presentations at cannabis conferences, both researchers and cannabis production companies were able to benefit from the work we generated. The publication of two refereed journal articles facilitated scientists working on cannabinoid biochemistry to obtain knowledge we generated during the project. What do you plan to do during the next reporting period to accomplish the goals?Our prior work indicates that the generation of CBG (cannabigerolic acid) is the rate-limiting step of cannbinoid biosynthesis. The substrates for CBG synthesis include terpenoids. This large family of compounds includes isoprenes (such are geranyl pyrophosphate (GPP)) which are aliphatic chaings. Isoprenes are precursors for not just the cannabinoids, but also mono, sequi, and diterpenes as well as key plant hormones such as gibberilic acid. How the regulation of GPP use as a substrate fpr these competing biosynthetic pathways in not characterized. Cannabis offeres and excellent model system to study this regulation of bifurcation of this important precurosor to different pathways in plants. Our new work will focus on generating new information about this regulation.
Impacts
What was accomplished under these goals?
1.Unlike model plants such as Arabidopsis, Cannabis is a short day crop; the mechanisms that initiate flowering during short days affect the cannabinoid levels and therefore commercial value of this crop. We monitored hormone and Phytochrome signaling effects on the molecular basis for flower initiation in cannabis. While discrete regulatory mechanisms have been identified, a unified model for the transcriptionalactivation of photoperiodic flowering in short-day (SD) plants has not been delineated. Although the GIGANTEA-CONSTANS-FLOWERING LOCUS T (GI-CO-FT) pathway appears to be highly conserved, each species may differ in its blueprint for activation of FT and floral meristem identity. In cannabis, an SD plant, PSEUDO-RESPONSE-REGULATOR 37 (PRR37) has a causal effect in determining photoperiodsensitivity. Our work identified a network of WITH NO LYSINE (K) kinases (WNK) which may be regulating the PRR proteins and downstream genes, including florigen (FT). CO-like genes have been identified in cannabis, but their function in regulating FT has not been determined. Our work shows that in cannabis, CO is critically-day-length-gated (CDL) and down-regulated by short days, likely to act as a repressor of FT. We found that CDL-gated gene expression can be manipulated in cannabis horticulture through modifying the Red: Far Red (R:FR) ratio of LED lighting, where increased expression of COL5 in response to light treatment is inversely related with expression of FT. Building on these insights, we propose a model for the CDL-gated regulation of FT expression in cannabis. 2. While the biochemical pathway for generation of the major cannabinoids in cannabis (THC, CBD, and CBC) are well characterized, little is known about the molecular basis for synthesis of the ~ 100 minor cannabinoids in this plant. A commercially valuable minor cannabinoid is the varin version of THC and CBD. We undertook work to determine the generation of varin moieties of cannabinoids from C3 (propyl) rather than the standard C5 (pentyl) fatty acid precursors. Current modelssuggest that two alleles, a truncated BETA-KETOACYL-REDUCTASE (BKR) mutant and an ACYL-LIPID-THIOESTERASE paralog (ALT4), are responsible for conferring propyl-cannabinoid biosynthesis. We evaluated several factors across an S1 progenysegregating for varin accumulation. We demonstrate that C3 and C5 dominant chemovars exhibit different patterns in total acid cannabinoid (TAC) accumulation, and that the ratio of C3:C5 cannabinoids in high C3 individuals follows a distinct pattern which correlates with ALT4 expression. In the high-C3 group, we discovered that the butanoic acid ratio was significantly higher than the high-C5 group, commensurate with a nearly 70-fold increase in varin ratio. We also show that the ratio of C3:C5 cannabinoids is much higher in vegetative leaves than mature flowers. Several genes that are critical to cannabinoid biosynthesis were differentially expressed across 6 weeks of flowering between the C3 and C5 groups, indicating that cannabinoid biosynthesis may be regulated via transcriptional control of FAS and cannabinoid synthases. Additionally, 97 DEGs were identified in the C3 trichome transcriptome when compared to that of the C5 genotype. We characterized the expression pattern of ALT4 and demonstrate that neither the overexpression of ALT4 nor the overexpression of a truncated, 7-exon BKR mutant yield an increased propyl cannabinoid ratio. Finally, we demonstrated that nearly all of the C3 individuals had a unique SNP pattern in the BKR locus, while almost none of the C5 individuals had any variation at the BKR locus.
Publications
- Type:
Peer Reviewed Journal Articles
Status:
Published
Year Published:
2025
Citation:
Haiden S, Johnson N, Berkowitz GA 2025 Transcriptomic analysis of CDL-gated photoperiodic flowering mechanisms in cannabis and their responsiveness to R: FR ratios in controlled environment agriculture. Scientific Reports. 15:17628.
- Type:
Peer Reviewed Journal Articles
Status:
Published
Year Published:
2024
Citation:
Sipahi H, Haiden S, and Berkowitz GA 2024 Genome-wide analysis of cellulose synthase (CesA) and cellulose synthase-like (Csl) proteins in Cannabis sativa L. PeerJ 12:e17821
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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.
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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.
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