Source: WEST VIRGINIA UNIVERSITY submitted to
CHARACTERIZATION OF THE TERPENE-CANNABINOID METABOLIC NETWORK AND ITS GENETIC REGULATION IN INDUSTRIAL HEMP
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
NEW
Funding Source
Reporting Frequency
Annual
Accession No.
1023211
Grant No.
2020-67014-30901
Project No.
WVA00925
Proposal No.
2019-05688
Multistate No.
(N/A)
Program Code
A1103
Project Start Date
Jun 1, 2020
Project End Date
May 31, 2022
Grant Year
2020
Project Director
Gutensohn, M.
Recipient Organization
WEST VIRGINIA UNIVERSITY
886 CHESTNUT RIDGE RD RM 202
MORGANTOWN,WV 26505-2742
Performing Department
Division of Plant and Soil Sciences
Non Technical Summary
Industrial hemp and marijuanaare bothone and the same plant speciesCannabis sativa,however, represent different varieties showing high genetic, morphological, and chemical diversity. While marijuana contains high levels (up to 20% of dry weight) of the psychoactive and intoxicating cannabinoid delta 9-tetrahydrocannabinol (THC), in contrast to be legally considered as industrial hemp in the US the crop may not contain more than 0.3% THC. Throughout human history hemp has been cultivated for food, fiber and medicine, and is presently used in over 25,000 products. Hemp seeds are pressed to obtain an oilwell suited for human diet and the residual seed cake can be used as animal feed. While hemp fibers are primarily used to produce papers and textiles, their use has recently been expanded towards the production of carbon nanosheets, plastics, absorbent materials and construction concrete. However, in addition to seeds and fibers significant revenues can also be achieved for some of the metabolites highly abundant in industrial hemp including non-intoxicating cannabinoids such as cannabidiol (CBD) and terpenes (up to 5% of dry weight) that are valued as pharmaceuticals, fragrances and chemical feedstocks.Despite the existence of large genetic diversity and in consequence a wide range of terpene and cannabinoid levelsin Cannabis sativa, the research and knowledge about the biosynthesis of these compoundsand the underlying geneticsis still quite limited due to previous legal restrictions. The recent lifting of these restrictions has not only lead to a revitalized interest in industrial hemp as a crop, but also to the urgent need to understand how the biosynthesis of specific terpenes and cannabinoids are regulated so that they can be preferentially overproduced or eliminated in hemp varieties grown for particular uses.Remarkably little is known about the regulation of these biosyntheticpathways by endogenous and environmental factors.This is relevant for the cultivation of industrial hemp considering on the one sidethe economic potential for the production of terpenes and CBD, and on the other sidelegal restrictions on THC content. In particular the frequent occurrence of environmental stress induced spikes in THC contentsurpassing legal limits represents a serious economic risk for growers. Thus, to address this critical need to establish a better understanding of the terpene-cannabinoid biosyntheticnetwork in industrial hemp and its regulation, in the first part of this project we will analyze the accumulation of terpenes and cannabinoids in industrial hemp under different environmental stresses. In the second part of the project we will analyze changes in the activity levels of key terpene and cannabinoid biosynthetic genes under these stresses, and identify novel regulatory factors. In the third part of the project we will engineer hemp lines to determine how inactivation or upregulation of two key biosynthetic genes in industrial hemp via a nanoparticle-based approach affects accumulation of terpenes and cannabinoids.The long-termgoal of this proposal is to establish a better understanding of the terpene-cannabinoid biosyntheticnetwork in industrial hemp and its genetic regulation which will ultimately result in the engineering and development of hemp varieties with improvedor eliminated productionof specific terpene and cannabinoid compounds. This project now provides the opportunity to obtain base-line data for seeking furtherresearch opportunities to investigate the hemp terpene-cannabinoid biosyntheticnetwork, its regulation and potential forengineering varieties with improved production of specific compounds.In addition, the plant lines generated in this project will also provide future opportunitiesto test the performance of hemp varieties with modified terpene and cannabinoid profiles under field conditions.
Animal Health Component
0%
Research Effort Categories
Basic
80%
Applied
(N/A)
Developmental
20%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
2031730100020%
2111730100010%
2121730100010%
2031730104010%
2111730104010%
2121730104010%
2011730108030%
Goals / Objectives
The long history of hemp breeding has resulted in numerous different varietieswith different levels and blends of cannabinoids and terpenes.Despite the existence of this large genetic diversity, the research and knowledge about the biosynthesis and genetics of these metabolites in hemp is still quite limited due to previous legal restrictions.Remarkably little is known about the regulation of these metabolic pathways by endogenous and environmental factors.This is particularly relevant for the cultivation of industrial hemp considering the economic potential for the production of terpenes and CBD, as well as the legal restrictions on THC content.The majorgoal of this proposal is to establish a better understanding of the terpene-cannabinoid metabolic network in industrial hemp and its genetic regulation which will serve as foundation towards the engineering of hemp varieties with increased or eliminated accumulation of specific terpene and cannabinoid products. Our central hypothesis for this proposal is that the individual branches of this metabolic network are highly linked via shared intermediates and thus preferential biosynthesis requires specific genetic regulation during different environmental conditions. Three specific objectives will be addressed in this proposal:Objective 1: Characterize the accumulation of terpenes and cannabinoids in industrial hemp under different abiotic and biotic stress conditions.Objective 2: Characterize the expression level and genetic regulation of key terpene and cannabinoid biosynthetic genes in industrial hemp under different abiotic and biotic stress conditions.Objective 3: Determine the effect of knockout and overexpression of key biosynthetic genes on the accumulation of metabolites in the terpene and cannabinoid biosynthetic network in industrial hemp.
Project Methods
To achieve the three specific objectives proposed in this project we will utilize the following methods:Objective 1:For all the analysis of theterpeneand cannabinoid accumulationunder different abiotic and biotic stress conditions, we will use several industrial hemp varieties including variety'Finola' for whichgenome and transcriptome data are available.We will grow hemp plants in growth chambers under controlled environmental conditions to test the effect of various stresses on the accumulation of terpenes and cannabinoids. Considering abiotic stresses that field grown hemp is potentially exposed to, we will perform the following treatments of hemp plants: i) drought stress using a sensory based automated watering system, ii) temperature stress using different day/night temperature regimes (22/16, 28/22, 34/28 oC), iii) light stress using different light intensities (400, 800, 1300 µm m-2 s-1), iv) nutrient stress applying different amounts of fertilizer via the automated watering system, v) salinity stress applying different amounts of NaCl via the automated watering system. In addition, we will treat hemp plants with jasmonic acid and salicylic acid, signaling compounds involved in the response to biotic stresses, to mimic potential attacks by pests and pathogens that field grown hemp will experience. Leaves of 4-6-week-old plants and flower buds of mature plants will be harvested from control and treated hemp plants, and subsequently used for solvent extraction (MTBE or ethanol/methanol) of metabolites. Extracts will be analyzed by GC-MS to verify the qualitative and quantitative composition of terpene and cannabinoid profiles. Individual compounds will be identified based on their mass spectrum and by comparison with available authentic standards, and will be quantified using internal standards. In addition, metabolite extracts will also be separated by liquid chromatography (UHPLC & LC-MS) which allows to analyze cannabinoids, their respective acids, and other pathway intermediates.Objective 2:Analysis of transcript levels of terpene and cannabinoid biosynthetic genes under stress: In parallel to the metabolite analysis (see Objective 1), tissue samples collected from control and stress treated hemp plants will be used to isolate total RNA. Subsequently we will perform quantitative RT-PCR to characterize potential changes in the transcript levels of terpene and cannabinoid biosynthetic genes upon exposure to various abiotic and biotic stresses. Gene specific primers for qRT-PCR analysis will be designed based on the publishedsequences of hempterpene biosynthetic genes (including MEP pathway genes, PTSs and TPSs), as well as cannabinoid biosynthetic genes (AAE1, TKS, OAC, GOT, THCAS and CBDAS). This analysis will allow us to verify if stress induced changes in metabolite profiles correlate with alterations in the expression level of respective biosynthetic genes.RNA-Seq analysis of hemp under stress conditions: To further study the underlying genetic mechanisms of stress-induced terpene and cannabinoid biosynthesis, we will perform RNA-Seq analysis to identify transcription factors responsible for gene regulation that are co-regulated with biosynthetic genes. Two stress treatments identified by qRT-PCR as having upregulated biosynthesis genes will be used for RNA-Seq. The libraries will be made with the KAPA Stranded mRNA-Seq Kit with Poly-A selection and run on an Illumina Hi-Seq to provide 100-bp paired-end reads, using 3 to 5 biological reps per sample. Up to 20 bar-coded samples will be pooled per run. RNA reads will be de-multiplexed by barcode and then mapped to the published 'Finola' genome. Only uniquely mapping reads will be retained for analyses of differential expression.Objective 3:Knock-out of THCA synthase: To determine whether knocking out the delta 9-tetrahydrocannabinolic acid synthase(THCAS) gene can eliminate THC biosynthesis, and affect accumulation of other cannabinoids and terpenes in hemp, we will microinject hemp seedling vegetative and adult inflorescence meristems with DNA-PEI-Au/SiO2 nanoparticles containing CRISPR/CAS9 constructs. These will encode CAS9 driven by double 35S or egg cell-specific promoters, respectively. The guide RNA (gRNA) will encode scaffolds that target two sites roughly located at the midpoint of the THCAS coding sequence. BLASTn of the 'Finola' genome suggested that off-target edits could occur in five other THCAS-like genes. Genome edits will be screened/genotyped by sequencing of PCR products in T0 generation tissues and in resulting seedlings upon selection on hygromycin. The six loci are distant enough that off-targeted edits could be removed by segregation. Terpene and cannabinoid content of THCAS-edited lines will be measured by GC-MS and UHPLC.Overexpression of GPP synthase small subunit (GPPS-SSU): To determine whetheroverexpression of a geranyl diphosphate synthase (GPPS) canchange terpene and cannabinoid yields in hemp, we will replace the endogenous THCAS coding sequencewith a codon-optimized version of thehemp GPPS small subunit (GPPS-SSU) gene. The pTC217 vector encodes CRISPR/CAS9 to make targeted genome cuts and gemini virus components to locally generate thousands of copies of DNA templates for homology-directed repair. Thus, pTC217 constructs encoding a THCAS gRNA and GPPS-SSUviral-like DNA flanked by THCAS homology arms will be grafted onto PEI-Au/SiO2 nanoparticles and microinjected as indicated above. A variant of pTC217 encoding an egg cell-specific promoter in place of the 35S for driving CAS9 expression will also be tested. Genome edits will be screened/genotyped and metabolite contents measured in homozygous plants as indicated above.