A new Cannabis genome assembly associates elevated cannabidiol (CBD) with hemp introgressed into marijuana.

Demand for cannabidiol (CBD), the predominant cannabinoid in hemp (Cannabis sativa), has favored cultivars producing unprecedented quantities of CBD. We investigated the ancestry of a new cultivar and cannabinoid synthase genes in relation to cannabinoid inheritance. A nanopore-based assembly anchored to a high-resolution linkage map provided a chromosome-resolved genome for CBDRx, a potent CBD-type cultivar. We measured cannabinoid synthase expression by cDNA sequencing and conducted a population genetic analysis of diverse Cannabis accessions. Quantitative trait locus mapping of cannabinoids in a hemp × marijuana segregating population was also performed. Cannabinoid synthase paralogs are arranged in tandem arrays embedded in long terminal repeat retrotransposons on chromosome 7. Although CBDRx is predominantly of marijuana ancestry, the genome has cannabidiolic acid synthase (CBDAS) introgressed from hemp and lacks a complete sequence for tetrahydrocannabinolic acid synthase (THCAS). Three additional genomes, including one with complete THCAS, confirmed this genomic structure. Only cannabidiolic acid synthase (CBDAS) was expressed in CBD-type Cannabis, while both CBDAS and THCAS were expressed in a cultivar with an intermediate tetrahydrocannabinol (THC) : CBD ratio. Although variation among cannabinoid synthase loci might affect the THC : CBD ratio, variability among cultivars in overall cannabinoid content (potency) was also associated with other chromosomes.

Validating a predictive model of cannabinoid inheritance with feral, clinical, and industrial Cannabis sativa.

PREMISE: How genetic variation within a species affects phytochemical composition is a fundamental question in botany. The ratio of two specialized metabolites in Cannabis sativa, tetrahydrocannabinol (THC) and cannabidiol (CBD), can be grouped into three main classes (THC-type, CBD-type, and intermediate type). We tested a genetic model associating these three groups with functional and nonfunctional alleles of the cannabidiolic acid synthase gene (CBDAS). METHODS: We characterized cannabinoid content and assayed CBDAS genotypes of >300 feral C. sativa plants in Minnesota, United States. We performed a test cross to assess CBDAS inheritance. Twenty clinical cultivars obtained blindly from the National Institute on Drug Abuse and 12 Canadian-certified grain cultivars were also examined. RESULTS: Frequencies of CBD-type, intermediate-type, and THC-type feral plants were 0.88, 0.11, and 0.01, respectively. Although total cannabinoid content varied substantially, the three groupings were perfectly correlated with CBDAS genotypes. Genotype frequencies observed in the test cross were consistent with codominant Mendelian inheritance of the THC:CBD ratio. Despite significant mean differences in total cannabinoid content, CBDAS genotypes blindly predicted the THC:CBD ratio among clinical cultivars, and the same was true for industrial grain cultivars when plants exhibited >0.5% total cannabinoid content. CONCLUSIONS: Our results extend the generality of the inheritance model for THC:CBD to diverse C. sativa accessions and demonstrate that CBDAS genotyping can predict the ratio in a variety of practical applications. Cannabinoid profiles and associated CBDAS segregation patterns suggest that feral C. sativa populations are potentially valuable experimental systems and sources of germplasm.

Gene duplication and divergence affecting drug content in Cannabis sativa.

Cannabis sativa is an economically important source of durable fibers, nutritious seeds, and psychoactive drugs but few economic plants are so poorly understood genetically. Marijuana and hemp were crossed to evaluate competing models of cannabinoid inheritance and to explain the predominance of tetrahydrocannabinolic acid (THCA) in marijuana compared with cannabidiolic acid (CBDA) in hemp. Individuals in the resulting F2 population were assessed for differential expression of cannabinoid synthase genes and were used in linkage mapping. Genetic markers associated with divergent cannabinoid phenotypes were identified. Although phenotypic segregation and a major quantitative trait locus (QTL) for the THCA/CBDA ratio were consistent with a simple model of codominant alleles at a single locus, the diversity of THCA and CBDA synthase sequences observed in the mapping population, the position of enzyme coding loci on the map, and patterns of expression suggest multiple linked loci. Phylogenetic analysis further suggests a history of duplication and divergence affecting drug content. Marijuana is distinguished from hemp by a nonfunctional CBDA synthase that appears to have been positively selected to enhance psychoactivity. An unlinked QTL for cannabinoid quantity may also have played a role in the recent escalation of drug potency.

The SAUR19 subfamily of SMALL AUXIN UP RNA genes promote cell expansion.

The plant hormone auxin controls numerous aspects of plant growth and development by regulating the expression of hundreds of genes. SMALL AUXIN UP RNA (SAUR) genes comprise the largest family of auxin-responsive genes, but their function is unknown. Although prior studies have correlated the expression of some SAUR genes with auxin-mediated cell expansion, genetic evidence implicating SAURs in cell expansion has not been reported. The Arabidopsis SAUR19, SAUR20, SAUR21, SAUR22, SAUR23, and SAUR24 (SAUR19-24) genes encode a subgroup of closely related SAUR proteins. We demonstrate that these SAUR proteins are highly unstable in Arabidopsis. However, the addition of an N-terminal GFP or epitope tag dramatically increases the stability of SAUR proteins. Expression of these stabilized SAUR fusion proteins in Arabidopsis confers numerous auxin-related phenotypes indicative of increased and/or unregulated cell expansion, including increased hypocotyl and leaf size, defective apical hook maintenance, and altered tropic responses. Furthermore, seedlings expressing an artificial microRNA targeting multiple members of the SAUR19-24 subfamily exhibit short hypocotyls and reduced leaf size. Together, these findings demonstrate that SAUR19-24 function as positive effectors of cell expansion. This regulation may be achieved through the modulation of auxin transport, as SAUR gain-of-function and loss-of-function seedlings exhibit increased and reduced basipetal indole-3-acetic acid transport, respectively. Consistent with this possibility, SAUR19-24 proteins predominantly localize to the plasma membrane.

A WD40 repeat protein from Medicago truncatula is necessary for tissue-specific anthocyanin and proanthocyanidin biosynthesis but not for trichome development.

WD40 repeat proteins regulate biosynthesis of anthocyanins, proanthocyanidins (PAs), and mucilage in the seed and the development of trichomes and root hairs. We have cloned and characterized a WD40 repeat protein gene from Medicago truncatula (MtWD40-1) via a retrotransposon-tagging approach. Deficiency of MtWD40-1 expression blocks accumulation of mucilage and a range of phenolic compounds, including PAs, epicatechin, other flavonoids, and benzoic acids, in the seed, reduces epicatechin levels without corresponding effects on other flavonoids in flowers, reduces isoflavone levels in roots, but does not impair trichome or root hair development. MtWD40-1 is expressed constitutively, with highest expression in the seed coat, where its transcript profile temporally parallels those of PA biosynthetic genes. Transcript profile analysis revealed that many genes of flavonoid biosynthesis were down-regulated in a tissue-specific manner in M. truncatula lines harboring retrotransposon insertions in the MtWD40-1 gene. MtWD40-1 complemented the anthocyanin, PA, and trichome phenotypes of the Arabidopsis (Arabidopsis thaliana) transparent testa glabrous1 mutant. We discuss the function of MtWD40-1 in natural product formation in M. truncatula and the potential use of the gene for engineering PAs in the forage legume alfalfa (Medicago sativa).

A new method for isolating large quantities of Arabidopsis trichomes for transcriptome, cell wall and other types of analyses.

A new procedure has been developed for the isolation of wild-type and mutant Arabidopsis trichomes. The isolated trichomes maintained enzymatic activity and were used for DNA, protein, and RNA isolation. The RNA was used to generate probes suitable for Affymetrix analysis. The validity of the Affymetrix results was confirmed by quantitative PCR analysis on a subset of genes that are preferentially expressed in trichomes or leaves. Sufficient quantities of trichomes were isolated to probe the biochemical nature of trichome cell walls. These analyses provide evidence for the presence of lignin in Arabidopsis trichome cell walls. The monosaccharide analysis and positive staining with ruthenium red indicates that the walls also contain a large portion of pectin. The 2.23-fold ratio of pectin-related sugars compared with potential cellulosic glucose suggests that the polysaccharides of the trichome cell walls are more like those of typical primary walls even though the wall becomes quite thick. Overall, these analyses open the door to using the Arabidopsis trichome cell wall as an excellent model to probe various questions concerning plant cell wall biosynthesis.

Identification of candidate genes affecting Delta9-tetrahydrocannabinol biosynthesis in Cannabis sativa.

RNA isolated from the glands of a Delta(9)-tetrahydrocannabinolic acid (THCA)-producing strain of Cannabis sativa was used to generate a cDNA library containing over 100 000 expressed sequence tags (ESTs). Sequencing of over 2000 clones from the library resulted in the identification of over 1000 unigenes. Candidate genes for almost every step in the biochemical pathways leading from primary metabolites to THCA were identified. Quantitative PCR analysis suggested that many of the pathway genes are preferentially expressed in the glands. Hexanoyl-CoA, one of the metabolites required for THCA synthesis, could be made via either de novo fatty acids synthesis or via the breakdown of existing lipids. qPCR analysis supported the de novo pathway. Many of the ESTs encode transcription factors and two putative MYB genes were identified that were preferentially expressed in glands. Given the similarity of the Cannabis MYB genes to those in other species with known functions, these Cannabis MYBs may play roles in regulating gland development and THCA synthesis. Three candidates for the polyketide synthase (PKS) gene responsible for the first committed step in the pathway to THCA were characterized in more detail. One of these was identical to a previously reported chalcone synthase (CHS) and was found to have CHS activity. All three could use malonyl-CoA and hexanoyl-CoA as substrates, including the CHS, but reaction conditions were not identified that allowed for the production of olivetolic acid (the proposed product of the PKS activity needed for THCA synthesis). One of the PKS candidates was highly and specifically expressed in glands (relative to whole leaves) and, on the basis of these expression data, it is proposed to be the most likely PKS responsible for olivetolic acid synthesis in Cannabis glands.

Transcriptome analysis of Arabidopsis wild-type and gl3-sst sim trichomes identifies four additional genes required for trichome development.

Transcriptome analyses have been performed on mature trichomes isolated from wild-type Arabidopsis leaves and on leaf trichomes isolated from the gl3-sst sim double mutant, which exhibit many attributes of immature trichomes. The mature trichome profile contained many highly expressed genes involved in cell wall synthesis, protein turnover, and abiotic stress response. The most highly expressed genes in the gl3-sst sim profile encoded ribosomal proteins and other proteins involved in translation. Comparative analyses showed that all but one of the genes encoding transcription factors previously found to be important for trichome formation, and many other trichome-important genes, were preferentially expressed in gl3-sst sim trichomes. The analysis of genes preferentially expressed in gl3-sst sim led to the identification of four additional genes required for normal trichome development. One of these was the HDG2 gene, which is a member of the HD-ZIP IV transcription factor gene family. Mutations in this gene did not alter trichome expansion, but did alter mature trichome cell walls. Mutations in BLT resulted in a loss of trichome branch formation. The relationship between blt and the phenotypically identical mutant, sti, was explored. Mutations in PEL3, which was previously shown to be required for development of the leaf cuticle, resulted in the occasional tangling of expanding trichomes. Mutations in another gene encoding a protein with an unknown function altered trichome branch formation.

E2F and retinoblastoma related proteins may regulate GL1 expression in developing Arabidopsis trichomes.

This is an addendum to our recent paper published in The Plant Journal (52:352-61). The major findings were: (1) trichomes on the leaves of gl3-sst sim double mutants developed as large multi-cellular clusters whereas wild type trichomes are composed of single cells; (2) ectopic CYCD3;1 expression in gl3-sst trichomes also resulted in trichome cluster formation; and (3) that GL1 expression is prolonged in the gl3-sst sim trichome clusters. This addendum shows that ectopic CYCD3;1 expression in gl3-sst also enhanced GL1 expression. An analysis of the GL1 promoter found two overlapping potential E2F binding sites in a region of the promoter known to be essential for GL1 function. This finding indicates that GL1 may be directly regulated by the activity of a CYCD3/CDKA complex that phosphorylates E2F-RB bound to the GL1 promoter.

Genetic interaction between glabra3-shapeshifter and siamese in Arabidopsis thaliana converts trichome precursors into cells with meristematic activity.

The identity of many genes required for trichome differentiation is known. This paper describes a novel interaction between mutant alleles of two such genes. One of the alleles, called gl3-sst, is derived from the GL3 locus, which encodes a basic helix-loop-helix type transcription factor. The mutation in the gl3-sst protein modifies its ability to form a complex with the GL1 protein (a MYB transcription factor required for trichome formation), leading to changes in gene expression compared with wild type during gl3-sst mutant trichome development. The other mutant allele, sim, is a likely loss of function allele derived from the SIM locus, which is predicted to encode a negative regulator of D-type cyclin activity. The gl3-sst sim double mutant exhibits mounds of cells derived from the proliferation of single trichome precursors. The ectopic expression of a D-type cyclin gene in gl3-sst mimics the double mutant phenotype. Thus, an interaction between altered trichome gene expression caused by the gl3-sst mutation and relaxed regulation of D-type cyclin activity in the double mutant converted a non-dividing cell into a novel highly proliferating cell type.