These findings differ from a proteomic study that aimed to identify genes expressed in Cannabis glands but failed to associate any highly expressed proteins with THCA synthesis . This difference reflects the much greater volume of genomic data enabling more robust identification of DNA sequences when compared to proteomics approaches based on the molecular weights of fragmented polypeptides. This is especially true for species such as Cannabis sativa for which there is little amino acid sequence data available to compare with peptide profiles. The present study highlights the utility of using isolated glands as starting material for making EST libraries to study gland metabolism, as was the case in other plant species . In this study more than 50% of the ESTs with NCBI matches were involved in metabolism or cellular activities such as transport and protein translation. Many other cannabinoids, in addition to THCA, have been identified in Cannabis , and it is likely that many of the genes identified in Supplementary Table 2 at JXB online are involved in the production of these other compounds. In addition to cannabinoids, many other classes of secondary compounds have been found in Cannabis . For example,vertical growing racks both monoterpenes and sesquiterpenes have been identified and candidate ESTs encoding activities to produce these compounds have been identified.Synthesis of olivetolic acid from malonyl-CoA and hexanoyl-CoA represents the first committed step toward the synthesis of THCA. Olivetolic acid synthesis is predicted to be mediated by a member of the type III PKS family through a series of three condensation reactions producing a triketide .
CAN24, represented by eight ESTs and one of the most highly expressed unigenes in our analysis, encodes a member of the PKS family. This gene was expressed 1600-fold higher in glands than in leaves. CAN1068, another PKS member represented by two ESTs, corresponds to a previously identified Cannabis CHS gene . A third PKS represented by a single EST, CAN383, was also identified. Analyses of PKS crystal structures indicate that the type III PKS enzymes are composed of a dimer with conserved reaction centres and a hollow reaction cavity . All three Cannabis PKS genes encoded polypeptides containing the conserved amino acids, Cys 167, His 307, and Asn 340, that are believed to constitute the reaction centre . In addition, two of the three amino acids that are important for defining the size of the reaction cavity in chalcone synthases are conserved. The third amino acid, Thr at position 300 that is conserved in all chalcone synthases, was missing in CAN24 and CAN383 . Instead CAN24 and CAN383 contained Leu and Iso, respectively, at position 300. Such differences might alter substrate specificity. It has been proposed that either olivetol or olivetolic acid are products of polyketide synthase in the THCA pathway . However assays of plant extracts found that olivetol, the decarboxylation product of olivetolic acid, was not a substrate in the pathway . Products of the three PKS genes identified in this study were tested for olivetolic acid synthesis in vitro. CAN24 and CAN383 yielded identical products according to HPLC analysis . Because CAN24 was more abundant, this PKS gene was analysed in detail, along with CAN1069, which had CHS activity as shown in Fig. 4.
The size of the product produced by the CAN24-encoded enzyme was smaller than olivetolic acid . Further, the absorption spectrum did not match olivetol. A sequence identical to CAN24 has been deposited in the NCBI database and was annotated as having olivetol synthase activity but without supporting data. The product generated in our analyses possibly represents a derailment product in which the enzyme catalyses two decarboxylative condensations instead of three . The failure of in vitro conditions to support the complete synthesis of native PKS products is well documented . The artificial nature of in vitro reaction products is reflected by the failure to find these products in vivo . This may indicate that reaction conditions were insufficient or that accessory proteins may be needed to produce olivetolic acid. Similar results were obtained in a study that characterized a gene identical to CAN1069 where the enzyme could use hexanoyl-CoA as a starter molecule, but only yielded a possible derailment product . That plant extracts used in in vitro assays have also not yielded olivetolic acid suggests that in vivo-like reaction conditions have yet to be imitated . The assay results with CAN1069 also highlight the permissiveness of substrate use by PKSs. CAN1069 clearly had CHS activity in that it could use coumaroyl-CoA as a substrate to produce naringenin. Given that CAN1069 was preferentially expressed in the glands and can act on hexanoyl-CoA, this PKS also may contribute to THCA production. Insights on THCA biosynthesis may be gained by comparison with the secondary metabolism of glandular trichomes in hop, the closest relative to Cannabis. Production of the bitter acid humulone in hop inflorescence glands requires a PKS called VPS. In two independent studies, ESTs representing VPS were among the most abundant in the collections and were at least 5-fold more abundant than other PKS encoding ESTs .
Accordingly, the role of CAN24 as olivetolic acid synthase remains tentative pending further biochemical support, however, it is the best candidate based on expression data.A large number of Cannabis gland unigenes encoded proteins similar to transcription factors . Several of these were analysed by qPCR and two were found to be preferentially expressed in glands. This is potentially significant as studies have shown that all but one of the 12 transcription factors required for Arabidopsis trichome formation are preferentially expressed in trichomes compared to whole leaves . In this study it was found that two R2R3-type Cannabis MYBs, encoded by CAN833 and CAN738, were preferentially expressed in isolated glands compared to leaves by 954-fold and 586-fold, respectively. CAN833 and CAN738 are most similar to the Arabidopsis MYBs related to AtMYB112 and AtMYB12, respectively . AtMYB112 corresponds to the BOTRYTIS SUSCEPTIBLE1 gene. bos1 mutants are more susceptible to pathogens such as Botrytis cinerea and Alternaria brassicicola, and have impaired tolerance to oxidative stress . A role for CAN883 in tolerance to oxidative stress in Cannabis glandular trichomes is logical, as the last enzymatic reaction in THCA synthesis releases hydrogen peroxide . AtMYB12 controls the synthesis of flavonol secondary metabolites in Arabidopsis and can induce the synthesis of similar compounds in tobacco . Flavonoids have been isolated from Cannabis leaves and flowers, but evidence is lacking for gland flavonoid production . Since flavonols are not predominant in Cannabis glands, it is possible that CAN738 instead plays a role in controlling the expression of genes required for other secondary metabolites in Cannabis such as THCA.The PKSs and many other genes identified in this study are closely related to those from hop . Humulus and Cannabis are monotypic sister genera in the family Cannabaceae .Glandular trichomes located on the inflorescence bracts of both Humulus and Cannabis are the location of unique PKS-derived secondary metabolism . Hop glands produce the bitter acid humulone, which is important for beer flavour, and the prenylated chalcone xanthohumol, which has several potential health beneficial properties . The biochemical pathways leading to THCA, xanthohumol, and humulone have common steps that include polyketide synthases and prenyltransferases. It is probable that these plants share other homologous biochemical pathways given their close ancestry. Information from Cannabis ESTs has the potential to improve the understanding of hop biochemical pathways as well.Meanwhile,vertical grow room design there is limited scientific evidence specifying which of the multitude of cannabinoid products offer the greatest therapeutic benefit and/or lowest risk of harm. Self-report studies that attempt to capture which products cannabis users choose and for what purpose are inherently biased by differences in ease of access to different products and preparations across US states and counties. Understanding how reducing barriers to access might impact cannabinoid choice and behavior among military veterans is of paramount importance. Emerging evidence suggests the therapeutic potential of cannabinoids for a range of conditions relevant to military veteran health , including chronic pain , traumatic brain injury , substance use disorder , and posttraumatic stress disorder .
Likely stemming from its actual or perceived therapeutic efficacy for many physical and psychological symptoms, many individuals are using cannabis as a substitute for more traditional pharmaceuticals , as well as licit and illicit substances . Epidemiological findings highlight this trend, documenting decreased opioid use within states that have transitioned to allow access to medicinal cannabis . Likewise, preliminary analysis from one longitudinal study found reductions in the use of prescription medications including opiates, antidepressants, mood stabilizers, and benzodiazepines three months after initiation of medical cannabis . Cannabis use, however, is also associated with a variety of negative effects, including risk of cannabis use disorder CUD . Relevant to veterans, CUD diagnoses have increased substantially in the past decade , specifically among veterans with PTSD . The contrast between therapeutic potential and increased problems may be attributable to the fact that “cannabis” represents a heterogeneous drug containing hundreds of cannabinoids, flavonoids, and terpenoids , which are associated with differential risk and potential therapeutic utility. For example, cannabis with higher delta-9-tetrahydrocannabinol relative to cannabidiol can be intoxicating and increase the risk of CUD , while cannabis with higher CBD relative to THC appears to have fewer of these properties . Preliminary evidence suggests that THC may be helpful for some physical conditions, such as neuropathic pain and spasticity due to multiple sclerosis , but may exacerbate mental health symptoms, such as PTSD and depression . In contrast, CBD could be helpful for anxiety-related symptoms , such as PTSD , social anxiety , and insomnia . Likewise, route of administration could impact both therapeutic potential and risk of problematic use. Bio-availability is directly impacted by whether cannabis products are smoked/vaporized, swallowed, absorbed sublingually, or administered topically , onset and duration of effects , and subjective experience of effects . Likewise, delivery of cannabis products that contain higher concentrations of THC is associated with greater risk of negative side effects , tolerance and withdrawal . In this complex cannabis landscape, patient guidance and oversight is desperately needed, yet seriously lacking. Owing to the designation of cannabis as a schedule I controlled substance, health-care providers do not have sufficient evidence from well-controlled studies to make recommendations to patients on choice of product, best method of administration, or cannabinoid profile. Moreover, most health-care providers, including those working through the Veteran Affairs Healthcare System, are prohibited from offering specific advice on choosing cannabinoid-based products to their patients. Veterans who choose to self-medicate with cannabis must “go it on their own” in choosing cannabinoid preparations. Moreover, the cost of cannabis must be entirely shouldered by the consumer, as no insurance reimbursement is available. This creates substantial barriers for most veterans in accessing cannabis to self-treat symptoms. To circumvent at least some of these barriers, veteran-focused cannabis collectives have begun to emerge across the US, offering free cannabis products to veterans with state approved cannabis cards. While a number of studies have documented characteristics of veteran cannabis users , these investigations have failed to address cannabis heterogeneity or the selection bias inherent in an environment with so many barriers to access. Findings may not generalize to the broader population of veterans who would initiate use if legal and financial barriers to access were reduced. The current study aimed to address some of these existing limitations by collecting data on cannabis preparation and administration preferences and potential substitution behavior in a sample of veterans with limited barriers to cannabis access.The current study collected data on cannabis preparation and administration preferences and drug substitution behavior in a sample of veterans with few barriers to cannabis access. There were several notable findings. First, participants reported a high degree of substitution behavior, particularly for alcohol. This finding is consistent with results of other surveys assessing medicinal cannabis use in veterans . Given that cannabis use is associated with a much lower dependency potential and risk of overdose compared to other substances with a risk of misuse , this finding could suggest a positive impact of reduced barriers to medicinal cannabis access among veterans. Meanwhile, nearly half the sample reported substituting cannabis for prescription medications. Cannabinoids tend to carry a significantly higher safety profile compared to opioids and several other controlled prescription medications . However, given the nature of this survey-based study, we do not know which specific medications these veterans were substituting cannabis for.