The activities of several housekeeping genes were also tested. A relatively modest increase in levels of histone H2A and beta tubulin expression in glands compared to leaves was detected. The increase in expression levels of these latter two genes might reflect a combination of the heightened metabolic activity and the unique cellular structure of glandular trichomes. Two different pathways could provide the hexanol required for olivetolic acid synthesis, as shown in Fig. 2. Expression levels provide support for the de novo pathway as a primary source, given that CAN498, CAN82, and CAN915 were much more highly expressed in glands than leaves , whereas the relative expression of genes encoding enzymes in the lipid breakdown pathway were depressed or modestly elevated in glands.The identities of the most abundant ESTs derived from the glandular trichomes of Cannabis sativa are consistent with the protective function of plant glands. For example, the most abundant ESTs encoded a protein closely related to type II metallothioneins. These proteins bind heavy metals such as Cd, Zn, and Cu, and their proposed primary function is the maintenance of Cu tolerance . The second most abundant class of ESTs encoded an RD22-like BURP domain containing protein. This class of proteins contains a hydrophobic Nterminal signal peptide,hydroponic grow systems and an N-terminal conserved region followed by a series of small repeats .
The BURP domain of approximately 230 amino acids is located in the C-terminal region. The function of RD22-like proteins is unknown but some members of this class of genes are induced by dehydration . The third most abundant ESTs encoded a protein containing a hevein domain. Hevein domains contain a conserved 43-amino acid motif that binds chitin and members of this protein class are known for anti-fungal activity . The unique secondary metabolism in Cannabis may also play a role in plant defence. Synthesis of THCA is extracellular and results in hydrogen peroxide production, which has general antimicrobial properties , and a recent report further indicates that THCA may directly inhibit microbial growth . The analysis of gland-derived ESTs has identified nearly all the candidate genes required for THCA synthesis from primary metabolic products. 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, 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,vertical grow table 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.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.Urbanization is a major driver of land cover change worldwide and affects the biophysical and socioeconomic landscape. It is estimated that by 2030 >60% of the global population will live in urban areas . Furthermore, in many parts of the world, human development is expanding rapidly at the edge of urban areas and the quality of rural habitat is declining owing to agricultural intensification . Thus, green spaces found within urban landscapes are quickly becoming important refuges for native biodiversity . Urban planners are increasingly interested in maintaining agriculture within and around cities due to food security concerns. Many cities contain ‘food deserts’, where access to fresh produce is limited due to reduced proximity to markets, financial constraints, or inadequate transportation . In response to food insecurity, urban agriculture has expanded rapidly. For example, in the US, UA has expanded by >30% in the past 30 years, especially in under-served communities . This is because urban agriculture can be very productive, providing an estimated 15–20% of the global food supply , and cities can provide good infrastructure, access to labor, and low transport costs for local food distribution . Additionally, interest in UA has escalated recently due to the desire to transform vacant land in post-industrial cities and to address nutrition and childhood obesity issues in disadvantaged urban neighborhoods . Though public and Scientific interest in UA has grown dramatically in the past two decades, there are still significant challenges for integrating UA in an increasingly spatially constrained urban landscape. Much of the debate is centered on land-use trade-offs of UA versus other types of urban development. Although there are a number of socioeconomic considerations that affect the development and proliferation of UA in cities, this review will focus on the ecological aspects of the UA system and how they can be designed to maximize the environmental and health benefits in order to increase acceptance of this particular land use in the urban sphere. One way to encourage the integration of UA is to better understand how planned and associated biodiversity within these systems contribute to urban ecosystem services. However, there are three major gaps in the literature regarding UA status and impacts that limit our ability to increase the range of benefits and ecosystem services that could come from UA systems. First, biodiversity patterns in urban agroecosystems have only recently been documented and require synthesis. Second, ecological communities within UA may translate to the delivery of valuable ecosystem services ; however, the availability of these services within UA has not been well-established. Finally, little is known about the role of UA in mediating resilience to major threats,specifically climate variability .