The top three unigenes representing the greatest number of ESTs encoded proteins related to metallothionein, RD22-like BURP domain-containing proteins, and chitin binding heve in-like proteins. All three of these proteins have functions related to biotic or abiotic stress responses.An analysis of biological function indicates that 27% of the unigenes encode proteins with metabolic activity. Unigenes with NCBI matches encoding proteins with unknown function comprise 14% of the total and another 28% are predicted to be involved in various cellular processes such as protein synthesis and protein degradation.The specific biochemical steps leading to THCA are pro posed to begin with a reaction involving a type III PKS enzyme that catalyses the synthesis of olivetolic acid from hexanoyl-CoA and three molecules of malonyl-CoA. Malonyl-CoA is derived from the carboxylation of acetyl-CoA. ESTs encoding acetyl-CoA carboxylase were identified. Hexanoyl-CoA could be produced by more than one pathway in the trichomes. One route to produce hexanoyl-CoA would involve the early termination of the fatty acid biosynthetic pathway, yielding hexanoyl-ACP. The hexanoyl moiety would then be transferred to CoA by the action of an ACP-CoA transacylase or it would be cleaved by the action of a thioesterase, yielding n-hexanol, which would then be converted into n-hexanoyl-CoA by the action of acyl-CoA synthase.
Most of the enzymes needed for this route are represented in the EST database,macetas de 20 litros except for thetransacylase and 2,3-trans-enoyl-ACP reductase. A second route to hexanoyl-CoA would involve the pro duction of hexanol from the breakdown of the fatty acid linoleic acid via the lipoxygenase pathway. A survey of the sequenced ESTs revealed candidate genes encoding the enzymes needed to synthesize linoleic acid from acetyl-CoA by the typical fatty acid biosynthetic pathway in plastids followed by the production of hexanol from linoleic acid via the LOX pathway. An third pathway related to the biosynthesis of branched chain amino acids has been proposed to be involved in the production of short-chain and medium-chain fatty acids. However, the enzymes in this pathway [2-isopropylmalate synthase, 3-isopropylmalate dehydratase, 3-isopropylmalate dehydrogenase, and 2- oxoisovalerate dehydrogenase ] were not repre sented in the Cannabis trichome EST library. After the formation of olivetolic acid, a prenyltransferase is proposed to add a prenyl group derived from geranyl diphosphate to create cannabigerolic acid. GPP is derived from the fusion of two isoprene units. Two different biochemical pathways support the synthesis of isoprenoids in plants. Within the list of unigenes all but one of the enzymatic activities needed to convert pyruvate and glycer aldehyde-3-phosphate into isopentenyl and dimethylallyl diphosphate via the methylerythritol 4-phosphate pathway were represented. This finding is consistent with isotopic studies showing that the GPP cannabinoid precursors are synthesized via this pathway.
The formation of GPP is mediated by GPP synthase. Several unigenes related to GPP synthase were identified , however, they were more closely related to other terpene synthases. In particular, CAN36 and CAN55, which possibly were de rived from the same gene, and the closely related CAN37, are most similar to hop sesquiterpene synthases HISTS1 and HISTS2 , with an average identity of 56% over the first 160 amino acid residues. CAN41 is most similar to hop monoterpene synthase HIMTS2.The nature of the prenyltransferase is unknown. How ever, previous studies identified a soluble aromatic geranyl pyrophosphate:olivetolate geranyltransferase in the extract of young leaves with the appropriate activity. The only EST encoding a predicted prenyltransferase was CAN121. However, the encoded protein is more closely related to members of the mem brane-bound chloroplast-localized family of prenyltrans ferases than to soluble prenyltransferases. The final step in the pathway is mediated by THCA synthase, which mediates the conversion of cannabigerolic acid to THCA. Two ESTs with sequences identical to the previous reportedly THCA synthase were identified.Whereas the nature of the prenyltransferase responsible for the synthesis of cannabigerolic acid is unknown, three unigenes, CAN24, CAN383, and CAN1069, comprising eight, one, and two ESTs, respectively, could encode the PKS activity needed to synthesize olivetolic acid. These were therefore characterized in more detail. All three unigenes were represented by individual ESTs encoding complete PKS polypetides. These were sequenced and compared to related PKS sequences. CAN1069 was identical to a previously identified Cannabis gene encoding a chalcone synthase, and is the most closely related of the PKS sequences to other known chalcone synthases from hop and Arabidopsis. The relationships of hop phlorisovalerophenone synthase , which mediates the conversion of malonyl-CoA and isovaleryl-CoA to phlorisovalerophenone, to CAN24 and CAN383 is less clear. CAN24 and CAN383 show 64.6% identity and are nearly equally similar to hop VPS at 71.2% and 72.0%, respectively. The enzymatic activities encoded by CAN24 and CAN1069 were explored in detail. The coding regions of the two genes were inserted into the pHis8 vector in frame with a His8 tag.
The tagged proteins were purified on a nickel-containing magnetic bead matrix and were assayed for chalcone and olivetol/olivetolic acid synthase activities. Recombinant protein from CAN1069, but not CAN24, produced reaction products when incubated with coumaroyl-CoA and malonyl-CoA. The reaction products were analysed by LC-MS and peak 2 was found to have a molecular mass and absorption spectrum consistent with naringenin , the major product of chalcone synthases. Both CAN24 and CAN1069 were capable of using malonyl-CoA and hexanoyl-CoA as re action substrates and LC-MS indicated that products of these enzymes were the same,maceta 5 litros but neither molecular mass nor the absorption spectrum of this product matched olivetol or olivetolic acid. Results similar to CAN24 were obtained using protein purified from CAN383.Genes required for THCA production are probably more highly expressed in glands of pistillate inflorescences be cause this is where THCA is most highly concentrated. To test this hypothesis, the relative expression levels in isolated glands versus young inflorescence-associated leaves of selected unigenes were compared using real-time qPCR. The identity of the genes assayed and the differences in relative expression levels are listed in Table 2 and in Supplementary Table 4A at JXB online. Consistent with this hypothesis, THCA synthase expression was 437 times higher in isolated glands than in leaves. CAN24 was expressed 1600 times higher in glands of the inflores cence than in associated leaves. CAN1069 encoding CHS was also more highly expressed in glands than leaves. The expression of a third PKS, CAN383, was expressed at similar levels in glands and leaves. These results are not explained by poor RNA isolation from leaves as unigene CAN219 encoding chlorophyll A/B binding protein showed elevated leaf expression levels. 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 re quired 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 N terminal signal peptide, 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 heve in 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 pro duce these compounds have been identified.Synthesis of olivetolic acid from malonyl-CoA and hex anoyl-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 olive tolic 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.