Arabidopsis RAD16 Homologues Are Involved in UV Tolerance and Growth.

In plants, prolonged exposure to ultraviolet (UV) radiation causes harmful DNA lesions. Nucleotide excision repair (NER) is an important DNA repair mechanism that operates via two pathways: transcription coupled repair (TC-NER) and global genomic repair (GG-NER). In plants and mammals, TC-NER is initiated by the Cockayne Syndrome A and B (CSA/CSB) complex, whereas GG-NER is initiated by the Damaged DNA Binding protein 1/2 (DDB1/2) complex. In the yeast Saccharomyces cerevisiae (S. cerevisiae), GG-NER is initiated by the Radiation Sensitive 7 and 16, (RAD7/16) complex. Arabidopsis thaliana has two homologues of yeast RAD16, At1g05120 and At1g02670, which we named AtRAD16 and AtRAD16b, respectively. In this study, we characterized the roles of AtRAD16 and AtRAD16b. Arabidopsis rad16 and rad16b null mutants exhibited increased UV sensitivity. Moreover, AtRAD16 overexpression increased plant UV tolerance. Thus, AtRAD16 and AtRAD16b contribute to plant UV tolerance and growth. Additionally, we found physical interaction between AtRAD16 and AtRAD7. Thus, the Arabidopsis RAD7/16 complex is functional in plant NER. Furthermore, AtRAD16 makes a significant contribution to Arabidopsis UV tolerance compared to the DDB1/2 and the CSB pathways. This is the first time the role and interaction of DDB1/2, RAD7/16, and CSA/CSB components in a single system have been studied.

Density-Dependent Prophylaxis in Freshwater Snails Driven by Oxylipin Chemical Cues.

While animal aggregations can benefit the fitness of group members, the behaviour may also lead to higher risks of parasite infection as group density increases. Some animals are known to moderate their investment in immunity relative to the risk of infection. These animals exhibit density-dependent prophylaxis (DDP) by increasing their immune investment as group density increases. Despite being documented in many taxa, the mechanisms of DDP remain largely unexplored. Snails are known to aggregate and experience large fluctuations in density and serve as required hosts for many parasites. Further, they are known to use chemical cues to aggregate. To test whether freshwater snails exhibit DDP and investigate the role that chemical signaling compounds may play in triggering this phenomenon, we performed four experiments on the freshwater snail Stagnicola elodes, which is a common host for many trematode parasite species. First, we tested if DDP occurred in snails in laboratory-controlled conditions (control vs snail-conditioned water) and whether differences in exposure to chemical cues affected immune function. Second, we used gas chromatography to characterize fatty acids expressed in snail-conditioned water to determine if precursors for particular signaling molecules, such as oxylipins, were being produced by snails. Third, we characterized the oxylipins released by infected and uninfected field-collected snails, to better understand how differences in oxylipin cocktails may play a role in inducing DDP. Finally, we tested the immune response of snails exposed to four oxylipins to test the ability of specific oxylipins to affect DDP. We found that snails exposed to water with higher densities of snails and raised in snail-conditioned water had higher counts of haemocytes. Additionally, lipid analysis demonstrated that fatty acid molecules that are also precursors for oxylipins were present in snail-conditioned water. Trematode-infected snails emitted 50 oxylipins in higher amounts, with 24 of these oxylipins only detected in this group. Finally, oxylipins that were higher in infected snails induced naïve snails to increase their immune responses compared to sham-exposed snails. Our results provide evidence that snails exhibit DDP, and the changes in oxylipins emitted by infected hosts may be one of the molecular mechanisms driving this phenomenon.

Evolutionary and biochemical characterization of a Chromochloris zofingiensis MBOAT with wax synthase and diacylglycerol acyltransferase activity.

Wax synthase (WS) catalyzes the last step in wax ester biosynthesis in green plants. Two unrelated sub-families of WS, including the bifunctional acyltransferase and plant-like WS have been reported, but the latter is largely uncharacterized in microalgae. Here, we functionally characterized a putative plant-like WS (CzWS1) from the emerging model green microalga Chromochloris zofingiensis. Our results showed that plant-like WS evolved under different selection constraints in plants and microalgae, with positive selection likely contributing to functional divergence. Unlike jojoba with high amounts of wax ester in seeds and a highly active WS enzyme, C. zofingiensis has no detectable wax ester but a high abundance of WS transcripts. Co-expression analysis showed that C. zofingiensis WS has different expression correlation with lipid biosynthetic genes from jojoba, and may have a divergent function. In vitro characterization indicated that CzWS1 had diacylglycerol acyltransferase activity along with WS activity, and overexpression of CzWS1 in yeast and Chlamydomonas reinhardtii affected triacylglycerol accumulation. Moreover, biochemical and bioinformatic analyses revealed the relevance of the C-terminal region of CzWS1 in enzyme function. Taken together, our results indicated a functional divergence of plant-like WS in plants and microalgae, and the importance of its C-terminal region in specialization of enzyme function.

Validated quantitative cannabis profiling for Canadian regulatory compliance - Cannabinoids, aflatoxins, and terpenes.

In response to the Canadian federal government's Cannabis Tracking and Licensing System compliance standards, a quantitative method was created for cannabis analysis, and validated using Eurachem V.2 (2014) guidelines. Cannabinol, cannabidiol, cannabigerol, cannabichromene, cannabidiolic acid, cannabigerolic acid, Δ-9-tetrahydrocannabinol, and Δ-9-tetrahydrocannabinolic acid A were all analysed by scheduled multiple reaction monitoring (MRM) via LC-MS/MS and isotope dilution. In addition, aflatoxins B1, B2, G1, and G2 were also analysed by scheduled MRM via LC-MS/MS and matrix matched calibration curves in order to achieve the reporting limits (≤2 µg kg-1) set out by the European Pharmacopoeia. The LODs/LOQs were 0.50/1.7, 2.0/6.7, 0.59/2.0, and 0.53/1.8 µg kg-1, for B1, B2, G1, and G2 respectively. Thirty one terpenes were analysed by selected reaction monitoring via GC-MS/MS and isotope dilution using ß-myrcene-d6 as a surrogate. All quantitative analyses can be accomplished using less than 1 g of material, with minimal solvent and consumable use, on low resolution instruments in less than 30 min of instrument time. Of important note is this method's power of selectivity, working ranges, and lack of need for extraction consumables such as SPE or QuEChERS, thereby minimising analytical costs and time.

A physical and genetic map of Cannabis sativa identifies extensive rearrangements at the THC/CBD acid synthase loci.

Cannabis sativa is widely cultivated for medicinal, food, industrial, and recreational use, but much remains unknown regarding its genetics, including the molecular determinants of cannabinoid content. Here, we describe a combined physical and genetic map derived from a cross between the drug-type strain Purple Kush and the hemp variety "Finola." The map reveals that cannabinoid biosynthesis genes are generally unlinked but that aromatic prenyltransferase (AP), which produces the substrate for THCA and CBDA synthases (THCAS and CBDAS), is tightly linked to a known marker for total cannabinoid content. We further identify the gene encoding CBCA synthase (CBCAS) and characterize its catalytic activity, providing insight into how cannabinoid diversity arises in cannabis. THCAS and CBDAS (which determine the drug vs. hemp chemotype) are contained within large (>250 kb) retrotransposon-rich regions that are highly nonhomologous between drug- and hemp-type alleles and are furthermore embedded within ∼40 Mb of minimally recombining repetitive DNA. The chromosome structures are similar to those in grains such as wheat, with recombination focused in gene-rich, repeat-depleted regions near chromosome ends. The physical and genetic map should facilitate further dissection of genetic and molecular mechanisms in this commercially and medically important plant.

Mediator Complex Subunits MED2, MED5, MED16, and MED23 Genetically Interact in the Regulation of Phenylpropanoid Biosynthesis.

The phenylpropanoid pathway is a major global carbon sink and is important for plant fitness and the engineering of bioenergy feedstocks. In Arabidopsis thaliana, disruption of two subunits of the transcriptional regulatory Mediator complex, MED5a and MED5b, results in an increase in phenylpropanoid accumulation. By contrast, the semidominant MED5b mutation reduced epidermal fluorescence4-3 (ref4-3) results in dwarfism and constitutively repressed phenylpropanoid accumulation. Here, we report the results of a forward genetic screen for suppressors of ref4-3. We identified 13 independent lines that restore growth and/or phenylpropanoid accumulation in the ref4-3 background. Two of the suppressors restore growth without restoring soluble phenylpropanoid accumulation, indicating that the growth and metabolic phenotypes of the ref4-3 mutant can be genetically disentangled. Whole-genome sequencing revealed that all but one of the suppressors carry mutations in MED5b or other Mediator subunits. RNA-seq analysis showed that the ref4-3 mutation causes widespread changes in gene expression, including the upregulation of negative regulators of the phenylpropanoid pathway, and that the suppressors reverse many of these changes. Together, our data highlight the interdependence of individual Mediator subunits and provide greater insight into the transcriptional regulation of phenylpropanoid biosynthesis by the Mediator complex.

The Genetic Structure of Marijuana and Hemp.

Despite its cultivation as a source of food, fibre and medicine, and its global status as the most used illicit drug, the genus Cannabis has an inconclusive taxonomic organization and evolutionary history. Drug types of Cannabis (marijuana), which contain high amounts of the psychoactive cannabinoid Δ9-tetrahydrocannabinol (THC), are used for medical purposes and as a recreational drug. Hemp types are grown for the production of seed and fibre, and contain low amounts of THC. Two species or gene pools (C. sativa and C. indica) are widely used in describing the pedigree or appearance of cultivated Cannabis plants. Using 14,031 single-nucleotide polymorphisms (SNPs) genotyped in 81 marijuana and 43 hemp samples, we show that marijuana and hemp are significantly differentiated at a genome-wide level, demonstrating that the distinction between these populations is not limited to genes underlying THC production. We find a moderate correlation between the genetic structure of marijuana strains and their reported C. sativa and C. indica ancestry and show that marijuana strain names often do not reflect a meaningful genetic identity. We also provide evidence that hemp is genetically more similar to C. indica type marijuana than to C. sativa strains.

Separation of isomeric short-chain acyl-CoAs in plant matrices using ultra-performance liquid chromatography coupled with tandem mass spectrometry.

Acyl coenzyme A (acyl-CoA) thioesters are important intermediates in cellular metabolism and being able to distinguish among them is critical to fully understanding metabolic pathways in plants. Although significant advances have been made in the identification and quantification of acyl-CoAs using liquid chromatography tandem mass spectrometry (LC-MS/MS), separation of isomeric species such as isobutyryl- and n-butyrl-CoA has remained elusive. Here we report an ultra-performance liquid chromatography (UPLC)-MS/MS method for quantifying short-chain acyl-CoAs including isomeric species n-butyryl-CoA and isobutyryl-CoA as well as n-valeryl-CoA and isovaleryl-CoA. The method was applied to the analysis of extracts of hop (Humulus lupulus) and provided strong evidence for the existence of an additional structural isomer of valeryl-CoA, 2-methylbutyryl-CoA, as well as an unexpected isomer of hexanoyl-CoA. The results showed differences in the acyl-CoA composition among varieties of Humulus lupulus, both in glandular trichomes and cone tissues. When compared with the analysis of hemp (Cannabis sativa) extracts, the contribution of isobutyryl-CoAs in hop was greater as would be expected based on the downstream polyketide products. Surprisingly, branched chain valeryl-CoAs (isovaleryl-CoA and 2-methylbutyryl-CoA) were the dominant form of valeryl-CoAs in both hop and hemp. The capability to separate these isomeric forms will help to understand biochemical pathways leading to specialized metabolites in plants.

Transcriptome analysis based on next-generation sequencing of non-model plants producing specialized metabolites of biotechnological interest.

Plants produce a vast array of specialized metabolites, many of which are used as pharmaceuticals, flavors, fragrances, and other high-value fine chemicals. However, most of these compounds occur in non-model plants for which genomic sequence information is not yet available. The production of a large amount of nucleotide sequence data using next-generation technologies is now relatively fast and cost-effective, especially when using the latest Roche-454 and Illumina sequencers with enhanced base-calling accuracy. To investigate specialized metabolite biosynthesis in non-model plants we have established a data-mining framework, employing next-generation sequencing and computational algorithms, to construct and analyze the transcriptomes of 75 non-model plants that produce compounds of interest for biotechnological applications. After sequence assembly an extensive annotation approach was applied to assign functional information to over 800,000 putative transcripts. The annotation is based on direct searches against public databases, including RefSeq and InterPro. Gene Ontology (GO), Enzyme Commission (EC) annotations and associated Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway maps are also collected. As a proof-of-concept, the selection of biosynthetic gene candidates associated with six specialized metabolic pathways is described. A web-based BLAST server has been established to allow public access to assembled transcriptome databases for all 75 plant species of the PhytoMetaSyn Project (www.phytometasyn.ca).

Identification of olivetolic acid cyclase from Cannabis sativa reveals a unique catalytic route to plant polyketides.

Δ(9)-Tetrahydrocannabinol (THC) and other cannabinoids are responsible for the psychoactive and medicinal properties of Cannabis sativa L. (marijuana). The first intermediate in the cannabinoid biosynthetic pathway is proposed to be olivetolic acid (OA), an alkylresorcinolic acid that forms the polyketide nucleus of the cannabinoids. OA has been postulated to be synthesized by a type III polyketide synthase (PKS) enzyme, but so far type III PKSs from cannabis have been shown to produce catalytic byproducts instead of OA. We analyzed the transcriptome of glandular trichomes from female cannabis flowers, which are the primary site of cannabinoid biosynthesis, and searched for polyketide cyclase-like enzymes that could assist in OA cyclization. Here, we show that a type III PKS (tetraketide synthase) from cannabis trichomes requires the presence of a polyketide cyclase enzyme, olivetolic acid cyclase (OAC), which catalyzes a C2-C7 intramolecular aldol condensation with carboxylate retention to form OA. OAC is a dimeric α+ß barrel (DABB) protein that is structurally similar to polyketide cyclases from Streptomyces species. OAC transcript is present at high levels in glandular trichomes, an expression profile that parallels other cannabinoid pathway enzymes. Our identification of OAC both clarifies the cannabinoid pathway and demonstrates unexpected evolutionary parallels between polyketide biosynthesis in plants and bacteria. In addition, the widespread occurrence of DABB proteins in plants suggests that polyketide cyclases may play an overlooked role in generating plant chemical diversity.

The hexanoyl-CoA precursor for cannabinoid biosynthesis is formed by an acyl-activating enzyme in Cannabis sativa trichomes.

The psychoactive and analgesic cannabinoids (e.g. Δ(9) -tetrahydrocannabinol (THC)) in Cannabis sativa are formed from the short-chain fatty acyl-coenzyme A (CoA) precursor hexanoyl-CoA. Cannabinoids are synthesized in glandular trichomes present mainly on female flowers. We quantified hexanoyl-CoA using LC-MS/MS and found levels of 15.5 pmol g(-1) fresh weight in female hemp flowers with lower amounts in leaves, stems and roots. This pattern parallels the accumulation of the end-product cannabinoid, cannabidiolic acid (CBDA). To search for the acyl-activating enzyme (AAE) that synthesizes hexanoyl-CoA from hexanoate, we analyzed the transcriptome of isolated glandular trichomes. We identified 11 unigenes that encoded putative AAEs including CsAAE1, which shows high transcript abundance in glandular trichomes. In vitro assays showed that recombinant CsAAE1 activates hexanoate and other short- and medium-chained fatty acids. This activity and the trichome-specific expression of CsAAE1 suggest that it is the hexanoyl-CoA synthetase that supplies the cannabinoid pathway. CsAAE3 encodes a peroxisomal enzyme that activates a variety of fatty acid substrates including hexanoate. Although phylogenetic analysis showed that CsAAE1 groups with peroxisomal AAEs, it lacked a peroxisome targeting sequence 1 (PTS1) and localized to the cytoplasm. We suggest that CsAAE1 may have been recruited to the cannabinoid pathway through the loss of its PTS1, thereby redirecting it to the cytoplasm. To probe the origin of hexanoate, we analyzed the trichome expressed sequence tag (EST) dataset for enzymes of fatty acid metabolism. The high abundance of transcripts that encode desaturases and a lipoxygenase suggests that hexanoate may be formed through a pathway that involves the oxygenation and breakdown of unsaturated fatty acids.

REF4 and RFR1, subunits of the transcriptional coregulatory complex mediator, are required for phenylpropanoid homeostasis in Arabidopsis.

The plant phenylpropanoid pathway produces an array of metabolites that impact human health and the utility of feed and fiber crops. We previously characterized several Arabidopsis thaliana mutants with dominant mutations in REDUCED EPIDERMAL FLUORESCENCE 4 (REF4) that cause dwarfing and decreased accumulation of phenylpropanoids. In contrast, ref4 null plants are of normal stature and have no apparent defect in phenylpropanoid biosynthesis. Here we show that disruption of both REF4 and its paralog, REF4-RELATED 1 (RFR1), results in enhanced expression of multiple phenylpropanoid biosynthetic genes, as well as increased accumulation of numerous downstream products. We also show that the dominant ref4-3 mutant protein interferes with the ability of the PAP1/MYB75 transcription factor to induce the expression of PAL1 and drive anthocyanin accumulation. Consistent with our experimental results, both REF4 and RFR1 have been shown to physically associate with the conserved transcriptional coregulatory complex, Mediator, which transduces information from cis-acting DNA elements to RNA polymerase II at the core promoter. Taken together, our data provide critical genetic support for a functional role of REF4 and RFR1 in the Mediator complex, and for Mediator in the maintenance of phenylpropanoid homeostasis. Finally, we show that wild-type RFR1 substantially mitigates the phenotype of the dominant ref4-3 mutant, suggesting that REF4 and RFR1 may compete with one another for common binding partners or for occupancy in Mediator. Determining the functions of diverse Mediator subunits is essential to understand eukaryotic gene regulation, and to facilitate rational manipulation of plant metabolic pathways to better suit human needs.

The draft genome and transcriptome of Cannabis sativa.

BACKGROUND: Cannabis sativa has been cultivated throughout human history as a source of fiber, oil and food, and for its medicinal and intoxicating properties. Selective breeding has produced cannabis plants for specific uses, including high-potency marijuana strains and hemp cultivars for fiber and seed production. The molecular biology underlying cannabinoid biosynthesis and other traits of interest is largely unexplored. RESULTS: We sequenced genomic DNA and RNA from the marijuana strain Purple Kush using shortread approaches. We report a draft haploid genome sequence of 534 Mb and a transcriptome of 30,000 genes. Comparison of the transcriptome of Purple Kush with that of the hemp cultivar 'Finola' revealed that many genes encoding proteins involved in cannabinoid and precursor pathways are more highly expressed in Purple Kush than in 'Finola'. The exclusive occurrence of Δ9-tetrahydrocannabinolic acid synthase in the Purple Kush transcriptome, and its replacement by cannabidiolic acid synthase in 'Finola', may explain why the psychoactive cannabinoid Δ9-tetrahydrocannabinol (THC) is produced in marijuana but not in hemp. Resequencing the hemp cultivars 'Finola' and 'USO-31' showed little difference in gene copy numbers of cannabinoid pathway enzymes. However, single nucleotide variant analysis uncovered a relatively high level of variation among four cannabis types, and supported a separation of marijuana and hemp. CONCLUSIONS: The availability of the Cannabis sativa genome enables the study of a multifunctional plant that occupies a unique role in human culture. Its availability will aid the development of therapeutic marijuana strains with tailored cannabinoid profiles and provide a basis for the breeding of hemp with improved agronomic characteristics.

Mutations in the cinnamate 4-hydroxylase gene impact metabolism, growth and development in Arabidopsis.

The initial reactions of the phenylpropanoid pathway convert phenylalanine to p-coumaroyl CoA, a branch point metabolite from which many phenylpropanoids are made. Although the second enzyme of this pathway, cinnamic acid 4-hydroxylase (C4H), is well characterized, a mutant for the gene encoding this enzyme has not yet, to our knowledge, been identified, presumably because knock-out mutations in this gene would have severe phenotypes. This work describes the characterization of an allelic series of Arabidopsis reduced epidermal fluorescence 3 (ref3) mutants, each of which harbor mis-sense mutations in C4H (At2g30490). Heterologous expression of the mutant proteins in Escherichia coli yields enzymes that exhibit P420 spectra, indicative of mis-folded proteins, or have limited ability to bind substrate, indicating that the mutations we have identified affect protein stability and/or enzyme function. In agreement with the early position of C4H in phenylpropanoid metabolism, ref3 mutant plants accumulate decreased levels of several different classes of phenylpropanoid end-products, and exhibit reduced lignin deposition and altered lignin monomer content. Furthermore, these plants accumulate a novel hydroxycinnamic ester, cinnamoylmalate, which is not found in the wild type. The decreased C4H activity in ref3 also causes pleiotropic phenotypes, including dwarfism, male sterility and the development of swellings at branch junctions. Together, these observations indicate that C4H function is critical to the normal biochemistry and development of Arabidopsis.

Independent origins of syringyl lignin in vascular plants.

Lycophytes arose in the early Silurian ( approximately 400 Mya) and represent a major lineage of vascular plants that has evolved in parallel with the ferns, gymnosperms, and angiosperms. A hallmark of vascular plants is the presence of the phenolic lignin heteropolymer in xylem and other sclerified cell types. Although syringyl lignin is often considered to be restricted in angiosperms, it has been detected in lycophytes as well. Here we report the characterization of a cytochrome P450-dependent monooxygenase from the lycophyte Selaginella moellendorffii. Gene expression data, cross-species complementation experiments, and in vitro enzyme assays indicate that this P450 is a ferulic acid/coniferaldehyde/coniferyl alcohol 5-hydroxylase (F5H), and is capable of diverting guaiacyl-substituted intermediates into syringyl lignin biosynthesis. Phylogenetic analysis indicates that the Selaginella F5H represents a new family of plant P450s and suggests that it has evolved independently of angiosperm F5Hs.

Semidominant mutations in reduced epidermal fluorescence 4 reduce phenylpropanoid content in Arabidopsis.

Plants synthesize an array of natural products that play diverse roles in growth, development, and defense. The plant-specific phenylpropanoid metabolic pathway produces as some of its major products flavonoids, monolignols, and hydroxycinnamic- acid conjugates. The reduced epidermal fluorescence 4 (ref4) mutant is partially dwarfed and accumulates reduced quantities of all phenylpropanoid-pathway end products. Further, plants heterozygous for ref4 exhibit intermediate growth and phenylpropanoid-related phenotypes, suggesting that these mutations are semidominant. The REF4 locus (At2g48110) was cloned by a combined map- and sequencing-based approach and was found to encode a large integral membrane protein that is unique to plants. The mutations in all ref4 alleles cause substitutions in conserved amino acids that are located adjacent to predicted transmembrane regions. Expression of the ref4-3 allele in wild-type and null REF4 plants caused reductions in sinapoylmalate content, lignin content, and growth, demonstrating that the mutant alleles are truly semidominant. Further, a suppressor mutant was isolated that abolishes a WW protein-protein interaction domain that may be important for REF4 function.

The hyper-fluorescent trichome phenotype of the brt1 mutant of Arabidopsis is the result of a defect in a sinapic acid: UDPG glucosyltransferase.

Sinapoylmalate is a major phenylpropanoid that is accumulated in Arabidopsis. Its presence causes the adaxial surface of leaves to fluoresce blue under UV light, and mutations that lead to lower levels of sinapoylmalate decrease UV-induced leaf fluorescence. The Arabidopsis bright trichomes 1 (brt1) mutant was first identified in a screen for mutants that exhibit a reduced epidermal fluorescence phenotype; however, subsequent examination of the mutant revealed that its trichomes are hyper-fluorescent. The results from genetic mapping and complementation analyses showed that BRT1 (At3g21560) encodes UGT84A2, a glucosyltransferase previously shown to be capable of using sinapic acid as a substrate. Residual levels of sinapoylmalate and sinapic acid:UDP-glucose glucosyltransferase activity in brt1 leaves suggest that BRT1 is one member of a family of partially redundant glycosyltransferases that function in Arabidopsis sinapate ester biosynthesis. RT-PCR analysis showed that BRT1 is expressed through all stages of plant life cycle, a result consistent with the impact of the brt1 mutation on both leaf sinapoylmalate levels and seed sinapoylcholine content. Finally, the compound accumulated in brt1 trichomes was identified as a sinapic acid-derived polyketide, indicating that when sinapic acid glycosylation is reduced, a portion of it is instead activated to its CoA thioester, which then serves as a substrate for chalcone synthase.