Stephen J. Ambrose

A New Abscisic Acid Catabolic Pathway

We report the discovery of a new hydroxylated abscisic acid (ABA) metabolite, found in the course of a mass spectrometric study of ABA metabolism in Brassica napus siliques. This metabolite reveals a previously unknown catabolic pathway for ABA in which the 9′-methyl group of ABA is oxidized. Analogs of (+)-ABA deuterated at the 8′-carbon atom and at both the 8′- and 9′-carbon atoms were fed to green siliques, and extracts containing the deuterated oxidized metabolites were analyzed to determine the position of ABA hydroxylation. The results indicated that hydroxylation of ABA had occurred at the 9′-methyl group, as well as at the 7′- and 8′-methyl groups. The chromatographic characteristics and mass spectral fragmentation patterns of the new ABA metabolite were compared with those of synthetic 9′-hydroxy ABA (9′-OH ABA), in both open and cyclized forms. The new compound isolated from plant extracts was identified as the cyclized form of 9′-OH ABA, which we have named neophaseic acid (neoPA). The proton nuclear magnetic resonance spectrum of pure neoPA isolated from immature seeds of B. napus was identical to that of the authentic synthetic compound. ABA and neoPA levels were high in young seeds and lower in older seeds. The open form (2Z,4E)-5-[(1R,6S)-1-Hydroxy-6-hydroxymethyl-2,6-dimethyl-4-oxo-cyclohex-2-enyl]-3-methyl-penta-2,4-dienoic acid, but not neoPA, exhibited ABA-like bioactivity in inhibiting Arabidopsis seed germination and in inducing gene expression in B. napus microspore-derived embryos. NeoPA was also detected in fruits of orange (Citrus sinensis) and tomato (Lycopersicon esculentum), in Arabidopsis, and in chickpea (Cicer arietinum), as well as in drought-stressed barley (Hordeum vulgare) and B. napus seedlings.

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.

Transcriptome analysis of bitter acid biosynthesis and precursor pathways in hop (Humulus lupulus)

BackgroundBitter acids (e.g. humulone) are prenylated polyketides synthesized in lupulin glands of the hop plant (Humulus lupulus) which are important contributors to the bitter flavour and stability of beer. Bitter acids are formed from acyl-CoA precursors derived from branched-chain amino acid (BCAA) degradation and C5 prenyl diphosphates from the methyl-D-erythritol 4-phosphate (MEP) pathway. We used RNA sequencing (RNA-seq) to obtain the transcriptomes of isolated lupulin glands, cones with glands removed and leaves from high α-acid hop cultivars, and analyzed these datasets for genes involved in bitter acid biosynthesis including the supply of major precursors. We also measured the levels of BCAAs, acyl-CoA intermediates, and bitter acids in glands, cones and leaves.ResultsTranscripts encoding all the enzymes of BCAA metabolism were significantly more abundant in lupulin glands, indicating that BCAA biosynthesis and subsequent degradation occurs in these specialized cells. Branched-chain acyl-CoAs and bitter acids were present at higher levels in glands compared with leaves and cones. RNA-seq analysis showed the gland-specific expression of the MEP pathway, enzymes of sucrose degradation and several transcription factors that may regulate bitter acid biosynthesis in glands. Two branched-chain aminotransferase (BCAT) enzymes, HlBCAT1 and HlBCAT2, were abundant, with gene expression quantification by RNA-seq and qRT-PCR indicating that HlBCAT1 was specific to glands while HlBCAT2 was present in glands, cones and leaves. Recombinant HlBCAT1 and HlBCAT2 catalyzed forward (biosynthetic) and reverse (catabolic) reactions with similar kinetic parameters. HlBCAT1 is targeted to mitochondria where it likely plays a role in BCAA catabolism. HlBCAT2 is a plastidial enzyme likely involved in BCAA biosynthesis. Phylogenetic analysis of the hop BCATs and those from other plants showed that they group into distinct biosynthetic (plastidial) and catabolic (mitochondrial) clades.ConclusionsOur analysis of the hop transcriptome significantly expands the genomic resources available for this agriculturally-important crop. This study provides evidence for the lupulin gland-specific biosynthesis of BCAAs and prenyl diphosphates to provide precursors for the production of bitter acids. The biosynthetic pathway leading to BCAAs in lupulin glands involves the plastidial enzyme, HlBCAT2. The mitochondrial enzyme HlBCAT1 degrades BCAAs as the first step in the catabolic pathway leading to branched chain-acyl-CoAs.

Disrupting Abscisic Acid Homeostasis in Western White Pine (Pinus monticola Dougl. Ex D. Don) Seeds Induces Dormancy Termination and Changes in Abscisic Acid Catabolites

To investigate the role of abscisic acid (ABA) biosynthesis and catabolism in dormant imbibed seeds of western white pine (Pinus monticola), ABA and selected catabolites were measured during a combined treatment of the ABA biosynthesis inhibitor fluridone, and gibberellic acid (GA). Fluridone in combination with GA effectively disrupted ABA homeostasis and replaced the approximately 90-day moist chilling period normally required to break dormancy in this species. Individually, both fluridone and GA treatments decreased ABA levels in the embryos and megagametophytes of white pine seeds compared to a water control; however, combined fluridone/GA treatment, the only treatment to terminate dormancy effectively, led to the greatest decline in ABA content. Fluridone treatments revealed that a high degree of ABA turnover/transport occurred in western white pine seeds during the initial stages of dormancy maintenance; at this time, ABA levels decreased by approximately two-thirds in both embryo and megagametophyte tissues. Gibberellic acid treatments, both alone and in combination with fluridone, suggested that GA acted transiently to disrupt ABA homeostasis by shifting the ratio between biosynthesis and catabolism to favor ABA catabolism or transport. Increases in phaseic acid (PA) and dihydrophaseic acid (DPA) were observed during fluridone/GA treatments; however, increases in ABA metabolites did not account for the reduction in ABA observed; additional catabolism and/or transport of ABA and selected metabolites in all probability accounts for this discrepancy. Finally, levels of 7′ hydroxy-ABA (7′OH-ABA) were higher in dormant-imbibed seeds, suggesting that metabolism through this pathway is increased in seeds that maintain higher levels of ABA, perhaps as a means to further regulate ABA homeostasis.

Using Gas Modifiers to Significantly Improve Sensitivity and Selectivity in a Cylindrical FAIMS Device

AbstractRecent reports describing enhanced performance when using gas additives in a DMS device (planar electrodes) have indicated that comparable benefits are not attainable using FAIMS (cylindrical electrodes), owing to the non-homogeneous electric fields within the analyzer region. In this study, a FAIMS system (having cylindrical electrodes) was modified to allow for controlled delivery of gas additives. An experiment was carried out that illustrates the important distinction between gas modifiers present as unregulated contaminants and modifiers added in a controlled manner. The effect of contamination was simulated by adjusting the ESI needle position to promote incomplete desolvation, thereby permitting ESI solvent vapor into the FAIMS analyzer region, causing signal instability and irreproducible CV values. However, by actively controlling the delivery of the gas modifier, reproducible CV spectra were obtained. The effects of adding different gas modifiers were examined using 15 positive ions having mass-to-charge (m/z) values between 90 and 734. Significant improvements in peak capacity and increases in ion transmission were readily attained by adding acetonitrile vapor, even at trace levels (≤0.1%). Increases in signal intensity were greatest for the low m/z ions; for the six lowest molecular weight species, signal intensities increased by ∼10- to over 100-fold compared with using nitrogen without gas additives, resulting in equivalent or better signal intensities compared with ESI without FAIMS. These results confirm that analytical benefits derived from the addition of gas modifiers reported with a uniform electric field (DMS) also are observed using a non-homogenous electric field (FAIMS) in the analyser region. Figureᅟ

Development of a fast extraction method and optimization of liquid chromatography-mass spectrometry for the analysis of phenolic compounds in lentil seed coats

A systematic set of optimization experiments was conducted to design an efficient extraction and analysis protocol for screening six different sub-classes of phenolic compounds in the seed coat of various lentil (Lens culinaris Medik.) genotypes. Different compounds from anthocyanidins, flavan-3-ols, proanthocyanidins, flavanones, flavones, and flavonols sub-classes were first optimized for use as standards for liquid chromatography mass spectrometry (LC-MS) with UV detection. The effect of maceration duration, reconstitution solvent, and extraction solvent were investigated using lentil genotype CDC Maxim. Chromatographic conditions were optimized by examining column separation efficiencies, organic composition, and solvent gradient. The results showed that a 1h maceration step was sufficient and that non-acidified solvents were more appropriate; a 70:30 acetone: water (v/v) solvent was ultimately selected. Using a Kinetex PFP column, the organic concentration, gradient, and flow rate were optimized to maximize the resolution of phenolic compounds in a short 30-min analysis time. The optimized method was applied to three lentil genotypes with different phenolic compound profiles to provide information of value to breeding programs.

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.

Genome re-sequencing and simple sequence repeat markers reveal the existence of divergent lineages in the Canadian Puccinia striiformis f. sp. tritici population with extensive DNA methylation: Population structure of the stripe rust pathogen in Canada

Wheat stripe rust, caused by Puccinia striiformis f. sp. tritici (Pst), is an important disease in Canada. The worldwide genetic structure of Pst populations have been characterized, excluding Canada. Here, we elucidated the genetic structure of the western Canadian Pst population using molecular markers, revealing the presence of four divergent lineages with predominantly clonal structure. In the worldwide context, two previously reported lineages were identified: PstS0 (22%), representing an old Northwestern-European and PstS1 (35%), an invasive warm-temperature adapted. Additionally, two new, unreported lineages, PstPr (9%) and PstS1-related (35%), were detected, which produced more telia than other lineages and had double the number of unique recombination events. The PstPr was a recent invasion, and likely evolved in a diverse, recombinant population as it was closely related to the PstS5, PstS7/Warrior, PstS8/Kranich, and PstS9 lineages originating from sexually recombining populations in the centre of diversity. The DNA methylation analysis revealed DNA-methyltransferase1-homologs, providing compelling evidence for epigenetic regulation and as a first report, an average of ∼5%, 5hmC in the Puccinia epigenome merits further investigation. The divergent lineages in the Canadian Pst population with the potential for genetic recombination, as well as epigenetic regulation needs consideration in the context of pathogen adaptation and management.