Microbial Diversity Impacts Non-Protein Amino Acid Production in Cyanobacterial Bloom Cultures Collected from Lake Winnipeg.

Lake Winnipeg in Manitoba, Canada is heavily impacted by harmful algal blooms that contain non-protein amino acids (NPAAs) produced by cyanobacteria: N-(2-aminoethyl)glycine (AEG), ß-aminomethyl-L-alanine (BAMA), ß-N-methylamino-L-alanine (BMAA), and 2,4-diaminobutyric acid (DAB). Our objective was to investigate the impact of microbial diversity on NPAA production by cyanobacteria using semi-purified crude cyanobacterial cultures established from field samples collected by the Lake Winnipeg Research Consortium between 2016 and 2021. NPAAs were detected and quantified by ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) using validated analytical methods, while Shannon and Simpson alpha diversity scores were determined from 16S rRNA metagenomic sequences. Alpha diversity in isolate cultures was significantly decreased compared to crude cyanobacterial cultures (p < 0.001), indicating successful semi-purification. BMAA and AEG concentrations were higher in crude compared to isolate cultures (p < 0.0001), and AEG concentrations were correlated to the alpha diversity in cultures (r = 0.554; p < 0.0001). BAMA concentrations were increased in isolate cultures (p < 0.05), while DAB concentrations were similar in crude and isolate cultures. These results demonstrate that microbial community complexity impacts NPAA production by cyanobacteria and related organisms.

Neurotoxic non-protein amino acids in commercially harvested Lobsters (Homarus americanus H. Milne-Edwards).

Cyanobacteria produce neurotoxic non-protein amino acids (NPAAs) that accumulate in ecosystems and food webs. American lobsters (Homarus americanus H. Milne-Edwards) are one of the most valuable seafood industries in Canada with exports valued at > $2 billion. Two previous studies have assessed the occurrence of ß-N-methylamino-L-alanine (BMAA) in a small number of lobster tissues but a complete study has not previously been undertaken. We measured NPAAs in eyeballs, brain, legs, claws, tails, and eggs of 4 lobsters per year for the 2021 and 2022 harvests. Our study included 4 male and 4 female lobsters. We detected BMAA and its isomers, N-(2-aminoethyl)glycine (AEG), 2,4-diaminobutyric acid (DAB) and ß-aminomethyl-L-alanine (BAMA) by a fully validated reverse phase chromatography-tandem mass spectrometry method. We quantified BMAA, DAB, AEG and BAMA in all of the lobster tissues. Our quantification data varied by individual lobster, sex and collection year. Significantly more BMAA was quantified in lobsters harvested in 2021 than 2022. Interestingly, more BAMA was quantified in lobsters harvested in 2022 than 2021. Monitoring of lobster harvests for cyanobacterial neurotoxins when harmful algal bloom events occur could mitigate risks to human health.

Predicted impacts of climate change on wild and commercial berry habitats will have food security, conservation and agricultural implications.

Climate change is now a reality and is altering ecosystems, with Canada experiencing 2-4 times the global average rate of warming. This will have a critical impact on berry cultivation and horticulture. Enhancing our understanding of how wild and cultivated berries will perform under changing climates will be essential to mitigating impacts on ecosystems, culture and food security. Our objective was to predict the impact of climate change on habitat suitability of four berry producing Vaccinium species: two species with primarily northern distributions (V. uliginosum, V. vitis-idaea), one species with a primarily southern distribution (V. oxycoccos), and the commercially cultivated V. macrocarpon. We used the maximum entropy (Maxent) model and the CMIP6 shared socioeconomic pathways (SSPs) 126 and 585 projected to 2041-2060 and 2061-2080. Wild species showed a uniform northward progression and expansion of suitable habitat. Our modeling predicts that suitable growing regions for commercial cranberries are also likely to shift with some farms becoming unsuitable for the current varieties and other regions becoming more suitable for cranberry farms. Both V. macrocarpon and V. oxycoccos showed a high dependence on precipitation-associated variables. Vaccinium vitis-idaea and V. uliginosum had a greater number of variables with smaller contributions which may improve their resilience to individual climactic events. Future competition between commercial cranberry farms and wild berries in protected areas could lead to conflicts between agriculture and conservation priorities. New varieties of commercial berries are required to maintain current commercial berry farms.

Mammalian Melatonin Agonist Pharmaceuticals Stimulate Rhomboid Proteins in Plants.

Melatonin is a human neurotransmitter and plant signalling metabolite that perceives and directs plant metabolism. The mechanisms of melatonin action in plants remain undefined. We hypothesized that roots have a melatonin-specific receptor and/or transporter that can respond to melatonin-mediating pharmaceuticals. To test this hypothesis Arabidopsis seedlings were grown with melatonin pharmaceutical receptor agonists: ramelteon and tasimelteon, and/or antagonists: luzindole and 4-P-PDOT. Ramelteon was found both to mimic and competitively inhibit melatonin metabolism in plants. Due to the higher selectivity of ramelteon for the MT1 receptor type in humans, a sequence homology search for MT1 in Arabidopsis identified the rhomboid-like protein 7 (RBL7). In physiological studies, Arabidopsis rbl7 mutants were less responsive to ramelteon and melatonin. Quantum dot visualizations of the effects of ramelteon on melatonin binding to root cell membranes revealed a potential mechanism. We propose that RBL7 is a melatonin-interacting protein that directs root architecture and growth in a mechanism that is responsive to environmental factors.

A Systematic Review of Melatonin in Plants: An Example of Evolution of Literature.

Melatonin (N-acetyl-5-methoxy-tryptamine) is a mammalian neurohormone, antioxidant and signaling molecule that was first discovered in plants in 1995. The first studies investigated plant melatonin from a human perspective quantifying melatonin in foods and medicinal plants and questioning whether its presence could explain the activity of some plants as medicines. Starting with these first handful of studies in the late 1990s, plant melatonin research has blossomed into a vibrant and active area of investigation and melatonin has been found to play critical roles in mediating plant responses and development at every stage of the plant life cycle from pollen and embryo development through seed germination, vegetative growth and stress response. Here we have utilized a systematic approach in accordance with the preferred reporting items for systematic reviews and meta-analyses (PRISMA) protocols to reduce bias in our assessment of the literature and provide an overview of the current state of melatonin research in plants, covering 1995-2021. This review provides an overview of the biosynthesis and metabolism of melatonin as well as identifying key themes including: abiotic stress responses, root development, light responses, interkingdom communication, phytohormone and plant signaling. Additionally, potential biases in the literature are investigated and a birefringence in the literature between researchers from plant and medical based which has helped to shape the current state of melatonin research. Several exciting new opportunities for future areas of melatonin research are also identified including investigation of non-crop and non-medicinal species as well as characterization of melatonin signaling networks in plants.

Metabolomics-Guided Hypothesis Generation for Mechanisms of Intestinal Protection by Live Biotherapeutic Products.

The use of live biotherapeutic products (LBPs), including single strains of beneficial probiotic bacteria or consortiums, is gaining traction as a viable option to treat inflammatory-mediated diseases like inflammatory bowel disease (IBD). However, LBPs' persistence in the intestine is heterogeneous since many beneficial bacteria lack mechanisms to tolerate the inflammation and the oxidative stress associated with IBD. We rationalized that optimizing LBPs with enhanced colonization and persistence in the inflamed intestine would help beneficial bacteria increase their bioavailability and sustain their beneficial responses. Our lab developed two bioengineered LBPs (SBT001/BioPersist and SBT002/BioColoniz) modified to enhance colonization or persistence in the inflamed intestine. In this study, we examined colon-derived metabolites via ultra-high performance liquid chromatography-mass spectrometry in colitic mice treated with either BioPersist or BioColoniz as compared to their unmodified parent strains (Escherichia coli Nissle 1917 [EcN] and Lactobacillus reuteri, respectively) or to each other. BioPersist administration resulted in lowered concentrations of inflammatory prostaglandins, decreased stress hormones such as adrenaline and corticosterone, increased serotonin, and decreased bile acid in comparison to EcN. In comparison to BioColoniz, BioPersist increased serotonin and antioxidant production, limited bile acid accumulation, and enhanced tissue restoration via activated purine and pyrimidine metabolism. These data generated several novel hypotheses for the beneficial roles that LBPs may play during colitis.

A Single Laboratory Validation for the Analysis of Underivatized ß-N-Methylamino-L-Alanine (BMAA).

ß-N-Methylamino-L-alanine (BMAA) is a non-protein amino acid produced by cyanobacteria that can accumulate in ecosystems and food webs. Human exposure to cyanobacterial and algal blooms may be a risk factor for neurodegenerative diseases such as Alzheimer's disease and amyotrophic lateral sclerosis. Analytical chemists have struggled to find reliable methods for BMAA analysis in complex sample matrices. Analysis of BMAA is complicated by at least 3 naturally occurring isomers: N-(2-aminoethyl)glycine (AEG), 2,4-diaminobutyric acid (DAB), and ß-aminomethyl-L-alanine (BAMA). More than 350 publications have reported detection and quantification of BMAA and its isomers, but varying results have led to controversy in the literature. The objective of this study was to perform a single laboratory validation (SLV) of a frequently published method for BMAA analysis using a ZIC-HILIC column. We investigated the selectivity, linearity, accuracy, precision, and sensitivity of the method and our data show that this HILIC method fails many of the criteria for a validated method. The method fails the criterion for selectivity as the chromatography does not separate BMAA from its isomer BAMA. Sensitivity of the method greatly decreased over the experimental period and it demonstrated a higher limit of detection (LOD) (7.5 pg on column) and a higher lower limit of quantification (LLOQ) (30 pg on column) than other published validated methods. The method demonstrated poor precision of repeated injections of standards of BMAA with % relative standard deviation (%RSD) values that ranged from 37 to 107% while HorRat values for BMAA had a fail rate of 80% and BAMA had a fail rate of 73%. No HorRat values between 0.5 and 2 were found for repeated injections of standards of AEG and DAB. Recovery of 13C3,15N2-BMAA in a cyanobacterial matrix was < 10% in experiments and we were also unable to accurately detect other protein amino acids including methionine, cysteine, or alanine, indicating matrix effects. The results of this study demonstrate that the ZIC-HILIC column is not fit for purpose for the analysis of BMAA in cyanobacterial matrices and further provides explanations for the high level of negative results reported by researchers using this method.

The Morphoregulatory Role of Thidiazuron: Metabolomics-Guided Hypothesis Generation for Mechanisms of Activity.

Thidiazuron (TDZ) is a diphenylurea synthetic herbicide and plant growth regulator used to defoliate cotton crops and to induce regeneration of recalcitrant species in plant tissue culture. In vitro cultures of African violet thin petiole sections are an ideal model system for studies of TDZ-induced morphogenesis. TDZ induces de novo shoot organogenesis at low concentrations and somatic embryogenesis at higher concentrations of exposure. We used an untargeted metabolomics approach to identify metabolites in control and TDZ-treated tissues. Statistical analysis including metabolite clustering, pattern and pathway tools, logical algorithms, synthetic biotransformations and hormonomics identified TDZ-induced changes in metabolism. A total of 18,602 putative metabolites with extracted masses and predicted formulae were identified with 1412 features that were found only in TDZ-treated tissues and 312 that increased in response to TDZ. The monomer of TDZ was not detected intact in the tissues but putative oligomers were found in the database and we hypothesize that these may form by a Diels-Alder reaction. Accumulation oligomers in the tissue may act as a reservoir, slowly releasing the active TDZ monomer over time. Cleavage of the amide bridge released TDZ-metabolites into the tissues including organic nitrogen and sulfur containing compounds. Metabolomics data analysis generated six novel hypotheses that can be summarized as an overall increase in uptake of sugars from the culture media, increase in primary metabolism, redirection of terpene metabolism and mediation of stress metabolism via indoleamine and phenylpropanoid metabolism. Further research into the specific mechanisms hypothesized is likely to unravel the mode of action of TDZ and to provide new insights into the control of plant morphogenesis.

Breadfruit flour is a healthy option for modern foods and food security.

Breadfruit is a traditional staple crop from Pacific islands with the potential to improve worldwide food security and mitigate diabetes. Flour produced from breadfruit is a gluten-free, low glycemic index, nutrient dense and complete protein option for modern foods but basic scientific knowledge of health impacts of a breadfruit-based diet in animals and humans was lacking. We designed a series of studies to provide basic and fundamental data on impacts of a breadfruit-based diet through an in vitro and in vivo model. Cooked breadfruit flour was digested through a multi-stage enzyme digestion model to estimate protein digestibility in comparison to wheat flour. Breadfruit protein was found to be easier to digest than wheat protein in the enzyme digestion model. The flour digestions were applied to Caco-2 cells to test the cytotoxicity and to measure the immunogenicity through cytokine expression. No significant differences were observed for immune factors and cytokines (IL-4, IL-10, IL-8, TNF-α, IFN-γ) on Caco-2 cells between the breadfruit and wheat groups. A breadfruit-based rodent chow was formulated by substitution of all of the wheat in the standard formulation with breadfruit. The diets were isocaloric, nutrient equivalent and used to feed male and female C57BL/6 mice for 21 days. No sign of malnutrition, discomfort, illness or death was observed among the mice because of the diet. The histology and the cytokine expression of the mice ileum from both groups were analyzed and showed similar results. The expression of major bacteria was measured in the colon and showed similar results. Mice fed the breadfruit diet had a significantly higher growth rate and body weight than standard diet fed mice. No negative health outcomes were observed in studies with in vitro or in vivo models and breadfruit flour is a healthy alternative to other starches for modern foods.

Exploring feature selection of St John's wort grown under different light spectra using 1 H-NMR spectroscopy.

INTRODUCTION: Nuclear magnetic resonance (NMR) spectroscopy combined with multivariate statistical analysis can provide tools to help detect differences in plant chemistry when grown under varying conditions. Hypericum perforatum, or Saint John's wort, plants are a suitable model to explore methods of discrimination between early stage plants grown in different conditions. OBJECTIVES: The purpose of this work was to develop a method for identifying differences in chemical profiles between young Hypericum perforatum plants grown under different lighting conditions. MATERIAL AND METHODS: Cuttings were grown for 3 weeks under different light conditions. Plant extracts were prepared in MeOD-d4 and analysed by 1 H-NMR. A multivariate analysis method of the NMR data was developed in an effort to determine variations in chemical profiles. RESULTS: The method identified specific metabolites as drivers of difference between the plants grown under different light conditions. STOCSY (statistical total correlation spectroscopy) and quantification of highlighted metabolites supported the findings of the multivariate analysis. Glutamine, sucrose and fructose were found to be chemical markers of light quality in this study. CONCLUSION: NMR metabolomics using a medium field instrument could find differences in plant chemistry when grown in different conditions. This method could easily be extended to benchtop instruments and be used for crop monitoring and growth condition optimisation.

Metabolomics and hormonomics to crack the code of filbert growth.

INTRODUCTION: Plants respond to changes in their environments through hormonal activation of a physiological cascade that redirects metabolic resources and growth. In filberts (Corylus sp.), chelated iron promotes the growth of new shoots but the mechanism(s) are not understood. OBJECTIVES: To use untargeted metabolomics and hormonomics approaches to generate novel hypotheses for the morphoregulatory role of ferric ethylenediamine-N,N'-di-(ortho-hydroxyphenyl) acetic acid (Fe-EDDHA) in filbert shoot organogenesis in vitro. METHODS: Data were generated using previously optimized standardized untargeted metabolomics protocols with time of flight mass spectrometry. Multivariate statistical tools (principal component and partial least squares discriminant analysis) did not detect significant differences. Discovery tools Significance Analysis of Microarrays (SAM), multiple linear regression analysis, Bayesian analysis, logical algorithms, machine learning, synthetic biotransformations, targeted hormonomics, and online resources including MetaboAnalyst were used. RESULTS: Starch/sucrose metabolism and shikimate pathway metabolites were increased. Dose dependent decreases were found in polyphenol metabolism, specifically ellagic acid and its methylated derivative 3,4,3'-tri-O-methylellagic acid. Hormonomics analysis revealed significant differences in phytohormones and their conjugates. FeEDDHA treatment reduced indole-3-acetic acid, abscisic acid, salicylic acid, jasmonic acid conjugates (JA-Trp, JA-Ile, OH-JA) and dihydrozeatinglucoside in regenerating explants. Serotonin (5HT) was decreased in FeEDDHA-treated regenerating tissues while the related metabolite melatonin was increased. Eight phenolic conjugates of 5HT and eight catabolites were affected by FeEDDHA indicating that metabolism to sequester, deactivate and metabolize 5HT was induced by Fe(III). Tryptophan was metabolized through kynurenine but not anthranilate. CONCLUSION: Seven novel hypotheses were generated to guide future studies to understand the regulatory control(s) of shoot organogenesis.

A systematic review of analytical methods for the detection and quantification of ß-N-methylamino-l-alanine (BMAA).

Neurodegenerative diseases are influenced by environmental factors such as exposure to toxins including the cyanotoxin ß-N-methylamino-l-alanine (BMAA) that can bioaccumulate in common food sources such as fish, mussels and crabs. Accurate and precise analytical methods are needed to detect and quantify BMAA to minimize human health risks. The objective of this review is to provide a comprehensive overview of the methods used for BMAA analysis from 2003 to 2019 and to evaluate the reported performance characteristics for each method to determine the consensus data for each analytical approach and different sample matrices. Detailed searches of the database Web of Science™ (WoS) were performed between August 21st, 2018 and April 5th, 2019. Eligible studies included analytical methods for the detection and quantification of BMAA in cyanobacteria and bioaccumulated BMAA in higher trophic levels, in phytoplankton and zooplankton and in human tissues and fluids. This systematic review has limitations in that only the English language literature is included and it did not include standard operating protocols nor any method validation data that have not been made public. We identified 148 eligible studies, of which a positive result for BMAA in one or more samples analyzed was reported in 84% (125 out of 148) of total studies, 57% of HILIC studies, 92% of RPLC studies and 71% of other studies. The largest discrepancy between different methods arose from the analysis of cyanobacteria samples, where BMAA was detected in 95% of RPLC studies but only in 25% of HILIC studies. Without sufficient published validation of each method's performance characteristics, it is difficult to establish each method as fit for purpose for each sample matrix. The importance of establishing methods as appropriate for their intended use is evidenced by the inconsistent reporting of BMAA across environmental samples, despite its prevalence in diverse ecosystems and food webs.

The Terroir of Cannabis: Terpene Metabolomics as a Tool to Understand Cannabis sativa Selections.

The phytochemical diversity of Cannabis chemovars is not well understood, and many chemovars were created in informal breeding programs without records of parentage or the criteria for selection. Key criteria for selection sometimes included aroma notes and visual cues, which some breeders associated with pharmacological activity. We hypothesized that the process of selection for scents believed to be related to specific tetrahydrocannabinol levels has resulted in modified terpene biosynthesis in these chemovars. Thirty-two cannabinoids, 29 monoterpenes and 38 sesquiterpenes were measured in 33 chemovars from 5 licensed producers. A classification system based on cannabinoid content was used with targeted metabolomic tools to determine relationships in the phytochemistry. Three monoterpenes, limonene, ß-myrcene, and α-pinene, and two sesquiterpenes, caryophyllene and humulene, were abundant in the majority of chemovars. Nine terpenes were present in tetrahydrocannabinol-dominant chemovars. Three monoterpenes and four sesquiterpenes were predominantly found in cannabidiol-containing chemovars. Low abundance terpenes may have been the aromatic cues identified by breeders. The medicinal activity of some of the terpenes is likely to contribute to the pharmacological effect of specific chemovars. Together, these data demonstrate the synergy of compounds in Cannabis chemovars and point to the need for additional research to understand the phytochemical complexity.

A virus-induced gene-silencing system for functional genetics in a betalainic species, Amaranthus tricolor (Amaranthaceae).

PREMISE OF THE STUDY: Research in Amaranthaceae could be accelerated by developing methods for targeted gene silencing. Most amaranths, including Amaranthus tricolor, produce betalains. However, the physiological and ecological roles of these pigments are uncertain. We sought to establish a virus-induced gene-silencing (VIGS) method for amaranths, using silencing of betalain pigments as a proof-of-principle. METHODS: We targeted AtriCYP76AD1, a putative cytochrome P450 component of the betalain biosynthetic pathway, using VIGS, and compared two different methods of introducing the VIGS construct into plants. We measured transcript abundance and concentrations of betalains and their l-DOPA precursor in VIGS-treated plants, and compared these to controls. RESULTS: We observed that when AtriCYP76AD1 was targeted by VIGS in normally red plants, AtriCYP76AD1 and the related genes AtriCYP76AD6 and AtriCYP76AD5 had diminished transcript abundance. Furthermore, newly emergent petioles and leaves of VIGS-treated plants appeared green, betacyanin accumulation was strongly reduced, and l-DOPA accumulation was increased. No betaxanthin could be detected in this variety of A. tricolor, either before or after VIGS treatment. DISCUSSION: These results help to establish the genetic basis of betalain synthesis in amaranths. Furthermore, this is the first report of VIGS in amaranths and demonstrates the potential of this technique for basic and applied research in these species.

Cyanobacterial neurotoxin BMAA and brain pathology in stranded dolphins.

Dolphin stranding events occur frequently in Florida and Massachusetts. Dolphins are an excellent sentinel species for toxin exposures in the marine environment. In this report we examine whether cyanobacterial neurotoxin, ß-methylamino-L-alanine (BMAA), is present in stranded dolphins. BMAA has been shown to bioaccumulate in the marine food web, including in the muscles and fins of sharks. Dietary exposure to BMAA is associated with the occurrence of neurofibrillary tangles and ß-amyloid plaques in nonhuman primates. The findings of protein-bound BMAA in brain tissues from patients with Alzheimer's disease has advanced the hypothesis that BMAA may be linked to dementia. Since dolphins are apex predators and consume prey containing high amounts of BMAA, we examined necropsy specimens to determine if dietary and environmental exposures may result in the accumulation of BMAA in the brains of dolphins. To test this hypothesis, we measured BMAA in a series of brains collected from dolphins stranded in Florida and Massachusetts using two orthogonal analytical methods: 1) high performance liquid chromatography, and 2) ultra-performance liquid chromatography with tandem mass spectrometry. We detected high levels of BMAA (20-748 µg/g) in the brains of 13 of 14 dolphins. To correlate neuropathological changes with toxin exposure, gross and microscopic examinations were performed on cortical brain regions responsible for acoustico-motor navigation. We observed increased numbers of ß-amyloid+ plaques and dystrophic neurites in the auditory cortex compared to the visual cortex and brainstem. The presence of BMAA and neuropathological changes in the stranded dolphin brain may help to further our understanding of cyanotoxin exposure and its potential impact on human health.

Direct visualization of location and uptake of applied melatonin and serotonin in living tissues and their redistribution in plants in response to thermal stress.

Melatonin and serotonin are important phytochemicals enabling plants to redirect growth in response to environmental stresses. Despite much research on their biosynthetic routes, localization of their biosynthetic enzymes and recent identification of a phytomelatonin receptor, localization of the molecules themselves has to date not been possible. Elucidation of their locations in living tissues can provide an effective tool to facilitate indolamine research across systems including both plants and animals. In this study, we employed a novel technique, quantum dot nanoparticles, to directly visualize melatonin and serotonin in axenic roots. Melatonin was absorbed through epidermal cells, travelled laterally, and accumulated in endodermal and rapidly dividing pericycle cells. Serotonin was absorbed by cells proximal to the crown with rapid polar movement toward the root tip. Thermal stress disrupted localization and dispersed melatonin and serotonin across cells. These data demonstrate the natural movement of melatonin and serotonin in roots directing cell growth and suggest that plants have a mechanism to disperse the indolamines throughout tissues as antioxidants in response to environmental stresses.

Single-Lab Validation for Determination of Kavalactones and Flavokavains in Piper methysticum (Kava).

Piper methysticum (Kava) is a plant whose roots are used in the preparation of traditional beverages with spiritual, medicinal, and social importance for the Pacific Islanders. Kava is also sold as a herbal supplement or recreational beverage consumed for its mild inebriating effect in Europe and North America. With an ongoing interest in the safety and quality of kava products, it is necessary to develop a validated method for determination of kava chemical composition to ensure confidence in quality assessment. Thus, an high-performance liquid chromatography with ultraviolet detection (HPLC-UV) method was developed, optimized, and validated for determining six major kavalactones and three flavokavains in kava raw materials and finished products based on AOAC single-laboratory validation guidelines. This is the first fully validated analytical method for measuring kavalactones and flavokavains in a single run. The separation of the analytes was achieved in 10 min with an Agilent Poroshell C18 column using gradient separation. The sample was extracted with methanol first and then acetone. The signals were detected at 240 nm and 355 nm. The limit of quantification was under 1.2 µg/mL (0.3 mg/g) for kavalactones and under 0.35 µg/mL (0.01 mg/g) for flavokavains. The Horwitz ratio values described ranged from 0.3 to 1.82. The spike recovery experiments showed an accuracy between 92 and 105% for all analytes. The results of the study demonstrate that the method is fit for the purpose of determining methysticin, dihydromethysticin, kavain, dihydrokavain, yangonin, desmethoxyyangonin, flavokavain A, flavokavain B, and flavokavain C in kava raw material and finished products (dry-filled capsule, liquid phytocaps, and tincture).

The metabolic fate of dietary nicotine in the cabbage looper, Trichoplusia ni (Hübner).

Cabbage looper (Trichoplusia ni) larvae are generalist herbivores that feed on numerous cultivated plants and weeds including crucifers, other vegetables, flowers, and field crops. Consuming plant material from a wide range of plant species exposes these larvae to a considerable variety of plant secondary metabolites involved in chemical defense against herbivory. The ability of the cabbage looper larvae to detoxify plant secondary metabolites, such as nicotine, has been attributed to the rapid induction of excretion via the Malpighian tubules. However, the role of metabolism in the detoxification of plant secondary metabolites in cabbage looper larvae is not well studied. We investigated nicotine metabolism in 4th larval instar cabbage looper using UPLC-MS/MS analysis to resolve the time course of nicotine metabolism, the kinetic distribution of nicotine, and the presence or absence of major metabolites of nicotine in larval tissue and excretions. The major metabolite found in our analysis was cotinine, with trace amounts of cotinine N-oxide and nicotine N-oxide. The nicotine metabolites detected are similar to those of the nicotine-tolerant Lepidopteran tobacco hornworm (Manduca sexta). The results of our study demonstrate that the 5'C-oxidation of nicotine to cotinine is the primary pathway for nicotine metabolism in cabbage looper larvae. This study showed that metabolism of nicotine and subsequent excretion of nicotine and its metabolites occurs in the larvae of the cabbage looper. Our results suggest that 5'C-oxidation in lepidopteran insects is a conserved metabolic pathway for the detoxification of plant secondary metabolites.

Melatonin in plant signalling and behaviour.

Melatonin is an indoleamine neurotransmitter that has recently become well established as an important multi-functional signalling molecule in plants. These signals have been found to induce several important physiological responses that may be interpreted as behaviours. The diverse processes in which melatonin has been implicated in plants have expanded far beyond the traditional roles for which it has been implicated in mammals, which include sleep, tropisms and reproduction. These functions, however, appear to also be important melatonin mediated processes in plants, though the mechanisms underlying these functions have yet to be fully elucidated. Mediation or redirection of plant physiological processes induced by melatonin can be summarised as a series of behaviours including, among others: herbivore defence, avoidance of undesirable circumstances or attraction to opportune conditions, problem solving and response to environmental stimulus. As the mechanisms of melatonin action are elucidated, its involvement in plant growth, development and behaviour is likely to expand beyond the aspects discussed in this review and hold promise for applications in diverse fundamental and applied plant sciences including conservation, cryopreservation, morphogenesis, industrial agriculture and natural health products.