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

Enhancement of specialized metabolism, regeneration efficiency and biological activity in lavandin (Lavandula x intermedia cv 'Grosso')

This study aimed to improve essential oil composition by modifying terpene production in Lavandula x intermedia cv Grosso via mutagenesis to more closely resemble the oil of the commercially valuable essential oil from L. angustifolia or the medicinally active essential oil from L. latifolia. Additionally this study aimed to identify genes that control essential oil production in lavenders, and to determine the effect of essential oil composition on biological activity, specifically insecticidal and insect repellent properties. This study resulted in an improved method for the efficient regeneration of Grosso lavender, and applied this method to generate ten unique mutants. The transcriptomes of some mutants were sequenced, and thirty seven differentially expressed transcripts were identified as being involved in the biosynthesis and production of essential oil terpenes. The transcript expression results were confirmed by real-time quantitative polymerase chain reaction analysis. The lavender essential oil showing greatest biological activity against an invasive pest, spotted wing drosophila, was identified as Lavandula latifolia cv Medikus and the active constituents were identified through fumigation and spray toxicity assays as the monoterpenes 1,8-cineole and linalool. These oils showed strong fumigation and contact toxicity. In all, this thesis presents the generation, screening and analysis of unique L. x intermedia essential oil mutants, which represent both potential new commercial cultivars and model organisms for the investigation of the regulation and biosynthesis of essential oil terpenes.Arts and Sciences, Irving K. Barber School of (Okanagan)Biology, Department of (Okanagan)Graduat

Auxin driven indoleamine biosynthesis and the role of tryptophan as an inductive signal in Hypericum perforatum (L.).

In the 60 years since Skoog and Miller first reported the chemical redirection of plant growth the underlying biochemical mechanisms are still poorly understood, with one challenge being the capacity for applied growth regulators to act indirectly or be metabolized to active phytohormones. We hypothesized that tryptophan is metabolized to auxin, melatonin or serotonin inducing organogenesis in St. John's wort (Hypericum perforatum L.). Root explants from two germplasm lines of St. John's wort with altered melatonin metabolism and wildtype were incubated with auxin or tryptophan for 24, 48 or 72 h to induce regeneration. In wildtype, tryptophan had little effect on the indoleamine pathway, and was found to promote primary growth, suggesting excess tryptophan moved quickly through various secondary metabolite pathways and protein synthesis. In lines 4 and 112 tryptophan was associated with modified morphogenesis, indoleamine and auxin levels. Incubation with tryptophan increased shoot organogenesis while incubation with auxin led to root regeneration. The established paradigm of thought views tryptophan primarily as a precursor for auxin and indoleamines, among other metabolites, and mediation of auxin action by the indoleamines as a one-way interaction. We propose that these processes run in both directions with auxin modifying indoleamine biosynthesis and the melatonin:serotonin balance contributing to its effects on plant morphogenesis, and that tryptophan also functions as an inductive signal to mediate diverse phytochemical and morphogenetic pathways

Enhancing the regeneration efficiency of lavandin (Lavandula x intermedia cv Grosso) : effects of light quality, medium strength, phenolic control agents, and polyamines

An efficient protocol for the regeneration of lavandin (Lavandula x intermedia cv. 'Grosso') is reported. Thiadazuron (9 μM), a plant growth-modulating phenylurea, was used to induce callus formation and shoot initiation from cultured leaf explants. Newly emerged shoots were maintained on media containing 0.05 μM naphthaleneacetic acid to allow maturation, and then transferred to media containing 2.9 μM indole-3-acetic acid to allow root formation. The phenolic control agents polyvinylpyrrolidone (PVP), ascorbic acid, 2-aminoindane-2-phosphonic acid, and activated charcoal were tested for their ability to prevent shoot browning and death in culture. All agents except PVP were found to be effective, with ascorbic acid being most consistent in promoting development of healthy mature shoots. The effect of light type (red light vs. white light) and culture medium composition (full- and half-strength Murashige and Skoog or Llyod and McCown’s woody plant medium (WPM)) on rooting efficiency was also evaluated. Cultures on half-strength WPM in white light were found to have the highest rooting efficiency. Additionally, application of the polyamines putrescine, spermine, and spermidine were tested for their effect on rooting. While rooting efficiency was not improved with any of the treatments, spermine and spermidine were found to have an inhibitory effect at concentrations greater than 10 μM.Arts and Sciences, Irving K. Barber School of (Okanagan)Biology, Department of (Okanagan)ReviewedFacult

Insecticidal and oviposition deterrent effects of essential oils and their constituents against the invasive pest Drosophila suzukii (Matsumura) (Diptera: Drosophilidae)

Spotted wing drosophila (SWD), Drosophila suzukii (Matsumura), is an important new invasive pest of stone and berry fruits in North America and current control methods require frequent application of synthetic pesticides. This has created a need for new and environmentally friendly biopesticides for the control of SWD. This paper investigated the potential of nine essential oils from avocado (Persea americana Mill.), neem (Azadirachta indica A. Juss), kukui nut (Aleurites moluccana L.), macadamia nut (Macadamia integrifolia Maiden & Betche), spike lavender (Lavandula latifolia Vill.), Grosso lavandin leaf and flower (Lavandula × intermedia cv 'Grosso'), and Provence lavandin leaf and flower (Lavandula × intermedia cv 'Provence') as well as three major monoterpene constituents of lavender essential oils: 1,8-cineole, 3-carene and linalool for their ability to control SWD through fumigation and contact toxicity assays as well as oviposition deterrent activity. Linalool was found to be the most effective monoterpene in fumigation assays (EC50 1.85 µL/L air) and spike lavender floral essential oil was found to be the most effective whole oil (EC50 3.79 µL oil/L air). In contact toxicity assays 1,8-cineole (EC50 0.67%) was the most effective monoterpene while avocado (EC50 0.54%) and spike lavender (EC50 0.69%) oils were the most effective whole oils. No significant oviposition deterrent activity was observed. This report indicates that L. latifolia essential oil and commercially available avocado oil are strong lead candidates for management of SWD. Furthermore the activity of L. latifolia essential oil is likely due to the high content of 1,8-cineole and linalool. This is the first report of significant insecticidal activity by these oils and compounds against SWD.Arts and Sciences, Irving K. Barber School of (Okanagan)Biology, Department of (Okanagan)ReviewedFacult

Preliminary assessment of the conservation status of medicinal plant species in Canada

Natural health products in North America are a 9.6 billion US dollar industry that has seen increased demands particularly in products for immune support and anti-viral supplements in response to the Covid-19 pandemic. It has been estimated that ≈40% of the plant raw materials used in natural health products are wild-harvested each year but exact data is missing for many species. Our data show that there are at least 1445 medicinal plant species harvested for commercial products in Canada and 1217 of these are native species. Medicinal plants and non-timber forest crops are also increasingly impacted by climate change as suitable growing areas migrate. In order to ensure long term species survival in the wild, it is necessary to generate accurate information about distribution of wild populations, need for conservation and sustainable method development. Our objective was to provide a preliminary assessment on the conservation status of Canadian medicinal plants by analysis of the available data resources.The accepted manuscript in pdf format is listed with the files at the bottom of this page. The presentation of the authors' names and (or) special characters in the title of the manuscript may differ slightly between what is listed on this page and what is listed in the pdf file of the accepted manuscript; that in the pdf file of the accepted manuscript is what was submitted by the author

Melatonin in Plants and Plant Culture Systems: Variability, Stability and Efficient Quantification

Despite growing evidence of the importance of melatonin and serotonin in the plant life, there is still much debate over the stability of melatonin, with extraction and analysis methods varying greatly from lab to lab with respect to time, temperature, light levels, extraction solvents and mechanical disruption. The variability in methodology has created conflicting results that confound the comparison of studies to determine the role of melatonin in plant physiology. We here describe a fully validated method for the quantification of melatonin, serotonin and their biosynthetic precursors: tryptophan, tryptamine and N-acetylserotonin by liquid chromatography single quadrupole mass spectrometry (LC-MS) in diverse plant species and tissues. This method can be performed on a simple and inexpensive platform, and is both rapid and simple to implement. The method has excellent reproducibility and acceptable sensitivity with percent relative standard deviation (%RSD) in all matrices between 1 and 10 % and recovery values of 82 to 113 % for all analytes. Instrument detection limits were 24.4 ng/mL, 6.10 ng/mL, 1.52 ng/mL, 6.10 ng/mL, and 95.3 pg/mL, for serotonin, tryptophan, tryptamine, N-acetylserotonin and melatonin respectively. Method detection limits were 1.62 µg/g, 0.407 µg/g, 0.101 µg/g, 0.407 µg/g and 6.17 ng/g respectively. The optimized method was then utilized to examine the issue of variable stability of melatonin in plant tissue culture systems. Media composition (Murashige and Skoog, Driver and Kuniyuki walnut or Lloyd and McCown’s woody plant medium) and light (16h photoperiod or dark) were found to have no effect on melatonin or serotonin content. A Youden trial suggested temperature as a major factor leading to degradation of melatonin. Both melatonin and serotonin appeared to be stable across the first ten days in media, melatonin losses reached a mean minimum degradation at 28 d of approximately 90 %; serotonin reached a mean minimum value of approximately 60 % at 28d. These results suggest that melatonin and serotonin show considerable stability in plant systems and these indoleamines and related compounds can be used for investigations that span over 3 weeks

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.Science, Faculty ofNon UBCChemistry, Department ofReviewedFacult

Selection and Micropropagation of an Elite Melatonin Rich Tulsi (Ocimum sanctum L.) Germplasm Line

Tulsi (Ocimum sanctum L.) is a sacred plant of medicinal and spiritual significance in many cultures. Medicinal properties of Tulsi are ascribed to its phytochemicals with antioxidant capabilities. The current study was undertaken to screen a large seed population of Tulsi to select germplasm lines with high antioxidant potential and to standardize protocols for micropropagation and biomass production to produce a phytochemically consistent crop. A total of 80 germplasm lines were established under in vitro conditions and screened for their antioxidant potential determined with the 2,2-diphenyl-1-picrylhydrazyl (DPPH) bioassay. The micropropagation of a selected line, named Vrinda, was established using nodal cultures grown on Murashige and Skoog medium containing benzylaminopurine (1.1 µM), gibberellic acid (0.3 µM), and activated charcoal (0.6%). The antioxidant phytohormones melatonin and serotonin were quantified in the field and greenhouse grown tissues of Vrinda and melatonin levels were found to be consistent in both conditions with higher serotonin levels under field conditions. This integrated approach combining the in vitro selection and propagation offers potential applications in the development of safe, effective, and novel natural health products of Tulsi, and many other medicinal plant species.Science, Irving K. Barber Faculty of (Okanagan)Non UBCReviewedFacultyResearche

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.Medicine, Faculty ofNon UBCReviewedFacult