Views and perspectives on the indoleamines serotonin and melatonin in plants: past, present and future.

In the decades since their discovery in plants in the mid-to-late 1900s, melatonin (N-acetyl-5-methoxytryptamine) and serotonin (5-methoxytryptamine) have been established as their own class of phytohormone and have become popular targets for examination and study as stress ameliorating compounds. The indoleamines play roles across the plant life cycle from reproduction to morphogenesis and plant environmental perception. There is growing interest in harnessing the power of these plant neurotransmitters in applied and agricultural settings, particularly as we face increasingly volatile climates for food production; however, there is still a lot to learn about the mechanisms of indoleamine action in plants. A recent explosion of interest in these compounds has led to exponential growth in the field of melatonin research in particular. This concept paper aims to summarize the current status of indoleamine research and highlight some emerging trends.

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.

HormonomicsDB: a novel workflow for the untargeted analysis of plant growth regulators and hormones.

Background: Metabolomics is the simultaneous determination of all metabolites in a system. Despite significant advances in the field, compound identification remains a challenge. Prior knowledge of the compound classes of interest can improve metabolite identification. Hormones are a small signaling molecules, which function in coordination to direct all aspects of development, function and reproduction in living systems and which also pose challenges as environmental contaminants. Hormones are inherently present at low levels in tissues, stored in many forms and mobilized rapidly in response to a stimulus making them difficult to measure, identify and quantify. Methods: An in-depth literature review was performed for known hormones, their precursors, metabolites and conjugates in plants to generate the database and an RShiny App developed to enable web-based searches against the database. An accompanying liquid chromatography - mass spectrometry (LC-MS) protocol was developed with retention time prediction in Retip. A meta-analysis of 14 plant metabolomics studies was used for validation. Results: We developed HormonomicsDB, a tool which can be used to query an untargeted mass spectrometry (MS) dataset against a database of more than 200 known hormones, their precursors and metabolites. The protocol encompasses sample preparation, analysis, data processing and hormone annotation and is designed to minimize degradation of labile hormones. The plant system is used a model to illustrate the workflow and data acquisition and interpretation. Analytical conditions were standardized to a 30 min analysis time using a common solvent system to allow for easy transfer by a researcher with basic knowledge of MS. Incorporation of synthetic biotransformations enables prediction of novel metabolites. Conclusions: HormonomicsDB is suitable for use on any LC-MS based system with compatible column and buffer system, enables the characterization of the known hormonome across a diversity of samples, and hypothesis generation to reveal knew insights into hormone signaling networks.

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.

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.

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.

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.

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.

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.

Melatonin and serotonin: Mediators in the symphony of plant morphogenesis.

Melatonin and serotonin are important signaling and stress mitigating molecules that play important roles across growth and development in plants. Despite many well-documented responses, a systematic investigation of the entire metabolic pathway (tryptophan, tryptamine, and N-acetylserotonin) does not exist, leaving many open questions. The objective of this study was to determine the responses of Hypericum perforatum (L.) to melatonin, serotonin, and their metabolic precursors. Two well-characterized germplasm lines (#4 and 112) created by mutation and a haploid breeding program were compared to wild type to identify specific responses. Germplasm line 4 has lower regenerative and photosynthetic capacity than either wild type or line 112, and there are documented significant differences in the chemistry and physiology of lines 4 and 112. Supplementation of the culture media with tryptophan, tryptamine, N-acetylserotonin, serotonin, or melatonin partially reversed the regenerative recalcitrance and growth impairment of the germplasm lines. Quantification of phytohormones revealed crosstalk between the indoleamines and related phytohormones including cytokinin, salicylic acid, and abscisic acid. We hypothesize that melatonin and serotonin function in coordination with their metabolites in a cascade of phytochemical responses including multiple pathways and phytohormone networks to direct morphogenesis and protect photosynthesis in H. perforatum.

Melatonin Natural Health Products and Supplements: Presence of Serotonin and Significant Variability of Melatonin Content.

STUDY OBJECTIVES: Melatonin is an important neurohormone, which mediates circadian rhythms and the sleep cycle. As such, it is a popular and readily available supplement for the treatment and prevention of sleep-related disorders including insomnia and jet lag. This study quantified melatonin in 30 commercial supplements, comprising different brands and forms and screened supplements for the presence of serotonin. METHODS: A total of 31 supplements were analyzed by ultraperformance liquid chromatography with electrochemical detection for quantification of melatonin and serotonin. Presence of serotonin was confirmed through analysis by ultraperformance liquid chromatography with mass spectrometry detection. RESULTS: Melatonin content was found to range from -83% to +478% of the labelled content. Additionally, lot-to-lot variable within a particular product varied by as much as 465%. This variability did not appear to be correlated with manufacturer or product type. Furthermore, serotonin (5-hydroxytryptamine), a related indoleamine and controlled substance used in the treatment of several neurological disorders, was identified in eight of the supplements at levels of 1 to 75 µg. CONCLUSIONS: Melatonin content did not meet label within a 10% margin of the label claim in more than 71% of supplements and an additional 26% were found to contain serotonin. It is important that clinicians and patients have confidence in the quality of supplements used in the treatment of sleep disorders. To address this, manufacturers require increased controls to ensure melatonin supplements meet both their label claim, and also are free from contaminants, such as serotonin. COMMENTARY: A commentary on this article appears in this issue on page 163.

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 82-113% 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 (16 h 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 10 days in media, melatonin losses reached a mean minimum degradation at 28 days of approximately 90%; serotonin reached a mean minimum value of approximately 60% at 28 days. 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.

Serotonin: An ancient molecule and an important regulator of plant processes.

Serotonin is an ancient indoleamine that was presumably part of the life cycle of the first prokaryotic life forms on Earth millions of years ago where it functioned as a powerful antioxidant to combat the increasingly oxygen rich atmosphere. First identified as a neurotransmitter signaling molecule in mammals, it is ubiquitous across all forms of life. Serotonin was discovered in plants many years after its discovery in mammals; however, it has now been confirmed in almost all plant families, where it plays important roles in plant growth and development, including functions in energy acquisition, seasonal cycles, modulation of reproductive development, control of root and shoot organogenesis, maintenance of plant tissues, delay of senescence, and responses to biotic and abiotic stresses. Despite its widespread presence and activity, there are many questions which remain unanswered about the role of serotonin in plants including the mode of signaling and receptor identity as well as the mechanisms of action of this important molecule. This review provides an overview of the role of serotonin in plant life and their ability to adapt.

Antifungal Screening of Lavender Essential oils and Essential Oil Constituents on three Post-harvest Fungal Pathogens.

A growing body of literature indicates that many synthetic pesticides have adverse effects on human, animal, and environmental health. As a result, plant-derived natural products are quickly gaining momentum as safer and less ecologically damaging alternatives due to their low toxicity, high biodegradability, and good specificity. Essential oils of Lavandula angustifolia, Lavandula x intermedia cv Grosso, and Lavandida x intermedia cv Provence as well as various mono- and sesquiterpene essential oil constituents were tested in order to assess their antifungal potential on three important agricultural pathogens: Botrytis cinerea, Mucor piriformis, and Penicillium expansum. Fungal susceptibility testing was performed using disk diffusion assays. The majority of essential oil constituents tested did not have a significant effect; however, 3-carene, carvacrol, geraniol, nerol and perillyl alcohol demonstrated significant inhibition at concentrations as low as 1 µ/mL. In vivo testing using strawberry fruit as a model system supported in vitro results and revealed that perillyl alcohol, carvacrol and 3-carene were effective in limiting infection by postharvest pathogens.

A new balancing act: The many roles of melatonin and serotonin in plant growth and development.

Melatonin and serotonin are indoleamines first identified as neurotransmitters in vertebrates; they have now been found to be ubiquitously present across all forms of life. Both melatonin and serotonin were discovered in plants several years after their discovery in mammals, but their presence has now been confirmed in almost all plant families. The mechanisms of action of melatonin and serotonin are still poorly defined. Melatonin and serotonin possess important roles in plant growth and development, including functions in chronoregulation and modulation of reproductive development, control of root and shoot organogenesis, maintenance of plant tissues, delay of senescence, and responses to biotic and abiotic stresses. This review focuses on the roles of melatonin and serotonin as a novel class of plant growth regulators. Their roles in reproductive and vegetative plant growth will be examined including an overview of current hypotheses and knowledge regarding their mechanisms of action in specific responses.

The biosynthetic origin of irregular monoterpenes in Lavandula: isolation and biochemical characterization of a novel cis-prenyl diphosphate synthase gene, lavandulyl diphosphate synthase.

Lavender essential oils are constituted predominantly of regular monoterpenes, for example linalool, 1,8-cineole, and camphor. However, they also contain irregular monoterpenes including lavandulol and lavandulyl acetate. Although the majority of genes responsible for the production of regular monoterpenes in lavenders are now known, enzymes (including lavandulyl diphosphate synthase (LPPS)) catalyzing the biosynthesis of irregular monoterpenes in these plants have not been described. Here, we report the isolation and functional characterization of a novel cis-prenyl diphosphate synthase cDNA, termed Lavandula x intermedia lavandulyl diphosphate synthase (LiLPPS), through a homology-based cloning strategy. The LiLPPS ORF, encoding for a 305-amino acid long protein, was expressed in Escherichia coli, and the recombinant protein was purified by nickel-nitrilotriacetic acid affinity chromatography. The approximately 34.5-kDa bacterially produced protein specifically catalyzed the head-to-middle condensation of two dimethylallyl diphosphate units to LPP in vitro with apparent Km and kcat values of 208 ± 12 µm and 0.1 s(-1), respectively. LiLPPS is a homodimeric enzyme with a sigmoidal saturation curve and Hill coefficient of 2.7, suggesting a positive co-operative interaction among its catalytic sites. LiLPPS could be used to modulate the production of lavandulol and its derivatives in plants through metabolic engineering.