Melatonin: plant growth regulator and/or biostimulator during stress?

89 downloads 77902 Views 600KB Size Report
Modifies the development pattern of stems and leaves (branching) ... inoculated apple (Malus prunifolia) trees, mitigating premature defoliation and pathogen ..... We thank Russel J. Reiter (University of Texas Health Science Center in.
Review

Melatonin: plant growth regulator and/or biostimulator during stress? Marino B. Arnao and Josefa Herna´ndez-Ruiz Department of Plant Biology (Plant Physiology), Faculty of Biology, University of Murcia, 30100 Murcia, Spain

Melatonin regulates the growth of roots, shoots, and explants, to activate seed germination and rhizogenesis and to delay induced leaf senescence. The antioxidant properties of melatonin would seem to explain, at least partially, its ability to fortify plants subjected to abiotic stress. In this Review we examine recent data on the gene-regulation capacity of melatonin that point to many interesting features, such as the upregulation of anti-stress genes and recent aspects of the auxin-independent effects of melatonin as a plant growth regulator. This, together with the recent data on endogenous melatonin biosynthesis induction by environmental factors, makes melatonin an interesting candidate for use as a natural biostimulating treatment for field crops. Melatonin in plants: discovery and roles Melatonin (N-acetyl-5-methoxytryptamine) was discovered in 1958 in the bovine pineal gland [1]. It is one of the best-studied biological molecules and its role has been explored in mammals, birds, amphibians, reptiles, and fish. Melatonin has many physiological roles in animals [2–5], influencing circadian rhythms, mood, sleep, body temperature, locomotor activity, food intake, retina physiology, sexual behavior, seasonal reproduction, and the immune system [6]. Melatonin acts as a signal of darkness, providing information to the brain and peripheral organs and serving as an endogenous synchronizer for physiological rhythms (e.g., sleep–wake cycles, seasonal reproduction, and endocrine release cycles). Alterations in rhythmic melatonin production have been associated with many disorders [2,7,8] such as Alzheimer’s and Parkinson’s syndrome [9], glaucoma, multiple sclerosis, depression, insomnia, chronic fatigue syndrome, schizophrenia, anxiety, metabolic syndrome, osteoporosis, and some types of cancer [10,11]. Thirty years after the discovery of melatonin in mammals, the detection of melatonin in the unicellular dinoflagellate Lingulodinium polyedrum (synonym Gonyaulax polyedra) completely altered the way in which this methoxyindole was regarded [12]. In 1993 melatonin was detected in Japanese morning glory (Pharbitis nil), although these results were not published extensively until 1995 [13]. The almost simultaneous publication of two Corresponding author: Arnao, M.B. ([email protected]). Keywords: antioxidant; melatonin; photosynthesis; phytomelatonin; plant growth regulator; plant stress; rhizogenesis. 1360-1385/ ß 2014 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.tplants.2014.07.006

papers on edible plants in 1995 unequivocally demonstrated the existence of melatonin in higher plants [14,15]. Since that time, successive studies have quantified the presence of melatonin in many plants [16], and it is now accepted that melatonin is present in all kingdoms, from prokaryotes to eukaryotes, from animals to plants [17–21]. The term ‘phytomelatonin’ was suggested in 2004 [22]. However, the presence of melatonin in lichens has not yet been investigated, and there is still only limited information about melatonin in fungi, bacteria, and nonvascular plants. In the past 2–3 years there has been much progress in unraveling the role of melatonin in plants, and the number of publications has undergone an exponential increase, underlining the growing interest in this topic. Melatonin functions as a chronoregulator in mammals and birds: its role in photoperiodic regulation had been demonstrated based on the duration and timing of the melatonin signal [3,5,23]. Initial investigations in plants explored whether melatonin played a similar chronoregulatory role in plants. Melatonin levels have been reported to oscillate in some species, such as goosefoot (Chenopodium rubrum) [24], water hyacinth (Eichhornia crassipes) [25], grape vine (Vitis vinifera) [26], sweet cherry (Prunus avium) [27], and the green macroalga Ulva sp. [28]. This rhythmic behavior, which is dependent on light–dark cycles, could be a regulatory element in photoperiodism such as flowering. Although the influence of light and other environmental factors on endogenous levels of melatonin has been demonstrated (discussed below), there are no conclusive data on its true role in the circadian rhythms of plants [29]. Various studies have suggested specific physiological actions for melatonin in plants, where it can act as a growth regulator [30,31], governing the growth of roots, shoots and explants [32–34], activate rhizogenesis [35], and delay-induced leaf senescence [36]. The natural antioxidant capacity of melatonin might explain some of its physiological actions: for example, its ability to fortify plants subjected to abiotic stresses such as drought, cold, heat, salinity, chemical pollutants, herbicides, and UV irradiation makes melatonin an interesting candidate for use as a natural biostimulating substance for treating field crops [16,37,38]. We review here recent studies on the gene-regulation capacity of melatonin and on the relationship between melatonin and chemical or environmental factors ([29,30,39–41] for further reviews), and discuss the potential for melatonin to be used as both a plant growth regulator and as a biostimulator in stress situations. Trends in Plant Science, December 2014, Vol. 19, No. 12

789

Review

Trends in Plant Science December 2014, Vol. 19, No. 12

Melatonin as a plant growth regulator A range of different functions of melatonin have been investigated in higher plants, some more thoroughly than others, but in all cases the data are scarce (Table 1). Among the studies that have tried to identity a specific role for melatonin in plants, its role as a possible growth regulator has been widely discussed. Melatonin acts as a growth promoter in etiolated lupin (Lupinus albus), acting in a similar way as auxin, indolyl-3-acetic acid (IAA), and induces active growth of hypocotyls at micromolar concentrations while having an inhibitory effect at high concentrations. The growth-promoting effect of melatonin is 63% that of IAA, which is a considerable auxinic effect [33]. In a similar study, using several monocots, oat (Avena sativa), wheat (Triticum aestivum), barley (Hordeum vulgare), and canary grass (Phalaris canariensis), the growthpromoting activity of melatonin ranged from 10% that of IAA in oat coleoptiles to 55% in barley coleoptiles. Furthermore, similarly to IAA, melatonin showed a concentration-dependent growth-inhibitory effect on the roots, ranging from 56% that of IAA in canary grass to 86% in wheat root [34]. The endogenous level of IAA and melatonin showed similar values, between 25 and 150 ng/g fresh weight; however, in the cases of barley and oat the coleoptile melatonin levels were significantly higher than those of IAA, suggesting possible co-action in some tissues [34]. These promoting and inhibitory effects of melatonin, depending on the concentration, have also been described in red cabbage (Brassica oleracea rubrum) [42]. In mustard (Brassica juncea) roots, 0.1 mM melatonin has been shown to have a stimulatory effect on root growth, whereas 100 mM has an inhibitory effect. Furthermore, endogenous free IAA levels increase at low melatonin concentrations. The authors suggested that the stimulation of root growth by low concentrations of melatonin was triggered by melatonin-stimulated IAA biosynthesis, although the specific relationship between IAA and melatonin is unclear [43]. Experiments with transgenic rice (Oryza sativa) seedlings overexpressing sheep serotonin N-acetyltransferase

(SNAT; the penultimate enzyme of the melatonin biosynthetic pathway: Box 1) showed enhanced (twofold) seminal root growth, which correlated with the production of 10fold higher levels of melatonin in their shoots relative to wild type seedlings, pointing to a direct relationship between the endogenous melatonin level and root growthrate [44]. The growth-promoting effect has also been observed in lupin cotyledons, which expanded in the presence of exogenous melatonin [45]. Another well-known physiological action of auxin is its capacity to induce changes in the organogenic pattern of plants. The role of melatonin in the induction of rhizogenesis was first demonstrated in 2007 [35]. Melatonin induced root primordials from pericycle cells in lupin, resulting in the generation of adventitious or lateral roots. This rhizogenic effect has recently been confirmed in cucumber (Cucumis sativus) [46], four cherry rootstocks (Prunus cerasus; P. cerasus  P. canescens; P. avium  P. mahaleb; P. avium  P. cerasus) [47,48], rice [44], and pomegranate (Punica granatum) [49]. In an analysis of the auxin-inducible gene expression marker DR5:GUS in roots of Arabidopsis (A. thaliana), transgenic lines treated with either IAA, the synthetic auxin 1-N-naphtaleneacetic acid (NAA), or melatonin, only IAA and NAA resulted in increased GUS activity throughout the primary and lateral root meristems. By contrast, DR5:GUS seedlings treated with three different melatonin concentrations did not show any increase in GUS expression, but did show a staining pattern similar to that obtained in control seedlings [50], which supports data showing that the early response activation of auxin signaling by IAA is not mimicked by melatonin [51]. Curiously, this inability of melatonin to induce auxin-responsive gene expression does not mean that it cannot activate the promotion of lateral roots. Whole-transcriptome sequencing (RNA-seq) analysis of a collection of cucumber roots with and without melatonin treatment generated 16 866 670 sequence reads aligned with 17 920 genes, which has provided abundant data for the analysis of lateral root formation. The expression of 121 genes was significantly

Table 1. Functions of phytomelatonin in higher plants Vegetative development

Reproductive development

Stress environment

790

Physiological action of melatonin Activates the growth of diverse seedlings Activates or inhibits the growth of primary roots Promotes lateral and adventitious rooting in several species Modifies the development pattern of stems and leaves (branching) Delays chlorophylls lost during leaf-induced senescence Enhances photosynthesis, CO2 uptake and biomass Promotes rhizogenesis and caulogenesis in explant cultures Cryopreserves callus or shoot tips for long-term storage Functions as a chronoregulator Affects different stages of floration in several species Levels are altered during fruit development and seed formation in several species Affects growth of plants subjected to stress situations, e.g., low and high temperatures, drought, UV irradiation, chemical stressors, and herbicides Provokes an increase in the proline level in stressed plants Increases the germination rate of stressed seeds Improves resistance to the fungal pathogen Diplocarpon mali in inoculated apple (Malus prunifolia) trees, mitigating premature defoliation and pathogen expansion

Refs [33,34,42,45] [34,43,44] [35,44,46–49,52] [54,55] [36,46,56,58,59] [28,46,48,56,60,81] [31,32,53] [73–75] [24–28] [81,88–91] [27,62,90,92–95]

[46,55,56,58,63,64,70,71,76,77,79–82,96,97] [48] [42,46,65,72,98]

[99]

Review

Trends in Plant Science December 2014, Vol. 19, No. 12

Box 1. Melatonin biosynthesis The melatonin biosynthetic pathway in vertebrates, including mammals, birds, and amphibians, is well known [100,101]. Tryptophan is converted into either 5-hydroxytryptophan by tryptophan 5-hydroxylase (T5H, E.C.1.14.16.4), or into tryptamine by tryptophan decarboxylase (TDC, E.C. 4.1.1.28) (Figure I). Although the pathway involving 5hydroxytryptophan is predominant in animals, and the tryptamine pathway has been shown to be more important in rice [102], both TDC and T5H are involved in the synthesis of serotonin. Indole-3-acetaldehyde (IAAld) is an intermediate product produced in the conversion of tryptamine to IAA. The N-acetylation of serotonin is catalyzed by the enzyme serotonin N-acetyltransferase (SNAT, E.C. 2.3.1.87). N-acetylserotonin is then methylated by hydroxyindole-O-methyltransferase (HIOMT, E.C.2.1.1.4) to generate melatonin. Two minor alternatives (broken blue arrows in Figure I) to this pathway have been described: (A) the synthesis of N-acetylserotonin from tryptamine catalyzed by SNAT and T5H, with N-acetyltryptamine as an intermediate, and (B) the production of melatonin directly from serotonin catalyzed by HIOMT and SNAT, with 5-methoxytryptamine as an intermediate. The melatonin biosynthetic pathway is not regulated in the same way in plants as it is in animals. In humans, SNAT is broadly considered to be a rate-limiting enzyme and the melatonin rhythmgenerating enzyme, exhibiting daily rhythms with nocturnal maxima in the pineal gland and in the retina of humans and many vertebrates. Nocturnal SNAT levels are 7- to 150-fold higher than daytime levels, but decrease rapidly following exposure to light, reflecting changes in the duration of the light period [101]. HIOMT in humans is generally constitutive, although a gentle rhythm of HIOMT activity has been described. In plants the biosynthetic pathway is more complex; however, to date, only limited data are available [21]. All the enzymes in the melatonin biosynthetic pathway have recently been characterized and localized in rice [103]. TDC and T5H transcript expression levels are regulated by light: high levels of expression occur under constant light and low levels under dark conditions [102,104,105]. Although the SNAT enzyme is localized in chloroplasts its activity is inhibited by chlorophylls [106]. HIOMT seems to be the rate-limiting enzyme in plants and its mRNA level is higher in the dark than under continuous light conditions [107]. Interestingly, TDC, SNAT, and HIOMT activities also seem to be regulated by high temperature, as is the ancestral cyanobacterial SNAT gene [80,108].

upregulated in melatonin-treated roots, and 196 genes were significantly downregulated, accompanied by an increased number of lateral roots [52]. The gene were categorized into 36 gene ontology (GO) functional groups; however, the expression pattern of the auxin-related genes exhibited minimal expression differences. The authors concluded that melatonin affected the root pattern in an auxin-independent manner, agreeing with previous data [50,51]. The possible role of melatonin as plant regulator in cell culture has also been investigated. Studies of in vitro culture explants of St. John’s wort (Hypericum perforatum) found that the endogenous level of melatonin in the culture medium modulated plant morphogenesis, changing the rate of rhizogenesis (auxin-induced) and caulogenesis (cytokinin-induced), thereby pointing to a possible role for melatonin as a plant regulator or auxin modulator [31,32]. In thidiazuron-treated leaf explants of purple coneflower (Echinacea purpurea), the application of auxin-transport or -action inhibitors provoked an increase in melatonin and serotonin levels. Moreover, supplementation with lidocaine, a sodium channel blocker, resulted in high levels of serotonin and melatonin, but not of auxin, and decreased the regeneration rate of explants. Given that not only did the levels of serotonin and melatonin increase as a result of

Tryptophan TDC

Tryptamine

T5H 5-Hydroxytryptophan

TDC

T5H

(A)

Serotonin (5-Hydroxytryptamine)

SNAT

IAAld

N-acetylserotonin

(B)

HIOMT

Melatonin IAA

(N-acetyl-5-methoxytryptamine) TRENDS in Plant Science

Figure I. Melatonin biosynthetic pathway.

exposure to thidiazuron (associated with the induction of regeneration), but also the level of auxin, the authors proposed that melatonin may act as a hormone independently or in concert with auxin [53]. The possible action of melatonin, acting as an auxin-like hormone, on the phenotypic changes in the apical dominance or branching response has also been studied. Transgenic tomato (Solanum lycopersicum) plants were modified by overexpressing the rice indoleamine 2,3-dioxygenase (IDO) gene, a well-documented enzyme that cleaves the indole ring of several indolic compounds such as indoleamines (tryptophan and related metabolites, including melatonin and serotonin) and also IAA [54]. IDO-transgenic tomato plants showed lower levels of melatonin in leaves than the corresponding wild type because of the action of the overexpressed-IDO protein. In the transgenic tomato plants there was a significant change in the lateral leaflet pattern (a decrease in the number of leaflets, and these were more flattened and less serrated than wild type) in plant lines overexpressing IDO. Nevertheless, the indiscriminate action of IDO on indolic compounds in plant tissues makes it difficult to draw conclusions as to whether the reduced level of melatonin is the cause of these morphological changes in tomato leaves. However, in a recent study [55] of tomato transgenic plants overexpressing 791

Review sheep SNAT and hydroxyindole-O-methyltransferase (HIOMT, the penultimate and ultimate enzymes of the melatonin biosynthetic pathway; Box 1), higher melatonin levels were observed in leaves (up to a sixfold increase versus wild type levels) accompanied by a substantial decrease in endogenous IAA levels (IAA levels in stems and leaves were only 25% and 14%, respectively, of wild type levels). Furthermore, apical dominance was diminished in transgenic lines, which showed a branching phenotype. The authors attributed this to reduced IAA levels as a consequence of the increase in melatonin and concomitant overconsumption of tryptophan, the common precursor of melatonin and IAA (Box 1). The authors concluded that in this case melatonin does not seem to replace IAA in the apical dominance function [55]. Melatonin as a biostimulator: anti-senescence and antistress effects Melatonin plays a significant role in the leaf senescence process. Melatonin at different concentrations slowed dark-induced senescence in barley leaves, delaying the total loss of chlorophyll with respect to control leaves incubated in water [36]. This unexpected effect of melatonin was confirmed using the opposite actions of kinetin and abscisic acid (ABA) on foliar senescence. In both cases the presence of melatonin during the kinetin or ABA treatment inhibited chlorophyll losses. This effect, discovered in 2009, was first associated with the excellent antioxidant properties of melatonin, even though the possible action of melatonin on chlorophyll-degrading enzyme genes was also suggested, and was later confirmed by other authors [56,57]. Based on these data, many studies have focused on the possible action of melatonin in photosynthesis. For example, exogenous melatonin has been shown to delay dark-induced senescence in apple (Malus domestica) leaves through the enhancement of reactive oxygen species (ROS)-scavenging enzyme activities, while maintaining the ascorbic acid and glutathione content at higher levels compared with those found in the control leaves [58]. Furthermore, following long-term application of melatonin to one-year-old apple trees under drought conditions, leaf senescence was delayed, with a significant reduction in chlorophyll degradation [56]. The suppression of the upregulation of the senescence marker, senescence-associated gene 12 (SAG12), and of the monooxygenase senescencerelated pheophorbide-a oxygenase (PaO), indicates that melatonin has a role as a regulating factor in induced foliar senescence [56,59]. In addition to this protective role of melatonin against leaf senescence, some interesting new data have been reported in recent years. Melatonin seems to increase the photosynthetic efficiency of plants, acting via an unusual biostimulatory pathway. For example, melatonin improves the efficiency of photosystem II under dark and light conditions in apple trees, alleviating the inhibition of photosynthesis caused by drought stress, and also allowing the leaves to maintain a higher capacity for CO2 assimilation and stomatal conductance [56]. Similar data were also obtained in a study of water-stressed cucumber seedlings. Melatonin treatment reduced chlorophyll degradation and increased the photosynthetic rate and 792

Trends in Plant Science December 2014, Vol. 19, No. 12

the activities of ROS-scavenging enzymes, thereby reversing the adverse effects of water stress [46]. Furthermore, in shoot-tip explants of cherry rootstock, the application of exogenous melatonin at low concentrations, in addition to its rhizogenic effects, slightly enhanced the content of photosynthetic pigments, total biomass, and total carbohydrates, and also increased the proline content of leaves, indicating a role for melatonin in plant stress metabolism [48]. Some preservative effect of melatonin on the chlorophyll content has also been observed in the macroalga Ulva sp. [28] and in a study of the freshwater Characeae Chara australis, which reported an increase in the efficiency of the reaction centers of photosystem II [60]. A primary function attributed to melatonin in plants is to act as an antioxidant [61], providing protection against environmental agents. This idea was suggested in early studies of melatonin in plants [15,62] but has only started to gather support in recent years. Barley [63] and lupin plants [64] treated with different chemical stressors such as zinc, hydrogen peroxide, or sodium chloride show an increase in endogenous melatonin levels. The increase in melatonin (up to 12-fold in lupin) is time- and concentration-dependent. Following exogenous melatonin application, plants such as barley and lupin have been reported to show improved vegetative development and survival in the presence of these chemical stressors [63,64]; likewise pea (Pisum sativum) plants and red cabbage seedlings in the presence of copper showed improved vegetative development and survival following melatonin application [42,65]. It has been suggested that melatonin improves the redox state of cells, decreasing ROS and reactive nitrogen species (RNS) levels, and stabilizing biological membranes, as it does in animal cells [5,66–68] ([69] for recent review). When 45-day-old apple (Malus hupehensis) seedlings that had been pretreated with melatonin were subjected to a high level of saline stress, shoot height, leaf number, chlorophyll content, and electrolyte leakage were less affected by the saline stress compared with untreated plants. Interestingly, hydrogen peroxide levels were halved, ROSmetabolizing enzymes (ascorbate peroxidase, catalase, and peroxidase activities) were induced, and Na+ and K+ transporters (NHX1 and AKT1) were upregulated, which would all help to alleviate saline-induced inhibition [70]. Similarly, a transgenic rice line overexpressing SNAT showed greater resistance to butafenacil (a singlet oxygen-generating herbicide) than did wild type plants [71]. Based on these observations, the authors proposed that endogenous melatonin is crucial as a ROS scavenger, acting as a first line of defense against oxidative stress. The relationship between water deficit/drought and melatonin has been examined. Water restrictions during the growth of lupin seedlings provoked an increase in endogenous melatonin, which was fourfold higher than that observed in well-irrigated plants, suggesting that melatonin plays a relevant signaling role [64]. In another study, water-stressed cucumber plants treated with melatonin had a higher seed-germination rate and showed improved root growth, indicating that the application of melatonin minimized induced water-stress [46]. A similar protective effect has been described in the case of long-term drought stress in apple. Exogenous melatonin treatment

Review through the roots provoked a marked reduction in leaf senescence, improved photosynthetic parameters such as photosystem II efficiency, and enhanced the antioxidative enzymes, leading the authors to propose the use of melatonin for agricultural purposes [56,58]. The shoot tips of cherry explants grown in vitro in medium containing melatonin showed a fivefold increase in proline levels and a three- to fourfold increase in total carbohydrate content, and this suggests that melatonin may have a role as a marker of stress tolerance, through an osmoregulatory response [48]. In a recent study of the resistance of tomato transgenic lines overexpressing sheep SNAT and HIOMT enzymes during 20 days of drought, the transgenic plants recovered completely following rewatering, whereas the wild type plants died. Furthermore, under drought conditions the leaves of wild type plants lost water more quickly than did the leaves of the transgenic lines [55]. Another abiotic stressor of plants is ambient temperature. Cucumber seeds pretreated with melatonin improved their germination rate during chilling stress compared with untreated seeds [72]. This protective role was also observed in the cryopreserved callus of Rhodiola crenulata, a Tibetan herb that grows under extreme conditions of cold, high altitude, and intense UV irradiation [73], and in mung bean (Vigna radiata) meristem cells after chilling [74]. The presence of 0.1–0.5 mM melatonin in both preculture and regrowth media enhanced the regrowth of frozen shoot explants of American elm (Ulmus americana), demonstrating the usefulness of melatonin for the longterm storage of germplasm for plant cell culture [75]. Furthermore, human SNAT transgenic rice seedlings have been shown to have greater cold-resistance than wild type [76]. Exactly as melatonin has a positive effect on chilled plants as regards their protection or resistance, cold-treatment has an effect on the biosynthetic rate of melatonin, increasing endogenous levels. Thus, lupin plants grown at 68C showed a 2.5-fold increase in their melatonin content compared with control plants grown at 248C [64]. In a recent study of Arabidopsis treated with melatonin and grown at 48C, the melatonin-treated plants had a significantly greater fresh weight, primary root length, and shoot height compared to the untreated plants, the effect being time- and concentration-dependent [77]. Treating Arabidopsis with melatonin upregulated the expression of some cold-signaling genes: the C-repeat-binding factors (CBFs), which control the expression of 100 genes, providing freezing tolerance to the plants; COR15a, a coldresponsive gene regulated by CBFs; CAMTA1, a transcription factor involved in freezing- and drought-stress tolerance that is related to the Ca2+/calmodulin proteins (wellknown in melatonin treatment in animal cells); and ZAT10 and ZAT12, two key transcription activators of ROS-related antioxidant genes. Thus, these data implicate melatonin in the upregulation of specific cold-responsive genes, supporting the hypothesis that melatonin plays a protective role against abiotic stresses [77,78]. Interesting data have been obtained comparing plants grown under controlled conditions (in culture chambers) with plants grown under field conditions. The melatonin contents (in different organs) of lupin and tomato plants

Trends in Plant Science December 2014, Vol. 19, No. 12

grown under field conditions were increased threefold and 10-fold, respectively, versus the levels in plants grown in culture chambers [64,79]. The influence of environmental factors on melatonin levels has also been shown in water hyacinth plants [25], grape berry skin [26], and cherry fruits [27]. Furthermore, a recent study in rice demonstrated that SNAT and HIOMT enzymes (Box 1) were influenced by temperature and light. Melatonin levels were higher at 558C and/or under dark conditions, and were associated with enzymatic activities but not with transcript levels [80]. Furthermore, melatonin-rich transgenic rice plants grown under field conditions were more robust and had greater height and biomass than wild type plants. Some phenotypic changes in flowering and grain yield were also observed in these transgenic plants [81]. Transgenic plants of woodland tobacco (Nicotiana sylvestris) expressing human SNAT and HIOMT enzymes had a higher melatonin content than the wild type plants, and the increase correlated with improved resistance to UV-B irradiation at the DNA level [82]. Studies of the effect of melatonin on flowering, fruit development, and ripening are listed in Table 1. Under field-conditions, multiple factors such as temperature oscillations, light–dark cycles, UV irradiation, and water availability, among others, are combined. In such a situation melatonin acts as direct antioxidant and also triggers an antioxidative response against a stressing environment [38]. In the most complete study to date of melatonin-mediated genetic functional analysis [57], mRNA-seq technology was used to analyze Arabidopsis plants subjected to a 16 h 100 pM (low) and 1 mM (high) melatonin treatment, and the expression profiles were analyzed to identify differentially expressed genes. The 100 pM melatonin treatment significantly affected the expression of 81 genes, with 51 being downregulated and 30 upregulated. However, the 1 mM melatonin treatment significantly altered the expression of 1308 genes, with 566 being upregulated and 742 downregulated. Not all the genes that showed altered expression when subjected to low melatonin were affected by high melatonin, suggesting that melatonin plays different roles in plant growth and development regulation at low and high concentrations. Furthermore, many of the genes whose expression was altered by melatonin were involved in plant stress defense: transcript levels for many stress receptors, mitogen-activated protein kinases, and stress-associated calcium signals were upregulated. Most of the transcription factors identified were also involved in plant stress defense and in the redox network. Interestingly, chlorophyllase and PaO, involved in chlorophyll degradation, were both downregulated, confirming preliminary studies on the ability of melatonin to delay senescence (discussed earlier) [36]. Furthermore, cell deathassociated genes were mostly downregulated by the 100 pM melatonin treatment. In total, 183 genes involved in hormone signaling were identified in this study [57]. Genes in the ABA, ethylene, salicylic acid, and jasmonic acid pathways were upregulated, whereas genes related to auxin responses, transport, homeostasis, and signaling, and those associated with cell wall synthesis, were mostly downregulated. Two 1-aminocyclopropane-1-carboxylate (ACC) synthases were also upregulated in response to 793

Review

Trends in Plant Science December 2014, Vol. 19, No. 12

melatonin, suggesting that melatonin may induce ethylene biosynthesis. One of these two ACC synthases is also auxin-inducible. None of the auxin biosynthesis pathway genes was significantly altered by the melatonin treatment, with the exception of an IAA–amino synthase that conjugates amino acids to IAA. In conclusion, the results indicate that during plant growth and development melatonin plays important physiological roles in many biological processes including abiotic stress defense, photosynthesis, cell wall modification, and redox homeostasis [57]. Concluding remarks and future perspectives The upregulation of some anti-stress genes (vs cold, drought, osmotic stress) in melatonin-treated plants, and the induction of endogenous melatonin by these stressors, has confirmed a central role for melatonin as a signaling molecule in abiotic stress. However, the action of melatonin as a possible plant growth regulator has not yet been confirmed. The role of melatonin in the growth response and in rooting has been demonstrated in various species, as well as its effect on the regulation of, for example, particular enzymes, promoters,

air al rep d mic n Che xifier na y o o za uidit det bili sta ane fl e br ran Direct aco acon mb mem Me

and hormone receptors. Perhaps melatonin plays a similar role to that of strigolactones, which play a key role in the fine-regulation of the root system [83]. Figure 1 summarizes the most relevant aspects of likely melatonin action as a plant growth regulator and biostimulator based on the available data. Further studies will be necessary to determine the relationship between melatonin, apical dominance, and the branching or flattened leaflets observed in transgenic plants: these auxin-like effects suggest that there is a possible relationship between melatonin and IAA. There are effects of auxin that are not mediated by the activation of gene expression through TRANSPORT INHIBITOR RESPONSE 1/AUXIN SIGNALING F-BOX: for example, roots, but not hypocotyls, require F-box receptors for their growth responses [84]. It appears that both the receptors and the downstream targets of auxin signaling in growth responses are different in roots and shoots. In this sense, melatonin and IAA might coexist as regulators, operating with different regulatory targets and pathways. There are insufficient data on how IAA and melatonin affect the endogenous levels of each other, although altering the

• Chlorophyll • PSII • Biomass • Carbohydrates • Yield

ROS and RN scaveng S ing

as anoxidant anoxida

Redox network Biosmulator

Abioc stressor

An-stress response Melatonin Hormone signaling genes: ABA, ET, SA, JA

Plant growth regulator

Growth response (concentraon-dependent)

• Cold or high temperature • Drought • UV irradiaon • Osmoc shock • Shoots (+) • Roots (+/–) • Cotyledons (+) • Cell culture (+) • Primary roots • Lateral roots • Advenous roots

Apical dominance

• Branching • Flaened leaflets

Auxin

IAA acon

Responsive genes

Roong Hormone signaling genes:

IAA level (↑/↓)

• Senescence marked genes • Cell death genes • Chlorophyllase gene • Pheophorbide -a oxygenase gene

TRENDS in Plant Science

Figure 1. Model of likely melatonin action as a plant growth regulator and biostimulator. Melatonin can act directly as an effective antioxidant, decreasing levels of reactive oxygen species (ROS) and reactive nitrogen species (RNS) and detoxifying diverse chemical contaminants. Melatonin can also act as a membrane stabilizer, playing an important role in membrane fluidity, and as an up- and downregulator of gene expression. As a biostimulator, melatonin modifies the expression of redox network genes involved in the anti-stress response, and fortifies plants by optimizing photosynthetic-related parameters and repressing senescence genes. The abiotic stressors that induce anti-stress genes also provoke an increase in endogenous melatonin levels. As a plant growth regulator, melatonin acts by regulating the expression of enzymes, promoters and hormonal receptors, among others, related with the growth response, rooting and branching. These actions may occur in a parallel pathway to the known pathway mediated by indolyl-3-acetic acid (IAA). Furthermore, melatonin may modify the IAA levels in plant tissues, altering the phenotypic response of plants. Up- and downregulatory actions are indicated by arrows and bar lines, respectively. Broken lines indicate hypothetical relationships that to date have not been demonstrated experimentally. (+) indicates activation of growth; ( ) indicates inhibition of growth. Abbreviations: ABA, abscisic acid; ET, ethylene; JA, jasmonic acid; PSII, photosystem II; SA, salicylic acid.

794

Review IAA:melatonin ratio had a phenotypic effect on the transgenic plants assayed [43,54,55]. Another possible effect to study is that of IAA and melatonin in their respective biosynthetic pathways, taking into account that tryptophan and tryptamine are common metabolites (Box 1). Although data for rice are available, further work will be necessary to isolate, characterize, and investigate the regulation of melatonin biosynthesis enzymes in other plants. With regard to melatonin catabolism (kynuric and indolic pathways), our broad knowledge of melatonin catabolism in animals should be gradually extended to include plants [25,85]. Another interesting area that could be investigated is whether, as in animals, membrane and nuclear melatonin receptor(s) are present in plants [86]. The effect of melatonin on the redox network can be used as a biostimulator to develop fortified plants with, for example, anti-senescence genes and improved photosynthesis, resulting in plants with greater strength and robustness. Although no conclusive data exist for many aspects of the action of melatonin, field trials are underway to verify the pleiotropic properties of this molecule as a plant regulator and as a protective agent against environmental stresses, with potential applications in crop improvement and protection. Some of these studies have focused on producing melatonin-rich plants for use as food nutraceuticals for human consumption, and also as a substitute for treatment by injection or dermal patches of exogenous melatonin in livestock as a sexual estrus inducer [16,22,87]. Another interesting possibility is whether melatonin-rich plants could be used to recover chemical-contaminated soils, complementing other phytoremediation practices. To conclude, the ability of melatonin to strengthen plants subjected to multiple abiotic (and biotic) stressors points to its promising potential for use as a natural biostimulating substance in the treatment of field crops. Acknowledgments We thank Russel J. Reiter (University of Texas Health Science Center in San Antonio, USA) and Juan Antonio Madrid (Department of Animal Physiology, Chronobiology Group at University of Murcia, Spain) for the initial stimulus to work on melatonin in plants and for their lectures on biological rhythms and melatonin.

References 1 Lerner, A. et al. (1958) Isolation of melatonin, a pineal factor that lightens melanocytes. J. Am. Chem. Soc. 80, 2587 2 Maronde, E. and Stehle, J. (2007) The mammalian pineal gland: known facts, unknown facets. Trends Endocr. Metabol. 18, 142–149 3 Pandi-Perumal, S. et al. (2008) Physiological effects of melatonin: role of melatonin receptors and signal transduction pathways. Prog. Neurobiol. 85, 335–353 4 Jan, J. et al. (2009) The role of the thalamus in sleep, pineal melatonin production, and circadian rhythm sleep disorders. J. Pineal Res. 46, 1–7 5 Hardeland, R. et al. (2012) Melatonin, the circadian multioscillator system and health: the need for detailed analysis of peripheral melatonin signal. J. Pineal Res. 52, 139–166 6 Carrillo-Vico, A. et al. (2013) Melatonin: buffering the immune system. Int. J. Mol. Sci. 14, 8638–8683 7 Wilhelmsen, M. et al. (2011) Analgesic effects of melatonin: a review of current evidence from experimental and clinical studies. J. Pineal Res. 51, 270–277 8 Hardeland, R. (2012) Melatonin in aging and disease. Multiple consequences of reduced secretion, options and limits of treatment. Aging Dis. 3, 194–225

Trends in Plant Science December 2014, Vol. 19, No. 12

9 Srinivasan, V. et al. (2005) Role of melatonin in neurodegenerative diseases. Neurotox. Res. 7, 293–318 10 Seely, D. et al. (2012) Melatonin as adjuvant cancer care with and without chemotherapy: a systematic review and meta-analysis of randomized trials. Integr. Cancer Ther. 11, 293–303 11 Di Bella, G. et al. (2013) Melatonin anticancer effects: Review. Int. J. Mol. Sci. 14, 2410–2430 12 Poeggeler, B. et al. (1991) Pineal hormone melatonin oscillates also in dinoflagellates? Naturwissenschaften 78, 268–269 13 van Tassel, D. et al. (1995) Melatonin from higher plants: isolation and identification of N-acetyl-5-methoxytryptamine. Plant Physiol. 108, 101 14 Dubbels, R. et al. (1995) Melatonin in edible plants identified by radioimmunoassay and by HPLC-MS. J. Pineal Res. 18, 28–31 15 Hattori, A. et al. (1995) Identification of melatonin in plants and its effects on plasma melatonin levels and binding to melatonin receptors in vertebrates. Biochem. Mol. Biol. Int. 35, 627–634 16 Arnao, M.B. (2014) Phytomelatonin: discovery, content, and role in plants. Adv. Bot. 2014, e815769 17 Hardeland, R. (1999) Melatonin and 5-methoxytrypyamine in nonmetazoans. Reprod. Nutr. Dev. 39, 399–408 18 Sprenger, J. et al. (1999) Melatonin and other 5-methoxylated indoles in yeast: presence in high concentrations and dependence on tryptophan availability. Cytologia 64, 209–213 19 Hardeland, R. and Poeggeler, B. (2003) Non-vertebrate melatonin. J. Pineal Res. 34, 233–241 20 Tilden, A.R. et al. (1997) Melatonin production in an aerobic photosynthetic bacterium: an evolutionarily early association with darkness. J. Pineal Res. 22, 102–106 21 Tan, D.X. et al. (2013) Mitochondria and chloroplasts as the original sites of melatonin synthesis: a hypothesis related to melatonin’s primary function and evolution in eukaryotes. J. Pineal Res. 54, 127–138 22 Blask, D.E. et al. (2004) Melatonin uptake and growth prevention in rat hepatoma 7288CTC in response to dietary melatonin: melatonin receptor-mediated inhibition of tumor linoleic acid metabolism to the growth signaling molecule 13-hydroxyoctadecadienoic acid and the potential role of phytomelatonin. Carcinogenesis 25, 951–960 23 Hardeland, R. et al. (2006) Melatonin. Int. J. Biochem. Cell Biol. 38, 313–316 24 Wolf, K. et al. (2001) Daily profile of melatonin levels in Chenopodium rubrum L. depends on photoperiod. J. Plant Physiol. 158, 1491–1493 25 Tan, D. et al. (2007) Novel rhythms of N-acetyl-N-formyl-5methoxykynuramine and its precursor melatonin in water hyacinth: importance for phytoremediation. FASEB J. 21, 1724–1729 26 Boccalandro, H. et al. (2011) Melatonin levels, determined by LC-ESIMS/MS, fluctuate during the day/night cycle in Vitis vinifera cv Malbec: evidence of its antioxidant role in fruits. J. Pineal Res. 51, 226–232 27 Zhao, Y. et al. (2012) Melatonin and its potential biological functions in the fruits of sweet cherry. J. Pineal Res. 55, 79–88 28 Tal, O. et al. (2011) Melatonin as an antioxidant and its semi-lunar rhythm in green macroalga Ulva sp. J. Exp. Bot. 62, 1903–1910 29 Tan, D. et al. (2012) Functional roles of melatonin in plants, and perspectives in nutritional and agricultural science. J. Exp. Bot. 63, 577–597 30 Arnao, M.B. and Herna´ndez-Ruiz, J. (2006) The physiological function of melatonin in plants. Plant Signal. Behav. 1, 89–95 31 Murch, S. and Saxena, P. (2002) Melatonin: a potential regulator of plant growth and development? In vitro. Cell. Dev. Biol. Plant 38, 531–536 32 Murch, S. et al. (2001) The role of serotonin and melatonin in plant morphogenesis. Regulation of auxin-induced root organogenesis in in vitro-cultured explants of Hypericum perforatum L. In vitro. Cell. Dev. Biol. Plant 37, 786–793 33 Herna´ndez-Ruiz, J. et al. (2004) Melatonin: growth-stimulating compound present in lupin tissues. Planta 220, 140–144 34 Herna´ndez-Ruiz, J. et al. (2005) Melatonin acts as a growthstimulating compound in some monocot species. J. Pineal Res. 39, 137–142 35 Arnao, M.B. and Herna´ndez-Ruiz, J. (2007) Melatonin promotes adventitious- and lateral root regeneration in etiolated hypocotyls of Lupinus albus L. J. Pineal Res. 42, 147–152 795

Review 36 Arnao, M.B. and Herna´ndez-Ruiz, J. (2009) Protective effect of melatonin against chlorophyll degradation during the senescence of barley leaves. J. Pineal Res. 46, 58–63 37 Janas, K. and Posmyk, M. (2013) Melatonin, an underestimated natural substance with great potential for agricultural application. Acta Physiol. Plant. 35, 3285–3292 38 Arnao, M.B. and Herna´ndez-Ruiz, J. (2014) Melatonin: possible role as light-protector in plants. In UV Radiation: Properties, Effects, and Applications. Physics Research & Technology Series (In: Radosevich, J.A., ed.), pp. 79–92, Nova Science Publishing 39 Paredes, S. et al. (2009) Phytomelatonin: a review. J. Exp. Bot. 60, 57–69 40 Posmyk, M. and Janas, K. (2009) Melatonin in plants. Acta Physiol. Plant. 31, 1–11 41 Park, W. (2011) Melatonin as an endogenous plant regulatory signal: debates and perspectives. J. Plant Biol. 54, 143–149 42 Posmyk, M.M. et al. (2008) Presowing seed treatment with melatonin protects red cabbage seedlings against toxic copper ion concentrations. J. Pineal Res. 45, 24–31 43 Chen, Q. et al. (2009) Exogenously applied melatonin stimulates root growth and raises endogenous IAA in roots of etiolated seedling of Brassica juncea. J. Plant Physiol. 166, 324–328 44 Park, S. and Back, K. (2012) Melatonin promotes seminal root elongation and root growth in transgenic rice after germination. J. Pineal Res. 53, 385–389 45 Herna´ndez-Ruiz, J. and Arnao, M.B. (2008) Melatonin stimulates the expansion of etiolated lupin cotyledons. Plant Growth Regul. 55, 29–34 46 Zhang, N. et al. (2013) Melatonin promotes water-stress tolerance, lateral root formation, and seed germination in cucumber (Cucumis sativus L.). J. Pineal Res. 54, 15–23 47 Sarropoulou, V. et al. (2012) Melatonin promotes adventitious root regeneration in in vitro shoot tip explants of the commercial sweet cherry rootstocks CAB-6P (Prunus cerasus L.), Gisela 6 (P. cerasus  P. canescens), and MxM 60 (P. avium  P. mahaleb). J. Pineal Res. 52, 38–46 48 Sarropoulou, V. et al. (2012) Melatonin enhances root regeneration, photosynthetic pigments, biomass, total carbohydrates and proline content in the cherry rootstock PHL-C (Prunus avium  Prunus cerasus). Plant Physiol. Biochem. 61, 162–168 49 Sarrou, E. et al. (2014) Melatonin and other factors that promote rooting and sprouting of shoot cuttings in Punica granatum cv. Wonderful. Turk. J. Bot. 38, 293–301 50 Koyama, F. et al. (2013) The structurally related auxin and melatonin tryptophan-derivatives and their roles in Arabidopsis thaliana and in the human malaria parasite Plasmodium falciparum. J. Eukaryot. Microbiol. 60, 646–651 51 Pelagio-Flores, R. et al. (2012) Melatonin regulates Arabidopsis root system architecture likely acting independently of auxin signaling. J. Pineal Res. 53, 279–288 52 Zhang, N. et al. (2014) The RNA-seq approach to discriminate gene expression profiles in response to melatonin on cucumber lateral root formation. J. Pineal Res. 56, 39–50 53 Jones, M. et al. (2007) The mode of action of thidiazuron: auxins, indoleamines, and ion channels in the regeneration of Echinacea purpurea L. Plant Cell Rep. 26, 1481–1490 54 Okazaki, M. et al. (2010) Lowering intercellular melatonin levels by transgenic analysis of indoleamine 2,3-dioxygenase from rice in tomato plants. J. Pineal Res. 49, 239–247 55 Wang, L. et al. (2014) Changes in melatonin levels in transgenic Micro-Tom tomato overexpressing ovine AANAT and ovine HIOMT genes. J. Pineal Res. 56, 134–142 56 Wang, P. et al. (2013) Long-term exogenous application of melatonin delays drought-induced leaf senescence in apple. J. Pineal Res. 54, 292–302 57 Weeda, S. et al. (2014) Arabidopsis transcriptome analysis reveals key roles of melatonin un plant defense systems. PLoS ONE 9, e93462 58 Wang, P. et al. (2012) Delayed senescence of apple leaves by exogenous melatonin treatment: toward regulating the ascorbate-glutathione cycle. J. Pineal Res. 53, 11–20 59 Wang, P. et al. (2013) Delay in leaf senescence of Malus hupehensis by long-term melatonin application is associated with its regulation of metabolic status and protein degradation. J. Pineal Res. 55, 424–434 60 Lazar, D. et al. (2013) Exogenous melatonin affects photosynthesis in characeae Chara australis. Plant Signal. Behav. 8, e23279 796

Trends in Plant Science December 2014, Vol. 19, No. 12

61 Tan, D. et al. (1993) Melatonin: a potent, endogenous hydroxyl radical scavenger. Endocr. J. 1, 57–60 62 Manchester, L. et al. (2000) High levels of melatonin in the seeds of edible plants. Possible function in germ tissue protection. Life Sci. 67, 3023–3029 63 Arnao, M.B. and Herna´ndez-Ruiz, J. (2009) Chemical stress by different agents affects the melatonin content of barley roots. J. Pineal Res. 46, 295–299 64 Arnao, M.B. and Herna´ndez-Ruiz, J. (2013) Growth conditions determine different melatonin levels in Lupinus albus L. J. Pineal Res. 55, 149–155 65 Tan, D. et al. (2007) Phytoremediative capacity of plants enriched with melatonin. Plant Signal. Behav. 2, 514–516 66 Fischer, T. et al. (2013) Melatonin enhances antioxidative enzyme gene expression (CAT,GPx,SOD), prevents their UVR-induced depletion, and protects against the formation of DNA damage in ex vivo human skin. J. Pineal Res. 54, 303–312 67 Galano, A. et al. (2011) Melatonin as a natural ally against oxidative stress: a physicochemical examination. J. Pineal Res. 51, 1–16 68 Catala´, A. (2007) The ability of melatonin to counteract lipid peroxidation in biological membranes. Curr. Mol. Med. 7, 638–649 69 Garcı´a, J.J. et al. (2014) Protective effects of melatonin in reducing oxidative stress and in preserving the fluidity of biological membranes: a review. J. Pineal Res. 56, 225–237 70 Li, C. et al. (2012) The mitigation effects of exogenous melatonin on salinity-induced stress in Malus hupehensis. J. Pineal Res. 53, 298–306 71 Park, S. et al. (2013) Melatonin-rich transgenic rice plants exhibit resistance to herbicide-induced oxidative stress. J. Pineal Res. 54, 258–263 72 Posmyk, M.M. et al. (2009) Melatonin applied to cucumber (Cucumis sativus L.) seeds improves germination during chilling stress. J. Pineal Res. 46, 214–223 73 Zhao, Y. et al. (2011) Melatonin improves the survival of cryopreserved callus of Rhodiola crenulata. J. Pineal Res. 50, 83–88 74 Szafranska, K. et al. (2013) Ameliorative effect of melatonin on meristematic cells of chilled and re-warmed Vigna radiata roots. Biol. Plant. 57, 91–96 75 Uchendu, E.E. et al. (2013) Melatonin enhances the recovery of cryopreserved shoot tips of American elm (Ulmus americana L.). J. Pineal Res. 55, 435–442 76 Kang, K. et al. (2010) Enhanced production of melatonin by ectopic overexpression of human serotonin N-acetyltransferase plays a role in cold resistance in transgenic rice seedlings. J. Pineal Res. 49, 176–182 77 Bajwa, V.S. et al. (2014) Role of melatonin in alleviating cold stress in Arabidopsis thaliana. J. Pineal Res. 56, 238–245 78 Shi, H. and Chan, Z. (2014) The cysteine2/histidine2-type transcription factor ZINC FINGER OF ARABIDOPSIS THALIANA 6-activated C-REPEAT-BINDING FACTOR pathway is essential for melatonin-mediated freezing stress resistance in Arabidopsis. J. Pineal Res. http://dx.doi.org/10.1111/jpi.12155 79 Arnao, M.B. and Herna´ndez-Ruiz, J. (2013) Growth conditions influence the melatonin content of tomato plants. Food Chem. 138, 1212–1214 80 Byeon, Y. and Back, K. (2014) Melatonin synthesis in rice seedlings in vivo is enhanced at high temperatures and under dark conditions due to increased serotonin N-acetyltransferase and N-acetylserotonin methyltransferase activities. J. Pineal Res. 56, 189–195 81 Byeon, Y. and Back, K. (2014) An increase in melatonin in transgenic rice causes pleiotropic phenotypes, including enhanced seedling growth, delayed flowering, and low grain yield. J. Pineal Res. 56, 408–414 82 Zhang, L. et al. (2012) Production of transgenic Nicotiana sylvestris plants expressing melatonin synthetase genes and their effect on UVB-induced DNA damage. In Vitro. Cell. Dev. Biol. Plant 48, 275–282 83 Rasmussen, A. et al. (2013) Strigolactones fine-tune the root system. Planta 238, 615–626 84 Scheitz, K. et al. (2013) Rapid auxin-induced root growth inhibition requires the TIR and AFB auxin receptors. Planta 238, 1171–1176 85 Tan, D. et al. (2007) One molecule, many derivates: a never-ending interaction of melatonin with reactive oxygen and nitrogen species? J. Pineal Res. 42, 28–42 86 Li, D. et al. (2013) Melatonin receptor genes in vertebrates. Int. J. Mol. Sci. 14, 11208–11223

Review 87 Feng, X. et al. (2014) Melatonin from different fruit sources, functional sources, and analytical methods. Trends Food Sci. Technol. 37, 21–31 88 Kolar, J. et al. (2003) Exogenously applied melatonin affects flowering of the short-day plant Chenopodium rubrum. Physiol. Plant. 118, 605–612 89 Murch, S. and Saxena, P. (2002) Mammalian neurohormones: potential significance in reproductive physiology of St. John’s wort (Hypericum perforatum L.)? Naturwissenschaften 89, 555–560 90 Murch, S.J. et al. (2009) Melatonin and serotonin in flowers and fruits of Datura metel L. J. Pineal Res. 47, 277–283 91 Park, S. et al. (2013) Transient induction of melatonin biosynthesis in rice (Oryza sativa L.) during the reproductive stage. J. Pineal Res. 55, 40–45 92 Okazaki, M. and Ezura, H. (2009) Profiling of melatonin in the model tomato (Solanum lycopersicum L.) cultivar Micro-Tom. J. Pineal Res. 46, 338–343 93 Murch, S. et al. (2010) Changes in the levels of indoleamine phytochemicals during veraison and ripening of wine grapes. J. Pineal Res. 49, 95–100 94 Lei, Q. et al. (2013) Identification of genes for melatonin synthetic enzymes in Red Fuji apple (Malus domestica Borkh.cv.Red) and their expression and melatonin production during fruit development. J. Pineal Res. 55, 443–451 95 Riga, P. et al. (2014) Melatonin content of pepper and tomato fruits: effects of cultivar and solar radiation. Food Chem. 156, 347–352 96 Turk, H. et al. (2014) The regulatory effect of melatonin on physiological, biochemical and molecular parameters in coldstressed wheat seedlings. Plant Growth Regul. http://dx.doi.org/ 10.1007/s10725-014-9905-0 97 Afreen, F. et al. (2006) Melatonin in Glycyrrhiza uralensis:response of plant roots to spectral quality of light and UV-B radiation. J. Pineal Res. 41, 108–115

Trends in Plant Science December 2014, Vol. 19, No. 12

98 Tiryaki, I. and Keles, H. (2012) Reversal of the inhibitory effect of light and high temperature on germination of Phacelia tanacetifolia seeds by melatonin. J. Pineal Res. 52, 332–339 99 Yin, L. et al. (2013) Exogenous melatonin improves Malus resistance to Marssonina apple blotch. J. Pineal Res. 54, 426–434 100 Falco´n, J. et al. (2009) Structural and functional evolution of the pineal melatonin system in vertebrates. Ann. N. Y. Acad. Sci. 1163, 101–111 101 Reiter, R. (1999) Pineal melatonin: cell biology of its synthesis and of its physiological interactions. Endocr. Rev. 12, 151–180 102 Park, S. et al. (2012) Tryptamine 5-hydroxylase-deficient Sekiguchi rice induces synthesis of 5-hydroxytryptophan and N-acetyltryptamine but decreases melatonin biosynthesis during senescence process of detached leaves. J. Pineal Res. 52, 211–216 103 Byeon, Y. et al. (2014) Cellular localization and kinetics of the rice melatonin biosynthetic enzymes SNAT and ASMT. J. Pineal Res. 56, 107–114 104 Park, S. et al. (2013) Transcriptional suppression of tryptamine 5hydroxylase, a terminal serotonin biosynthetic gene, induces melatonin biosynthesis in rice (Oryza sativa L.). J. Pineal Res. 55, 131–137 105 Byeon, Y. et al. (2014) Elevated production of melatonin in transgenic rice seeds expressing rice tryptophan decarboxylase. J. Pineal Res. 56, 275–282 106 Byeon, Y. et al. (2013) Microarray analysis of genes differentially expressed in melatonin-rich transgenic rice expressing a sheep serotonin N-acetyltransferase. J. Pineal Res. 55, 357–363 107 Park, S. et al. (2013) Functional analyses of three ASMT gene family members in rice plants. J. Pineal Res. 55, 409–415 108 Byeon, Y. et al. (2013) Molecular cloning and functional analysis of serotonin N-acetyltransferase from the cyanobacterium Synechocystis sp. PCC 6803. J. Pineal Res. 55, 371–376

797