2.1 Melatonin biosynthesis

Looking at the work by Reiter (1991) and Falcón et al. (2009) in vertebrates, the biosynthesis pathway of melatonin, which takes place in the pineal gland, is quite known. Falcón et al. (2009) discovered that the amino acid tryptophan is transformed by the enzyme tryptophan 5-hydroxylase (T5H) to 5-hydroxytryptophan, which is then transformed into serotonin by the enzyme tryptophan decarboxylase. This pathway is also involved in the production of serotonin in the Hypericum perforatum L. plants (Murch et al., 2000; Murch & Saxena, 2006). However, Park and Back (2012) revealed that in rice, the synthesis is more effectively caused by the tryptamine pathway (tryptophan-tryptamine-serotonin). This pathway has been found in many plant species. In both animals and plants, serotonin is transformed to N-acetylserotonin via the enzyme SNAT. After that, HIOMT (hydroxyIndole-O-methyl transferase), also known as ASMT (acetyl serotonin methyl transferase), methylates N-acetyl serotonin to form melatonin. Tryptamine produces N-acetylserotonin in plants, especially rice, with the facilitation of N-acetyltryptamine, where SNAT and tryptophan 5-hydroxylase catalyzes the pathway. Serotonin synthesizes melatonin with the intermediation of 5-methoxytryptamine, SNAT, and HIOMT/ASMT acting as catalysts (Arnao & Hernandez-Ruiz, 2014). Tryptamine also produces an important auxin named indole acetic acid (IAA) with the intermediation of indole-3-acetyl aldehyde (Arnao & Hernandez-Ruiz, 2014) (Figure 1).

As described by Arnao and Hernandez-Ruiz (2014), melatonin in plants is synthesized in higher concentrations, and its biosynthesis is more intricate in plants compared to animals. Since the information pertaining to melatonin biosynthesis is very limited in plants; its distinctive pathway is yet to be finalized (Byeon, Tan, et al., 2015). A recent study revealed that the genes for all the enzymes involved in melatonin biosynthesis are present in transgenic rice (Byeon et al., 2013), which may contribute to the description of the precise details of the biosynthetic pathway of melatonin in plants. Contrary to the biosynthesis, melatonin also undergoes the process of degradation. N1-acetyl-N2-formyl-5methoxykynuramine (AFMK) is a secondary metabolite present in vascular plants that degrades melatonin either through an enzymatic or non-enzymatic reaction (Tan, Manchester, Mascio, et al., 2007). Among metabolites of melatonin, 2-hydroxymelatonin (99%) and 4-hydroxymelatonin (0.05%), to a much lesser extent, are being documented in rice (Byeon, Tan, et al., 2015).

2.2 Effects of melatonin on plant growth and development

Murch et al. (1997) described the regulatory role of melatonin in Hypericum perforatum L. They detected induced root growth with increased concentration of melatonin and promoted shoot growth with serotonin (melatonin precursor) accumulation in vitro cultures. However, inhibitors of melatonin and serotonin transport were shown to block auxin-promoted root formation. On the other hand, substances inhibiting the conversion of serotonin into melatonin stimulate the formation of the cytokinin-induced shoot growth, as shown by Murch and Saxena (2002). So, the morphogenetic abilities of cultures of Hypericum perforatum L. seem to be dependent on a proper ratio of serotonin/melatonin, similarly like changes in the ratio of phytohormones like cytokinins to auxins cause modification in plant growth (Jones et al., 2007; Murch & Saxena, 2002).

Many studies have explained the regulation of the biological function of melatonin in plants, like the growth of explants, shoots, roots, and seed germination is generally improved by melatonin (Arnao & Hernández-Ruiz, 2019b; Murch et al., 2001). In 2005, Hernández-Ruiz et al. reported a direct melatonin involvement in plant growth stimulation; they observed the extension of coleoptiles (10–55%) due to melatonin in wheat, oat, barley, and canary grass (monocots). Later, Park and Back (2012) explained that enhancement in the length of initial seminal roots, root biomass, and growth of transgenic varieties of rice was observed by 0.5–1 μM application of melatonin. It is now known that melatonin is involved in altering many plant characteristics, including grain yields, variation in time of flowering, senescence, and seedling growth (Wang, Sun, Chang, et al., 2013) and germination (Zhang et al., 2014). Overexpressing sheep SNAT in transgenic rice, induced early seedling growth by melatonin, but flowering was delayed, and grain yield was reduced, as found by Byeon and Back (2014b). Wang, Sun, Chang, et al. (2013) showed that the metabolic status was altered and protein degradation was delayed in apple plants (Malus hupehensis Rehd.) due to the long term application of melatonin (100 μM) in soil. They also found an increase in photosynthetic end products (sorbitol, sucrose, and starch), photosynthetic rates, and chlorophyll contents compared with control plants. All such changes are linked with improved preservation capacity of proteins. In line with these findings, it was reported by Wang, Sun, Li, et al. (2013) that drought-induced leaf senescence was delayed in Hanfu apple (Malus domestica Borkh) by long term application of melatonin (100 μM), by suppressing upregulation of SAG-12 (senescence-associated gene 12) and PAO (pheophorbide an oxygenase) and reducing oxidative stress. Also, in Arabidopsis, natural leaf senescence showed a delay by exogenous application of melatonin (Shi, Reiter, et al., 2015).

As stated by the FAO (Food and Agricultural Organization of the United Nation), the fruit and vegetable post-harvest losses are very high (20–40%). In 2009 out of all food produced, 32% was lost or wasted, as shown by Lipinski et al. (2013). Keeping in view the interval of senescence and aging owing to melatonin pre as well as post-application, there exists a possibility that in an extension of fruits and vegetable shelf life, melatonin may play a significant role and prove useful in fruits' on-tree storage, both of which may decrease post-harvest losses and thus, increase production in fresh horticultural produce. In sweet cherry, Zhao, Tan, et al. (2012) detected that melatonin concentration during the second stage of fruit development was much higher. At the same time, it was comparatively much lower during the first and third stages. Seed and embryo development, cell elongation, and cell expansion occur during the second stage.

2.3 Action of melatonin as a bio stimulator and plant growth regulator

Melatonin is an influential antioxidant that abates the concentrations of ROS (reactive oxygen species) and RNS (reactive nitrogen species) and detoxifies a wide array of chemical pollutants. Melatonin stabilizes the membranes, facilitates its fluidity, and regulates gene expression. Melatonin also acts as a bio-stimulator by modifying the redox network gene expression responsible for triggering an anti-stress response in plants by ameliorating the photosynthetic activity and inhibiting genes responsible for senescence. The endogenous levels of melatonin enhance the activation of anti-stress genes caused by abiotic stress. Melatonin, as a growth regulator in plants, regulates enzymes' expression, promoters, and receptors of hormones pertaining to growth response such as branching and rooting (Arnao & Hernandez-Ruiz, 2014; Debnath et al., 2019). Moreover, melatonin may enhance the IAA levels in plants by changing their phenotypic response. Despite the thorough scrutiny of the role of melatonin in higher plants, limited studies are available on its overall role (Table 2). Figures 2 and 3 show the action of melatonin in plants as a plant growth regulator and bio-stimulator, based on the available information.

2.4 Exogenous application of melatonin to plants

After describing melatonin in animals and its detection in plants, studies concerning the effect of exogenous treatment of melatonin in different abiotic stresses were soon conducted. In this regard, both recent, as well as pioneer studies, are discussed in this section.

From the initial studies where the cold-induced apoptosis in culture cells of carrot (Daucus carota) was attenuated by the presence of exogenous melatonin (Lei et al., 2004), the protective role of melatonin against biotic and abiotic stresses in plants was postulated (Arnao & Hernández-Ruiz, 2019c, 2019d; Nawaz et al., 2016). Thus, the analysis of many stress situations and the effect of melatonin has been done. Most studies are on pea plant (Pisum sativum), where the survival and tolerance of plants in copper contaminated soil were enhanced with the application of exogenous melatonin (Tan, Manchester, & Helton, 2007). Also, in seedlings of red cabbage (Brassica oleracea rubrum), copper toxicity was reduced after the pretreatment of seeds with melatonin (Posmyk et al., 2008). During chilling stress, the germination rate was improved by pretreatment of cucumber seeds with melatonin compared to untreated seed (Posmyk et al., 2009). Similarly, an apparent increase in root growth and seed germination was shown in melatonin-treated cucumber plants under water stress indicated the minimization of water stress due to melatonin application (Zhang et al., 2013).

In apple species (Malus hupehensis) under salt stress, pretreatment of seedlings with melatonin showed that plants were less affected than untreated plants. Moreover, saline-induced inhibition would be reduced by induction of ROS-metabolizing enzymes (peroxidase, ascorbate peroxidase, and catalase activities), upregulation of K⁺, and Na⁺ transporters (AKT1 and NHX1), and halved hydrogen peroxide levels. (Szafranska et al., 2013). In meristem cells of mung bean (Vigna radiata), the protective role of melatonin applied exogenously after chilling was also observed (Szafranska et al., 2013). In plants, for long term storage of germplasm of cell culture, the usefulness of melatonin was demonstrated by the enhancement of cold shoot explants growth in Ulmus americana (American elm) due to the presence of both pre-culture and regrowth melatonin media (Uchendu et al., 2013). In a recent study of Arabidopsis grown at 4°C and treated with melatonin, fresh weight, shoot height, and primary root length was significantly greater in treated than untreated plants; the effect was dependent on both concentration and time (Bajwa et al., 2014).

Another study obtained similar data for soybean plants (Glycine max). Many parameters were optimized in seeds imbibed with melatonin, such as leaf size, seedling growth, biomass, pod number, seed number, and plant height. Also, in this species, both drought and salt tolerance were improved by melatonin treatment, which demonstrates the potential for field crops' improvement (Wei et al., 2015). Bermuda grass (Cynodon dactylon), a widely used turfgrass, showed similar results, as cold, drought, and salt stress situations were actively protected with melatonin applied exogenously as compared to untreated grass. A lower electrolyte leakage, cell damage and ROS burst and higher height and weight of plants, high levels of organic acids, amino acids, sugar alcohols, and sugars were shown in stressed melatonin treated plants than untreated plants, thus clearly affecting nitrogen and carbohydrates metabolism which are the main solutes having involvement in osmotic-stress response (Shi, Jiang, et al., 2015).

Another recent studies obtained similar results under salt stress in seedlings of Citrus aurantium (Kostopoulou et al., 2015); in cell cultures of Chara australis (Beilby et al., 2015); in cucumber seeds (Zhang et al., 2014) and seedlings, cotyledons and roots of sunflower (Mukherjee et al., 2014). Also, underwater stress in Vitis vinifera cuttings, under alkaline stress in tomato plants (Liu, Jin, et al., 2015), and under high-temperature stress in cucumber seedlings (Zhao, Sun, et al., 2012a), the importance of melatonin was revealed.

Special consideration is needed concerning the effects of melatonin on the photosynthetic process. The pioneering work of Arnao and Hernández-Ruiz (2009a) showed a delay in the loss of chlorophylls and retarding of induced senescence in leaves of barley as compared to untreated leaves, by exogenous melatonin. This effect of melatonin was contrasted with the retardant effect of kinetin and inductive effect of ABA (a synthetic cytokinin and abscisic acid, respectively) on foliar senescence. Later studies also confirmed this in other species, including Arabidopsis (Weeda et al., 2014), cucumber (Zhang et al., 2013), rice (Byeon et al., 2012, 2013), apple (Wang et al., 2012; Wang, Sun, Li, et al., 2013) and cherry (Sarropoulou et al., 2012). In apple leaves, through the development of some ROS activities, dark-induced senescence was delayed by exogenous melatonin, which contributed to the maintenance of higher glutathione and ascorbic acid contents and elimination of the excess of H₂O₂ generated in stressed leaves as compared to control leaves (Wang et al., 2012).

In apple trees, better efficiency of photosystem II was proved under both dark and light conditions. Photosynthetic efficiency may also be improved by melatonin in plants, besides its protective role against leaf senescence. Under drought stress, melatonin alleviates the inhibition in photosynthesis and allows leaves to maintain capacity for stomatal conductance and CO₂ assimilation (Wang, Sun, Li, et al., 2013). Similarly, data were obtained in cucumber seedlings under water stress. Adverse effects of water stress were reversed by melatonin treatment, as activities of ROS scavenging enzymes and photosynthetic rates were increased, and chlorophyll degradation was reduced (Zhang et al., 2013). Also, in cherry, the role of melatonin in plant stress metabolism has been indicated, as the content of the photosynthetic pigment, total carbohydrates, and total biomass was enhanced. Proline contents of roots were reduced by the exogenous application of less concentrated melatonin in shoot tip explants of the rootstock of cherry (Sarropoulou et al., 2012).

Several previous studies have also described preservative effects of melatonin on chlorophyll content, in freshwater C. australis (Lazar et al., 2013) and macroalga Ulva sp. (Tal et al., 2011), where the efficiency of the reaction centers of photosystem II was increased. Recently, similar research has been done regarding the protective role of melatonin on photosynthetic pigments under alkaline stress in tomato plants; in sunflower under salt-stress (Mukherjee et al., 2014); in wheat under cold-stress (Turk et al., 2014); and in citrus under salt-stress (Kostopoulou et al., 2015).

A clear relationship has been described in some cases between the beneficial effects of exogenous melatonin and leaf anatomical and morphological changes. Thus, underwater deficiency stress, oxidative damage was alleviated by melatonin in grape cuttings, by increasing antioxidative enzymes' activity (SOD, CAT, POX) and decreasing malondialdehyde content and ROS burst. Moreover, there was an increase in levels of antioxidant metabolites like ascorbic acid and glutathione, as well as proline content.

Direct evidence has been provided by a recent study of Wei et al. (2015), where melatonin coated seeds of soybean plants showed a significant increase in plant height, seeds, and pods per plant, fatty acid substances, and leaf area. New paths for enhancing the plant yield and revolutionizing the seed industry has been suggested by this study, as for a considerable number of essential horticultural and agronomic plants, melatonin-coated seeds could be used commercially. As described in Table 3, the application of melatonin resulted in substantial alleviation in growth reduction, photosynthetic inhibition, chlorophyll loss, and enhance antioxidant activities in response to biotic and abiotic stresses in many plants is shown in several studies.

2.5 Endogenous application of melatonin to plants

As described in the above section, the protective role of exogenously applied melatonin in plants is pointed out by many studies. However, recently it has been demonstrated that environmental conditions can vary the endogenous melatonin levels. In the study of Afreen et al. (2006), after UV-B radiation treatment, endogenous melatonin content of roots of Glycyrrhiza uralensis multiplied to about 80 L g⁻¹ FW, which is about a sevenfold increase as compared to control plants. These data suggest the accumulation of melatonin as a protective molecule in the plant tissue, toward different abiotic stressors like chemical agents, UV radiation, cold, water deficiency, and light–dark cycle.

Recently, under salt-stress conditions in sunflower seedlings, melatonin levels have been determined (Mukherjee et al., 2014). A sixfold increase in melatonin in cotyledons and a twofold increase in roots were shown by seedlings grown by adding NaCl compared to untreated plants, despite very high quantification of melatonin (on the order of L g g⁻¹ FW). Salt-treated plants also have a higher level of serotonin. In two Malus species, drought conditions upregulated four biosynthetic genes of melatonin, i.e., T5H, HIOMT, TDC, and SNAT (Li et al., 2015). All up to date available data showed evidence that the melatonin biosynthesis is induced by abiotic stressors, and in response to the abiotic stressor, melatonin plays the role of signal intermediate.

In freezing tissues or cold conditions, the protective role of melatonin has been demonstrated by a number of studies (Bajwa et al., 2014; Zhao et al., 2011). On the contrary, limited studies worked on heat stress. On germination, the high temperature’ inhibitory effect was reversed by melatonin treated seeds of thermos-sensitive Phacelia tanacetifolia (Tiryaki & Keles, 2012). Also, the resistance against heat stress in cucumber seedlings was improved, as activities of enzymes related to nitrogen metabolism at high temperatures were significantly increased by exogenous melatonin, thus restricting the ammonium content and increasing the nitrate content.

Recently, in Arabidopsis, the effect of high temperatures on thermo-tolerance factors and endogenous melatonin levels has been studied (Shi, Tan, et al., 2015). In Arabidopsis seedlings, endogenous melatonin content was increased by twofold to fivefold, provoked by heat stress (37°C). Also, under heat stress (45°C for 120 min), the survival rate of plants was enhanced (~50%) by exogenous treatment of melatonin (20 μM) as compared to untreated plants in which the survival rate was 5%.

A range of transgenic plants having an expression for ectopic genes, primary genes for melatonin synthesis from animal origin, has been utilized, to study the potential role of melatonin in the physiology of plants. As a rule, in overexpressing plants enriched with melatonin, a higher resistance toward abiotic stressors like UV-B, cold, and drought can be observed compared to wild type plants. It is demonstrated by these results that endogenous melatonin plays its role in defense against stressors and is critical for scavenging of ROS, even at lower concentrations (nano or picograms measured per gram of fresh weight). In transgenic modified plants, higher root biomass, root growth rate, and robustness were observed.