Review Article | Open Access
Marino B. Arnao, "Phytomelatonin: Discovery, Content, and Role in Plants", Advances in Botany, vol. 2014, Article ID 815769, 11 pages, 2014. https://doi.org/10.1155/2014/815769
Phytomelatonin: Discovery, Content, and Role in Plants
Melatonin (N-acetyl-5-methoxytryptamine) is an indolic compound derived from tryptophan. Usually identified as a neurotransmitter or animal hormone, this compound was detected in plants in 1995. Interest in knowing the melatonin content of plants and its possible role therein is growing, as indicated by the increasing number of related publications. Melatonin is present in all plant species studied, with large variations in its level depending on the plant organ or tissue. It seems to be more abundant in aromatic plants and in leaves than in seeds. Regarding its physiological function in plants, melatonin shows auxin activity and is an excellent antioxidant, regulating the growth of roots, shoots, and explants, activating seed germination and rhizogenesis (lateral- and adventitious-roots), and delaying induced leaf senescence. Its ability to strengthen plants subjected to abiotic stress such as drought, cold, heat, salinity, chemical pollutants, herbicides, and UV radiation makes melatonin an interesting candidate for use as a natural biostimulating substance for treating field crops.
Melatonin is a molecule endowed with a multitude of functions, particularly in mammals. This indoleamine is a hormone that acts in many physiological aspects, influencing mood, sleep, body temperature, the retina, and sexual behavior, among others. Most of these aspects are regulated through or in conjunction with the circadian clock present in animals [1–7]. In addition, melatonin is involved in numerous cellular actions as an antioxidant, acting as an excellent in vitro and in vivo free radical scavenger [8–15].
Chemically, melatonin (N-acetyl-5-methoxytryptamine) is an indolic compound derived from serotonin (5-hydroxytryptamine) (Figure 1). Both biogenic amines are synthesized from the amino acid tryptophan in a well-characterized biosynthetic pathway in animals and with some particular characteristics in plants. In plants, the auxin, indolyl-3-acetic acid (IAA), bears some resemblance to melatonin since both are indole compounds and have a common biosynthetic pathway through the compound tryptamine in the tryptophan-dependent IAA biosynthetic pathway (Figure 1).
2. Discovery of Melatonin in Plants
The presence of melatonin was described for the first time in the bovine pineal gland by Lerner and coworkers . The isolated substance was able to stimulate melanin aggregation, lightening the skin of frogs and other amphibians but not of mammals. In 1959, melatonin was identified as N-acetyl-5-methoxytryptamine and, soon after, its biosynthetic pathway from tryptophan, with serotonin as intermediary, was discovered [17–19]. In 1959, melatonin was detected in humans  and in the 1960s and 70s, its presence was described in many mammals and vertebrates such as birds, amphibians, and fish [21–23]. In the 1980s, melatonin was discovered in invertebrates such as planarians, annelids, molluscs, insects, and crustaceans, among others [24–29].
In higher plants, the first identification of endogenous melatonin was described in 1993 by van Tassel and O’Neill in a congress communication . The authors had detected melatonin by radioimmunoassay (RIA) and gas chromatography with mass spectrometry (GC-MS) in the Convolvulaceae ivy morning glory (Pharbitis nil L., syn. Ipomoea nil L.) and in tomato fruits (Solanum lycopersicum L.), although the results were not published extensively until 1995 . Also in 1995, two papers published simultaneously demonstrated the presence of melatonin in higher plants. Dubbels and coworkers used RIA and HPLC-MS to measure the melatonin levels in extracts of Nicotiana tabacum L. and in five edible plants (see Table 1) . Two months later another publication appeared, in which the presence of melatonin in extracts of a large number of edible plants was quantified by RIA and liquid chromatography (HPLC) with fluorescence detection (Table 1) . Another communication appeared in the same year, in which a Czech research group identified the presence of melatonin in Chenopodium rubrum L. using liquid chromatography with mass identification (LC-MS/MS) . Successive studies have quantified the presence of melatonin in many plants and it is now accepted that melatonin is present in animals, plants, and in all the other kingdoms.
3. Extraction, Detection, and Quantification of Phytomelatonin
The first challenge in phytomelatonin quantification was its correct extraction from plant extracts. However, the extraction procedures used for different matrices may pose a problem as regards the measurement of melatonin levels, whatever the subsequent quantification method followed. In higher plants, melatonin levels have been shown to vary from a few picograms up to micrograms per gram of material analyzed. One of the problems concerning the reliable measurement of phytomelatonin that has been mentioned by several authors is the difficulty involved in the extraction and recovery of melatonin in plants [35–39]. At present, several methods of phytomelatonin extraction are used. Generally, it is extracted from liquid nitrogen treated-plant tissue using organic solvents such as methanol, chloroform, or ethyl acetate. However, when aqueous extraction is used, variable or low recovery rates have been reported [37, 40, 41], and due to the amphiphilicity of the melatonin molecule, direct sample extraction procedures (without homogenization of fresh tissues) using only organic solvent are recommended. Hardeland and coworkers mentioned that extraction efficiency is a decisive factor in the global estimation of melatonin content in photoautotrophic organisms . The level of melatonin in different samples was seen to be directly affected by the respective recovery rates of each extraction procedure applied. Thus, the reliability of the melatonin level data will depend on the recovery rate. According to our data, the inclusion of an ultrasonic treatment in the direct sample extraction procedure resulted in different levels of efficiency (2–20%), depending on the sample, although the inclusion of this treatment in the extraction procedure may lead to a significant increase in phytomelatonin extraction [38, 42]. Other possible problems, such as false-negative and false-positive results, overestimations, losses by destruction, coelution in chromatographic methods, cross-reactivities in immunological procedures, and so forth, have been described in the determination of melatonin in complex matrices . Analysis by liquid chromatography and identification by mass spectrometry (LC-MS/MS) are the most used and recommended techniques for the detection and quantification of phytomelatonin. In this respect, LC-MS/MS with positive electrospray ionization (ESI+) and multiple reaction monitoring (MRM) is widely used. The innovative technique of liquid chromatography with time-of-flight/mass spectrometry (LC-TOF/MS) has also been applied to phytomelatonin detection. However, less specific but very sensitive techniques such as LC with electrochemical or fluorometric detection are also excellent if accompanied by identification by LC-MS/MS [36, 38, 42–49]. Unlike in animals, immunological techniques such as RIA or ELISA present serious problems in plants due to cross-reactivity with coextractives [37, 39, 50].
No exhaustive studies of the melatonin content of edible plants, taking into account variables such as soil, cultivar, growing, post-harvest conditions, and so forth have been made and the lack of contrasted results is one of the main problems in any attempt to explain the meaning of melatonin in plant material and foodstuffs. Interest has focused on measuring its levels because of possible implications for human food consumption, since melatonin from plant foods is absorbed from the gastrointestinal tract and incorporated in the blood stream. Melatonin also crosses the blood-brain barrier and the placenta, being incorporated at subcellular level in the nucleus and mitochondria. Thus, the possibility of modulating blood melatonin levels in mammals and avians through the ingestion of plant-derived foods containing high levels of phytomelatonin could be of potential interest [35, 51]. Table 1 shows the phytomelatonin content of edible plant organs such as fruits, seeds, and roots of the species analyzed to date. In general, seeds present the highest level of phytomelatonin and fruits the lowest. Thus, levels of below 50 pg·g−1 of tissue have been seen in the fruits of kiwi, cucumber, banana, apple, and strawberry, while higher levels have been observed in the seeds of mustard, alfalfa, fenugreek, and sunflower, among others (see Table 1). It is interesting to note that the analytical technique used in studies strongly influences the quantitative results. For example, studies made in the 1990’s tended to find lower levels of melatonin in plants [32, 33, 52] than is possible with more recent analyses, as can be observed in Table 1 in the case of strawberry and white radish, where quantitative differences reach up to three orders of magnitude or in the case of tomato fruit, where the values found range from 0.5 to 114,500 pg·g−1 of tissue. The explanation is the lack of control in the phytomelatonin extraction process, leading to poor recoveries, and also, in some cases, due to the use of immunological detection techniques, which give rise to large interferences and false negatives. Whatever the case, data for the melatonin content of plant foods are very scarce and no comparisons are possible, especially bearing in mind the large number of variables such as growing conditions (soil, irrigation, photoperiod, irradiance, and harvest), postharvest preservation methods, subspecies, and/or cultivars.
Although more data exist for wild plants, they are still scarce given the great diversity of plants. Table 2 shows the phytomelatonin content of the different wild species studied and in different organs (leaf, fruit, stem, root, flower, and seed). As can be seen, the phytomelatonin content ranges from 6 pg·g−1 in the shoots of Ipomoea nil L. to 34 μg·g−1 in the root of Glycyrrhiza uralensis Fisch, a difference of 106!
|L: leaves; F: fruits; S: shoots; R: roots; Fl: flowers; Se: seeds.|
These great differences in phytomelatonin content have also been described in Chinese medicinal herbs, as part of a wide-ranging study of 108 common Chinese herbs, where phytomelatonin levels range from 12 ng·g−1 DW in Gardenia jasminoides Ellis to 2.3 μg·g−1 DW in Viola philippica Cav. . Usually, the phytomelatonin content follows the pattern: leaves > seeds > roots > flowers > fruits, but in most cases data are missing for at least one organ of the plants studied. However, some studies have shown that the melatonin content in different plant organs cannot be considered homogenous. For example, Hernández-Ruiz and Arnao showed that phytomelatonin formed a concentration gradient in Lupinus albus L., following the pattern apical > central > basal zone for both roots and hypocotyls . Curiously, this gradient pattern was similar to that observed by IAA, which suggests that both indolic compounds could play similar physiological roles in plants .
4. Physiological Roles of Phytomelatonin
Our studies on melatonin were initiated as a natural continuation of our research line on antioxidants in plants and plant foods. The development of new antioxidant estimation methods that can be applied to many types of plant food allowed us to ascertain the characteristics of metabolites possessing interesting antioxidant qualities, such as organic acids, phenolic acids, flavonoids (flavanols, flavanones, and anthocyanins), tocopherols, and carotenoids, among others. Some of these studies on antioxidants can be consulted in our works [55–59]. One of the most interesting groups is the indolic compounds, substances derived mainly from the amino acid tryptophan. These are very diverse and numerous in many plants, which are considered to be of medicinal interest in some cases. They are particularly of high interest because they include the compound indolyl-3-acetic acid (IAA), the most widespread auxin in plants that acts as a growth promoter, among its other physiological functions. So, in a study on the antioxidant properties of various indolic compounds, we found that melatonin had a high antioxidant capacity compared with standard antioxidants such as ascorbic acid or trolox (synthetic analog of vitamin E). Melatonin was seen to have double the antioxidant activity of ascorbic acid or trolox and approximately double that of other indoles such as indolyl-3-acetic acid, indole-3-methanol, indole-3-propionic acid, indole-3-butyric acid, and tryptophan .
Parallel to these studies and thanks to a collaboration with Professor J.A. Madrid of the Department of Animal Physiology at the University of Murcia, with whom we studied the influence of various factors on plasma antioxidant levels in rats and their relation with the melatonin content, our group developed a methodology to estimate melatonin in plants and to study their possible physiological role. In our first work on phytomelatonin published in 2004, we hypothesised that melatonin plays a role as plant growth regulator. Generally, the relationship between the molecular structure and biological activity is close. If we observe the molecular structure of melatonin and IAA, both are seen to have a common indole ring but their substituent on the ring is very distinct (see Figure 1). IAA has an acid group in position 3 of the indole ring, while melatonin has an N-acetyl group in position 3 and a methoxy group in position 5. However, the atomic distances between the indole ring (delocalized positive charge) and the acidic group (negative charge) of IAA or between the indole ring and the carbonyl group of melatonin are quite similar (~0.50 nm). This suggested that melatonin might simulate the action of auxin under certain conditions. There is a similar relationship between auxinic activity and synthetic compounds such as 2,4-dichlorophenoxy acetic acid and naphthalene acetic acid. Melatonin stimulates the growth of etiolated hypocotyls of Lupinus albus L. in a similar way as in the assay made with IAA . The growth response was melatonin concentration-dependent and exogenous melatonin could replace the auxin stimuli when aerial meristem was excised. This was the first experimental data that clearly demonstrated the auxinic role of melatonin, although, several authors had previously proposed the possible role of melatonin as a plant regulator [61–63]. Our group also reported similar effects of melatonin in the Gramineae Avena sativa, Triticum aestivum, Hordeum vulgare, and Phalaris canariensis . In these cases, the growth-promoting activity of melatonin reached up to 55% with respect to IAA, while showing a concentration-dependent growth inhibitory effect on the roots, in a similar way to IAA. The growth promoting effect was also observed in lupin cotyledons, which expanded in the presence of exogenous melatonin . Later, the stimulatory growth effect of melatonin on Brassica sp. was also demonstrated [66, 67].
Melatonin was also seen to have the capacity to modulate the morphogenesis of plants, in the same way as auxin. For example, melatonin was able to induce rhizogenesis in pericycle cells from Lupinus albus L. This physiological role, mainly attributed to IAA, whereby lateral roots can be generated from primary roots or adventitious roots from stems, was also provoked by melatonin at a similar concentration to IAA . Later, this organogenetic effect of melatonin was also demonstrated in Cucumis sativus, Oryza sativa, and in Prunus cerasus [69–71]. In this sense, recent works demonstrated that melatonin does not mimic the auxin signaling response [72, 73].
Our research group was also the first to suggest that melatonin could play a significant role in the foliar senescence processes. The application of exogenous melatonin delays dark-induced senescence in leaves of Hordeum vulgare L. . The action of melatonin decreasing the rate of chlorophyll degradation in senescent leaves may be related with their antioxidant capacity, as we and other authors have suggested, but may also be due to the possible regulation of certain markers of senescence, as demonstrated in leaves of apple (Malus domestica L.). In melatonin-treated leaves, certain ROS scavenging enzyme activities were enhanced and the upregulation of some senescence-related genes was suppressed, which indicated that melatonin does indeed act as a regulating factor in induced leaf senescence [75, 76]. In addition to its protective effect against senescence, melatonin is also able to promote photosynthetic efficiency, as has been seen in Malus domestica , Cucumis sativus L. seedlings , and in Prunus sp. . Also, an increase in the efficiency of Photosystem II was observed in melatonin-treated cells of the fresh water Characeae Chara australis .
The possible role of melatonin as a regulator of light-dark cycles in plants has also been studied. This function has been clearly established in mammals and birds, where its role in photoperiodic regulation has been demonstrated based on the duration and timing of the melatonin signal [8, 79]. In plants, some oscillations in endogenous melatonin levels have been described in Chenopodium rubrum L. [34, 80, 81], Eichhornia crassipes (hyacinth) , Vitis vinifera cv. Malbec , and Prunus , pointing to its possible role as light-dark regulator. Also in the green macroalgae Ulva sp., a melatonin maximum at night in a long-photoperiod day has been described .
However, one of the most interesting aspects was the possible role of melatonin as a protective agent against abiotic stress situations in plants. The excellent properties of melatonin as a natural antioxidant against ROS/RNS and the absence of prooxidant effects have been the subject of a great deal of research. Arnao and Hernández-Ruiz demonstrated that treatment with chemical agents, such as hydrogen peroxide, sodium chloride, or zinc sulphate, stimulates the endogenous level of melatonin in Hordeum vulgare roots in a time- and concentration-dependent manner . This increase in melatonin levels probably plays an important role in the antioxidative defense against chemical-induced stress. Also in Lupinus albus plants, chemical stressors such as zinc sulphate increased endogenous melatonin levels twelve-fold, possibly through the induction of some enzyme(s) of its biosynthetic pathway . The negative/deleterious effects on plant of other stressors such as copper in soil [67, 88], cold [89–92], herbicides , drought [69, 76, 87], light/dark, and UV radiation [44, 82, 87, 94–96] can be reduced or minimized by the presence of melatonin. Generally, these stressor agents provoke an increase in endogenous melatonin levels, suggesting that melatonin may have a significant physiological role in many stress situations, strengthening the cell redox-regulated network [75, 77].
5. Conclusions and Future Perspectives
Melatonin has been seen to be involved in several physiological aspects in plants, where it acts as a circadian regulator, cytoprotector and growth promotor. It also acts in rhizogenesis, cellular expansion and stress-protection. In this respect, several reviews with summarized data can be consulted [39, 43, 45, 46, 97–99]. Currently, two aspects of phytomelatonin arouse the most interest: (i) its application as biostimulator in agriculture and (ii) its use as human natural nutraceutical. As regards the first aspect, trials with melatonin have shown that the application of exogenous melatonin to plants produces an improvement in important aspects of their development, including better adaptation to stress situations such as drought, salinity, pollutants, cold, heat, and radiation, among others. Melatonin also enhances the rate of germination and growth and plant productivity. It acts as a retardant in stress-induced leaf senescence. All this leads us to the idea that exogenous melatonin-treatment of cultivated plants or obtaining melatonin-overproducing plants might help crops resist more easily many adverse environmental conditions from which they normally suffer throughout their development. Another proposal concerns the use of plants rich in melatonin as a tool in phytoremediation techniques for the recovery of contaminated soils such as mining operations, industrial waste, or soils containing high levels of phytochemicals due to agricultural use.
The second of the above-mentioned aspects refers to the possibility of introducing melatonin-rich plants foods into our diet. Contrary to what frequently occurs with dietary supplements, increasing blood melatonin levels through eating natural foods such as plants could be considered a healthy habit. An oral dose of melatonin of up to 1 gram per day produces no adverse effects in humans. In addition, melatonin is easily absorbed via the gastrointestinal tract. So, its use as a nutraceutical product through the intake of melatonin-rich plants seems to have a promising future as a healthy phytochemical. It would, therefore, seem a worthwhile task to search for plants with high levels of endogenous melatonin that could be used as a natural source of nutraceuticals.
Conflict of Interests
The author declares that there is no conflict of interests regarding the publication of this paper.
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