Study on Plant Melatonin

Melatonin is a substance belonging to the indole class, initially extracted from the pineal gland of cows. It causes the skin of tadpoles to change from black to light white, hence the name melatonin [1]. Melatonin has been extensively studied in animals and possesses various physiological functions, including regulation of circadian rhythms, improvement of sleep quality, scavenging of free radicals, enhancement of immune function, and delay of aging, making it important for health maintenance.With the detection of melatonin in various plants [2], research in the plant field has gradually begun.

 Studies have identified the melatonin synthesis pathways in plants and found that melatonin can act as a growth regulator to promote cell enlargement, root development, and participate in flowering regulation, among other functions.Melatonin can also enhance plants' tolerance to abiotic stresses such as drought, salinity, low temperature, and disease, and its mechanisms of action have been elucidated from both plant physiological and molecular biological perspectives. This review summarizes the current research progress on melatonin synthesis pathways, growth and development regulation, and stress response mechanisms in plants, aiming to provide references for future studies on melatonin in plants.

1  Melatonin in Plants

After melatonin was discovered in animals, researchers began to identify and isolate it in plants. In 1995, melatonin was detected in 24 plant species [2]. Although plants do not have pineal glands, they can still synthesize melatonin, making the melatonin synthesis pathway a research hotspot.By referencing the melatonin synthesis pathway in animals, researchers have gradually identified the key components of melatonin synthesis in plants, and the melatonin synthesis pathway in plants has become increasingly clear [3]. Researchers have also cloned the first melatonin receptor in plants, providing strong support for melatonin functional studies [4].

1.1  Endogenous melatonin content in plants

Melatonin is present in many organs of higher plants, such as roots, stems, leaves, flowers, fruits, and seeds, with concentrations typically ranging from pg to μg per gram of tissue.Crops such as rice, barley, and corn from the Poaceae family have relatively high melatonin content, while some medicinal plants like St. John's wort and white mandrake, as well as many fruits including strawberries, kiwifruit, pineapple, banana, and grape, also contain melatonin.

Common methods for determining melatonin include liquid chromatography, radioimmunoassay, ultra-high-performance liquid chromatography-mass spectrometry, and gas chromatography-mass spectrometry.Studies have reported melatonin content in rice seeds of different varieties ranging from 20 to 200 ng/g, with higher levels in brown rice compared to white rice, and higher in unpolished rice than in polished rice [6].Using liquid chromatography, melatonin content in barley seeds was determined to be approximately 82.3 ng/g, and in wheat seeds, it was 124.7 ng/g.Melatonin content in fruits was 0.02 ng/g in kiwifruit, 0.16 ng/g in apples, and relatively high in green tea, reaching 2.12 μg/g.Melatonin content not only varies among different species but also among different varieties of the same species. Additionally, melatonin content differs under various environmental conditions, particularly under adverse conditions, and at different stages of plant development [7].

1.2  Melatonin Synthesis Pathway in Plants

Since melatonin was first discovered in plants, its synthesis pathway has been a focus of research. The melatonin synthesis pathway in animals is well understood, with tryptophan serving as the precursor, undergoing four enzymatic reactions to ultimately produce melatonin.The first step is the conversion of tryptophan by tryptophan hydroxylase (TPH) to L-serotonin, which is then converted by aromatic amino acid decarboxylase (AADC) to N-acetylserotonin. The second step involves the conversion of N-acetylserotonin by aralkylamine N-acetyltransferase (AANAT), also known as acetyl transferase,SNAT), to form N-acetylserotonin; finally, N-acetylserotonin is converted into melatonin by N-acetylserotonin O-methyltransferase (ASMT),also known as hydroxyindole O-methyltransferase (HIOMT), to form melatonin.

The melatonin synthesis pathway in plants is generally similar to this, but the specific processes differ. In plants, tryptophan is first converted into tryptamine by tryptophan decarboxylase (TDC),and then tryptamine is hydroxylated by tryptamine 5-hydroxylase (tryptamine 5-hydroxylase, TDH) in the second step. The melatonin synthesis steps are the same [3].

Genes related to melatonin synthesis in plants have been identified one after another, and research on the melatonin synthesis pathway in rice has progressed rapidly.and all melatonin synthesis-related genes have been fully cloned. Kang et al. [8] cloned TDC1/AK069031 and TDC2/AK103253 from rice, and overexpressing TDC1 and TDC2 genes in rice increased serotonin content in leaves and seeds. Byeon et al. [9] cloned TDC3/NM_001067504 from rice. Overexpression of TDC3 in rice not only increased melatonin content in leaves and seeds,but also increased the content of 5-hydroxytryptophan synthesis intermediates. Fujiwara et al. [10] cloned T5H from a rice sekiguchilesion mutant that accumulates tryptophan precursors for serotonin synthesis through map-based cloning. In vitro recombinant expression of this protein exhibited tryptophan 5-hydroxylase activity,catalyzing the conversion of tryptophan to serotonin.

Kang et al. [11] cloned SNAT, the serotonin N-acetyltransferase responsible for the third step of melatonin synthesis, from rice. By analyzing the homologous enzyme acetyl transferase superfamily (GCN5-related N-acetyltransferase superfamily) in plants, they identified GNAT5 as the plant SNAT, which can rapidly catalyze the conversion of serotonin to N-acetyl serotonin. The final step of melatonin synthesis in rice is catalyzed by O-methyltransferase (OMT), which converts the hydroxyl group of N-acetyl serotonin to an oxygen-methyl group,Researchers analyzed all OMT genes in the rice genome and found that MT15 possesses ASMT enzyme activity capable of catalyzing melatonin synthesis [12].Subsequently, two additional ASMT genes were cloned, designated ASMT2 and ASMT3. Studies indicated that both possess O-demethylation activity. ASMT2 exhibits higher expression levels in stems and flowers, while ASMT3 shows low expression throughout the entire plant. The expression of these three genes is induced by abiotic stress and jasmonic acid [13].

1.3 Plant melatonin signal receptors

In 2018, Wei et al. [4] identified the first plant melatonin receptor candidate, G protein-coupled receptor 2 (CAND2), in Arabidopsis. Binding of melatonin to CAND2 triggers the dissociation of the βγ subunit from the α subunit of G proteins, thereby activating the G protein-coupled signal transduction pathway, which ultimately leads to the activation of the cAMP pathway.

cand2 mutants were insensitive to melatonin-induced stomatal closure. Melatonin binding to CAND2 triggers the dissociation of the βγ subunit from the α subunit of G proteins, thereby activating NADPH oxidase-dependent H₂O₂ production, enhancing Ca²⁺ influx, and promoting K⁺ efflux, ultimately leading to stomatal closure.Compared with traditional plant hormones, melatonin has been studied less as a plant hormone and requires further investigation.

2  Physiological functions of plant melatonin

As an indoleamine, melatonin has strong reducing power and can scavenge superoxide anions, hydrogen peroxide, and other free radicals produced in plants, thereby exerting antioxidant effects.Melatonin and indole-3-acetic acid (IAA) share similar physiological functions in the biosynthetic pathway within plants [15]. Studies have shown that melatonin not only acts as a signaling molecule to regulate plant growth and development but also participates in plant responses to abiotic and biotic stresses as both a signaling molecule and an antioxidant [16].

2.1 Melatonin regulation of plant growth and development

It has been demonstrated that melatonin promotes the enlargement and growth of the hypocotyl of isolated white lupine seedlings. The concentration distribution of melatonin and IAA in the hypocotyl is similar, with the highest levels at the apex, followed by the middle, and the lowest at the base; their modes of action are also similar, both promoting growth by increasing cell enlargement [17].

Melatonin promotes the growth of seed roots after germination. In rice overexpressing sheep SNAT, melatonin content increased, and compared with the wild type, the number of seed roots after germination in transgenic rice remained unchanged but root length was longer, and biomass also significantly increased.Under continuous light conditions, low concentrations of melatonin (0.5–1.0 μmol·L⁻¹) promote seedling root elongation in wild-type rice [18]. Melatonin regulates plant growth by modulating sugar metabolism pathways, and exogenous melatonin addition exhibits both promoting and inhibitory effects on corn seedlings.

Initial studies indicated that melatonin content is positively correlated with the expression of genes related to sucrose synthesis and hydrolysis, enzyme activity, and sucrose metabolism.Further studies revealed that exogenous application of low concentrations of melatonin (10 μmol·L⁻¹) increased photosynthetic rate and hexokinase activity, up-regulated the expression of photosynthesis-related genes and hexokinase-related genes, promoted corn growth, and enhanced sucrose metabolism, photosynthesis, and sucrose phloem loading.However, high concentrations of melatonin (100 μmol·L⁻¹) accelerated the accumulation of sucrose, hexoses, and starch, while inhibiting photosynthesis and sucrose phloem loading [19].

Melatonin also influences flowering time; the melatonin synthesis gene SNAT2 in Arabidopsis is highly expressed in flowers,In SNAT2 inactivating mutants, the expression levels of genes related to the gibberellin synthesis pathway, such as ent-kaurene synthase (KS), and the flowering control gene FLOWERING LOCUS T (FT) are downregulated, leading to a late-flowering phenotype [20].

The above studies indicate that melatonin can act as a small-molecule active substance to regulate plant physiological processes such as cell enlargement, root growth, and carbohydrate metabolism. Different concentrations have different effects, and whether melatonin functions as an independent signaling molecule or merely as an analog of IAA requires further investigation.

2.2 Melatonin enhances plant resistance to abiotic stress

2.2.1 Drought stress

Drought stress is a common abiotic stress, and oxidative stress is considered one of the primary damages caused by abiotic stress to plants. Drought stress primarily causes damage to the membrane system due to an imbalance between the production and removal of reactive oxygen species within cells [21].Melatonin is an endogenous antioxidant. Exogenous application of melatonin can increase the content and activity of antioxidant substances such as peroxidase, superoxide dismutase, and catalase in plants [22].Exogenous melatonin significantly reduces chlorophyll degradation in apple leaves under drought stress, improves photosynthetic efficiency, alleviates oxidative stress-induced damage, and effectively delays leaf senescence [23].

Exogenous melatonin effectively alleviates the inhibitory effect of polyethylene glycol-induced osmotic stress on cucumber seed germination, promotes rooting, enhances root system vitality, and increases the root-to-shoot ratio [24].Increasing endogenous melatonin content in plants can also enhance drought tolerance. Overexpression of apple ASMT in Arabidopsis resulted in melatonin levels in transgenic plants that were 2–4 times higher than those in wild-type plants. Under drought stress, ROS accumulation in transgenic plants was significantly lower than in wild-type plants, and survival rates were higher than those of wild-type plants [25].Exogenous melatonin addition enhances water retention, reduces ion leakage, and stabilizes chlorophyll content in apple seedlings, thereby improving drought tolerance.

Melatonin treatment downregulates the expression of the ABA synthesis gene 9-cis-epoxycarotenoid P450 family 707 subfamily A polypeptide 1 (CYP707A1) and reduces ABA content; it also upregulates the expression of melatonin synthesis-related genes, increasing endogenous melatonin synthesis, thereby improving drought tolerance. , CYP707A1) and CYP707A2, thereby reducing ABA content; it also upregulates the expression of melatonin synthesis-related genes, increasing endogenous melatonin synthesis, and improves apple drought tolerance by enhancing stomatal function and removing hydrogen peroxide [26].

2.2.2 Salt stress

Salt stress inhibits plant growth and development. Excessive salt ions primarily harm plants through osmotic stress, ion toxicity, and oxidative stress.

Exogenous application of 1 μmol·L⁻¹ melatonin alleviates the inhibitory effect of salt stress on apple growth, delays chlorophyll degradation, and reduces oxidative damage; melatonin treatment also induces the expression of sodium-hydrogen ion exchange protein 1 (Na+/H+ exchanger 1,MdNHX1) and potassium ion channel protein (Arabidopsis K+ transporter 1, MdAKT1), transporting Na+ into the vacuole and thereby reducing Na+ concentration in the cytoplasm, mitigating Na+ toxicity, and enhancing plant salt tolerance [27].

Zhang et al. [28] found that exogenous melatonin significantly improved cucumber seed germination rates under stress conditions. On one hand, melatonin enhanced the activity of cucumber seed peroxidase, catalase, and superoxide dismutase, reducing oxidative damage caused by salt stress.while also inducing the expression of ABA metabolism-related cytochrome oxidase CYP707A1 and CYP707A2, accelerating ABA degradation, and simultaneously inducing the expression of GA synthesis-related genes, thereby alleviating the inhibitory effect of salt stress on seed germination.Exogenous application of melatonin regulates the expression of a series of redox enzyme genes in rice, inhibits the accumulation of reactive oxygen species in photopigmentation-deficient leaf mutants, delays cell death, effectively removes reactive oxygen species accumulated under aging and stress conditions, reduces cellular damage, thereby delaying leaf aging in rice and enhancing salt stress tolerance [29].

Melatonin pretreatment maintained the growth and net photosynthetic rate of tomato seedlings under salt stress, increased the effective quantum yield of photosystem II and the photochemical quenching coefficient under physiological saline conditions, thereby enhancing the tolerance of tomato seedlings to salt stress [30].Salt stress reduced potassium ion absorption and increased hydrogen peroxide accumulation in apples, while melatonin treatment up-regulated potassium ion 3-cyclo-1] treatment enhanced salt tolerance in grape seedlings,The mechanism of action is that melatonin acts as a signaling molecule to induce the transcription factor myeloid cell leukemia virus oncogene homolog 108A (MYB108A),MYB108A), which regulates 1-aminocyclopropane-1-carboxylic acid synthase (1-melatonin also enhances salt tolerance in rice seedlings. On one hand, melatonin treatment reduces salt-induced potassium ion efflux, maintaining the balance between sodium and potassium ions,while melatonin itself, as an antioxidant, clears hydroxyl free radicals generated by salt stress, reducing free radical damage [33].

2.2.3  Low-temperature stress

Low-temperature environments affect plant cell membrane  fluidity and enzyme activity, inhibiting photosynthesis and nutrient  transport,causing plant damage and reducing crop yields. Lei et al. [34] found that melatonin could maintain cell membrane integrity in carrot suspension cells subjected to low-temperature stress; melatonin treatment also enhanced the low-temperature tolerance of Rhodiola rosea callus tissue and the regeneration of American elm frozen buds [35–36].

Exogenous addition of melatonin significantly increased plant height, root length, and fresh weight of seedlings under low-temperature stress in Arabidopsis, while the expression levels of cold adaptation-related transcription factors such as C-repeat binding factor (CBF), dehydration response element-binding protein (DREB), zinc finger protein 10 (ZAT10) [37]. Shi et al. [38] found that melatonin synthesis increased in Arabidopsis under low-temperature stress,while the expression levels of the zinc finger structural transcription factor ZAT6 gene also increased. Further studies revealed that ZAT6 directly regulates the expression of CBF genes, which is essential for melatonin-induced plant resistance to cold stress; however, ZAT6 does not regulate changes in melatonin content during cold stress treatment.

2.2.4 High-temperature stress

Exogenous melatonin treatment significantly enhances the heat tolerance of Arabidopsis, with class A1 heat shock factors (HSFA1s) participating in melatonin-induced heat tolerance.When Arabidopsis is subjected to heat stress, endogenous melatonin levels are rapidly and significantly induced, and the increased melatonin further upregulates the expression of AtHSFA1s.Subsequently, the activation of AtHSFA1s increases the transcriptional levels of heat-responsive genes in Arabidopsis, such as heat shock protein 90 (HSP90) and HSP101, thereby enhancing the plant's heat tolerance [39].In tomatoes, melatonin also enhances heat stress tolerance, alleviating photoinhibition under heat stress and reducing electrolyte leakage.At the gene expression level, melatonin enhances protein folding by upregulating HSPs and increases the expression of autophagy-related genes (ATG) to degrade aggregated proteins. By folding or degrading denatured proteins, it promotes cellular protein protection and enhances heat stress tolerance [40].

2.2.5 Heavy metal stress

Exogenous melatonin treatment enhances plant tolerance to heavy metals. In tomato seedlings subjected to cadmium stress, melatonin treatment significantly enhanced plant tolerance to cadmium. On one hand, it enhanced the plant's antioxidant capacity,reducing the impact of cadmium stress on photosynthesis; on the other hand, it enhanced the compartmentalization of cadmium ions during their transport from roots to aboveground parts, as evidenced by unchanged cadmium ion content in roots after melatonin treatment, while cadmium ion content in leaves decreased [41]. Melatonin treatment of watermelon seedlings enhanced plant tolerance to vanadium.Exogenous melatonin enhances the synthesis of endogenous melatonin in watermelon, while also inducing the expression of genes encoding superoxide dismutase,peroxidase, catalase, and glutathione transferase, reducing the accumulation of hydrogen peroxide and malondialdehyde; additionally, it reduced the transport of vanadium from roots to stems and leaves, lowering vanadium accumulation in stems and leaves [42].

The above results indicate that melatonin enhances plant non-biotic stress resistance primarily through two mechanisms: first, melatonin acts as an antioxidant to scavenge free radicals generated by non-biotic stress in plants, thereby reducing oxidative stress damage; second, it functions as a signaling molecule to activate the expression of non-biotic stress response genes, thereby enhancing non-biotic stress resistance.

2.3 Melatonin enhances plant biotic stress resistance

Melatonin not only enhances plants' tolerance to abiotic stress but also enhances their defense against biotic stress. In rice suspension culture medium, exogenous application of serotonin induces the expression of defense genes and cell death, increasing rice's resistance to rice blast infection. These results indicate that serotonin plays an important role in rice's innate immune system [43].Melatonin participates in the defense against the highly pathogenic bacterium Pseudomonas syringae DC3000. Application of melatonin at a concentration of 10 μmol·L⁻¹ on Arabidopsis and tobacco leaves induces the expression of multiple pathogenesis-related genes (PRGs) and a series of defense genes activated by salicylic acid (SA) and ethylene (ET). The Arabidopsis pathogen-related gene non-expressor of pathogenesis-related gene 1 (npr1) mutant

SA) and ethylene (ET)-activated defense genes. In the Arabidopsis pathogenesis-related gene non-expressor 1 mutant npr1 (nonexpressor of pathogenesis-related gene 1, NPR1) and the ethylene-insensitive mutant ein2 (ethylene-insensitive 2, EIN2), melatonin-induced PR gene expression was almost completely or partially suppressed.

This indicates that SA and ET depend on melatonin-induced plant defense signals, and also suggests that melatonin may be a new defense signal in plant-pathogen interactions. In Arabidopsis melatonin synthesis pathway gene SNAT mutants, the expression of disease-related genes was affected.When inoculated with tomato bacterial leaf spot pathogen DC3000, melatonin synthesis levels in snat were reduced to 50% of wild-type levels, resulting in increased susceptibility to pathogen infection and reduced expression of defense genes PR1, isochorismate synthase 1 (ICS1), and plant defensin 1. 2 (plant defensin 1.2, PDF1.2) decreased. Exogenous melatonin treatment of salicylate hydroxylase (nahG) transgenic Arabidopsis plants failed to induce defense gene expression but restored the induced expression of defense genes in snat mutant plants.This indicates that melatonin-induced expression of defense genes is SA pathway-dependent [45].

Melatonin enhances pathogen resistance by inducing the expression of some plant defense-related genes. Studies have shown that this induction is associated with mitogen-activated protein kinase (MAPK).Melatonin treatment of Arabidopsis and tobacco rapidly activated mitogen-activated protein kinase 3 (MPK3) and MPK6, and melatonin precursors could also induce this activation, although to a lesser extent than melatonin itself.When melatonin was applied to mitogen-activated protein kinase kinase (MKK) mutants, it was found that MKK4, MKK5, MKK7, and MKK9 were involved in the activation of MPK3 and MPK6. This suggests that melatonin-mediated immune responses are achieved through the MAPK cascade signaling pathway [46].

Melatonin treatment enhances apple plants' resistance to apple scab. Leaves treated with melatonin exhibit lower hydrogen peroxide content and enhanced activity of plant defense-related enzymes, potentially improving apple disease resistance through these two mechanisms [47].Exogenous melatonin enhances potato tolerance to late blight. On one hand, melatonin inhibits the growth of late blight fungal hyphae, alters cellular ultrastructure, and reduces the pathogen's tolerance to stress, significantly suppressing fungal growth.On the other hand, melatonin influences the amino acid metabolism, stress tolerance, fungicide resistance, and expression of virulence-related genes in potato late blight pathogens.Research has also found that melatonin and fungicides have a synergistic effect; when used together, they reduce fungicide usage and enhance the fungicide's efficacy against potato late blight [48].

The above studies indicate that melatonin enhances plant biostress resistance primarily through three mechanisms:First, melatonin acts as an inhibitor to restrict bacterial or fungal growth; second, melatonin exerts its antioxidant function to reduce oxidative stress induced by pathogen invasion; third, melatonin serves as a signaling molecule, inducing the expression of defense genes through the SA or MAPK pathway to enhance plant resistance to biotic stress.

3  Outlook

Melatonin, as a new small-molecule growth regulator, is gradually emerging as a hot topic in plant research. In terms of melatonin biosynthesis, researchers have cloned key enzymes involved in plant melatonin synthesis by referencing the animal melatonin synthesis pathway and elucidated the biosynthetic pathway of plant melatonin.In terms of physiological functions, melatonin contains an indole ring, and whether it can exert similar effects to IAA, such as promoting cell enlargement, regulating lateral root growth, and modulating sugar transport pathways, remains to be further investigated.

In response to abiotic stress, melatonin acts as an antioxidant to scavenge free radicals generated by stress, and also serves as a signaling molecule to induce the expression of abiotic stress response genes, thereby enhancing plant stress tolerance. In response to biotic stress, melatonin can inhibit the growth of bacteria and fungi, and also induce the expression of plant defense genes through SA to resist diseases.

Although research on plant melatonin has made significant progress, several issues remain. First, while there has been extensive research on methods for determining melatonin content in plants, studies on breeding crop varieties with high melatonin content are limited.

How to utilize existing research findings on melatonin, combined with traditional breeding theories and experience, to select and breed crop varieties with high melatonin content is an attractive and challenging research direction; second, the role of melatonin in signal perception and transmission remains unclear. Although a melatonin plant receptor has been cloned, research on the direct downstream signals activated by the receptor after binding to melatonin is limited;and whether there are other melatonin receptors remains an urgent scientific question; third, melatonin can induce the expression of stress-tolerant genes in plants under non-biotic stress, but the specific signal pathways involved remain unclear; fourth, melatonin can induce the expression of defense genes in plants under biotic stress through the SA or MAPK signaling pathways,while the potential interaction between melatonin and jasmonic acid, another plant hormone closely associated with defense responses, also requires further investigation.

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