Plant melatonin: roles and regulatory mechanisms in plant growth, development, and responses to biotic and abiotic stresses

ABSTRACT

Melatonin, a key indoleamine compound intrinsic to life, is ubiquitously distributed across the plant kingdom in plants. As a plant growth regulator and biostimulant, melatonin plays a pivotal role in both increasing plant growth and bolstering resilience to stress. This review provides a comprehensive analysis of regulatory mechanisms plant growth and development and addresses biotic and abiotic stressors. We dissect the biosynthetic and metabolic pathways of melatonin in plants and elaborate on its roles in catalyzing plant growth, development, and antioxidant activities. Furthermore, our discussion delves into the ways in which melatonin manipulates plant morphology, physiology, redox systems, ion homeostasis, biomolecular content levels, and the expression of stress resistance genes or proteins. We additionally highlight its cooperative interaction with other endogenous hormones in mitigating the deleterious impacts of challenging environments. In essence, melatonin, as a multifunctional biological signaling molecule, offers the potential to increase crop yield even under adverse conditions by plant seed germination rates and promoting robust growth. Consequently, it as a compelling candidate for ecofriendly crop production strategies. This review is intended to serve as a theoretical guide to unravel the multifaceted regulatory mechanisms of melatonin in governing plant growth, development, and stress resistance.

Introduction

Melatonin (MT), chemically referred to as N-acetyl−5-methoxytryptamine, is an indoleamine compound of decreased size that is ubiquitous in animals, plants, and microorganisms. recognized as pineal to to lighten the pigmentation of specific species of fish, reptiles, and amphibians. The initial discovery of MT in animals, where it was found to regulate circadian rhythms and immunological functions, led to the presumption that MT is an exclusive neurohormone of the animal kingdom.^1-3 This perception transformed when two pioneering teams led by Hattori and Dubbels revealed the presence of MT in plants in 1995.^4,5 This revolutionary finding the emergence of plant MT research as a standalone scientific field.

In recent years, we have witnessed substantial advancements in the exploration of MT in plants. Endogenous MT is associated primarily with maintaining the equilibrium of reactive oxygen species metabolism within cells. Investigations into exogenous MT have underscored its role in modulating seed germination, root growth, overall plant development, and fruit maturation and in attenuating damage caused by abiotic stressors such as heavy metals, extreme temperatures, drought, salinity, as well as biotic stressors such as pathogens and pests.^6-10 By enhancing plant resilience against unfavorable stress conditions and promoting plant survival and reproduction, MT represents a promising field of study.

The emergence of advanced fields such as metabolomics, transcriptomics, and proteomics has converged with plant MT studies, consequently attracting an increasing number of researchers to this evolving domain. For instance, the application of exogenous MT Carya cathayensisin drought resistance, facilitated by the restoration of plant growth, improved photosynthetic efficiency, and augmented antioxidant defense systems. These mechanisms increase the ability of plants to scavenge reactive oxygen species (ROS). Studies integrating metabolomics also revealed that MT mediates crucial metabolic pathways in plants under drought stress, such as phenylpropane, chlorophyll, and carotenoid biosynthesis, along with biochemical and sugar metabolism. Moreover, MT considerably the transcription levels of key genes associated with these metabolic pathways, such as chlorophyllase (CHLASE), superoxide dismutase (SOD), catalase (CAT), ascorbic acid peroxidase (APX), peroxidase (POD) and phenylalanine ammonia lyase (PAL). Additionally, MT has been to interact with other hormones (e.g., zeatin, gibberellin, jasmonic acid, and abscisic acid) to govern plant physiological processes.¹¹ These findings indicate the regulatory ability of MT at the metabolic and molecular levels to counteract nonbiotic stress. Concurrently, Yang et al. reported at the transcriptional level that MT can be involved in plant hormone regulation, thereby modulating drought tolerance in tomatoes.¹²

Through a systematic review of the recent literature on melatonin in plants, this article summarizes current key research areas into the following five themes: (1) The biosynthesis and metabolic pathways of melatonin, encompassing the identification and functional characterization of key synthases, metabolic routes, and associated regulatory genes;^13-16 (2) The mechanisms of melatonin signal perception and transduction, involving the identification of putative receptors, downstream signaling networks, and crosstalk with other plant hormones;^17-22 (3) The regulatory roles of melatonin in plant growth and development, covering critical processes such as seed germination, root system establishment, leaf senescence, flowering time, and fruit maturation;^23,24 (4) The functions and underlying mechanisms of melatonin in plant responses to abiotic and biotic stresses, including physiological and molecular adaptations to drought, salinity, extreme temperatures, and pathogen infection;^18,25-28 (5) The antioxidant function of melatonin, focusing on its capacity as an endogenous antioxidant to scavenge reactive oxygen species (ROS), alleviate oxidative damage, and its synergistic relationship with the plant's intrinsic antioxidant system.^24,29,30 These research themes collectively define the multifaceted functions of melatonin in plant physiology and provide a crucial theoretical foundation for elucidating its comprehensive regulatory networks and future application prospects.

As a multifaceted biological signaling molecule, melatonin demonstrates immense potential in enhancing crop resilience and yield, particularly under adverse environmental conditions. This review has synthesized evidence of its efficacy in promoting seed germination, plant growth, and overall stress tolerance, positioning it as a promising, eco-friendly candidate for sustainable agricultural practices.³¹ Future research should prioritize elucidating the precise transport mechanisms of melatonin within plants, identifying the key receptors responsible for signal perception and the subsequent transduction pathways, and deepening our understanding of its intricate interactions with other hormonal networks. By addressing these fundamental questions, we can further elucidate the comprehensive regulatory mechanisms governed by melatonin, thereby solidifying the theoretical foundation for its strategic application in modulating plant growth, development, and stress resistance.

Biosynthesis of MT

While the synthesis and secretion pathways of animal MT have been thoroughly investigated, our understanding of the synthesis pathways and effects of plant MT is still evolving. Early research reported that the precursor substance for plant MT synthesis, 5-hydroxytryptamine, was first identified in the 1950s. The seminal concept of in-plant MT synthesis was introduced by Murch through isotope labeling studies conducted on Hypericum monogynum.¹³ Follow-up experiments in vitro 14 C-labeled tryptophan to plants such as Hypericum perforatum L. and Hypericum monosynum led to the detection of radioactive intermediate products such as indole−3-acid (IAA), tryptamine (TAM), 5-hydroxytryptophan (5-HTP), and serotonin (5-HT). These products mirror those detected during MT synthesis in animals, suggesting that the MT synthesis pathways in plants may closely resemble those in animal MT synthesis processes, with tryptophan (Trp) acting as a precursor substance for synthesis in both cases.^13,32,33

One notable distinction is the inherent ability to synthesize tryptophan, as opposed to that of animals, which relies on dietary intake of this amino acid.³⁴ This discrepancy implies that the pathway for plant MT synthesis could be more complex.^32,33 Current research suggests that at least four biosynthetic pathways in plants, including both classical and nonclassical methods, exist. The initial substance for synthesis is tryptophan, and the process four sequential enzyme-catalyzed steps, necessitating at least six enzymes, including tryptophan decarboxylase (TDC), tryptophan hydroxylase (TPH), tryptamine 5-hydroxylase (T5H), SerotonSection 6.1n N-acetyltransferase (SNAT), N-acetylserotonin methyltransferase (ASMT) and caffeic acid O-methyltransferase (COMT).³⁵ Moreover, N-acetylserotonin deacetylase (ASDAC) is involved in maintaining the steady-state level of MT synthesis.³⁶ Each synthesis pathway includes a common intermediate product, serotonin, and the site of synthesis for these intermediates varies across different MT synthesis pathways.

In the classical pathway, TDC catalyzes the first step of plant MT synthesis, transforming tryptophan into tryptamine. TDC is recognized as the rate-limiting enzyme of the MT synthesis pathway.^13,14 In the subsequent step, T5H catalyzes the formation of 5-hydroxytryptamine, a process contrary to MT synthesis in animals.^37,38 In the third step, SNAT facilitates the conversion of 5-hydroxytryptamine into N-acetyl−5-hydroxytryptamine.³⁹ Finally, either ASMT or COMT catalyzes the transformation of N-acetyl−5-hydroxytryptamine into MT (Figure 1).⁴⁰ As the intermediates of TDC, T5H, SNAT, and ASMT/COMT in MT biosynthesis are synthesized in subcellular components such as the cytoplasm, endoplasmic reticulum, and chloroplast, the efficiency of exchange and transport between these organelles also determines the rate and level of MT synthesis.²⁵

Figure 1.

Biosynthetic pathway of MT. The black arrow represents the classic pathway of MT biosynthesis, while the dashed arrow represents a nonclassical pathway.

Metabolic pathways of MT

Research on the metabolic pathways of plant MT lags behind that of its biosynthesis, with corresponding molecular mechanisms still to be elucidated. In contrast, animal MT metabolism has been extensively studied, revealing metabolic pathways, mainly through enzymatic catalysis, pseudoenzyme reactions, and free radical cascade interactions. These pathways result in a variety of metabolites, such as 2-hydroxymelatonin, 4-hydroxymelatonin, 6-hydroxymel, cyclo-3-hydroxymel, N¹-acetyl-N²-formyl-5-methoxycaninuramide (AFMK), N¹-acetyl-5-methoxycaninuramide (AMK), and others. 6-Hydroxymelatonin is considered the primary metabolite of animal MT, while 2-HydroxyMelatonin is the main MT metabolite in plants (Figure 2).^26-28

Figure 2.

Metabolic pathway of MT.

Enzyme catalysis

Current research reveals that the enzyme catalysis involved in plant MT metabolism parallels the enzymatic processes experienced by vertebrates and other eukaryotes in MT metabolism.⁴¹ AFMK, first identified in Calla palustris, is the initial MT metabolite recognized in plants, and it exhibits circadian rhythm characteristics akin to those of MT.⁴² The indoleamine 2,3-dioxygenase (IDO) gene, extracted from rice, can also catalyze the conversion of MT into AFMK in animals. Concurrently, transgenic tomatoes overexpressing this gene a decrease in MT content, implying that IDO might play a role in the metabolic process of plant MT.⁴³ Notably, AFMK can also be synthesized from cyclic 3-hydroxymelatonin through nonenzymatic synthesis.⁴¹

Analogous to the metabolic pathway of MT in animals and other eukaryotes, plant MT might undergo hydroxylation, similar to other secondary metabolites, typically facilitated by CYP and 2-oxoglutarate-dependent dioxygenase (2-ODD).^43,44 A study by Byeon et al. involving the heterologous expression of 35 2-ODD genes from rice in E. coli demonstrated that the proteins encoded by four of these genes (2-ODD11, 2-ODD19, 2-ODD21, and 2-ODD33) can metabolize MT to produce 2-hydroxymelatonin. This study led to the first cloning of the enzyme gene M2H, which is responsible for catalyzing the decomposition of MT into 2-hydroxymelatonin in rice. The M2H gene is a member of the 2-ODD gene family.⁴⁵ Interestingly, the 2-hydroxymelatonin content in rice seedling roots is higher than that in the buds, with the 2-ODD21 gene exhibiting significant expression, which is correlated with the high activity of M2H in the roots. Enzyme activity kinetics research reveals that the activity of M2H in plants greatly surpasses that of the MT synthesis enzymes SNAT and ASMT, sometimes resulting in 2-hydroxymelatonin concentrations exceeding those of MT in plants.^42,46 Like MT, both endogenous induction and exogenous application of 2-hydroxymelatonin can enhance plant tolerance to various individual stress conditions, such as cold, drought, metal stress, and pathogen attack.^42,47,48 However, a notable distinction is that can enable plants to resist various combined abiotic stresses, a feature that MT does not possess, and the underlying reason for this phenomenon remains unclear.⁴¹

Non-enzymatic reactions

The diverse metabolic pathways and multifarious metabolites of animal MT have been authenticated through extensive research. In addition to enzyme-catalyzed processes, MT and its metabolites can also decompose gradually via a sequence of cascade reactions, either through pseudoenzyme reactions or interactions with a range of free radicals. Concurrently, these mechanisms scavenge free radicals, providing antioxidant protection and defending organisms from oxidative stress.^48-51

Nonenzymatic hydroxylation is a universal occurrence across all organisms. The cyclo−3-hydroxymelatonin generated through this reaction has also been identified in plants.⁵² For non-enzymatic degradation of MT, nitrosation can occur on the nitrogen atom of its indole ring, thereby forming N-nitrosylmelatonin.⁵² Byeon et al. that MT first undergoes degradation by M2H into 2-hydroxymelatonin, which may then further decompose into AFMK through a cascade reaction with free radicals, mirroring animal mechanisms.⁵³ However, the exact metabolic mechanism still requires more comprehensive study. Singh et al. that N-nitroso melatonin could serve as an intracellular nitric oxide (NO) reservoir, chiefly engaged in long-distance redox signal transmission.⁴¹ However, given the instability of N-nitroso melatonin, the hypothesis that it might act as a long-distance NO carrier in plants has been met with skepticism. As MT undergoes nonenzymatic degradation under the influence of free radicals and environmental conditions, it produces active metabolites, thereby increasing the pool of indoleamine substances. These nonenzymatic processes might be even more critical in plants, particularly when they face harsh environmental conditions such as intense light, ultraviolet radiation, and soil pollution. Nevertheless, the signal transduction pathways of MT metabolites in plants have not been fully elucidated. As such, confirmation of the metabolic pathway of plant MT is needed to thoroughly comprehend the dynamic accumulation pattern and action mechanism of plant MT.

Melatonin signaling perception and transduction mechanisms in plants

Receptors and early signaling events

In plants, the melatonin signal perception mechanism is fundamentally different from that in animals. Current research suggests that melatonin may be recognized through specific proteins identified as CAND2/PMTR1.²¹ PMTR1 has been confirmed to bind melatonin, thereby activating downstream MAPK cascades and triggering early signaling events.²² Additionally, melatonin can rapidly induce the production of intracellular second messengers such as calcium ions (Ca²⁺), reactive oxygen species (H₂O₂), and nitric oxide (NO), forming a signal amplification network (Figure 3). These signaling molecules collectively regulate downstream gene expression and physiological responses, constituting the core framework of melatonin signal transduction.

Figure 3.

Cellular mechanism of melatonin signaling in plants.

Crosstalk with other biomolecules

Melatonin in plants, far from being a mere signaling molecule, resides at the heart of an exceptionally complex molecular network. Through intricate crosstalk mechanisms, it orchestrates virtually every aspect of plants, from growth and development to stress survival. Central to this network are its three primary identities: a potent direct antioxidant, an activator of the endogenous antioxidant system, and an integrator of multiple hormonal signals.

Upon exposure to environmental stresses, reactive oxygen species (ROS) rapidly accumulate within plant cells, leading to oxidative damage. Melatonin not only serves as a shield by directly neutralizing highly destructive hydroxyl radicals but also strategically enhances overall plant defense.⁵⁴ It upregulates the expression and activity of key antioxidant enzymes such as SOD and CAT, thereby maintaining ROS levels within signaling ranges and preventing them from reaching toxic thresholds. Crucially, melatonin maintains a dynamic and balanced relationship with nitric oxide (NO), another pivotal signaling molecule.⁵⁵ Melatonin and NO often act in concert to initiate systemic resistance and induce stomatal closure, among other defense responses. Simultaneously, melatonin fine-tunes this interplay by scavenging peroxynitrite derived from NO, thereby mitigating nitrosative stress and exemplifying its sophisticated regulatory capacity within complex signaling networks.⁵⁶ Beyond its role in oxidative stress mitigation, melatonin functions as a “central integrator” of the phytohormone network, engaging in extensive crosstalk with classical hormone pathways. Its interaction with auxin (IAA), a key regulator of plant morphogenesis, is particularly nuanced. Both molecules share tryptophan as a biosynthetic precursor, creating potential metabolic competition. More importantly, melatonin modulates the spatial distribution of auxin efflux carriers in root tissues, thereby reshaping root architecture under stress. This enables melatonin to inhibit primary root elongation—avoiding unfavorable zones—while promoting lateral root and root hair development to increase nutrient and water absorption, representing a strategic survival adaptation.^57,58 Under abiotic stresses such as drought and salinity, melatonin and abscisic acid (ABA) exhibit strong synergistic effects. Melatonin enhances ABA biosynthesis and signaling, collectively promoting stomatal closure via guard cell regulation—a key mechanism for reducing water loss and ensuring survival.⁵⁹ In immune responses, melatonin acts as a “versatile amplifier”: it strengthens salicylic acid (SA)-mediated signaling against biotrophic pathogens and enhances jasmonic acid (JA)-dependent defenses against necrotrophs, thereby broadening the plant’s immune repertoire.^17,18 Additionally, melatonin suppresses excessive stem elongation by antagonizing gibberellin (GA) signaling, promoting a more compact plant architecture and optimizing resource allocation.⁶⁰ The rapid transmission of signals within this intricate network often relies on second messengers. Melatonin can rapidly induce fluctuations in cytosolic Ca²⁺, which serve as an “activation switch” for downstream events. These Ca²⁺ signals trigger cascades involving calcium-dependent protein kinases (CDPKs), ultimately translating the initial stimulus into specific gene expression and physiological responses.⁶¹

In summary, through its potent antioxidant activity and role as a signaling hub that interacts extensively with ROS, Ca²⁺, auxin, ABA, SA, JA, and GA, plant melatonin forms a sophisticated and robust regulatory network. It functions not only as a sensor and amplifier of stress signals but also as a decision maker that balances growth and defense, guiding the plant toward adaptive responses that maximize survival and reproductive success.

The regulatory role of MT in plant growth and development

MT exerts a pivotal influence on various stages of plant growth and development. Owing to effects, MT has been posited a significant regulator plant growth and in the modulation of various physiological processes in plants. These include regulating seed germination, stimulating plant growth, root development, fruit maturation, and delaying leaf senescence.^8,62-68 In addition, MT also mediates its function by regulating components linked to redox networks or interfering with the activity of other plant hormones.^69-71

Regulating seed germination

Seeds serve as a distinct reproductive mechanism in plants and are instrumental in the perpetuation of species. Seed germination is a critical phase in the continuum of plant growth and development, indicating the commencement of a fresh life cycle and determining the timeline of plant incorporation into both natural and agricultural ecosystems.²³ In 2009, Posmyk et al. were the first to identify a role for MT in seed germination.⁷² The authors evaluated the germination rate of Brassica oleracea seeds using three distinct concentration gradients of MT upon water induction. They concluded that low-concentration MT solutions (1 and 10 μM) could significantly increase the germination rate, while a relatively high concentration (100 μM) had an inhibitory effect. These findings demonstrated that exogenous MT a significant dose-dependent effect on seed germination. Posmyk later corroborated this finding with cucumber seeds, theorizing that this might be attributable to the antioxidant nature of MT, which, owing to its hydrophilic and lipophilic properties, can easily infiltrate the seeds, increasing their antioxidant capacity and thereby increasing their vitality and germination rate.²⁹ MT can directly or indirectly suppress excessive accumulation of H₂O₂, thereby enabling seeds to withstand various stress conditions and their vitality and germination rate.⁷³ Multiple studies have indicated that pretreatment with an optimal dose of MT can also increase the germination rate of oat and watermelon seeds under adverse conditions.^74,75 Lv Yan explored the mechanism impact on seed germination.⁷⁶ After treating seeds with high concentrations of exogenous MT, RNA-Seq was employed to probe for germination-related candidate genes. MT primarily modulates seed germination by controlling sugar metabolism and cell-associated synthesis, metabolism, and repair processes. MT may also influence seed germination by cooperating with other plant hormones to regulate hormone-related genes. For example, MT enhances the seed germination rate by endogenous GA and ABA levels. It upregulates ABA catabolism genes such as CYP707As and 8 'hydroxylase genes, as well as GA-associated biosynthesis genes such as GA20ox and GA3ox, and downregulates ABA biosynthesis genes such as NCEDs, LpZEP, and LpNCED1, thereby increasing the seed germination rate. The activities of α-AMS and β-GAL hydrolases provide the requisite energy for seed germination, counteracting seed coat and embryo dormancy.⁷⁷ Furthermore, when the seeds of A. thaliana were treated with exogenous MT and other plant hormones, the presence of auxin mitigated the inhibitory effect of high concentrations of MT on seed germination, indicating potential interactive regulation between the two, thereby influencing plant seed germination.

In summary, the promotion of seed germination by MT might be attributed to its unrestricted cellular entry, which leverages its inherent antioxidant capabilities to protect the seed's lipids from oxidative stress. This process propels seed germination by governing cell-related processes such as synthesis and repair, along with primary and secondary metabolic activities, and promotes energy production during growth (Figure 4).

Figure 4.

Regulatory roles of MT in plant growth and development.

Stimulating plant growth

Recently, research exploring the role of MT in facilitating crop growth and development has studies have demonstrated that MT mediates plant growth and development through its potent antioxidant activity and activation of the MAPK cascade.²⁴ Hernández-Ruiz et al. first proposed that MT acts as a plant growth regulator.⁷⁸ These authors discovered that MT and the plant growth hormone IAA have certain functional similarities, both of which are capable of promoting the growth of albino lupin tissue hypocotyls in vitro. Similar outcomes have been reported for various plants, such as canary grass, wheat, barley, and oat.⁷⁹ Moreover, MT can also increase seedling growth by optimizing the photosynthetic capacity of plants. The administration of exogenous MT to sugar beet seedlings augmented their photosynthesis, water status, ion homeostasis, and antioxidant defense system to mitigate salt stress, thereby stimulating the growth, root yield, and sugar content of the seedlings.³⁰ MT treatment has also been found to facilitate the growth, development, and yield of soybeans, Arabidopsis, and yellow-flower white feather fan beans.^80-82 In a subsequent study, Agathokleous et al. found that the growth- and developmental-promoting effects of exogenous MT on plants may be intricately associated with its concentration.⁸³ Similarly, Kolar et al.'s research revealed that varying concentrations of MT have differential regulatory effects on Chenopodium rubrum, resulting in a low-concentration enhancement and high-concentration inhibitory effect.⁸⁴

MT also plays a role in various flowering pathways, including the environmental temperature, vernalization, photoperiod, GA, and AGE pathways.⁸⁵ In Antirrhinum majus, different concentrations of MT enhance the quantity, size, and quality of treatment with blue light and MT can effectively improve the postharvest quality of flowers and prolong their life.⁸⁵ These results further elucidate the contribution of MT to plant growth and development, including but not limited to roots, stems, and leaves, which are susceptible to nutrient absorption and resource competition (Figure 4). Hence, encouraging the formation of MT is likely to play a significant role in facilitating plant growth.

Promoting plant rooting

The root system, a nutrient organ developed by plants to adapt to terrestrial environments over time, aids in absorbing water and nutrients from the soil while concurrently anchoring plants into the soil.⁸⁶ Plant MT and auxin share the common substrate tryptophan in their biosynthetic pathways. Owing to the chemical similarities between MT and the plant growth hormone IAA, the effects of MT on plant root growth and development have garnered extensive interest. Studies have revealed that lower concentrations of MT can promote elongation of the main root, while higher concentrations of MT the same effect because of a significant decrease in the transcription levels of IAA synthesis and transport genes.^87,88 MT profoundly impacts the growth of the main roots of plants. For instance, when exogenous MT at different concentrations was used to treat Triticeae Dumort,⁷⁹ Arabidopsis,⁵⁷ and Rorippa indicaseedlings,⁵⁷ it was observed that MT primarily exerts an inhibitory effect on the main roots of plants, while a minority of the main roots of plants are not sensitive to MT, which is principally associated with crop type and MT concentration. Moreover, MT is also involved in the formation of plant lateral roots.^89,90 These findings suggest that MT seed length and biomass in rice seedlings to regulate root growth and development. MT governs the expression levels of the cell cycle-related genes SlCDKA1, SlCYCD3, and SlKRP2 by stimulating SlPAO1-H2O2 and SlRboh3/4-O2, resulting in the initial development of lateral root primordia.

Based on preceding research, we assert that MT is a critical regulator of root morphology that can function by indirectly activating the IAA signaling pathway and may influence the polar transport and perception of IAA. In conclusion, the appropriate concentration of MT can significantly promote the growth of plant roots, facilitate nutrient absorption, and increase the stress resistance of plants (Figure 4).

Regulating fruit development

Fruit represents an organ arising from the pollination and fertilization of the pistil of angiosperms, encompassing the development of the ovary and other parts of the flower (such as the receptacle, sepals, etc.). The functionality of fruits primarily includes two aspects, namely, plant reproduction and human consumption, and they carry significant economic value. In recent years, an increasing number of scholarly articles have highlighted the role of MT in the regulation of fruit development (Figure 4).

In plants, MT primarily enhances crop yield by increasing the weight of individual fruits and modifying the nutritional For instance, Liu Jianlong that MT treatment can promote the expansion of tomato fruits under early dry stress, substantially increasing single-fruit weight but reducing the content of soluble solids in the fruit.²⁵ Some studies have reported that MT treatment of fruits such as apples⁹¹, peas⁹², and blackberries⁹³ can stimulate fruit enlargement, increase fruit weight, and increase the content of soluble solids, sucrose, and other unique components in the fruit. Furthermore, MT treatment also enhances the quality, quantity, and weight of fruits. MT regulates fruit not only through seedling irrigation but also by applying exogenous MT through seed soaking and leaf spraying to increase fruit growth and improve fruit quality. For example, soaking tomato seeds with a low concentration of MT prior to germination can increase fruit yield; increase the content of ascorbic acid (AsA), lycopene, and Ca in fruits; and decrease the contents of N, Mg, Cu, Zn, Fe, Mn, and other elements.²⁵ Owing to the similarity of their chemical structure to that of IAA, MT has physiological functions akin to the effects of IAA. At present, studies have demonstrated that low concentrations of MT can induce the synthesis of IAA and synergistically regulate plant fruit growth.⁵⁷ Moreover, MT treatment postfruit harvesting can also promote fruit ripening. For instance, when exogenous MT was used to treat grapefruits, it was discovered that MT can increase the ethylene content in the fruit, participate in signal regulation, foster fruit ripening, and induce the accumulation of polyphenols in the fruit;⁹⁴ MT also has unique advantages in maintaining fruit storage tolerance and quality.⁹⁵ In summary, MT treatment has superior impacts on various aspects of fruit development.

Delaying leaf aging

The leaf serves as the primary organ for photosynthesis in plants, with leaf senescence marking the ultimate phase of leaf development, culminating in apoptosis. Premature leaf senescence can inhibit and decelerate plant growth and development, thereby impacting crop yield and quality. Consequently, the postponement of early leaf senescence significant implications for increasing light efficiency during plant growth and improving crop yield. In recent years, the role of MT in delaying leaf senescence has garnered increasing interest.^7,16,96 Arnao and Hernández-Ruiz et al. were the first to report the role of MT in delaying leaf senescence.⁹⁷ These authors incubated barley leaves in darkness for a period of time and subsequently treated them with MT, markedly slowing the aging process of barley leaves. Further research revealed that this is due to the capacity of exogenous MT to safeguard chlorophyll from degradation, thereby accomplishing the goal of delaying barley leaf senescence.

Presently, the majority of studies investigating the promotion of leaf senescence by chlorophyll have focused on the potential role of MT in photosynthesis. For instance, after prolonged application of MT to Malus pumila under drought conditions, the senescence of Malus pumila leaves was delayed, and chlorophyll degradation was significantly diminished.⁹² In addition, MT can also regulate ROS levels, decrease membrane damage, and ultimately attenuate tomato leaf senescence induced by methyl jasmonic acid.⁹⁷ MT, owing to its potent antioxidant properties, can also play a critical role in delaying leaf aging. For example, exogenous MT postpones dark-induced aging in Malus domestica leaves by increasing ROS-scavenging enzyme activity while maintaining relatively high levels of ascorbic acid and glutathione content.⁹⁸ A study examining the whole-genome expression profile of Arabidopsis treated with MT demonstrated that MT can also delay leaf senescence by regulating genes involved in promoting or inducing senescence through various plant hormone signal transduction pathways and interacting with plant hormones.⁹⁹ Therefore, MT is anticipated to become a bioactive factor that postpones crop aging in agricultural practices (Figure 4).

The antioxidant effect of MT in plants

An increase in reactive oxygen species (ROS) can cause damage to plant somatic cells and other key physiological process systems, leading to apoptosis and eventual death.¹⁰⁰ The primary function of MT in plants is to serve as an antioxidant, offering protection against environmental stressors.⁷ Plant hormones can interact directly with ROS to mitigate damage caused by oxidative stress on cells and stabilize cell membranes. Both endogenous and exogenous MT alleviate oxidative stress induced by abiotic stressors (such as high temperature, cold, drought, and salt stress) through the removal of reactive oxygen species.^86-88

MT can also activate enzymes within the antioxidant system to increase plant tolerance to various stresses. For instance, under certain abiotic stress conditions, exogenous MT can induce the action of antioxidant enzymes such as catalase (CAT), superoxide dismutase (SOD), peroxidase (POD), and polyphenol oxidase (PPO) to counter the excessive accumulation of ROS.¹⁰¹ This reduction in oxidative stress in plants has been substantiated in numerous studies.^102-104

Several investigations have demonstrated that MT can also amplify plant stress resistance by regenerating endogenous antioxidants such as glutathione (GSH) and ascorbic acid (AsA). Specifically, MT increases the AsA/DHA and GSH/GSSG ratios by mediating key enzymes (DHAR, APX, and MDAHR) produced during the ascorbic acid-glutathione (AsA-GSH) cycle, thereby increasing the plant's antioxidant capacity.^26,105,106

In addition to regulating enzyme activity for antioxidant functions, MT can also counteract oxidative stress by modulating genes associated with carbohydrate metabolism and repairing damaged biofilms and proteins.¹⁰⁷ These findings suggest that MT can activate a comprehensive antioxidant system in plants, shielding cells from oxidative stress damage induced by various stresses and thereby increasing cellular redox homeostasis.¹⁰⁸

In summary, MT plays a pivotal role in antioxidative processes in plants, and endogenous MT serves as a vital ROS scavenger, functioning as the primary line of defense against oxidative stress damage in plants.

The function and regulatory mechanism of MT in plant response to stress environment

Function and regulation mechanism of MT in plant response to abiotic stress

Owing to their inability to evade adverse environmental pressures through mobility, plants are required to rapidly perceive and respond to these complex and ever-evolving conditions to ensure their survival and reproduction. In recent years, a growing body of research has highlighted the systematic temporal and spatial effects of MT under various stress conditions, such as drought and salt stress.^109,110 The molecular signaling and regulatory transduction pathways of MT under different stress conditions remain an area of active investigation, attracting considerable interest and exploration among researchers.

Efforts have been made to elucidate how plants perceive endogenous signals and effectively utilize MT and its intricate signal transduction to handle varying environmental pressures. Research has shown that endogenous MT can mitigate abiotic stress in plants, including drought stress, salt stress, high and low temperatures, and heavy metal stress (Figure 5).

Figure 5.

Regulatory role of MT in plant response to abiotic stress.

The role of MT in drought stress

Drought is a major stressor that hampers global agricultural production and development, severely plant growth and crop yield and quality.^111,112 It disrupts plant water metabolism, undermines photosynthesis, and poses significant challenges to the synthesis and transportation of nutrients, thereby stunting plant growth. Severe and prolonged stress can also compromise natural defense mechanisms, leading to physiological abnormalities such as reductions in photosynthetic efficiency, stomatal conductance, leaf area, growth rate, plant height, and water potential.¹¹³ As a potent antioxidant and plant growth regulator, MT has attracted significant research attention for its role in alleviating plant damage from drought stress.¹¹⁴

Drought stress in plants can damage the membrane system, leading to an imbalance in redox homeostasis and alteration in physiological and biochemical processes.¹¹⁵ Reports indicate that the application of exogenous MT can modulate these processes, enhancing plant resistance to stress responses.¹¹⁶ Ectopic expression of MzSNAT5 in Arabidopsis can bolster mitochondrial MT synthesis, minimize oxidative damage, and consequently improve plant drought resistance.¹¹⁷ MT can also activate the activities of antioxidant enzymes, thus restraining the accumulation of reactive oxygen species in arid environments.¹¹⁸ Furthermore, according to proteomics studies, MT under drought stress can promote the expression of glycolytic proteins and trigger autophagy-related metabolic cascade reactions in wheat, playing a critical regulatory role in cell autophagy, protease expression, and ubiquitin-protein degradation.¹¹⁹

In conclusion, MT plays a pivotal role in plants' response to drought stress. Through its antioxidant, osmoregulatory, and growth-promoting properties, MT effectively mitigates the adverse effects of drought stress on plants, enabling their better adaptation to drought environments (Figure 5).

The role of MT in salt stress

Soil salinization significantly hampers plant growth and development, with excessive salt ions inflicting damage to plants through ion toxicity, osmotic stress, and oxidative stress, resulting in decreased crop yield, particularly in arid and semiarid regions.¹²⁰ Recently, a surge in studies attests to the potential of MT in assisting plants in thwarting salt stress-induced damage.

Excessive salt concentration and ion stress disrupt the oxidative stress balance, triggering a range of abnormal stress responses in plants. MTs provide plants with the capacity for salt tolerance through multiple mechanisms. Studies have shown that MT can amplify the osmotic regulation level of plants to respond to stress, including salt stress. For instance, MT treatment can increase the transcription level of the key proline gene P5CS in alfalfa under salt stress conditions while curbing the expression of the proline degradation gene ProDH to increase the proline content, thereby increasing the resistance of alfalfa to salt.^110,121 Additionally, MT application can increase the amount of endogenous MT substances, thereby increasing leaf osmotic potential, improving water absorption, and fundamentally driving the photosynthetic process of plants under salt stress.^110,122

Furthermore, research has shown that the ectopic expression of MzASMT9 in Arabidopsis can increase the endogenous MT content, increase salt tolerance in transgenic plants, reduce the ROS and MDA contents, and thus increase photosynthesis.¹²³ Concurrently, MT treatment significantly enhances the K⁺ content under salt stress and reduces the Na⁺ content, thus increasing the ion ratio to maintain ion homeostasis in plants.¹²⁴ MT treatment can also increase the quality of plant fruits. In high-salt environments, MT treatment markedly improved tomato yield while also increasing soluble sugar, AsA, and β-carotene contents and decreasing the concentrations of organic acids and nitrates, thereby increasing fruit quality.

In summary, MT has multiple intricate signal transduction mechanisms and plays a pivotal role in mitigating plant damage from salt stress in various ways under such stress (Figure 5). Moreover, MT can alleviate osmotic stress through a multitude of metabolic pathways and increase the levels of osmoregulatory substances.

The role of MT in high-temperature stress

In recent years, rising global temperatures have garnered increasing attention worldwide. High-temperature stress can provoke a sequence of metabolic alterations in plants, including the excessive production of ROS, photoinhibition, protein denaturation, synthesis inhibition, and the impairment of biofilm structure and function, consequently inflicting damage on plant growth and development.⁸

Plant heat shock proteins (HSPs) possess the ability to refold compromised proteins and prevent the formation of polymers, which to a degree, equips plants with resistance to high-temperature stress.¹²⁵ MT treatment has been found to upregulate the expression of the heat stress protein gene HSP, thereby mending proteins that were denatured or harmed following high-temperature stress.⁸ In Arabidopsis, MT treatment can also substantially amplify the expression of heat shock factor HSFA1s to activate heat-responsive genes such as HSFA2, HSA32, HSP90, and HSP101, consequently augmenting plant heat tolerance.¹²⁶

These research findings provide fresh insights into the regulation of plant high-temperature tolerance by MT (Figure 5), but further in-depth exploration is required to elucidate its molecular mechanism.

The role of MT in low-temperature stress

Low-temperature stress can be severely detrimental to crop growth and production. It can impair the fluidity and enzymatic activity of plant cell membranes, inhibit plant photosynthesis and nutrient transportation, and inflict damage on the plant body, thereby resulting in reduced crop yield. However, extremely cold conditions often instigate an accumulation of MT as a protective response to shield plants from catastrophic harm. Therefore, increasing the level of endogenous MT can effectively improve the cold resilience of plants (Figure 5). There is evidence that MT can be transported from the roots to the aerial parts of plants, increasing their cold tolerance via roots to branch conveyance.¹²⁷

Moreover, MT treatment can maintain the quality of fruits, vegetables, and freshly cut flowers by increasing their cold resilience under refrigeration conditions. For example, prestorage treatment with MT can lead to an accumulation of phenolic compounds and a reduction in lignin, thereby diminishing the loss of flavor compounds and nutrients triggered by cold storage conditions.¹²⁸ MT treatment can also increase the accumulation levels of secondary metabolites, including amino acids, organic acids, carbohydrates, and sugars, thereby improving plant cold tolerance.¹²⁹ MT treatment enhances the ability of tomatoes to resist cold stress, which is attributed to the accumulation of arginine-dependent carbon monoxide after MT treatment. Additionally, MT treatment can regulate the expression of genes related to circadian rhythms to modulate plant growth and development and enhance cold resistance.²⁷

The role of MT in heavy metal stress

The issue of soil contamination due to heavy metals has attracted substantial interest owing to their high concentration, challenging degradation, and potent toxicity. Elevated levels of heavy metals in soil can result in stunted plant growth, damage to the photosynthetic system and roots, disruption of mineral dynamics, and overproduction of reactive oxygen species (ROS), thus diminishing plant tolerance to a range of heavy metal toxins.¹³⁰ Research has revealed that treating seedlings with MT can increase the accumulation of secondary metabolites primarily involved in metal chelation and impede the production of ROS, thereby mitigating the inhibitory effects of heavy metals on plant growth.¹³¹

Under different heavy metal stresses (including Cd, Pb, Zn, etc.), the MT content can notably increase, thus demonstrating the mitigating effects of endogenous MT on heavy metal toxicity, as well as the feasibility of applying exogenous MT to alleviate heavy metal stress in plants.¹³² For instance, under Cd stress conditions, the interplay between exogenous MT and Cd stress induces the accumulation of endogenous MT, which positively influences plant growth, ROS scavenging, antioxidant capacity, and the biosynthesis of thiol compounds. Additionally, Cd stress can stimulate the expression of HsfA1a, which can directly bind to the Caffeic acid O-methyltransferase 1 (COMT1) promoter to enhance its expression, thereby increasing the biosynthesis of MT. Hence, HsfA1a serves as a positive regulatory factor for COMT1 transcription and promotes MT accumulation.¹³⁰ In Arabidopsis, heterologous expression of MsSNAT can also diminish Cd content in root tissue and enhance plant Cd stress tolerance.¹²⁶

Research has found that under Cd stress conditions, leaf spraying with MT on Chinese cabbage can eliminate the excessive accumulation of ROS in seedlings, bolster oxidase activity and antioxidant content, thus slowing down oxidative stress, and reduce the transcription of Cd absorption and transport-related genes IRT1/2, Nramp1/3, HMA2/4, thereby decreasing Cd accumulation in seedlings.¹³³ Cd stress can also induce the expression of HSFA1a and HSPs, thereby increasing MT biosynthesis, promoting GSH and PC biosynthesis, and chelating with Cd²⁺ to transport to vacuoles, reducing the toxic impact of Cd on plants.¹³⁴

Studies also discovered that the modulatory effect of MT on Cd stress might be related to nitric oxide (NO). As a downstream signaling molecule of MT, NO participates in regulating the expression of IRT1 and IRT2 to decrease the accumulation and absorption of Cd in plant seedlings, thus improving plant tolerance to Cd stress.¹³⁵ Furthermore, MT can interact synergistically with salicylic acid (SA) to reduce Cd absorption, increase the photosynthetic pigment content, accelerate the AsA‒GSH cycle, and regulate the glyoxalase system, thereby enhancing plant tolerance to Cd stress.¹³⁶

Under Pb stress conditions, 0.2 μmol·L⁻¹ MT can significantly augment the proliferation of Nicotiana tabacum BY−2 cells and preserve cell activity.¹³⁷ Moreover, MT is also involved in modulating the stress of other heavy metals, such as V, Ni, As; promoting improvements in the photosynthetic efficiency, SOD and CAT activities, and soluble protein content; and enhancing seed germination and seedling growth. It reduces the content of H₂O₂ and MDA and alleviates lipid peroxidation damage, thus reducing the toxic effect of heavy metal stress on plants (Figure 5).

In summary, substantial progress has been made concerning the application of MT in bolstering plant resistance to heavy metal stress. However, additional molecular and genetic studies are needed to elucidate the regulatory mechanism of MT in heavy metal tolerance, as well as the mechanisms underlying the induction of MT biosynthesis and the enhancement of heavy metal stress tolerance under various heavy metal stresses.

Function and regulation mechanism of MT in plant response to Biotic stress

MT plays a pivotal role not only in mitigating abiotic stress in plants but also in addressing biotic stress. MT significantly amplifies plant tolerance under biotic stress, employing diverse regulatory mechanisms in its response. Given that MT is an eco-friendly derivative of tryptophan, it has the potential to make plants more economical and environmentally sustainable in combating biotic stress (including fungi, bacteria, and viruses).^138,139

As an inhibitor to inhibit the growth of plant pathogenic microorganisms

Pathogenic microorganism infection is a principal cause of plant disease. MT can deter the proliferation of these pathogenic microorganisms, thereby diminishing damage to plant life under fungal stress (Figure 6). Chili anthracnose, caused by various pathogenic microorganisms, can be mitigated with the application of exogenous MT to Colletotrichum gloeosporioides, Colletotrichum oxysporum, and two strains of Phytophthora capsici (PC and HX−9). This significantly impedes the mycelial growth of all four fungi under diverse concentration conditions.¹⁴⁰

Figure 6.

Regulation Mechanism of MT in Plant Response to Biotic stress: (a) MT as an inhibitor, inhibits the growth of pathogenic microorganisms that infect plants and reduces their plaque; (b) MT can directly reduce the content of reactive oxygen species in plants after Biotic stress, or reduce the content of reactive oxygen species by increasing the content of antioxidant enzymes; (c) MT as a signal molecule, can induce the up-regulation of transcription factor expression in plants, enhance the expression of related genes in the MAPK signaling pathway, and enhance the expression of defense genes in various pathways such as salicylic acid resistance pathway, jasmonic acid signaling pathway, PAMP and ETI in plants.

Tea plants are susceptible to gray mold disease caused by infection with extremely fine spore fungi. The application of exogenous MT to diseased tea plants results in a gradual reduction in the fungal biomass on the leaves as the MT concentration increases, thereby inhibiting the proliferation of fine spore fungi.¹⁴¹ Similarly, rice plants infected by Xanthomonas bacteria can develop severe bacterial streak disease. When MT is applied to the leaves of affected plants, the biomass of bacteria is reduced, and the disease symptoms of the rice are mitigated.¹⁴² Exogenous MT can alleviate plant disease by inhibiting the growth of pathogenic microorganisms and controlling the spread of disease spots.

As a reactive oxygen species scavenger to improving plant antioxidant capacity

When plants are invaded by pathogens, the host triggers allergic reactions, thereby impeding the growth of the pathogens within the plant body. For instance, when wheat is infected by the fungus Leaf Rust, a significant amount of reactive oxygen species are generated. The application of exogenous MT to diseased plants can significantly eliminate these reactive oxygen species, effectively improving resistance to rust.¹⁴³ When apple fruit is afflicted by brown spot fungus, severe apple brown spot disease can occur. Following the application of MT to the affected fruit, the cells manage the content of intracellular reactive oxygen species by maintaining H₂O₂ stability.¹⁴⁴

Upon pathogenic microorganism infection, a plant body generates a significant amount of reactive oxygen species. MT can eradicate these reactive oxygen species (ROS) by increasing the activity of various antioxidant enzymes in plants, maintaining the steady state of ROS, and alleviating oxidative stress during pathogen invasion, thus playing a crucial role in resisting biotic stress (Figure 6).

As a signaling molecule to inducing defense gene expression

Under biotic stress, both exogenous and endogenous MT can stimulate the expression of defense genes within plants through their signaling molecules, thereby bolstering the capacity of plants to resist biotic stress. In Arabidopsis, knockout of the Serotonin N-acetyltransferase (SNAT) gene, the key enzyme in the MT biosynthesis pathway, results in susceptibility to pathogenic microorganisms. In these SNAT mutants, the decrease in MT levels leads to a reduction in salicylic acid levels as well as a decrease in the expression of genes such as PR1, ICS1, and PDF.¹⁷ When Botrytis cinerea infects tomato fruits, there is a significant increase in the expression levels of both the endogenous MT gene and the gene encoding a key enzyme in the MT biosynthesis pathway (SISNAT1). When exogenous MT is applied to tomato fruits, the expression of SlLoxD, SlAOC, and SlPI Ⅱ genes in the jasmonic acid pathway is markedly upregulated.¹⁸

Similarly, when watermelon plants infected with powdery mildew are sprayed with MT, transcriptome analysis of these plants indicates that MT modulates the expression of defense-related genes via PAMP and ETI.¹⁴⁵ Bananas, which are prone to infection by Bacillus anthracis, exhibit significant upregulation of the MAPK5 gene and several WRKY transcription factors in the mitogen-activated protein kinase (MAPK) signaling pathway after MT treatment.¹⁴⁶

These observations underscore the role of MT as a signaling molecule. MT can upregulate transcription factors in plants and simultaneously increase the expression of genes related to the MAPK signaling pathway. It also regulates the expression of defense genes in various pathways, such as the salicylic acid resistance pathway, jasmonic acid signaling pathway, and plant-mediated defense mechanisms (PAMP and ETI), thereby increasing the ability of plants to resist biotic stress (Figure 6).

Conclusion

Both domestic and international researchers have explored melatonin (MT) in plants, yielding significant insights. This body of work has not only elucidated the biosynthetic pathway of plant MT but also revealed the breadth of its biological functions. As a plant growth regulator, MT is involved in various physiological processes, including seed germination, growth, rooting, fruit development, senescence, and photosynthesis, and has effects similar to those of indole−3-acetic acid (IAA). Moreover, MT serves as a signaling molecule that controls the expression of genes related to the stress response, antioxidant activity, and plant hormone pathways (including IAA, BAB, CK, JA, and SA). It also collaborates with nitric oxide (NO) to maintain redox homeostasis. The intricate regulatory network formed by the synergy between MT and various hormones can increase photosynthesis and the accumulation of assimilates in plants under stressful conditions, inhibit reactive oxygen species (ROS) production, increase the amount of antioxidant substances, decrease oxidative stress damage, reduce osmolyte content, maintain ion balance, regulate the expression of stress response-related genes such as DREB and HSF, and thereby increase plant stress resistance.^28,92,93,122

Moreover, exogenous MT can promote plant growth and development, demonstrating a certain concentration dependency on enhancing the function of plant stress resistance. Research has revealed that both plant growth and development, as well as external stress environments, can instigate the biosynthesis of endogenous MT in plants. These findings indicate that MT plays crucial roles in regulating a myriad of physiological and biochemical processes, such as plant growth and development and the stress response. Multiple studies have proven that appropriate concentrations of MT can effectively mitigate oxidative stress triggered by adverse environments, thereby safeguarding plants from stress-induced damage.

However, despite the multitude of studies on MT and the discovery of its biosynthetic pathways and various physiological and biochemical functions, there are still numerous unexplored areas that require further investigation and elucidation.

First, the content of MT varies across different stages of plant development and tissues and can also be triggered by the external environment.^147,148 Nonetheless, we need to further understand how MT is transported within plants, how MT content transitions from low to high levels, and what receptors or related proteins contribute to these alterations in MT content. Hence, it is imperative to conduct comprehensive research on the metabolism and regulatory pathways of endogenous MTs. By integrating multiomics with CRISPR/Cas9 technology, we can expedite the identification of key genes involved in MT biosynthesis, degradation, and signal transduction.¹⁴⁴ Concurrently, the receptor or protein related to the signal transduction pathway involved in MT catabolism and content changes is also worth further exploration.

Second, as a multifaceted regulatory factor, MT can effectively manage plant growth, development, and stress resistance. However, its specific role as a signaling molecule in stress resistance reactions, intermediate products, and metabolic pathways remains unclear. Future studies should focus on unraveling how MT directly or indirectly regulates physiological and biochemical reactions in plants, including signal perception, signal transduction receptors, and transportation modes of MT in plants.

Last, MT can stimulate the biosynthesis of other hormones and work synergistically to modulate plant growth and stress resistance. MT can participate in the synthesis and degradation pathways of gibberellic acid (GA) and abscisic acid (ABA). MT can increase the GA content while reducing the ABA content during early seed germination, promoting more robust seed germination and growth.⁷³ MT can also upregulate the expression of IAA signal transduction genes to manage root growth, while ABA can act as a downstream molecular signal of MT in the stress response. Furthermore, MT can participate in the synthesis of JA, SA, ethylene (ETH), and NO to collectively improve plant stress resistance.⁶⁹ However, understanding how MT influences the contents of other endogenous hormones through signal transduction mechanisms, thereby facilitating alterations in plant physiology and biochemistry and the molecular regulatory network of interactions between MT and other plant hormones, remains a topic for future research. Additionally, comparatively less research has been conducted on crops whose MT content increases, making it imperative to employ existing biological techniques and MT research findings to study crop varieties with high MT This area of investigation will be a vital direction for future studies.

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Funding Statement

We are grateful for the 2023 Autonomous Region Key R&D and Achievement Transformation Plan Project – Science and Technology Support for Ecological Protection and High-Quality Development of the Yellow River Basin, which supports this article.

Disclosure statement

The authors declare no conflict of interest.

Author contributions

Taiyang Chen organized the overall framework of the article, collated and wrote Sections 3 and 7, drew images in the article and organized the entire content. Ling Xu wrote Section 6.1 and drew images in the article. Ping Yang collated and wrote Sections 4 and 5. Jiachen Tong collated and wrote Sections 1 and 2. Yanyan Liu collated and wrote Section 6.2. Shuying Sun and Youla Su edited and directed writing of the manuscript.

Data availability statement

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