Changes in melatonin levels in transgenic ‘Micro‐Tom’ tomato overexpressing ovine AANAT and ovine HIOMT genes

In animals, the melatonin biosynthesis pathway has been well defined after the isolation and identification of the four key genes that are involved in the conversion of tryptophan to melatonin. In plants, there are special alternative catalyzing steps, and plant genes share very low homology with the animal genes. It was of interest to examine the phenotype of transgenic Micro‐Tom tomato plants overexpressing the homologous sheep oAANAT and oHIOMT genes responsible for the last two steps of melatonin synthesis. The oAANAT transgenic plants have higher melatonin levels and lower indoleacetic acid (IAA) contents than control due to the competition for tryptophan, the same precursor for both melatonin and IAA. Therefore, the oAANAT lines lose the ‘apical dominance’ inferring that melatonin likely lacks auxin activity. The significantly higher melatonin content in oHIOMT lines than oAANAT lines provides new proof for the important role of ASMT in plant melatonin synthesis. In addition, the enhanced drought tolerance of oHIOMT lines will also be an important contribution for plant engineering.     

Melatonin enhances cold tolerance in drought‐primed wild‐type and abscisic acid‐deficient mutant barley

Melatonin is involved in multiple plant developmental processes and various stress responses. To explore the roles of melatonin played as well as its association with abscisic acid (ABA) in a process of drought priming‐induced cold tolerance (DPICT), a wild‐type barley and its ABA‐deficient mutant Az34 counterpart were selected for comparison, in which the effects of melatonin application (either foliarly or rhizospherically) and/or drought priming on the cold tolerance of both types of barleys were systematically investigated. It was demonstrated that the early drought priming induced an increase of endogenous melatonin production, which is not ABA dependent. In addition, exogenously applied melatonin resulted in higher ABA concentration in the drought‐primed plants than in the nonprimed plants when exposed to cold stress, indicating that ABA responded in a drought‐dependent manner. The interplay of melatonin and ABA leads to plants maintaining better water status. Drought priming‐induced melatonin accumulation enhanced the antioxidant capacity in both chloroplasts and mitochondria, which sustained the photosynthetic electron transport in photosynthetic apparatus of the plants under cold stress. These results suggest that the exogenous melatonin application enhances the DPICT by modulating subcellular antioxidant systems and ABA levels in barley.     

Exogenous melatonin improves Malus resistance to Marssonina apple blotch

We examined whether exogenously applied melatonin could improve resistance to Marssonina apple blotch (Diplocarpon mali) by apple [Malus prunifolia (Willd.) Borkh. cv. Donghongguo]. This serious disease leads to premature defoliation in the main regions of apple production. When plants were pretreated with melatonin, resistance was increased in the leaves. We investigated the potential roles for melatonin in modulating levels of hydrogen peroxide (H₂O₂), as well the activities of antioxidant enzymes and pathogenesis‐related proteins during these plant–pathogen interactions. Pretreatment enabled plants to maintain intracellular H₂O₂ concentrations at steady‐state levels and enhance the activities of plant defence‐related enzymes, possibly improving disease resistance. Because melatonin is safe and beneficial to animals and humans, exogenous pretreatment might represent a promising cultivation strategy to protect plants against this pathogen infection.     

Glutathione‐dependent induction of local and systemic defense against oxidative stress by exogenous melatonin in cucumber (Cucumis sativus L.)

Melatonin is involved in defending against oxidative stress caused by various environmental stresses in plants. In this study, the roles of exogenous melatonin in regulating local and systemic defense against photooxidative stress in cucumber (Cucumis sativus) and the involvement of redox signaling were examined. Foliar or rhizospheric treatment with melatonin enhanced tolerance to photooxidative stress in both melatonin‐treated leaves and untreated systemic leaves. Increased melatonin levels are capable of increasing glutathione (reduced glutathione [GSH]) redox status. Application of H₂O₂ and GSH also induced tolerance to photooxidative stress, while inhibition of H₂O₂ accumulation and GSH synthesis compromised melatonin‐induced local and systemic tolerance to photooxidative stress. H₂O₂ treatment increased the GSH/oxidized glutathione (GSSG) ratio, while inhibition of H₂O₂ accumulation prevented a melatonin‐induced increase in the GSH/GSSG ratio. Additionally, inhibition of GSH synthesis blocked H₂O₂‐induced photooxidative stress tolerance, whereas scavenging or inhibition of H₂O₂ production attenuated but did not abolish GSH‐induced tolerance to photooxidative stress. These results strongly suggest that exogenous melatonin is capable of inducing both local and systemic defense against photooxidative stress and melatonin‐enhanced GSH/GSSG ratio in a H₂O₂‐dependent manner is critical in the induction of tolerance.     

Melatonin enhances the occurrence of autophagy induced by oxidative stress in Arabidopsis seedlings

The beneficial effect that melatonin has against mitochondrial dysfunctioning seems to be linked to mitophagy. Roles for melatonin have been demonstrated in promoting health and preventing disease, as well as activating the process of autophagy in general. However, no reports have been made about how the application of melatonin regulates that process when plants are exposed to oxidative stress. We investigated the influence of different concentrations of melatonin (0.0, 0.5, 5.0, 10.0, or 50.0 μm ) on autophagy under methyl viologen (MV)‐induced oxidative stress. Arabidopsis seedlings that were pretreated with 5 or 10 μm melatonin underwent relatively strong induction of autophagy, as evidenced by the number of monodansylcadaverine (MDC)‐stained autophagosomes in root samples. Pretreatment with 10 μm melatonin also alleviated MV‐induced photo‐oxidation damage and significantly reduced the accumulation of oxidized proteins. Those responses might have been due to the strong upregulation of genes that involved in AtATG8‐PE conjugation pathway, which enhanced the capacity for autophagy. Histochemical staining revealed that both and H₂O₂ were highly accumulated upon MV exposure, although the response did not differ significantly between control and melatonin‐pretreated seedlings. By contrast, exogenous melatonin upregulated the expression of two genes for H₂O₂‐scavenging enzymes, that is, AtAPX1 and AtCATs. The activation of autophagy by melatonin without an alteration in ROS production may be part of a survival mechanism that is enhanced by melatonin after cellular damage. Therefore, it represents a second level of defense to remove damaged proteins when antioxidant activities are compromised.     

Cloning of Arabidopsis serotonin N‐acetyltransferase and its role with caffeic acid O‐methyltransferase in the biosynthesis of melatonin in vitro despite their different subcellular localizations

Serotonin N‐acetyltransferase (SNAT) is the penultimate enzyme in melatonin biosynthesis. We cloned SNAT from Arabidopsis thaliana (AtSNAT) and functionally characterized this enzyme for the first time from dicotyledonous plants. Similar to rice SNAT, AtSNAT was found to localize to chloroplasts with peak enzyme activity at 45°C (K_m, 309 μm ; V_max, 1400 pmol/min/mg protein). AtSNAT also catalyzed 5‐methoxytryptamine (5‐MT) into melatonin with high catalytic activity (K_m, 51 μm ; V_max, 5300 pmol/min/mg protein). In contrast, Arabidopsis caffeic acid O‐methyltransferase (AtCOMT) localized to the cytoplasm. Interestingly, AtCOMT can methylate serotonin into 5‐MT with low catalytic activity (K_m, 3.396 mm ; V_max, 528 pmol/min/mg protein). These data suggest that serotonin can be converted into either N‐acetylserotonin by SNAT or into 5‐MT by COMT, after which it is metabolized into melatonin by COMT or SNAT, respectively. To support this hypothesis, serotonin was incubated in the presence of both AtSNAT and AtCOMT enzymes. In addition to melatonin production, the production of major intermediates depended on incubation temperatures; N‐acetylserotonin was predominantly produced at high temperatures (45°C), while low temperatures (37°C) favored the production of 5‐MT. Our results provide biochemical evidence for the presence of a serotonin O‐methylation pathway in plant melatonin biosynthesis.     

Melatonin alleviates low PS I‐limited carbon assimilation under elevated CO2 and enhances the cold tolerance of offspring in chlorophyll b‐deficient mutant wheat

Melatonin is involved in the regulation of carbohydrate metabolism and induction of cold tolerance in plants. The objective of this study was to investigate the roles of melatonin in modulation of carbon assimilation of wild‐type wheat and the Chl b‐deficient mutant ANK32B in response to elevated CO₂ concentration ([CO₂]) and the transgenerational effects of application of exogenous melatonin (hereafter identified as melatonin priming) on the cold tolerance in offspring. The results showed that the melatonin priming enhanced the carbon assimilation in ANK32B under elevated [CO₂], via boosting the activities of ATPase and sucrose synthesis and maintaining a relatively higher level of total chlorophyll concentration in leaves. In addition, melatonin priming in maternal plants at grain filling promoted the seed germination in offspring by accelerating the starch degradation and improved the cold tolerance of seedlings through activating the antioxidant enzymes and enhancing the photosynthetic electron transport efficiency. These findings suggest the important roles of melatonin in plant response to future climate change, indicating that the melatonin priming at grain filling in maternal plants could be an effective approach to improve cold tolerance of wheat offspring at seedling stage.     

Melatonin induction and its role in high light stress tolerance in Arabidopsis thaliana

In plants, melatonin is a potent bioactive molecule involved in the response against various biotic and abiotic stresses. However, little is known of its defensive role against high light (HL ) stress. In this study, we found that melatonin was transiently induced in response to HL stress in Arabidopsis thaliana with a simultaneous increase in the expression of melatonin biosynthetic genes, including serotonin N ‐acetyltransferase1 (SNAT 1 ). Transient induction of melatonin was also observed in the flu mutant, a singlet oxygen (¹O₂)‐producing mutant, upon light exposure, suggestive of melatonin induction by chloroplastidic ¹O₂ against HL stress. An Arabidopsis snat1 mutant was devoid of melatonin induction upon HL stress, resulting in high susceptibility to HL stress. Exogenous melatonin treatment mitigated damage caused by HL stress in the snat1 mutant by reducing O₂^? production and increasing the expression of various ROS ‐responsive genes. In analogy, an Arabidopsis SNAT 1 ‐overexpressing line showed increased tolerance of HL stress concomitant with a reduction in malondialdehyde and ion leakage. A complementation line expressing an Arabidopsis SNAT 1 genomic fragment in the snat1 mutant completely restored HL stress susceptibility in the snat1 mutant to levels comparable to that of wild‐type Col‐0 plants. The results of the analysis of several Arabidopsis genetic lines reveal for the first time at the genetic level that melatonin is involved in conferring HL stress tolerance in plants.     

Mitogen‐activated protein kinase pathways are required for melatonin‐mediated defense responses in plants

Melatonin enhances pathogen resistance by inducing the expression of a number of plant defense‐related genes. To examine whether the melatonin‐mediated pathogen resistance is associated with mitogen‐activated protein kinase (MAPK) cascades, Arabidopsis and tobacco leaves were treated with melatonin and investigated for MAPK activation using an antiphospho‐p44/42 MAPK (Erk1/2) monoclonal antibody. Two MAPKs, MPK3 and MPK6, were activated rapidly and transiently by 1 μm melatonin treatment in Arabidopsis. Its tobacco ortholog MAPKs were also activated. The activation of MPK3 and MPK6 by 2‐hydroxymelatonin and N‐acetylserotonin was also observed, albeit to a lesser degree than that by melatonin. Furthermore, MAPK activation by melatonin was uncoupled from G‐protein signaling, because melatonin efficiently activated two MAPKs in a G‐protein β knockout mutant (agb1). Suppression of both MPK3 and MPK6 in transgenic Arabidopsis exhibited significant decreases in the induction of defense‐related gene expression and pathogen resistance relative to wild‐type plants. Using an array of MAP kinase kinase (MKK) knockout mutants, we found that four MKKs, namely MKK4, MKK5, MKK7, and MKK9, are responsible for the activation of MPK3 and MPK6 by melatonin, indicating that melatonin‐mediated innate immunity is triggered by MAPK signaling through MKK4/5/7/9‐MPK3/6 cascades.     

Melatonin regulates carbohydrate metabolism and defenses against Pseudomonas syringae pv. tomato DC3000 infection in Arabidopsis thaliana

Melatonin has been reported to promote plant growth and development. Our experiments with Arabidopsis thaliana showed that exogenous applications of this molecule mediated invertase inhibitor (C/VIF)‐regulated invertase activity and enhanced sucrose metabolism. Hexoses were accumulated in response to elevated activities by cell wall invertase (CWI) and vacuolar invertase (VI). Analyses of sugar metabolism‐related genes revealed differential expression during plant development that was modulated by melatonin. In particular, C/VIF1 and C/VIF2 were strongly down‐regulated by exogenous feeding. We also found the elevated CWI activity in melatonin‐treated Arabidopsis improved the factors (cellulose, xylose, and galactose) for cell wall reinforcement and callose deposition during Pseudomonas syringae pv. tomato DC3000 infection, therefore, partially induced the pathogen resistance. However, CWI did not involve in salicylic acid (SA)‐regulated defense pathway. Taken together, this study reveals that melatonin plays an important role in invertase‐related carbohydrate metabolism, plant growth, and pathogen defense.     

Long‐term exogenous application of melatonin delays drought‐induced leaf senescence in apple

To examine the potential roles of melatonin in drought tolerance, we tested the effects of its long‐term exogenous application on ‘Hanfu’ apple (Malus domestica Borkh.). When 100 μm melatonin was added to soils under drought conditions, the resultant oxidative stress was eased and leaf senescence was delayed. This molecule significantly reduced chlorophyll degradation and suppressed the up‐regulation of senescence‐associated gene 12 (SAG12) and pheophorbide a oxygenase (PAO). Such treatment also alleviated the inhibition of photosynthesis brought on by drought stress. We also investigated quenching and the efficiency of Photosystem II (PSII) photochemistry under dark and light conditions and found that melatonin helped to maintain better function of PSII under drought. The addition of melatonin also controlled the burst of hydrogen peroxide, possibly through direct scavenging and by enhancing the activities of antioxidative enzymes and the capacity of the ascorbate–glutathione cycle. Thus, understanding this effect of melatonin on drought tolerance introduces new possibilities to use this compound for agricultural purposes.     

Caffeic acid O‐methyltransferase is involved in the synthesis of melatonin by methylating N‐acetylserotonin in Arabidopsis

Although a plant N‐acetylserotonin methyltransferase (ASMT) was recently cloned from rice, homologous genes appear to be absent in dicotyledonous plants. To clone an ASMT de novo from a dicotyledonous plant, we expressed eight Arabidopsis thaliana O‐methyltransferase (OMT) cDNAs in Escherichia coli and screened for ASMT activity by measuring melatonin production after the application of 1 mm N‐acetylserotonin (NAS). Among the eight strains harboring the full‐length cDNAs, the OMT3 strain produced high levels of melatonin, suggesting that OMT3 encodes an active ASMT. OMT3 is already known as caffeic acid OMT (COMT), suggesting multiple functions for this enzyme. The purified recombinant A. thaliana COMT (AtCOMT) showed high ASMT activity, catalyzing the conversion of NAS to melatonin. The K_m and V_max values for ASMT activity were 233 μm and 1800 pmol/min/mg protein, while the K_m and V_max values for COMT activity were 103 μm and 564,000 pmol/min/mg protein, respectively. The catalytic efficiency (V_max/K_m) for ASMT activity was 709‐fold lower than for COMT. In vitro, ASMT activity was dramatically decreased by the addition of caffeic acid in a dose‐dependent manner, but the activity of COMT was not altered by NAS. Lastly, the Arabidopsis comt knockout mutant exhibited less production of melatonin than the wild type when Arabidopsis leaves were infiltrated with 1 mm NAS, suggestive of in vivo role of COMT in melatonin biosynthesis in plants.