Plants depend on sunlight absolutely as an overall energy source so that they develop multiple protein complexes to accomplish photosynthesis. These protein complexes include Photosystem II (PSII), Photosystem I (PSI), cytochrome b₆f complex, and so on (Jarvis and Lopez-Juez, 2013). When light energy is insufficient, plants capture more light energy through changing the location of chloroplast (Salgado-Luarte and Gianoli, 2011). When the absorbed light energy of plants exceeds their demand, the photosynthetic complexes will be injured, leading to the decrease of photosynthetic efficiency (Demmig-adams and Adams, 1992). At the same time, reactive oxygen species (ROS) bursts (Jarvis and Lopez-Juez, 2013), and the resultant ROS is toxic to plants (Nishiyama et al., 2001). Although the damage mechanism of photosynthetic apparatus caused by high light is still controversial, it is indisputable that the high light finally leads to injure D1 subunit of PSII (Allakhverdiev and Murata, 2004). The photodamage of D1 occurs at all light intensities, but the photoinhibition occurs only when the balance between the photodamage and repair of D1 is broken (Allakhverdiev and Murata, 2004). Fortunately, plants had established an elaborate protective mechanism, including chloroplast avoidance movement which could minimize light exposure, ROS scavenging systems that are composed of SOD, POD, APX, etc. (Apel and Hirt, 2004), and PSII repair cycle (Kirchhoff, 2014). Although this multi-level photoprotective mechanism helps plants to minimize the injury on the photosynthetic machinery, the damage is unavoidable. Even the damage would affect plant growth and development, resulting in yield reduction and death.

Melatonin (N-acetyl-5-methoxytryptamine), a kind of indoleamine which widely exists in organism, was discovered in plants in 1995 and numerous studies proved that melatonin has involved in multiple processes in plants, including the development of flower (Lee et al., 2019), the architecture of root (Yang et al., 2021), the ripening of fruit (Wang et al., 2020), the senescence of leaf (Wang et al., 2013), the regulation of circadian rhythms, and the protective effect on chlorophyll and photosynthesis (Arnao and Hernandez-Ruiz, 2015). Melatonin alleviated oxidative damage through effectively scavenging ROS and reactive nitrogen species (RNS) (Arnao and Hernandez-Ruiz, 2015). And its metabolites, such as 2-hydroxylmelatonin and N1-acetyl-N2-formyl-5-methoxykynuramine, could also directly and efficiently scavenge ROS (Tan et al., 2007). Besides, melatonin also inspired antioxidant activity by stimulating antioxidant enzymes and could augment the ascorbate-glutathione (AsA-GSH) cycle to scavenge excess ROS (Li et al., 2015). And melatonin helped plants to defend against multiple abiotic stresses, such as cold, heavy metals, salt, drought, and so on (Arnao and Hernandez-Ruiz, 2015). Exogenous melatonin relieved the photoinhibition of tomato seedlings by improving non-photochemical quenching under cold stress (Ding et al., 2017). Similarly, the accumulation of melatonin in water hyacinth under sunlight was significantly higher than that under artificial low-light (Tan et al., 2007). This implies that melatonin can be induced by high light. Supporting these results, the expression of the melatonin-synthesis-related gene ASMT in apple had been up-regulated by high light, leading to the accumulation of melatonin (Zheng et al., 2017). In addition, melatonin enhanced the tolerance to high light in Arabidopsis thaliana (Lee and Back, 2018). However, the underlying physiological and molecular mechanism of the elevated tolerance to high light by melatonin remains unclear in plants.

Plants need light for photosynthesis and thus gain energy for their growth, but excessively high light does harm to photosynthetic apparatus. There were many researches on the high light stress in plants, but the role of melatonin under high light had been less explored. Lee and Back (2018) found that high light led to the brust of ROS, and the synthesis of melatonin was induced by chloroplastidic singlet oxygen and promoted the accumulation of melatonin. At the same time, melatonin increased the activity of antioxidant enzymes, thus enhancing the tolerance of plants to high light. In addition, Yao et al. (2020) reported the synthesis of melatonin was induced by UV-B. The wavelength of light spectrum also affected the synthesis of melatonin. Afreen et al. (2006) reported that the melatonin concentrations were highest in red-light-exposed plants and followed the blue light and white light. A lot of study showed that high light inhibited photosynthesis, but the role of melatonin in this physiological process is still unclosed.

Based on the reported relationship between melatonin and light intensity, we suggested that melatonin decreased the level of ROS by regulating antioxidant system to protect the photosynthesis under high light. To test this hypothesis, we measured ROS accumulation, membrane lipid peroxide, photosynthetic parameters, antioxidant enzyme, and PSII protein after the melatonin pretreatment under high light. The results demonstrated that melatonin provided effective ROS scavenging ability for plants and preserved the integrity of the photosynthetic protein, and then enhanced the tolerance to high light.

Light is a source of energy and signal for plant growth. Plant has to go through a dark - low light - high light - low light - dark cycle every day. High light often causes destruction of the photosynthetic system and even cell death. Melatonin is a multitasking biomolecule, and it is involved in numerous physiological processes in plants, including redox reactions, biosynthesis, circadian clock, and stress defenses (Arnao and Hernandez-Ruiz, 2015). According to recent research, high light seriously destroyed the photosynthetic structure of chloroplasts and weakened its photosynthesis, and finally inhibited the growth of plants (Ding et al., 2017). In the present study, high light caused ROS burst and the reduction of photosynthesis. However, the application of exogenous melatonin significantly alleviated the damage caused by high light. Also, stress triggered endogenous melatonin response. Melatonin is effective in striving against stress, but the reception of stress signal, the activation of endogenous melatonin biosynthesis and the action process of melatonin were all restricted by many factors. At the cellular level, a stress signal is firstly received by the cell membrane, and then transferred to the nucleus. These starts to activate the melatonin biosynthesis pathway in mitochondria and chloroplasts by upregulating the melatonin-biosynthesis genes (Moustafa-Farag et al., 2020). Melatonin is effective in striving against stress, but the reception of stress signal, the activation of endogenous melatonin biosynthesis and the action process of melatonin were all restricted by many factors. In addition to ROS brust caused by high light to promote endogenous melatonin response, it seems that there are other pathways to promote the synthesis of melatonin. Transcription factors like MYB, bHLH, bZIP, ERF, NAC, and WRKY are major players in stress signaling and some constitute major hubs in the signaling webs (Tripathi et al., 2014). Hu et al. (2019) reported that high light up-regulated constantly the expression of 29 transcriptional factors, which could regulate the expression of genes associated with photosynthesis and ROS scavenging-related genes. Zhao et al. (2010) found that UV-B radiation induced OsWRKY89 to participate in light responses. Transcription factor regulatory networks are also involved in the regulation of melatonin synthesis. Wei et al. (2017) reported that MeWRKY79 and MeHsf20 of cassava could act with W-box and thermal stress element HSEs (Heat-stress elements) in the promoter of MeASMT to induce its expression. And previous research found one cysteine2/histidine2-type zinc finger transcription factor, ZAT6, was involved in melatonin-mediated stress response in Arabidopsis (Shi and Chan, 2014). Maybe melatonin participated in light response through some of these transcription factors. In addition, transcription factor could directly affect the expression of melatonin synthesis gene, and also indirectly affect the effect on antioxidant system.

Reactive oxygen species is the byproducts of photosynthesis, respiration, and other normal metabolism, and it plays an important role in the resistance and tolerance to stress (Chen et al., 2016). Excessive ROS was toxic to cells and organisms, and involved in the programmed response to abiotic stress (Manchester et al., 2015). Yuan et al. (2020) found that high light triggered the accumulation of ROS. Han et al. (2017) found that exogenous melatonin decreased the level of ROS under cold stress. In the present study, H₂O₂ and O2− increased significantly under high light, and they decreased with melatonin pretreatment.

Excessive ROS could stimulate membrane lipid peroxidation, and then led to the damage of cell membrane, loss of cellular integrity, and cell death (Chen et al., 2018). MDA and EL are regarded as important indicators of oxidative damage and previous study indicated that melatonin decreased the high level of EL and MDA caused by water stress (Zhang et al., 2013). Consistently, our research suggested that exogenous melatonin decreased the level of EL and MDA and reduced cell death under high light. It is worthwhile to note that the situation of snat-1 and snat-2 was worse than WT under high light. Exogenous melatonin application maintained a relatively low level of ROS and reduced the degree of cell damage, further conferring plant resistance to high light.

Enzymatic antioxidant system and non-enzymatic antioxidant system were evolved in response to oxidative stress in plants (Apel and Hirt, 2004). Melatonin alleviated oxidative damage caused by salinity, drought and cold perhaps by directly enhancing antioxidative enzyme activities, like SOD, POD and APX (Apel and Hirt, 2004). Chen et al. (2018) also found that the application of melatonin increased the activities of antioxidant enzymes in maize seedlings under salt stress. In the present research, melatonin increased the activity of SOD under high light. Exogenous melatonin downregulated the activity of POD and upregulated the activity of APX and GPX. They are all the converter for H₂O₂ but work in different ways. Melatonin inhibited the pathway of POD but promoted the pathway of APX and GPX to scavenge H₂O₂. Previous works showed that exogenous melatonin resulted in higher content of AsA and GSH under salt stress (Chen et al., 2018). But our study suggested that exogenous melatonin had little effect on them and the lack of endogenous melatonin weakened their levels. AsA-GSH cycle is a vital antioxidant system against oxidative stress in plants (Zhang et al., 2015). APX and GPX are the key enzymes of the glutathione ascorbic acid cycle, and melatonin effectively increased their activity. The glutathione-ascorbic acid cycle might play a key role in alleviating the high light stress. Melatonin did not only remove ROS as a scavenger but also regulated the activity of antioxidants in plants. Our results showed that melatonin reduced the accumulation of ROS but decreased the activity of POD. So melatonin was not overkill to ROS. In addition, Li et al. (2020) found that low concentration of melatonin induced the production of ROS and ROS worked as a key signal in many physiological processes. Maybe melatonin not only improves the defense capabilities of plant, but also ensures the role of ROS as a message transmitter in stress depending on its regulation role to the antioxidant system.

Photosynthetic pigments are susceptible to environmental stress. Melatonin effectively alleviated the degradation of chlorophyll and carotenoid under stress and made it with a certain level. Wu et al. (2021) found that melatonin suppressed the activities of chlorophyll catabolic enzymes such as chlorophyllase (CLH), pheophytinase (PPH), pheophorbide a oxygenase (PAO) and down-regulated the expressions of BoNYC1, BoNOL, BoCLH, BoPPH, BoPAO, BoRCCR and BoSGR1 which involved in chlorophyll catabolism. In addition, Jahan et al. (2020) found melatonin upregulated the expression of chlorophyll synthesis genes, i.e., POR, CAO, CHL G.

The decrease of photosynthesis efficiency in plants after being exposed to adverse environmental might be a key reason for the reduction of crop. The previous studies showed abiotic stress induced irreversible damage to PSII in tomato, oat seeds, Ligustrum vicaryi and maize seedlings thereby decreasing photosynthetic rate (Ding et al., 2017; Chen et al., 2018; Alyammahi and Gururani, 2020; Kanwar et al., 2020; Yuan et al., 2020). The decrease of stomatal conductance could result in a declined P_n and reduced assimilation products, thus causing an inhibited growth and a lower yield (Rao and Chaitanya, 2016). In this research, the P_n and g_s reduced under high light, and exogenous melatonin increased P_n. These findings were in line with the report of maize under salt stress (Chen et al., 2018). Hu et al. (2021) suggested that the reduction of Chl a may be one of the reasons for the decrease of P_n in acid rain stressed barley plants. And this was similar with our results (Figures 2A, 3C). The increase of P_n by melatonin under high light might be due to its protective effect on chlorophyll. The g_s was mainly controlled by guard cell through regulating the opening and closing of stomata (Assmann, 1999; Vavasseur and Raghavendra, 2005). Erland et al. (2019) employed a novel technique, quantum dot nanoparticles, to visualize the location of melatonin and found melatonin-QD aggregated in guard cells. It is possible that melatonin exerted an effect on g_s through this pathway.

In nature, as soon as there is light, it will cause photooxidative damage to photosynthetic apparatus and then photoinhibition is unavoidable (Allakhverdiev and Murata, 2004). The extent of photoinhibition depends on the balance between photodamage and the repairing cycle (Allakhverdiev and Murata, 2004). Melatonin had been found to protect PSII proteins from oxidative injuries (Han et al., 2017; Huang et al., 2019). In previous work, the protective role of melatonin was confirmed on photosynthetic proteins in maize and tomato under drought and high light stress (Ding et al., 2017; Huang et al., 2019). Among ROS, H₂O₂ in chloroplast is an important inhibitor of the Calvin cycle. It might inhibit the activities of enzymes possessing sulfhydryl groups and reduced the photosynthetic CO₂ assimilation (Hancock et al., 2005). In addition, the photooxidative damage products (especially H₂O₂) firstly stimulated the apparent photoinhibition of PSII by inhibiting the repair of PSII instead of accelerating photodamage to PSII (Nishiyama et al., 2001). In present study, we found that the change in the content of H₂O₂ in the chloroplast showed that the photosynthetic system was suffering from huge oxidative pressure, the proteins of the photosystem were destroyed under high light, and this situation was relieved by exogenous melatonin. Therefore, these results suggested that melatonin significantly inhibited ROS burst under high light. Numerous works had indicated that D1 protein is the key target under environmental stress and the D1 protein remained a relatively high level with melatonin pretreatment in our study.

Taken together, our research evaluated the effect and mechanism of melatonin on Arabidopsis under high light. Melatonin effectively protected photosynthesis in response to high light. Melatonin mainly worked through two aspects. On the one hand, melatonin was involved in cellular REDOX regulation. Melatonin directly removed ROS as antioxidants (Zhao et al., 2021). At the same time, melatonin regulated the activity of antioxidant enzyme as a signal molecule (Zhao et al., 2021). Therefore, melatonin protected photosynthetic pigments and proteins through redox homeostasis, and contributed to photosynthesis. On the other hand, melatonin gathered in guard cells (Erland et al., 2019), and might participate in stomatal movement. Simultaneously, the role of endogenous melatonin in plants was indispensable for the responses of plants to stress.

Our findings provided the evidence for melatonin to relieve high light stress, and extended new uses for melatonin as a plant growth regulator. At the same time, endogenous melatonin played an important role to against stress, and its potential mechanism needs further study. Our results and other reports suggested that melatonin might also be involved in stomatal movement (Assmann, 1999; Vavasseur and Raghavendra, 2005; Erland et al., 2019), but the mechanism is still unclear. Given the key role of melatonin in tolerance against various abiotic stresses, it is of interest to explore the mechanism of melatonin in plant.

The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding author.

MY designed the experiments. S-JY, BH, Y-QZ, DH, TC, and C-BD performed the experiments and data analysis. S-JY and MY wrote the manuscript. All authors contributed to the article and approved the submitted version.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.