Crosstalk between Melatonin and Reactive Oxygen Species in Plant Abiotic Stress Responses: An Update

Melatonin acts as a multifunctional molecule that takes part in various physiological processes, especially in the protection against abiotic stresses, such as salinity, drought, heat, cold, heavy metals, etc. These stresses typically elicit reactive oxygen species (ROS) accumulation. Excessive ROS induce oxidative stress and decrease crop growth and productivity. Significant advances in melatonin initiate a complex antioxidant system that modulates ROS homeostasis in plants. Numerous evidences further reveal that melatonin often cooperates with other signaling molecules, such as ROS, nitric oxide (NO), and hydrogen sulfide (H2S). The interaction among melatonin, NO, H2S, and ROS orchestrates the responses to abiotic stresses via signaling networks, thus conferring the plant tolerance. In this review, we summarize the roles of melatonin in establishing redox homeostasis through the antioxidant system and the current progress of complex interactions among melatonin, NO, H2S, and ROS in higher plant responses to abiotic stresses. We further highlight the vital role of respiratory burst oxidase homologs (RBOHs) during these processes. The complicated integration that occurs between ROS and melatonin in plants is also discussed.

Moreover, any deviation from ROS balance can be thought of as a reaction to ROS signaling [8,23]. Numerous studies have revealed that ROS signaling plays a dual role, and is also beneficial to plants at specific cellular compartments during the abiotic stress process [8,23]. Multiple enzymatic systems, such as POD and plasma membrane-bound NADPH oxidase, generate ROS [24]. The respiratory burst oxidase homolog (RBOH) NADPH oxidases are the primary source of ROS production at the apoplast [25]. Superoxide is generated by NADPH oxidase and dismutated to H 2 O 2 [26]. It has been observed that AtrbohD and AtrbohF can regulate sodium (Na) and potassium (K) transport, thus limiting Na concentrations and enhancing salinity tolerance [27,28]. AtrbohF also plays a vital role in mediating cadmium (Cd) uptake, chelation, and translocation [29]. Moreover, the ROS wave is required for a plant's high light, cold, and heat tolerance as well [30][31][32].
To date, several papers have confirmed the crosstalk between melatonin and signaling molecules, such as nitric oxide (NO), hydrogen sulfide (H 2 S), and hydrogen gas (H 2 ); therefore, the present paper does not elucidate it in detail [9,10,18,19,[33][34][35]. Here, we systematically review the updated literature on the crosstalk between melatonin and ROS in plants upon abiotic stresses, highlight the role of RBOHs, and give perspectives for future research.

Melatonin Acts as an Antioxidant to Establish Redox Homeostasis through the Antioxidant System in Plants under Abiotic Stresses
As a master regulator, melatonin plays an important role in plant tolerance to abiotic stresses, such as salinity, cold, heat, drought, and heavy metals [9][10][11][12][13][14][15][16][17]. Our previous review systematically summarized the melatonin biosynthesis and catabolism in plants [19]. We also showed that Cd stress strongly induced melatonin accumulation via regulating the expression of genes encoding tryptophan decarboxylase (TDC), tryptamine 5-hydroxylase (T5H), N-acetylserotonin methyltransferase (ASMT), caffeic acid O-methyltransferase (COMT), and serotonin N-acetyltransferase (SNAT, also called arylakylamine N-acetyltransferase (AANAT)) [21]. Moreover, salinity stress up-regulated the expression SNAT genes and improved melatonin levels in Arabidopsis and Brassica napus [35,36]. In cucumber, cold treatment induced the expression of TDC, T5H, SNAT, and COMT genes, and thus enhanced melatonin levels [37]. Heat stress also improved the transcripts of T5H and ASMT genes and melatonin levels in tomato seedlings [38]. Similarly, drought stress up-regulated the expression of TDC1, T5H, AANAT2, and ASMT1 in Malus hupehensis and maize plants [39]. These studies mainly found that melatonin levels were significantly induced via up-regulating the transcriptional level of melatonin biosynthesis genes in response to abiotic stress in plants. Interestingly, MzASMT9 protein levels were enhanced by salinity stress in leaves of Malus zumi [40]. Moreover, abiotic stress also tightly regulated the activities of melatonin biosynthesis enzymes, such as T5H, TDC, SNAT, and ASMT enzymes [41,42]. For example, high temperature elevated SNAT and ASMT activity, and increased melatonin levels in rice seedlings [41]. Salinity stress induced serotonin accumulation and N-acetylserotonin O-methyltransferase (HIOMT) activity in vascular bundles and the cortex, leading to melatonin accumulation in sunflower (Helianthus annuus) plants [42].
In general, these stresses caused endogenous melatonin accumulation, indicating that melatonin might be involved in a plant's tolerance to abiotic stress. A series of studies found that the application of exogenous melatonin increased the level of endogenous melatonin, thereby improving plant tolerance to abiotic stress [20,21,36,43,44]. For example, the endogenous melatonin content was increased in maize by application of exogenous melatonin upon both control and Al stress conditions [43]. Moreover, this increase significantly mitigated Al-induced oxidative stress [43]. Similar results were found in the role of exogenous melatonin application in the alleviation of Cd-induced growth inhibition of mallow (Malva parviflora) plants [20]. Pharmacological studies also revealed that exogenous melatonin application improved resistance to salinity and drought stresses via the modulation of photosynthesis and starch/sucrose metabolism in soybean [21]. The application of melatonin partly counteracted salinity-induced seedling growth inhibition in rapeseed (Brassica napus L.) [36]. The role of exogenous melatonin in reducing the severity induced by heat stress in wheat seedlings was also evaluated [44].
Recently, several general genetic studies have been conducted in plants [10][11][12]35,[45][46][47]. In these studies, it was demonstrated that both the TDC-silenced mutant and COMT1silenced mutant showed a lower level of melatonin in tomato plants [10]. In Arabidopsis, we found that the atsnat mutant showed a low content of melatonin, and appeared hypersensitive to salinity stress in comparison with the wild-type seedlings [12,35]. It was also observed that both atsnat-1 and atsnat-1 showed sensitivity to high levels of light [46]. The transgenic Arabidopsis seedlings overexpressing alfalfa SNAT enhanced melatonin accumulation and exhibited more resistance to Cd stress than wild-type plants [11]. In tomato plants, overexpressing AANAT or HIOMT enhanced melatonin accumulation and improved drought tolerance [45]. Heterologous expression of HIOMT in apple leaves showed higher melatonin levels and improved salinity stress tolerance [46]. Nevertheless, there is still much to be learned about the post-translational modulation of melatonin biosynthesis genes and the regulation of related proteins, which should be further studied in the future.
Abiotic stresses cause endogenous ROS (mainly O 2 •-, H 2 O 2, and MDA) accumulation in plants, which generates in different various organelles including chloroplast, peroxisome, mitochondria, and the cell membrane during abiotic stresses ( Figure 1) [4]. For example, O 2 •acts as the by-product of oxygen reduction by the electron transport chain (ETC) in chloroplast and mitochondria [48,49]. They also generate by photorespiration and fatty acid oxidation in peroxisome [50,51]. Then, H 2 O 2 is produced from O 2 •by the activity of SOD or glycolate oxidases. Furthermore, NADPH oxidases, cell-wall-bound peroxidases (POX), and polyamine oxidases (PAO) result in ROS generation in the cell membrane, cell wall, and apoplast, respectively [4,52]. As toxic byproducts, ROS could cause damages to the RNA, DNA, and proteins of plants (oxidative stress situations) [8].

Plant Abiotic Stress Tolerance Is Mediated by the Crosstalk between Melatonin and Signal Molecules (NO, H 2 S, and ROS)
Melatonin was shown to be a crucial regulator occupying extensive roles in many physiological and biochemical processes throughout plant life, especially plant responses to abiotic stress [9,10,19]. Furthermore, melatonin was shown to function with other signal molecules in order to manipulate environmental damages, such as NO, H 2 S, ROS, and H 2 [9,10,18,19,[33][34][35]. The latest reviews systematically revealed the inter-relationship between melatonin and gasotransmitters (including NO, CO, H 2 S, CH 4 , and H 2 ) in resistance to plant abiotic stress [10,[81][82][83]. For example, melatonin altered the endogenous NO accumulation, and reduced reactive nitrogen species (RNS) (ONOO-, and peroxynitrous acid), which was generated by stress [81,82]. Nevertheless, melatonin regulated the expression of the nitric oxide synthase (NOS) gene, and triggered endogenous NO accumulation [84]. Ample evidence manifested that NO acted as the downstream signal of melatonin to regulate plant tolerance to salinity, drought, heat, cold, Cd, and Al stresses [33,34,36,[85][86][87][88]. Zhao et al. found that melatonin enhanced rapeseed seedling tolerance via NO signaling against salinity stress, and similar results were obtained for sunflower seedlings as well [36,85]. Melatonin down-regulated the NO accumulation, thus promoting soybean tolerance to drought stress [86]. Moreover, positive and antagonistic interactions between melatonin and NO might exist in plant responses to stress caused by heavy metals [19,87,88].
It was also shown that H 2 S plays a vital role in enhancing plant tolerance to abiotic stress and alleviating its detrimental effects [89][90][91][92]. Melatonin increased the activities of the H 2 S-produced enzymes (D-cysteine desulfhydrase (DCD), L-cysteine desulfhydrase (LCD)), thus improving H 2 S accumulation [93][94][95]. Further, application of hypotaurine (HT, H 2 S scavenger) reversed the contribution of melatonin in alleviation of the salinity and heat damages by reestablishing the redox homeostasis in tomato, cucumber, and wheat seedlings [44,94,95]. Kaya et al. found that the interactive effect of NO and H 2 S improved the wheat's resistance to Cd stress via enhancing the antioxidative defense system and reducing the damage induced by oxidative stress [96]. Moreover, the H 2 S and NO jointly were involved in melatonin-regulated salinity tolerance in cucumbers [95]. They were also involved in melatonin-mediated resistance to iron deficiency and salinity stress in pepper seedlings [97]. Until now, the interactions among melatonin, NO, and H 2 S in plant responses to abiotic stress were not largely explored.
In recent years, apart from NO and H 2 S, great efforts were made in studies conducted on ROS-directed plant abiotic stress responses [4][5][6][7][8]. In this review, Section 2 shows that ROS are inevitably produced by adverse environmental conditions, and thereby significantly cause damages to the structural and functional integrity of the whole plant seedling. More importantly, ROS instantly produced in chloroplast, peroxisome, mitochondria, and cell membrane organelles often modulate signaling pathways when maintained at a moderate concentration [4][5][6][7][8]. Recent studies have further shed new light on the role of ROS in melatonin-regulated tolerance to abiotic stresses in plants. In early responses to cold stress, melatonin was found to stimulate H 2 O 2 accumulation in watermelon [98]. Chen et al. found that endogenous melatonin rapidly induced ROS accumulation under short-term salinity treatment in Arabidopsis [12]. Then, ROS triggered SOS-mediated Na + efflux and intensified the increased antioxidant defense [12]. Similarly, melatonin triggered an ROS burst that enhanced the expression of K + uptake transporters to enable K + retention under salinity stress in rice [53]. H 2 O 2 scavengers negated the effects of melatonin-mitigated abiotic stress, such as drought, heat, and cold stress in tomato plants [99]. Collectively, these studies preliminarily revealed that ROS signaling acts downstream of melatonin in alleviation of abiotic stress in plants.
Several articles have also shown the mechanisms underlying the complexity of ROS with NO and/or H 2 S signaling in plant tolerance against abiotic stress [2][3][4]89,90,92]. Zeng et al. reviewed the crosstalk among melatonin, NO, and ROS in plant tolerance to bacterial, fungal, and viral diseases [100]. This phenomenon was also shown to promote fruit ripening [9]. However, more advances should be made to provide new insights on the understanding of the crosstalk among melatonin, NO, H 2 S, and ROS in plant abiotic tolerance using genetic, pharmacological, genomic, and proteomic approaches.

The Roles of RBOH-Involved ROS Signaling in Melatonin-Modulated Plant Processes
ROS produced in several organs (the cell membrane, chloroplast, peroxisome, mitochondria, and apoplast) are implicated in signaling pathways (Figure 1). Respiratory burst oxidase homolog (RBOH) proteins are the NADPH oxidases localized on the plasma membrane [101]. They are the key proteins associated with the signal transduction event [101].
There is a C-terminal FAD/NADP(H)-binding domain, a N-terminal regulatory domain, six transmembrane domains, and several potential phosphorylation sites in RBOHs [24,25]. The NADPH oxidases are modulated by phosphorylation and/or binding of calcium ions at the cytosol, and then produce O 2 •− at the apoplast ( Figure 1). O 2 •− is converted into H 2 O 2 via the action of SOD, or spontaneously [8,101]. Afterwards, H 2 O 2 could enter various types of cells and trigger different signaling responses. There are ten or eight RBOH genes encoding NADPH oxidase in Arabidopsis or tomato, respectively [24,25]. In recent years, many studies have shed light on ROS-directed plant growth and stress responses in Arabidopsis and other crops. In this review, we emphasize the roles of RBOH-involved ROS signaling in melatonin-mediated plant abiotic stress tolerance.
Much more is now known about how RBOH genes are regulated in response to abiotic stresses. Recent evidence suggests that ROS function as signaling molecules in relation to hormone responses, and the mechanistic bases are complicated. Clearly, melatonin has complex crosstalks with signaling molecules and other phytohormones [19,111]. In our studies, melatonin-triggered lateral root formation was H 2 O 2 -dependent in alfalfa [13]. Further, melatonin induced PAO and RBOH-derived ROS accumulation to facilitate the lateral root development in tomatoes [112,113]. The roles of PuRBOHF-dependent H 2 O 2 were also essential for melatonin-induced anthocyanin accumulation in red pear fruit [114,115]. Moreover, a RbohF-dependent ROS burst was required for melatonin-triggered salinity tolerance in Arabidopsis and rice [12,53]. So far, the ways that most RBOHs function in melatonin-regulated processes in response to abiotic stress are still elusive, and it should be clarified in future studies.

How Melatonin Directs with the RBOH-Regulated ROS Signaling in Plant Tolerance to Abiotic Stress
Several studies take the important stance that the transmembrane receptor of melatonin (PMTR1/CAND2) is found in Arabidopsis, tobacco, alfalfa, and maize [116][117][118][119]. It is also located in the plasma membrane, and can interact with G-protein α subunits, thereby activating RBOHs to promote stomatal closure in Arabidopsis ( Figure 2) [116,119]. Melatonin inhibits endogenous NO accumulation and reduces the S-nitrosylation of RBOH to activate the ROS signaling pathway [99]. ROS signaling induces the expression of defensive genes to enhance plant tolerance to oxidative stress [120,121]. However, whether or how the interaction between PMTR1/CAND2 and G-protein α subunits directly regulates RBOHs in plant response to abiotic stress remains to be deciphered. tonin (PMTR1/CAND2) is found in Arabidopsis, tobacco, alfalfa, and maize [116-1 also located in the plasma membrane, and can interact with G-protein α subunits, activating RBOHs to promote stomatal closure in Arabidopsis ( Figure 2) [116,119 tonin inhibits endogenous NO accumulation and reduces the S-nitrosylation of R activate the ROS signaling pathway [99]. ROS signaling induces the expression o sive genes to enhance plant tolerance to oxidative stress [120,121]. However, wh how the interaction between PMTR1/CAND2 and G-protein α subunits directly r RBOHs in plant response to abiotic stress remains to be deciphered. Increasing evidence shows that melatonin induces NO generation and enhances R tivity through denitrosylation, thereby activating ROS signaling in tomatoes (blue arrow) tion between CAND2/PMTR1 (the melatonin receptor) and G-protein α subunits activates resulting in stomatal closure (green arrow). The direct relationship between the melato RBOHs in plant tolerance to abiotic stress is still largely unknown (red arrow, yet largely un NO, nitric oxide; ROS, reactive oxygen species; RBOHs, respiratory burst oxidase homolog Recent data imply that melatonin connects with the multiple elements, inclu hormone and signaling molecules. Moreover, it was found that H2S modulates t translational modification of protein cysteine residues in plants [122][123][124]. Our p review further suggested that ROS interacted with H2S by regulating transcript post-translational modifications in response to oxidative stress [125]. Therefore, tion of melatonin with ROS and H2S in regulating abiotic stress also has signific portance and remains to be identified in future. Increasing evidence shows that melatonin induces NO generation and enhances RBOH activity through denitrosylation, thereby activating ROS signaling in tomatoes (blue arrow). Interaction between CAND2/PMTR1 (the melatonin receptor) and G-protein α subunits activates RBOHs, resulting in stomatal closure (green arrow). The direct relationship between the melatonin and RBOHs in plant tolerance to abiotic stress is still largely unknown (red arrow, yet largely unknown). NO, nitric oxide; ROS, reactive oxygen species; RBOHs, respiratory burst oxidase homologs.
Recent data imply that melatonin connects with the multiple elements, including the hormone and signaling molecules. Moreover, it was found that H 2 S modulates the posttranslational modification of protein cysteine residues in plants [122][123][124]. Our previous review further suggested that ROS interacted with H 2 S by regulating transcriptional or post-translational modifications in response to oxidative stress [125]. Therefore, interaction of melatonin with ROS and H 2 S in regulating abiotic stress also has significant importance and remains to be identified in future.

Conclusions and Perspectives
Much more is now known about the regulatory mechanism of melatonin-mediated tolerance to abiotic stresses, especially the cooperation between melatonin and ROS, NO, and/or H 2 S. To further promote this research in plants, our review summarizes the ROSinvolved regulatory roles and mechanisms of melatonin-mediated abiotic stress resistance. Melatonin confers oxidative stress tolerance mainly through the reestablishment of redox homeostasis. Moreover, ROS act as signaling molecules that regulate melatonin-modulated protective effects. In particular, the vital role of RBOHs during these processes was shown. However, there are still many questions that should be characterized to understand the signal transduction pathway of melatonin in plants in response to abiotic stress. For example, it is necessary to focus more attention on the signaling role of ROS produced by photosystem II (PSII) and photorespiration in melatonin-alleviated abiotic stress in future studies.
As melatonin is an important regulatory element of phytohormones, it collaborates with multiple elements (such as the discovered signaling molecules NO, H 2 S, and ROS) and hormones (such as auxin, ethylene, salicylic acid, gibberellin, and abscisic acid signaling). Importantly, most studies do not provide solid in vivo evidence. Future studies using related mutants produced by gene editing technology and plastid transformation technology [126,127] should aim to illustrate how melatonin functions with these signaling molecules in plants under stressful situations.