Soil salinization is a growing severe global problem as salinity can impede plant growth and development and reduce crop yields (van Zelm et al., 2020). There is approximately one billion ha of salt-affected soils all over the world (Li et al., 2019). The higher salinity levels have detrimental effects on soil physicochemical and biological characteristics and plant metabolism (Sahab et al., 2021). Salt stress has both osmotic and ionic or ion-toxicity effects on plants (Zhu, 2016). Under salt stress conditions, salt ions accumulate excessively in old leaves through transpiration, causing cytotoxicity and changes in ion homeostasis, resulting in ionic disorders (Debnath et al., 2019; Zhan et al., 2019). Salt stress can also increase the intracellular osmotic pressure and can cause the accumulation of sodium to toxic levels (Zhao et al., 2021). Moreover, salt stress triggers the overdose accumulation of reactive oxygen species (ROS) in crop plants, leading to severe oxidative damage to living tissues (Cheng X. et al., 2021).

Melatonin is an indolic compound derived from tryptophan (Arnao, 2014). It is widespread in animals, plants, and microorganisms. In animals, melatonin plays an essential role in chronobiology, regulating circadian rhythm, improving sleep quality, and positively affecting mood disorders and cancer (Cardinali et al., 2012; Lanfumey et al., 2013; Majidinia et al., 2018; Lok et al., 2019). Phytomelatonin was first discovered in tomatoes and a few other edible crops in 1995 (Dubbels et al., 1995). Research has shown that melatonin is a hormone that acts in many physiological aspects of plants, including seed germination, root organogenesis, flowering, fruit ripening, and stomatal immunity (Park and Back, 2012; Zhang et al., 2015; Yang et al., 2021b; Wang K. et al., 2022). Melatonin also plays an indispensable role in alleviating plants’ bacterial, fungal, and viral diseases (Yang et al., 2021a; Zeng et al., 2022). Moreover, melatonin and its metabolites are powerful antioxidants and free radical scavengers resulting in plant resistance improvement (Sharif et al., 2018; Debnath et al., 2019). These functions improve plant photosynthesis and ionic homeostasis and activate a series of downstream signals, such as hormones, nitric oxide (NO), and polyamine metabolism (Zhan et al., 2019; Niu et al., 2022).

Exogenous melatonin has many positive effects on plants. In the first half of 2022, there were about 250 papers related to the positive effects of exogenous melatonin on plants (www.webofscience.com). However, the use of exogenous melatonin in the horticultural industry continues to face numerous challenges, including identifying effective melatonin levels and developing economically viable melatonin sources (Gao et al., 2022). Previous studies have demonstrated that increasing the endogenous synthesis of the plant is an effective alternative. Overexpression of apple melatonin synthesis gene MzASMT1 significantly improved drought tolerance of transgenic Arabidopsis plants (Zuo et al., 2014). Ectopic overexpression of grape melatonin synthesis gene VvSNAT1 in Arabidopsis resulted in increased melatonin production and salt tolerance (Wu et al., 2021). The number and size of vascular bundles in the culms and leaves were significantly increased in the rice overexpressed melatonin synthesis gene OsCOMT (Huangfu et al., 2022).

The classical biosynthesis pathway of melatonin in plants has been elucidated. It begins with tryptophan and consists of four enzymatic steps, and five enzymes are known to be involved. Tryptophan decarboxylase (TDC), Tryptamine-5-hydroxylase (T5H), Serotonin N-acetyltransferase (SNAT), N-acetylserotonin methyltransferase (ASMT), Caffeic acid-O-methyltransferase (COMT). TDC enzymatic conversion of tryptophan to tryptamine. T5H catalyzes the conversion of tryptamine to serotonin. SNAT is the penultimate enzyme that catalyzes serotonin into N-acetylserotonin. COMT and ASMT catalyze N-acetylserotonin to melatonin (Back et al., 2016; Byeon et al., 2016; Zheng et al., 2021b). T5H is the second enzyme in melatonin biosynthesis. It belongs to the cytochrome P450 monooxygenase family, which is a multigene family in plants (Fujiwara et al., 2010; Park et al., 2013a; Park et al., 2013b). The identification of gene T5H in mulberry trees has been previously reported and seven MnT5Hs have been identified in the mulberry genome, with MnT5H2 at the highest expression (Zheng et al., 2021a).

T5H is an indispensable enzyme in melatonin biosynthesis. However, some studies have reported a negative correlation between the content of serotonin, the catalytic product of T5H, and melatonin content in rice and tomato (González-Gómez et al., 2009; Park et al., 2012; Commisso et al., 2022). And serotonin is also implicated in several physiological roles, such as flowering, morphogenesis, and adaptation to environmental changes (Kang et al., 2007). The mechanism of the T5H effects in plants is intricate, especially in woody plants, with limited reporting.

Mulberry is an economically important food crop for the domesticated silkworm (Jiao et al., 2020). Mulberry is also planted for their fruit, which is often eaten as fresh food or made into juice, jam, and wine because of its tasty and nutritious characteristics (Lee et al., 2013; Shen et al., 2014). Furthermore, mulberry can adapt well to stress, such as metal exposure, high salinity, drought, and flooding (Zeng et al., 2020; Rao et al., 2021). Therefore, mulberry is an important economic forest tree species in many developing countries such as China. In our previous studies, it has been found that mulberry has high efficiency of melatonin synthesis. Amongst the 49 mulberry varieties, the Chuansang mulberry (Morus notabilis) has the highest melatonin content. However, the significance of the efficient melatonin synthesis for stress resistance of mulberry has not been analyzed (Zheng et al., 2021a; Zheng et al., 2021b). In order to explore this question, we transformed MnT5H2, a crucial gene for melatonin synthesis in mulberry, into tobacco (Nicotiana tabacum L.) and examined the changes in the content of each substance in the melatonin synthesis pathway in transgenic tobacco. The ectopic overexpression of MnT5H2 increases melatonin content in transgenic tobacco. Furthermore, MnT5H2 enhanced the salt tolerance of transgenic tobacco by improving ROS and ion homeostasis. Overall, our study initially reported the positive regulatory role of mulberry MnT5H gene in salt stress and its mechanism, which provides some new knowledges for understanding the significance of the efficient melatonin synthesis for stress resistance of mulberry and a new target for improving plant salt tolerance.

The transgenic tobacco treated with salt stress had a more significant growth advantage than the WT under 300 mM salt stress. In multiple replicates, both WT and transgenic tobacco exhibited significant leaf yellowing and growth retardation, but transgenic tobacco performed significantly better. Similar outcomes were observed in VvSNAT-overexpression Arabidopsis and NtCOMT-overexpression tobacco (Wu et al., 2021; Yao et al., 2022). Extensive studies have shown that melatonin plays a vital role in increasing salt tolerance in various plant species (Jalili et al., 2022; Song et al., 2022b). The exogenous addition of melatonin increases the resistance of plants to salt stress, and we consider that this increase in resistance in MnT5H2 transgenic plants is most likely related to their increased melatonin content. Unfortunately, this complex mechanism is not addressed in our work. Melatonin has similar physiological functions to auxin, and its specific mechanism in plants is complex and needs further study (Hernandez-Ruiz et al., 2005; Friml et al., 2022).

Extraction of melatonin synthesis pathway-related substances from transgenic tobacco and WT tobacco after salt stress, and further validation of the content of each substance by UPLC-MS/MS. The levels of tryptophan and tryptamine, precursors of serotonin synthesis, were significantly elevated. The same was observed under exogenous addition, which may be related to the positive feedback regulation of melatonin (Song et al., 2022a).

Among these substances, we extra focus on serotonin the products of MnT5H2, and melatonin. Compared to WT, serotonin and melatonin were significantly increased in transgenic tobacco. Interestingly, the amount of serotonin in it rose to about 0.2 mg/g, a 2-fold increase. This trend was further enlarged under salt stress. Serotonin has been confirmed in almost all plant families, where it plays important role in plant growth and development (Erland et al., 2016). It also an analogue of IAA, is involved in the regulation of lateral root growth in Arabidopsis (Pelagio-Flores et al., 2011; Wan et al., 2018). Some studies have reported that overexpression of T5H will lead to increased serotonin content and decreased melatonin content in transgenic plants (Park et al., 2012; Park et al., 2013a). In this study, we found both melatonin and serotonin increased in MnT5H2 transgenic plants. This provides new insights into the relationship between serotonin and melatonin. Furthermore, the increase in serotonin in transgenic tobacco may also be positively related to its increased salt tolerance.

The classic melatonin synthetic pathway has been described in this article ( Figure 2A ). However, in this study, it was found that MnT5H2 overexpression resulted in increased melatonin synthesis, but in the absence of N-acetylserotonin changes. This indicates that there are other pathways for melatonin synthesis in tobacco. Previous studies have shown that the sequence of roles of SNAT and ASMT in the melatonin synthesis pathway can be altered (Lee et al., 2014; Tan et al., 2016). It has also been reported that these two enzymes have different substrate selection preferences at different temperatures and that ASMT is the rate-limiting enzyme for melatonin synthesis (Reiter et al., 2007; Kang et al., 2013). Our result suggests that there may be a more preferential synthetic pathway for tobacco melatonin that differs from the classical pathway ( Figure 8 ). The significance of this different pathway synthesis mechanism needs to be further investigated.

Referring to our previous study, we also counted tobacco seeds germinating and growing at specific salt concentrations (Liu et al., 2018). Interestingly, transgenic tobacco T1 seeds did not exhibit higher germination rates, but after germination transgenic tobacco seedlings grew significantly better than WT under salt stress, as evidenced by the fact that transgenic tobacco seedlings could grow slowly in salt-stressed media, but WT seedlings almost stopped growing ( Figure 7 ). Exogenous melatonin has been reported to promote seedling growth and to promote seed germination (Park and Back, 2012; Nabaei and Amooaghaie, 2019; Li et al., 2022; Wang J. et al., 2022). This study found that the promotion of seedling growth by MnT5H2 may be related to the increased synthesis of endogenous melatonin. However, the absence of effect on seed germination may be related to the specific localization of other enzymes, such as SNAT whose main activity is concentrated in chloroplasts (Byeon et al., 2014).

Consuming melatonin from plant-based foods or supplementing with melatonin may promote health by virtue of its many properties (Iriti et al., 2010; Favero et al., 2017). Medicinal herbs are often rich in melatonin, while many vegetables and fruits are low in melatonin and vary greatly between species (Cheng G. et al., 2021). Overexpression of highly efficient melatonin synthesis genes MnT5H resulted in a dramatic increase in melatonin levels in tobacco. This provides useful ideas and references for improving melatonin accumulation in vegetables.

When plants are stressed, the dynamic balance of ROS production and elimination in plant cells is disrupted. Excess ROS can disrupt the cell membrane system and negatively affect the growth of plants (Pardo-Hernandez et al., 2021). The indicators of ROS were examined in tobacco overexpression MnT5H2 after salt stress, and it was found that CAT enzyme and POD enzyme activities involved in antioxidant were higher in transgenic tobacco. And the content of MDA was decreased, the content of proline was increased. The result indicated that MnT5H2 enhances salt resistance in tobacco through antioxidant properties and ion homeostasis. The conclusion was further supported by expression analysis by real-time PCR of NtSOD, NtCAT, and NtERD10C (Liu et al., 2013). Under salt stress, the expression of those genes in transgenic tobacco was significantly higher than that in WT tobacco. MnT5H2 enhances plant tolerance to saline environments by up-regulating the expression of related genes, enhancing the activity of antioxidant enzymes, and increasing the content of protective organisms and antioxidant-related substances.

In this study, after overexpression of the MnT5H2 gene, melatonin content was significantly increased in transgenic tobacco, and melatonin level was still significantly higher in transgenic tobacco than in WT tobacco under salt stress treatment. As melatonin can act as a strong antioxidant in the plant body to eliminate the reactive oxygen species stress caused by the adverse environment, the physiological and biochemical indicators of transgenic tobacco showed a more positive trend than WT tobacco. In contrast to the results of previous studies (Park et al., 2012; Park et al., 2013a), we believe that the improved salt tolerance of transgenic tobacco is due to the overexpression of MnT5H2, which ultimately increases the amount of melatonin synthesis in the tobacco and the increased level of melatonin enhances the salt tolerance of transgenic tobacco. This reveals that the MnT5H gene is of great value in both the vegetable industry and horticulture.

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

BZ, SZ, and AZ conceived and designed the experiments. BZ, MZ, and XC performed the experiments. BZ, WF, ZX, and AZ wrote the article. All authors approved the final manuscript. All authors contributed to the article and approved the submitted version.

This research was funded by the China Agriculture Research System of MOF and MARA, grant number CARS-18-ZJ0201.

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.