Exogenous application of a low concentration of melatonin enhances salt tolerance in rapeseed (Brassica napus L.) seedlings

ZENG Liu, CAI Jun-song, LI Jing-jing,, LU Guang-yuan, LI Chun-sheng, FU Gui-ping, ZHANG Xue-kun, MA Hai-qing, LIU Qing-yun, ZOU Xi-ling, CHENG Yong

1 Key Laboratory of Biology and Genetic Improvement of Oil Crops, Ministry of Agriculture/Oil Crops Research Institute, Chinese Academy of Agricultural Sciences, Wuhan 430062, P.R.China

2 Hubei Province Oilseed Rape Office, Wuhan 430060, P.R.China

3 Hubei Engineering University, Xiaogan 432000, P.R.China

4 The Agricultural Bureau of Xishui County, Huanggang 438200, P.R.China

1. Introduction

Salinity is one of the major abiotic factors limiting crop yield and threatening food security worldwide (Sahet al. 2016). In plants, salt stress can result in the production of excessive reactive oxygen species (ROS), and can also cause the peroxidation of membrane lipids or proteins and destroy the normal structure of cell membranes, possibly leading to cell death. Additionally, high concentrations of salt can cause osmotic stress with a reduction of water potential in plant roots, subsequently impedes water and nutrient uptake, and severely inhibits plant growth and development, possibly resulting in the wilting and death of plants (Julkowska and Testerink 2015).

Developing crops to grow successfully under salt stress has been a concern for a long time (Munns 2002). Plant growth regulators are extensively used to regulate plant growth and to enhance plant stress tolerance. Therefore,exploring potential growth regulators and their mechanisms is highly important for improving salt tolerance in crops.Melatonin (N-acetyl-5-methoxytryptamine) is an indole hormone widely presenting in plants and animals (Barrattet al. 1977; Dubbelset al. 1995; Reiteret al. 2011; Nawazet al. 2015; Shiet al. 2016). Exogenous melatonin has been reported to improve salt tolerance effectively in certain plants. Liet al. (2012) found that pretreatment with melatonin attenuated the inhibitory effects of salt stress on plant growth signifcantly, including retarding the degradation and loss of chlorophyll, maintaining relatively high photosynthetic efficiency, and reducing the oxidative damage caused by salt stress inMalus hupehensis. Under salt stress, the expression of the ferredoxin genePetFwas decreased in soybean seedlings and could be effectively increased by exogenous melatonin through modulating the ascorbate content and inhibiting chlorophyll degradation(Zhanget al. 2014; Weiet al. 2015). Zhanget al. (2014)also found that pretreatment with exogenous melatonin enhanced the expression of genes encoding antioxidant enzymes and significantly improved the activities of superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT) in cucumber seeds, thereby attenuating the oxidative damage and improving the germination rate of cucumber seeds under salt stress. SOD, POD, CAT, and APX are important antioxidant enzymes in plant, as they can help maintain the stability and integrity of the cell membrane by scavenging hydroxyl peroxide and hydrogen peroxide,which reduce the damage caused by ROS (Liet al. 2012;Kostopoulouet al. 2015).

Rapeseed (Brassica napusL.) as a major resource for oil production, is moderately sensitive to salt stress, and the yield is affected by salt stress, especially in arid and semiarid regions (Musgrave 2000). Despite previous reports on melatonin regarding salt stress, to the best of our knowledge,no relevant study has been conducted on salt stress in rapeseed. Therefore, in the present study, we examined the adaptability of rapeseed seedlings in salt stressviathe exogenous application of melatonin by evaluating several phenotypic and physiological indices, aiming to exploring the possible mechanism of salt tolerance.

2. Materials and methods

2.1. Plant materials

The rapeseed variety ZS11 was supplied by the Oil Crops Research Institute, Chinese Academy of Agricultural Sciences.

2.2. Methods

Healthy seeds were selected and disinfected by soaking in 3% NaOCl solution for 10 min. After being rinsed with distilled water, the seeds were sown on fine gauze and cultured in a 24°C culture chamber with a 16 h/8 h light-dark photoperiod for 7 d.

Preliminary testThe uniform seedlings with two leaves were transferred into modified Hoagland’s nutrient solution(Dunet al. 2016) with different concentrations for selecting the optimal NaCl salt stress. The concentrations of the Hoagland’s nutrient solution were as follows: 0 NaCl (CK),0.25% NaCl (T1), 0.5% NaCl (T2), 0.75% NaCl (T3), 1.0%NaCl (T4), and 1.25% NaCl (T5). Each treatment consisted of eight seedlings, and three replications were performed.After 7 d treatment, the dry weight and fresh weight of shoot and root for each seedling was determined.

Main testAfter the optimal NaCl concentration was decided,the main experiment was continued. Seedlings were prepared as the preliminary test. Then, uniform seedlings were transferred into solutions with the optimal NaCl and different melatonin concentrations. The treatments were as follows: 0 NaCl and 0 melatonin (CK1), optimal NaCl and 0 melatonin (CK2), optimal NaCl and 30 μmol L–1melatonin(30MT), optimal NaCl and 45 μmol L–1melatonin (45MT),optimal NaCl and 60 μmol L–1melatonin (60MT), optimal NaCl and 75 μmol L–1(75MT), and optimal NaCl and 100 μmol L–1melatonin (100MT). After 9 d treatment, root length, stem length (height from cotyledons to the growing point), dry weight and fresh weight of shoot and root, and leaf area for each seedling was determined.

Data collection methodsThe dry weight was measured at 105°C for 30 min and kept at 80°C to a constant weight.

Additionally, fresh samples of the third leaf were collected from each seedling and stored at –80°C until analysis. The leaf samples were used for the determination of biochemical indices, including POD, APX, CAT, H2O2, proline, water soluble protein (WSP), and water soluble glucan (WSG).All biochemical indices of leaves were determined using commercial kits according to the manufacturer’s instructions(Nanjing Jiancheng Bioengineering Institute, China).

2.3. Statistical analysis

Data were processed using Microsoft Excel. One-way analysis of variance (ANOVA) was conducted at the 0.05 level using SPSS 20.0 statistical software (International Business Machines Corporation, USA). Graphs were plotted using Origin 8.0 (OriginLab Corporation, USA).

3. Results

3.1. Selection of the optimal NaCl concentration for salt stress

Various concentrations of NaCl were used for the stress treatment of rapeseed plants. The results indicated that the growth of rapeseed plants was significantly inhibited by salt stress, and obvious symptoms of salt stress were observed (Table 1). As the salt concentration increased, the growth of the rapeseed plants was suppressed. The shoot fresh weight, shoot dry weight, root fresh weight, and root dry weight of plants under T3 treatment were 1.187, 0.076,0.232, and 0.014 g, respectively. Compared with that of CK, these parameters were decreased by 46.2, 39.2, 35.5,and 30.0%, respectively, showing significant differences.Few differences were observed in the shoot dry weight,root fresh weight, and root dry weight among T3, T4 and T5 treatments, and these differences were not significant among the three treatments. The shoot fresh weight of plants under T3 treatment was significantly different from that of CK, T1, T2, T4 and T5 treatments. No significant difference in the shoot fresh weight was observed between T4 and T5 treatments, and the shoot fresh weight was decreased more than 50% due to the strong inhibitory effects of salt stress. Thus, treatment at the intermediate level of 0.75% NaCl was selected as the salt concentration for further experimentation.

3.2. Effects of exogenous melatonin on the growth parameters of seedlings under salt stress

Various concentrations of melatonin were applied to rapeseed seedlings stressed with 0.75% NaCl, which concentration was selected in the preliminary experiment.As shown in Table 2, the biomass of plants under CK2 treatment was markedly lower than that under CK1. Under 30MT treatment, the shoot fresh weight and dry weight, as well as the root fresh weight and dry weight were significantly higher than that in CK2, and the increased value was 28.2,42.9, 22.2, and 24.2%, respectively. Under the treatments of 30MT, 45MT, 60MT, 75MT and 100MT, the root length of plants was all slightly decreased compared with that of CK2, regardless of the melatonin concentration, and no significant differences in the root length were observed between different concentrations of melatonin. Neither saltstress nor the application of melatonin strongly affected the shoot length of plants, which ranged form 3.77 to 4.37 cm.The shoot fresh weight of plants was markedly increased under 30MT, 45MT, 60MT, 75MT and 100MT treatments,compared with that of CK2, ranging from 5.9 to 28.2%.However, the increase in shoot fresh weight diminished with increasing concentrations of melatonin. The shoot dry weight, root fresh weight, and root dry weight showed similar trends to those of the shoot fresh weight, and compared to CK2, the three parameters were changed by 23.6 to 42.9%, –6.7 to 22.2% and 15.2 to 24.2%, respectively. The root fresh weight of plants under 60MT, 75MT, and 100MT treatments was all lower than that in CK2. Interestingly, the shoot dry weight and root fresh weight were increased by 18.3 and 18.9%, respectively, under 30MT treatment when compared with that of CK1. The leaf area of plants under 30MT treatment did not differ significantly from that in CK2.Nonetheless, a higher concentration (45MT, 60MT, 75MT and 100MT treatments) of exogenous melatonin resulted in the decreased leaf surface area compared with that of CK2.The leaf surface area of plants under 100MT treatment was 24.7% smaller than that of CK2. No significant difference was found in the leaf surface area for 30MT, 45MT, 60MT,75MT and 100MT treatments.

Table 1 Effects of different concentrations of NaCl on growth parameters in rapeseed seedlings

Table 2 Effects of different concentrations of melatonin on various physiological indicators in rapeseed seedlings under NaCl stress

3.3. Effect of melatonin on H2O2 content

H2O2is ROS produced by cellular metabolism in plants that has toxic effects on cells. In this study, the H2O2content was increased by 10.6% under CK2 treatment compared with that of CK1 (Fig. 1). In contrast, under 30MT treatment,the H2O2content was decreased by 11.4% compared with that of CK2 and decreased by 2.0% compared with that of CK1. However, there was an upward trend in the H2O2content with increasing concentrations of melatonin. Under 45MT and 60MT treatments, the H2O2contents decreased by 7.9 and 1.1%, respectively, compared with that of CK2.But, the H2O2contents of plants under 75MT and 100MT treatments were increased by 1.3 and 3.3%, respectively,compared with that of CK2.

3.4. Effect of melatonin on the antioxidant system

As shown in Figs. 2-4, the activities of CAT, POD, and APX in the leaves of rapeseed seedlings were markedly enhanced by 5.3, 2.6, and 10.9%, respectively, under CK2 treatment when compared with that of CK1. That is, the application of exogenous melatonin significantly affected the CAT, POD, and APX activities in the leaves of rapeseed seedlings. Under 30MT treatment, the activities of antioxidant enzymes were significantly different from those in CK2. The activities of CAT, POD, and APX were increased by 16.5, 19.3, and 14.2%, respectively, compared with those of CK2, and their activities were increased by 22.7, 22.5, and 26.7%, respectively compared with those of CK1. However,the activities of all the three enzymes showed a downward trend with increasing concentrations of melatonin. Under the treatments of 60MT, 75MT and 100MT, the activities of CAT,POD, and APX were all decreased when compared to that of CK2, and CAT activity was decreased by 1.1, 6.9, and 9.0%,respectively (Fig. 2); POD activity was decreased by 10.4,24.5, and 25.0%, respectively (Fig. 3); and APX activity was decreased by 8.6, 10.8, and 24.2%, respectively (Fig. 4).

Fig. 1 Effects of different concentrations of melatonin on H2O2 content under salt stress. CK1, CK2, 30MT, 45MT, 60MT,75MT, and 100MT represent the seedlings were treated by 0 NaCl and 0 melatonin, 0.75% NaCl and 0 melatonin, 0.75%NaCl and 30 μmol L–1 melatonin, 0.75% NaCl and 45 μmol L–1 melatonin, 0.75% NaCl and 60 μmol L–1 melatonin, 0.75% NaCl and 75 μmol L–1 melatonin, and 0.75% NaCl and 100 μmol L–1 melatonin, respectively. And the 0.75% NaCl is optimal for growing. FW, fresh weight. Values are means±standard error(n=6). Different letters indicate significant differences according to Duncan’s multiple range test (P＜0.05).

Fig. 2 Effects of different concentrations of melatonin on catalase (CAT) activity under salt stress. CK1, CK2, 30MT,45MT, 60MT, 75MT, and 100MT represent the seedlings were treated by 0 NaCl and 0 melatonin, 0.75% NaCl and 0 melatonin, 0.75% NaCl and 30 μmol L–1 melatonin, 0.75%NaCl and 45 μmol L–1 melatonin, 0.75% NaCl and 60 μmol L–1 melatonin, 0.75% NaCl and 75 μmol L–1 melatonin, and 0.75% NaCl and 100 μmol L–1 melatonin, respectively. And the 0.75% NaCl is optimal for growing. FW, fresh weight. Values are means±standard error (n=6). Different letters indicate significant differences according to Duncan’s multiple range test (P＜0.05).

3.5. Effect of melatonin on osmoregulatory substances

The solute contents were significantly increased under CK2 treatment, and the contents of WSP (Fig. 5), protein(Fig. 6), and WSG (Fig. 7) were increased by 21.6, 650.0,and 10.1%, respectively, compared with that of CK1. Under 30MT treatment, the solute contents were further increased;the contents of WSP, protein, and WSG were increased by 58.7, 26.8, and 15.1%, respectively, compared with that of CK2. As shown in Fig. 5, the content of WSP in plants treated with 30 to 100 μmol L-1melatonin was significantly higher than that of CK1 and CK2; this parameter was increased by 58.7, 42.2, 41.2, 40.1, and 39.6%, respectively,compared with that of CK2 and by 92.9, 72.8, 71.6, 70.3,and 69.6%, respectively, compared with that of CK1. In contrast, the content of WSG decreased with increasing concentrations of melatonin. Under the treatments of 30MT and 45MT, the content of WSG was increased by 15.1 and 11.6%, respectively, compared with that of CK2. Under the treatments of 60MT, 75MT and 100MT, the content of WSG was even lower than that in CK2, with decreases of 2.2,3.2, and 8.2%, respectively. A similar trend was observed in the content of protein. Under 30MT treatment, the highest protein content was observed, and with increasing concentrations of melatonin, the protein content decreased.

Fig. 3 Effects of different concentrations of melatonin on peroxidase (POD) activity under salt stress. CK1, CK2, 30MT,45MT, 60MT, 75MT, and 100MT represent the seedlings were treated by 0 NaCl and 0 melatonin, 0.75% NaCl and 0 melatonin, 0.75% NaCl and 30 μmol L–1 melatonin, 0.75%NaCl and 45 μmol L–1 melatonin, 0.75% NaCl and 60 μmol L–1 melatonin, 0.75% NaCl and 75 μmol L–1 melatonin, and 0.75% NaCl and 100 μmol L–1 melatonin, respectively. And the 0.75% NaCl is optimal for growing. FW, fresh weight. Values are means±standard error (n=6). Different letters indicate significant differences according to Duncan’s multiple range test (P＜0.05).

Fig. 4 Effects of different concentrations of melatonin on ascorbate peroxidase (APX) activity under salt stress. CK1,CK2, 30MT, 45MT, 60MT, 75MT, and 100MT represent the seedlings were treated by 0 NaCl and 0 melatonin, 0.75%NaCl and 0 melatonin, 0.75% NaCl and 30 μmol L–1 melatonin,0.75% NaCl and 45 μmol L–1 melatonin, 0.75% NaCl and 60 μmol L–1 melatonin, 0.75% NaCl and 75 μmol L–1 melatonin,and 0.75% NaCl and 100 μmol L–1 melatonin, respectively.And the 0.75% NaCl is optimal for growing. FW, fresh weight.Values are means±standard error (n=6). Different letters indicate significant differences according to Duncan’s multiple range test (P＜0.05).

Fig. 5 Effects of different concentrations of melatonin on water soluble protein (WSP) under salt stress. CK1, CK2, 30MT,45MT, 60MT, 75MT, and 100MT represent the seedlings were treated by 0 NaCl and 0 melatonin, 0.75% NaCl and 0 melatonin, 0.75% NaCl and 30 μmol L–1 melatonin, 0.75%NaCl and 45 μmol L–1 melatonin, 0.75% NaCl and 60 μmol L–1 melatonin, 0.75% NaCl and 75 μmol L–1 melatonin, and 0.75% NaCl and 100 μmol L–1 melatonin, respectively. And the 0.75% NaCl is optimal for growing. FW, fresh weight. Values are means±standard error (n=6). Different letters indicate significant differences according to Duncan’s multiple range test (P＜0.05).

Fig. 6 Effects of different concentrations of melatonin on proline (Pro) under salt stress. CK1, CK2, 30MT, 45MT, 60MT,75MT, and 100MT represent the seedlings were treated by 0 NaCl and 0 melatonin, 0.75% NaCl and 0 melatonin, 0.75%NaCl and 30 μmol L–1 melatonin, 0.75% NaCl and 45 μmol L–1 melatonin, 0.75% NaCl and 60 μmol L–1 melatonin, 0.75% NaCl and 75 μmol L–1 melatonin, and 0.75% NaCl and 100 μmol L–1 melatonin, respectively. And the 0.75% NaCl is optimal for growing. FW, fresh weight. Values are means±standard error(n=6). Different letters indicate significant differences according to Duncan’s multiple range test (P＜0.05).

4. Discussion

In our study, the growth of rapeseed plants was markedly inhibited when the plants were subjected to salt stress. However, the fresh weight and dry weight of rapeseed seedlings were significantly increased under 30MT treatment. Our results are similar to those of a previous study reporting that melatonin plays a role in alleviating salt stress and promoting plant growth (Liet al. 2012). As shown in Table 2, the root length of rapeseed seedlings, measured as a character index, was not increased due to the application of melatonin but was instead slightly decreased. However, the root fresh weight and dry weight were significantly increased by the application of melatonin. Zhanget al. (2013) reported that the exogenous application of melatonin effectively alleviated the inhibitory effect of polyethylene glycol stress on the seed germination of cucumber through enhancing root activity and improving the root/shoot ratio. Even under normal conditions, exogenously applied melatonin stimulates root growth in the etiolated seedlings ofB. juncea(Chenet al. 2009) and promotes adventitious root regeneration in the shoot tip explants of sweet cherry (Sarropoulouet al.2012). Therefore, we inferred that exogenous melatonin promotes the root growth of rapeseed seedlings in our experiment primarily by increasing the number or diameter of the roots rather than promoting root elongation. Moreover,our results showed that shoot length and leaf area were not significantly increased by the application of exogenous melatonin in rapeseed plants under salt stress. In fact, high concentrations of melatonin decreased leaf surface area.On the other hand, the shoot fresh weight and dry weight of plants were significantly increased by the treatment of plants under salt stress with exogenous melatonin. This indicated that melatonin might increase the leaf thickness and stem diameter of rapeseed seedlings, thereby increasing the shoot weight. It has been reported that under salt stress, the leaf surface area and plant height of soybean were increased by coating the seeds with melatonin (Weiet al. 2015). Our results differ from their findings, and the discrepancy might be due to differences in the experimental methods. In the present study, we applied exogenous melatonin using a hydroponic method, whereas the previous study used seed coating. Moreover, the differences in our results might be attributed to plant species, and this possibility will require further study.

Fig. 7 Effects of different concentrations of melatonin on water soluble glucan (WSG) under salt stress. CK1, CK2, 30MT,45MT, 60MT, 75MT, and 100MT represent the seedlings were treated by 0 NaCl and 0 melatonin, 0.75% NaCl and 0 melatonin, 0.75% NaCl and 30 μmol L–1 melatonin, 0.75%NaCl and 45 μmol L–1 melatonin, 0.75% NaCl and 60 μmol L–1 melatonin, 0.75% NaCl and 75 μmol L–1 melatonin, and 0.75% NaCl and 100 μmol L–1 melatonin, respectively. And the 0.75% NaCl is optimal for growing. FW, fresh weight. Values are means±standard error (n=6). Different letters indicate significant differences according to Duncan’s multiple range test (P＜0.05).

One of the injuries in plants caused by salt stress is related to oxidative stress. Under normal conditions, the production and elimination of ROS in plants are in a state of dynamic equilibrium that is mainly regulated by antioxidant enzymes and antioxidants, and salt stress can result in the production of a large amount of ROS, which causes injury to plants (Mittler 2002). Plants remove accumulated ROS through the synergistic actions of antioxidant protection enzymes such as SOD, POD, and CAT. The results of our study showed that the H2O2content of rapeseed plants was significantly increased under 0.75% NaCl treatment which indicated that a large amount of ROS was produced due to salt stress. In contrast, the H2O2content was markedly decreased under 30MT treatment (Fig. 1). In our study,the activities of antioxidant enzymes (POD, APX, and CAT)increased under 0.75% NaCl treatment, while the activities of POD, CAT, and APX were further significantly enhanced under 30MT treatment, consistent with previous research(Liet al. 2012; Zhanget al. 2014; Shiet al. 2015). It is generally believed that melatonin serves as an effective endogenous free radical scavenger that directly removes ROS, such as H2O2, that are produced by salt stress (Wanget al. 2013; Zhanget al. 2013), or as an antioxidant to improve the activity of enzymes related to anti-oxidative stress(Bonnefont-Rousselotet al. 2011), as a factor to regulate the transcription levels of genes related to the antioxidant system, thereby alleviating salt stress injury (Zhanget al.2014; Shiet al. 2015). Furthermore, our results showed that the beneficial effect of high concentrations of melatonin on salt stress mitigation was gradually weakened, and the higher concentrations of melatonin even had a negative effect.

Salt stress can increase the osmotic potential of the soil and thus decrease water uptake by the roots. The accumulation of compatible solutes is one of the strategies that plants have developed to tolerate salt stress (Julkowska and Testerink 2015). In our study, the contents of protein,WSG, and WSP were increased in rapeseed plants under 0.75% NaCl treatment. These parameters were further increased under 30MT treatment. Protein is one of the main metabolites that accumulate in various species of higher plants in response to salt stress (Al Hassanet al. 2016).Kostopoulouet al. (2015) reported that the addition of 1 μmol L-1melatonin toCitrus aurantiumL. seedlings cultivated in clay loam and regularly irrigated with Hoagland’s nutrient solution resulted in no significant difference in protein content of leaves under salt stress, whereas the protein content of roots was significantly decreased. Our result differs from these findings, and further study is necessary to determine whether this discrepancy is associated with the different species tested and the different treatments used. Under normal conditions, WSG can be used as a carbon skeleton or energy source by plants to synthesize other organic matter. Under salt stress, WSG can be used as an osmotic regulator, and it can protect important enzymatic activities at high concentrations of intracellular inorganic ions (Na+ and Cl-). In the leaves ofC. aurantiumL. seedlings, melatonin promoted the accumulation of carbohydrates under salt stress, and also increased the expression of the trehalose synthesis related genes, which encodes an important carbohydrate that helps plants preserve their cellular integrity in various stresses (Kostopoulouet al. 2015). Therefore,we believe that melatonin may regulate the WSG content by influencing the expression of sugar synthesis related genes. Most WSPs in plants are involved in various metabolic pathways, and the WSP content is an important parameter for understanding the overall metabolic state of plants. The application of exogenous melatonin is beneficial for maintaining the enzymatic activities required by plants under salt stress and for improving salt tolerance in plants.

Notably, in our study statistical analysis showed that a lower concentration of melatonin (30 μmol L-1) promoted seedling development compared with the control treatment.However, higher concentrations (＞50 μmol L-1) of melatonin could dampen the beneficial effects on seedling development or even have inhibitory effects. Similar results were reported in rice. The pretreatment of rice seedlings with a range of melatonin concentrations for 10 days revealed that 10 or 20 μmol L-1melatonin had the most beneficial effect,whereas higher concentrations had negative effects (Lianget al. 2015). This indicates that the effect of exogenously applied melatonin is closely related to its concentration.A higher concentration of melatonin is not conducive to the mitigation of salt stress, and it is necessary to select an appropriate concentration of melatonin based on the specific situation.

5. Conclusion

In summary, exogenously applying a low concentration of melatonin can improve the H2O2-scavenging capacity of rapeseed plants under salt stress by enhancing the activities of antioxidant enzymes such as POD, CAT, and APX. Additionally, melatonin treatment can alleviate osmotic stress by promoting the accumulation of osmoregulatory substances such as WSP, protein, and WSG. Ultimately,exogenous melatonin could facilitate root development and can improve the biomass of rapeseed seedlings under salt stress, thereby alleviating salt stress in rapeseed seedlings.

Acknowledgements

This study was supported by the Agricultural Science and Technology Innovation Program of Chinese Academy of Agricultural Sciences (CAAS), the Hubei Agricultural Science and Technology Innovation Center, China, and the Canola Key Industrial Innovation Team of Xiaogan, China.

Al Hassan M, Pacurar A, López-Gresa M P, Donat-Torres M P, Llinares J V, Boscaiu M, Vicente O. 2016. Effects of salt stress on three ecologically distinctPlantagospecies.PLOS ONE, 11, e0160236.

Barratt G F, Nadakavukaren M J, Frehn J L. 1977. Effect of melatonin implants on gonadal weights and pineal glandfine structure of the golden hamster.Tissue and Cell, 9,335–345.

Bonnefont-Rousselot D, Collin F, Jore D, Gardès-Albert M.2011. Reaction mechanism of melatonin oxidation by reactive oxygen speciesin vitro.Journal of Pineal Research,50, 328–335.

Chen Q, Qi W B, Reiter R J, Wei W, Bao M W. 2009.Exogenously applied melatonin stimulates root growth and raises endogenous indoleacetic acid in roots of etiolated seedlings ofBrassica juncea.Journal of Plant Physiology,166, 324–328.

Dubbels R, Reiter R J, Klenke E, Goebel A, Schnakenberg E,Ehlers C, Schlwara H W, Schloot W. 1995. Melatonin in edible plants identified by radioimmunoassay and by high performance liquid chromatography-mass spectrometry.Journal of Pineal Research, 18, 28–31.

Dun X L, Tao Z S, Wang J, Wang X F, Liu G H, Wang H Z.2016. Comparative transcriptome analysis of primary roots ofBrassica napusseedlings with extremely different primary root lengths using RNA sequencing.Frontiers in Plant Science, 7, 1238.

Julkowska M M, Testerink C. 2015. Tuning plant signaling and growth to survive salt.Trends in Plant Science, 20, 586–594.

Kostopoulou Z, Therios I, Roumeliotis E, Kanellis A K,Molassiotis A. 2015. Melatonin combined with ascorbic acid provides salt adaptation inCitrus aurantiumL. seedlings.Plant Physiology and Biochemistry, 86, 155–165.

Li C, Wang P, Wei Z W, Liang D, Liu C H, Yin L H, Jia D F, Fu M Y, Ma F W. 2012. The mitigation effects of exogenous melatonin on salinity-induced stress inMalus hupehensis.Journal of Pineal Research, 53, 298–306.

Liang C Z, Zheng G Y, Li W Z, Wang Y Q, Hu B, Wang H R,Wu H K, Qian Y W, Zhu X G, Tan D X, Chen S Y, Chu C C. 2015. Melatonin delays leaf senescence and enhances salt stress tolerance in rice.Journal of Pineal Research,59, 91–101.

Mittler R. 2002. Oxidative stress, antioxidants and stress tolerance.Trends in Plant Science, 7, 405–410.

Munns R. 2002. Comparative physiology of salt and water stress.Plant,Cell & Environment, 25, 239–250.

Musgrave M E. 2000. Realizing the potential of rapid-cyclingBrassicaas a model system for use in plant biology research.Journal of Plant Growth Regulation, 19, 314–325.

Nawaz M A, Huang Y, Bie Z L, Ahmed W, Reiter R J, Niu M L, Hameed S. 2015. Melatonin: Current status and future perspectives in plant science.Frontiers in Plant Science,6, 1230.

Reiter R J, Coto-Montes A, Boga J A, Fuentes-Broto L, Rosales-Corral S, Tan D X. 2011. Melatonin: New applications in clinical and veterinary medicine, plant physiology and industry.Neuroendocrinology Letters, 32, 575–587.

Sah S K, Reddy K R, Li J X. 2016. Abscisic acid and abiotic stress tolerance in crop plants.Frontiers in Plant Science,7, 571.

Sarropoulou V N, Therios I N, Dimassi-Theriou K N. 2012.Melatonin promotes adventitious root regeneration inin vitroshoot tip explants of the commercial sweet cherry rootstocks CAB-6P (Prunus cerasusL.), Gisela 6 (P.cerasus×P.canescens), and MxM 60 (P.avium×P.mahaleb).Journal of Pineal Research, 52, 38–46.

Shi H T, Chen K L, Wei Y X, He C Z. 2016. Fundamental issues of melatonin-mediated stress signaling in plants.Frontiers in Plant Science, 7, 1124.

Shi H T, Jiang C, Ye T T, Tan D X, Reiter R J, Zhang H, Liu R Y,Chan Z L. 2015. Comparative physiological, metabolomic,and transcriptomic analyses reveal mechanisms of improved abiotic stress resistance in bermudagrass(Cynodon dactylon(L). Pers.) by exogenous melatonin.Journal of Experimental Botany, 66, 681–694.

Wang P, Sun X, Li C, Wei Z W, Liang D, Ma F W. 2013.Long-term exogenous application of melatonin delays drought-induced leaf senescence in apple.Journal of Pineal Research, 54, 292–302.

Wei W, Li Q T, Chu Y N, Reiter R J, Yu X M, Zhu D H, Zhang W K, Ma B, Lin Q, Zhang J S, Chen S Y. 2015. Melatonin enhances plant growth and abiotic stress tolerance in soybean plants.Journal of Experimental Botany, 66,695–707.

Zhang H J, Zhang N, Yang R C, Wang L, Sun Q Q, Li D B,Cao Y Y, Weeda S, Zhao B, Ren S X, Guo Y D. 2014.Melatonin promotes seed germination under high salinity by regulating antioxidant systems, ABA and GA(4) interaction in cucumber (Cucumis sativusL.).Journal of Pineal Research, 57, 269–279.

Zhang N, Zhao B, Zhang H J, Weeda S, Yang C, Yang Z C,Ren S X, Guo Y D. 2013. Melatonin promotes water-stress tolerance, lateral root formation, and seed germination in cucumber (Cucumis sativusL.).Journal of Pineal Research,54, 15–23.