In Vitro Responses of Alfalfa Root (Medicago Sativa L.) to Salt stress and Melatonin

Alfalfa (Medicago sativa L.) is the most cultivated forage legume. The growth and development of alfalfa roots are limited by salt stress. Growth regulator is an essential role of melatonin in plants, especially in root involved in stress tolerance. In this study, alfalfa roots under in vitro salt stress (150 and 200 mM NaCl) were pretreated with different concentrations of melatonin (0.1, 10 and 15 μM). principal component analysis to nd out the best targets for melatonin and salinity. Exogenous utilization of melatonin in alfalfa roots signicantly elevated the compatible solutes particularly glycin betaein (GB), soluble carbohydrate and K + content. Principal component analysis revealed that, Indol acetic acid (IAA) content, GB, avonoids, carbohydrate and Na + were the best melatonin targets. Furthermore, salinity affected ROS, H 2 O 2 , O 2- , malondialdehyde (MDA), phenylalanine ammonia-lyase (PAL), K + /Na + ratio and tyrosine ammonia-lyase (TAL) enzymes were augmented, and electrolyte leakage, Na + accumulation decreased in the saline medium. Our study presented, exogenous melatonin caused changes in the concentration of endogenous free IAA in alfalfa root and leaf. Salt stress supplemented with melatonin, the amount of melatonin and IAA in alfalfa leaf and root were higher in treated plants compared with control plants. However, melatonin content in leaf and root decreased in high salt treatment when no melatonin was applied. It seems that melatonin promoted growth and development of alfalfa roots through an IAA-dependent pathway. In contrast, in overproducing melatonin plants, a signicant decrease in IAA levels has occurred possibly by inhibiting expression of PIN1, 3, 7 and decreasing IAA levels as Arabidopsis roots Therefore, it can be postulated that, it may manage auxin synthesis and polar auxin transport in plants. We present a new perspective on the relationship between auxin, melatonin and alfalfa root growth against salinity stress. Exogenous application of melatonin improved growth of alfalfa roots in tissue culture by inducing of some antioxidant defense systems.

regulating phenolic content, carbohydrate metabolism, compatible osmolytes and lipid biosynthesis under environmental stress (Arnao and Hernández-Ruiz 2014). Melatonin may be effective as a rst line defensive factor against ROS production under stress conditions (Tan et al. 2002). The phenylpropanoid pathway is one of the critical signaling pathways for producing many important compounds, such as phenols, avonoids and anthocyanin (Pourcel et al. 2007). phenylalanine ammonia-lyase (PAL) and tyrosine ammonia-lyase (TAL) are the key enzymes in the phenylpropanoid pathway. The relationship between activity of TAL and PAL in roots treated with melatonin under salt stress has not been well understood.
This study, presents evidence for signi cant dissection of the regulatory mechanism and physiological function of melatonin-mediated salt tolerance. Understanding of root responses of alfalfa could bring a novel conception into melatonin function in root plants using in vitro tissue culture technology.

Materials And Methods:
Plant material and culture conditions: Seeds of Medicago sativa cv. Isfahani from the Pakan Bazr Company in Isfahan, Iran was used for this research. Alfalfa seeds were surface-sterilized with 70% (v/v) ethanol for 1 min then 10% (v/v) bleach for 20 min and washed three times with sterile distilled water. Seeds then were germinated in sterile (MS) (Murashige and Skoog 1962) medium adjusted pH of 5.8, supplemented with lter-sterilized melatonin from solarbio life sciences, Beijing, China (0, 0.1, 10,15 µM), and NaCl (150, 200 mM). Medium without melatonin and NaCl used as control. All cultures were transferred to the culture room (25±1•C under 16/8 h (44 μmol phot.m -2 s -1 ) light /dark photoperiod. After 10 days, physiological and biochemical factors were measured.

Measurement of Growth Factors:
Lateral Root Number, Root length and fresh weight (FW) of the roots were estimated after 10 days. For measuring dry weight, samples were dry at 70°C for 24 h.

Determination of Soluble Carbohydrates Content:
Dubois method was used for determining of Soluble carbohydrates (1956). Dry root was homogenized by deionized water. The extracts mixed with 0.5 mL of phenol (5 %) and 2.5 mL sulfuric acid (96 %). This mixture was vortexed and absorbance was recorded at 490 nm (Synergy HTX multi-mode reader, BioTek, USA) after 30 min.

Determination of Total Anthocyanin Content:
Total anthocyanin was measured based on Wagner (Wagner 1979) method with a slight modi cation. Fresh roots were grinded in acidi ed methanol solution and incubated in dark place for 24 h. Samples were centrifuged for 10 min at 4000 rpm and the absorbance of the supernatant was measured at 550 nm. The anthocyanin content measured by extinction coe cient (33000 M -1 cm -1 ) and results were reported as μmol.g −1 FW.

Determination of Total Phenolic Content:
The Folin-Ciocalteu test was determined total phenolic content (Singleton et al. 1999). 0.5 mL methanolic extract of the root was added to 1/5 mL Folin-Ciocalteu reagent (10%) and 1 mL Na 2 CO 3 (7.5%). The liquid mixture was let to standing for 30 minutes and phenolic content was measured at 760 nm.

Determination of Total Flavonoid Content:
Flavonoid contents in the root extracts were determined by aluminium chloride. 0.1 mL methanolic extract of the root was mixed with 0.2 mL of 80% methanol, 0.2 mL of aluminum chloride (10%), 0.2 mL of sodium acetate. samples were allowed to standing for 30 min, Absorbance of the reaction mixtures was recorded at 415 nm. Results were represented as mg. g -1 FW (Chang et al. 2002).
Measurement of PAL and TAL enzymes activity: 300 mg of root samples were grinded at 4ºC with 4 mL buffer (50 mmol/L Tris pH 8.5, 14.4 mmol/L 2-mercaptoethanol, 5% w/v polyvinyl polypyrrorolidone) and the homogenate was centrifuged at 6,000 g for 10 min at 4°C. The total protein content was measured by Bradford (1976) method. The reaction mixture contained 500 μmol Tris-HCl buffer (pH 8), 100 μL of enzyme extracts and either or 5.5 μmol of L-tyrosine (Sigma, USA) for measuring TAL activity (EC 4.3.1) or 6 μmol of Lphenylalanine for measuring PAL activity (EC 4.3.1.5). The contents of trans-cinnamic and p-coumaric acids were determined by measuring absorbance at 290 and 333 nm, respectively (Beaudoin-Eagan and Thorpe 1985).
Membrane permeability (electrolyte leakage) assay: 0.2 g fresh roots were incubated with 25 mL double-distilled water for 24h at room temperature on shaker. Digital conductometer was used for measuring The initial electrical conductivity (EC1). Bottles containing some roots were also autoclaved for 15 min at 120°C, then conductivity (EC2) was measured again (Lutts et al. 1995). Relative electrolyte leakage (REL) was estimated as: Malondialdehyde (MDA) assay: Thiobarbituric acid-reactive substance (TBARS) was measured as MDA. Root sample (0.2 g) was homogenized in 2 mL of 0.1% (w/v) trichloroacetic acid (TCA) that follow by centrifuged at 10000 rpm for 20 min. Then, 4 mL of 20% TCA containing 0.5% (w/v) TBA was added to 1 mL of supernatant. The mixture was warmed at 95•C for 25 min and immediately cooled to stop the reaction. The concentration of MDA was calculated by an extinction co-e cient of 155 mM.cm -1 . (Heath and Packer 1968) Proline concentration: Free proline amount in alfalfa roots was measured by ninhydrin assay at 520 nm by colorimetric determinations, according to the method of Bates et al. (1973). The proline content was represented as µmol.g -1 FW. Na + and K + content: Dry root was grinded into a ne powder, and nally digested in a solution of sulfosalicylic acid 3% for 24 h in the refrigerator.
Subsequently, the K + and Na + ions were measured by ame photometer (Halstead Essex-corning-England) using standard solutions (Shi et al. 2012).

Measurement of Glycine betaine (GB).
Glycine betaine was extracted from the oven dried and grinded roots using Grieve and Grattan method (1983).
Staining and quanti cation of H 2 O 2 : 50 mg fresh roots were ground with 2 mL of 0.1% trichloroaceticacid (TCA) and The homogenates were centrifuged and after that, added to 0.5 mL of 10 mM potassium phosphate buffer (pH 7.0) with 1 mL of 1 M KI (potassium iodide). Absorbance was recorded at 390 nm using a multi-mode reader. The H 2 O 2 content was calculated by extinction coe cient (0.28 µM -1 cm -1 ) and results were expressed as μmol.g −1 FW (Velikova et al. 2000). Accumulations of the hydrogen peroxide (H 2 O 2 ) were observed by histochemical staining methods (Thordal-Christensen et al. 1997). H 2 O 2 was stained using 3,3′diaminobenzidine (DAB) staining. Excised roots were immersed in a solution of 0.1% DAB (PH=3.8) for 12 h in darkness. The brown color on the roots indicated presence of hydrogen peroxide.
Staining and quanti cation of O 2-: Presence of the superoxide ion O −2 was measured by Nitroblue tetrazolium (NBT) staining. Roots were incubated in 50 mM phosphate buffer (pH 6.4) containing 0.1% NBT and 10 mM sodium azid. Samples were shaken in darkness for 12 h (80 rpm/min). Blue formazan compounds were visualized at the position of NBT precipitation (Fryer et al. 2002). O 2content was measured according to the method of Rook et al. (1985) with a slight modi cation.

Statistical analysis:
All experiments were analyzed by two-way ANOVA and Duncan's multiple range test (P < 0.05). Each sample value was at least in three replicates and was reported as mean ± standard deviation (SD). PCA was applied for determining the best target between several physiological factors for melatonin under salinity.

Result:
Effects of melatonin treatment on root growth of alfalfa under salinity stress: Increasing NaCl concentration negatively affected the fresh weight, dry weight, root length and lateral root number in alfalfa roots ( Table 1). The results indicated that the dry weight and fresh weight were signi cantly increased by melatonin treatments with 150 mM salt treatment. Salt stress (150 and 200 mM NaCl) noticeably inhibited the root growth of alfalfa seedlings. Application of 0.1 μM melatonin could also effectively alleviated root growth parameters, speci cally under salt stress. This feature was proved by increasing of the root length (Table 1). The lateral root number was decreased by increasing salinity, but 0.1 and 10 μM melatonin improved that compare to the control. The results indicated that applied melatonin increase salt tolerance of alfalfa roots.
Effect of melatonin on Phenolic compounds, Phenylalanine ammonia-lyase (PAL) and tyrosine ammonia-lyase (TAL) activities in alfalfa root: Salt stress noticeably caused decrease content of Total Phenol, Anthocyanin and Flavonoids in alfalfa root compared with untreated root. On the other hand, melatonin pretreatment had no effect on Anthocyanin content either with or without salt treatment. According to our results, melatonin as a single treatment is able to increasing Total Phenol and Flavonoids.
Moreover, melatonin improved phenol and Flavonoids contents in the medium supplemented with salt. As the results show in Table 2, PAL and TAL activities decreased notably as the rate of salinity increased. In the presence of melatonin without salt treatment, PAL and TAL activity signi cantly raised compared with control. Salt stress induced by NaCl in combination with melatonin treatments dramatically increased PAL and TAL activities with 10 and 15 µM Melatonin having a more pronounced effect on PAL and TAL activity under salt stress ( Table 2).
Effect of Melatonin on soluble carbohydrate, proline and Glycine Betaine content: Soluble sugar, Proline and Glycine Betaine usually act as important osmotic regulators in root. Results show that application of 150 and 200 mM NaCl declined the content of soluble carbohydrate by 44% and 55% respectively compared with control root. Melatonin treatment signi cantly increased soluble sugar in the root under salt stress speci cally in 200 mM NaCl. It seems that, 10 µM melatonin had the positive effect on this osmolyte (Fig. 1A). Figure 1B shows proline contents in alfalfa root under salt stress treatment with melatonin. Salinity caused signi cant increase in proline. Our results presented that the amount of proline increased from 100.18 in control sample to 396.89 and 530.67 µmol.g -1 FW in 150 and 200 mM NaCl.
Application of 0.1 µM melatonin dramatically raised proline content in 200 mM salt treated roots (Fig. 1B). Glycine betaine accumulation in salt stress is considered as a sign of salinity tolerance. The results in gure 1C show that, the two applied salinity levels (150 and 200 mM NaCl) caused gradual increases in glycine betaine relative to control root. Melatonin concentrations (0.1, 10 and 15µM) elevated glycine betaine content relative to their corresponding controls. Effects of Melatonin on K + and Na + content: As the result show (Fig. 3A, B, C), the Na + content in root with salt stress treatment was signi cantly higher than that in nonstressed root, and melatonin pretreatment reduced Na + accumulation under saline conditions. Salt stress treatment reduced K + uptake, and melatonin pretreatment increased K + uptake under salt stress conditions speci cally in 200 mM salt. 0.1 μM melatonin dramatically enhanced the concentrations of K + and decreased Na + content under salinity stress in alfalfa root.

Quantitation of endogenous melatonin and auxin content in Alfalfa seedlings:
To examine the role of melatonin in plants' response to salt stress, the endogenous melatonin production in alfalfa leaf and root was measured exposure to salt stress. Endogenous melatonin content was approximately 20 ng.g −1 FW at control leaf.
As the results show (Fig. 4A) 15 µM melatonin had the highest melatonin index (without salinity). Salt stress substantially decreased melatonin content in alfalfa leafs. However, pretreatment with exogenous melatonin signi cantly increased melatonin content for the control and salt treated leafs. As can be seen in Figure 4A, 10µM melatonin+150 mM NaCl increased melatonin content by 4 folds as compared to the control. Figure 4B shows that exogenous application of melatonin had a considerable effect on auxin levels in alfalfa leaf. Pretreatment with 10 µM melatonin under 150 mM NaCl induced salinity increased auxin content and signi cantly improved the accumulation of endogenous melatonin in alfalfa leafs. As the result shown in gure 4C, melatonin concentration increased in non-salt treated root compared to the control.
Supplemented of the medium with 10 µM melatonin showed the highest melatonin content under salt stress. In Figure 4D, high salt concentrations increased root auxin content but exogenous melatonin enhanced the auxin content. The application of 15 µM melatonin dramatically raised amount of auxin in treated roots with 200 mM salt. As shown in gures 4E and 4F, salinity caused signi cant increase in IAA / melatonin ratio but, melatonin treatment reduced IAA / melatonin ratio under salt stress in alfalfa shoot but this ration was increased in alfalfa roots, in 200 mM salinity.
Identi cation of Melatonin and NaCl targets: principal component analysis (PCA) was performed to identifying the major objectives for melatonin and salinity in the alfalfa root. PCA1 and PCA2 were reported from the root growth parameters as well as physiological and biochemical factors (Fig. 5A). In fact, PCA presented the best objectives of melatonin and NaCl on alfalfa root under tissue culture condition (Fig. 5B). The PCA results determined root growth parameters, which might separate into two separate groups. Figure 5B, summarizes a simple model of the major objectives for melatonin and salt. We found that glycine betaine, auxin content, soluble carbohydrate and avonoid were the main melatonin objectives in alfalfa. In conclusion, the main targets of NaCl were H 2 O 2 , O 2-, ROS, MDA, PAL and K + /Na + in PCA analysis.

Discussion
Salinity is one of the most serious abiotic stresses that limit root growth. Higher plants have developed several strategies in reply to various stresses. Melatonin is thought to improving plant tolerance to environmental stresses responses (Arnao and Hernández-Ruiz 2014). Plants can also absorb and accumulate exogenous melatonin in their tissue (Tan et al. 2012).
Responses to melatonin in regulating root growth and development are dose dependent even in the same species under stress conditions. In the present study, salinity signi cantly decreased root physiological and growth parameters; however, the positive protective role of melatonin improved root growth of alfalfa seedlings. We showed that 0.1 μM melatonin increased the dry and fresh weight of alfalfa root treatment with salt. Since melatonin as an indoleamine shares structural similarity with Indole-3-acetic-acid (IAA), and endogenous IAA also is increased by exogenous melatonin it may play a role similar to auxins consequently, due to enhancement of the root growth, the fresh and dry weight of alfalfa root was increased. Furthermore, melatonin-triggered IAA synthesis also increased IAA / melatonin ratio in alfalfa root indicating the similar impact of IAA and melatonin under salt stress (Chen et al. 2009). It seemed that, the balance among endogenous hormones might be responsible for root growth regulation under stress condition (Arnao and Hernández-Ruiz 2018).
Previous studies reported, melatonin augmented other antioxidant agents such as phenolic compounds, avonoids and anthocyanins. Similar effect on phenolic compounds was observed in alfalfa root, when plants exposed to salt stress conditions supplemented with 10 and 15µM melatonin compared with only salt treatment. Phenolic compounds with antioxidant property could affect alfalfa root either by capturing unpaired electrons or by prohibiting the function of the radical oxygen species. In agreement with our data, Olenichenko reported that, (Olenichenko and Zagoskina 2005) cold stress increased phenolic content in wheat, and salt stress in Vigna radiate (Dawood and El-Awadi 2015). Interestingly, PCA results showed, avonoid was strongly correlated with melatonin whereas anthocyanin was not affected under salt stress. Since salinity increased PAL and TAL enzymes' activities in alfalfa root, and based on Zhang, (Zhang et al. 2016) ndings in other plant species, it can be implied that these two key enzymes were target of salinity in the present study.
Lipid peroxidation assessment by MDA content is a widely used sign of membrane damage due to increasing in free radicals, which causes peroxidation of polyunsaturated fatty acids, and enhancement of the content of MDA. Oxidative damage affects on the membrane's integrity under salt stress and the best indicator for this case is electrolyte leakage (Wang et al. 2016). Salt stress affected the MDA content as an objective of salinity in alfalfa root and melatonin with antioxidant ability could reduce lipid peroxidation, neutralizing the effect of salinity on membrane permeability. Melatonin could attenuate oxidative damage caused by salt stress in alfalfa root with decreasing amount of superoxide anion and H 2 O 2 . This can be partially due to the fact that melatonin with broad-spectrum antioxidant could remove H 2 O 2 produced by salt stress (Arnao and Hernández-Ruiz 2015). In the cell membranes, melatonin can be located in the polar heads of lipids and so, can alleviate the lipid peroxidation and preserve membrane permeability. The positive effects of melatonin on the MDA and membrane damage under salt and water-stress have been expressed (Zhang et al. 2013) supporting our observations that antioxidant ability of melatonin reduced MDA content and percentage of electrolyte leakage.
In alfalfa root, 0.1 µM melatonin was more effective than other concentrations in terms of ROS, H 2 O 2 and O 2reduction under high level of salinity. ROS content reduced by melatonin pretreatment can be related to induction of antioxidant factors.
Melatonin by reducing radical generation in mitochondria and neutralizes free radicals, recognized as a mitochondrial antioxidant (Tan et al, 2002). For removing free radicals, melatonin speeds up the electron ow through the ETC (Electron Transport Chain) and slightly activates the MPTP (Mitochondrial Permeability Transition Pore). protection of mitochondria against oxidative damages is one of the important role of melatonin. (Tan et al, 2012). Our results and staining of H 2 O 2 and O 2are consistent with those reported by other authors (Shi et al. 2015;Wei et al. 2015). Our data suggested that, application of exogenous melatonin as a crucial antioxidant alleviated ROS accumulation in alfalfa root.
Glycine betaine (GB), proline and carbohydrate contents are the major organic osmolytes which are accumulated in plants in response to different stresses. These have an osmoprotectant function preventing dehydration of cytoplasm, protecting water around proteins and maintaining turgor pressure in cells (Smirnoff 1993). Glycine betaine has an important role in the adjustment of cells to various adverse environmental stresses through increasing osmotic pressure in the cytoplasm, stabilizing membranes and proteins, and preserving the relatively water content required for root development and cellular function. This investigation showed that, salt stress improved proline and GB content in root, whereas exogenous melatonin led to more increase in GB content but did not signi cantly affect proline content. Our work on alfalfa is supported by Sarropoulou et al. (2012) who reported, in the roots of cherry rootstock PHL-C (Prunus avium × Prunus cerasus), melatonin enhanced the carbohydrate contents by 1.5 times, while signi cantly reduced the proline content.
When alfalfa roots were cultured in the medium containing NaCl, salinity reduced carbohydrates content in root compared to control, whereas exogenous melatonin led to signi cant increase in carbohydrate levels under salt stress. The present study indicates that, perhaps melatonin increases the carbohydrate synthesis by expression of trehalose synthesis gene as described by Jain and Roy. Furthermore, carbohydrates may act as free radical scavengers and contribute to enhancement in membrane stabilization (Shi et al. 2015;Jain and Roy 2009).
Since PCA indicated that soluble carbohydrate was an important target for melatonin in alfalfa root, we can conclude that compatible solutes might be a part of an adaptive process contributing to osmotic adjustment under severe salinity. However, further studies may be needed to clarify the relationship among melatonin, proline, Glycine betaine and carbohydrates.
It has been shown that melatonin signi cantly decreases accumulation of Na + and markedly increases K + contents under salinity stress. Our data showed an increase of Na + contents and a signi cant decrease of K + contents in the root when root were exposed only to salinity stress and no melatonin. While, melatonin enabled root to maintain signi cantly higher K + content compared to control. Under salt stress in alfalfa root maintaining ion homeostasis using melatonin might be possible by regulating the gene expression and activity of K + and Na + transporters as reported in Arabidopsis thaliana (Shi et al. 2003).
Plant hormones are critical component for improving growth of roots and shoots. Melatonin widely takes part in the metabolism of some plant hormones, such as indole-3-acetic acid (IAA), abscisic acid (ABA), gibberellic acids (GA), cytokinins (CK), and ethylene (Arnao and Hernández-Ruiz 2018). Our study presented, exogenous melatonin caused changes in the concentration of endogenous free IAA in alfalfa root and leaf. Salt stress supplemented with melatonin, the amount of melatonin and IAA in alfalfa leaf and root were higher in treated plants compared with control plants. However, melatonin content in leaf and root decreased in high salt treatment when no melatonin was applied. It seems that melatonin promoted growth and development of alfalfa roots through an IAA-dependent pathway. In contrast, in overproducing melatonin plants, a signi cant decrease in IAA levels has occurred possibly by inhibiting expression of PIN1, 3, 7 and decreasing IAA levels as reported in Arabidopsis roots (Wang et al. 2016). Therefore, it can be postulated that, it may manage auxin synthesis and polar auxin transport in plants. We present a new perspective on the relationship between auxin, melatonin and alfalfa root growth against salinity stress. Exogenous application of melatonin improved growth of alfalfa roots in tissue culture by inducing of some antioxidant defense systems.

Concluding Remarks:
Roots use several methods to deal with salt stress; such as elimination of ions, production of compatible solutes, stimulation of antioxidant enzymes, and induction of phytohormones. The present results showed that melatonin might behave as excellent free radical scavengers by changes of IAA content, GB, avonoids and soluble carbohydrates. On the other hand, ROS, H 2 O 2 , O 2-, MDA and PAL seemed to be targets of salinity stress based on PCA. Application of exogenous melatonin increased phenol, endogenous melatonin and K + content. Furthermore, melatonin with antioxidant capacity decreased electrolyte leakage and reduced Na + accumulation in alfalfa root.