Introduction
In arid and semi-arid regions, lack of rainfall and high temperatures have increased soil salinity. Because most plants are sensitive to high salt contents, their growth and yield are affected by the osmotic and oxidative stresses induced by saline soils (Li et al. 2011, Wu et al. 2012). Production of reactive oxygen species (ROS) due to oxidative stress is an inevitable process in plants exposed to salt and other environmental stresses (Allen 1995, Garg and Manchanda 2009). Increased ROS levels under salinity stress damage plant metabolism and destroy cell membrane lipids and proteins as well as other biomacromolecules (Bor et al. 2003, Gill and Tuteja, 2010), which subsequently alters selective membrane permeability and causes the material to leak out of the cell (Weckx and Clijsters 1997). In saline areas, one of the best ways to reduce the harmful effects of salinity and increase productivity is to cultivate salt-resistant plants (Oba et al. 2001). Many halophyte plants can grow in saline soils due to changes in their energy metabolism (Winicovand Bastola 1997). The salinity tolerance mechanisms of halophytes are not well understood but may be due in part to the synthesis of proline and other osmolytes, ion homeostasis, and the activity of the antioxidant defense system to scavenge ROS (Hasegawa et al. 2000).
L-arginine(Arg) is one of the proteinogenic amino acids of plant cells, which is a precursor to the synthesis of proline, polyamines, and nitric oxide (Liu et al. 2006). Arg treatment improved chlorophyll synthesis and photosynthesis and prevented chlorophyll degradation and ageing (Zeid 2009, Mostafa et al. 2010). Exogenously applying Arg in plants exposed to environmental stresses upregulated antioxidant enzymes (Barand et al. 2015) and induced the accumulation of compatible solutes (Ramadan et al. 2019).
Salicornia europea L. is a halophyte plant that is widely distributed in coastal areas and salt marshes (Davy et al. 2001). Because it can survive in salt concentrations that are toxic to most plants, Salicornia is known as a salt-tolerant plant species (Flowers and Colmer 2008, Patel 2016). It is taken not only as a model species for salinity research, but it has also recently been considered as a potential food and pharmaceutical plant due to the accumulation of suitable nutrients such as polyphenols, fibres, and flavonoids(Patel 2016). Despite some studies on salt stress and the role of Arg in its tolerance (Nejadalimoradi et al. 2014, Ramadan et al. 2019), there is no report on the possible effects of this amino acid on salt stress tolerance in Salicornia. Therefore, the present work aimed to evaluate the impact of exogenous Arg on the growth and control of osmotic and oxidative stresses caused by salinity in S.europea.
Material and methods
Plant materials and experimental design
Uniform seeds of Salicornia europaea L. were obtained from the Pakan Bazr Company at Esfahan. Seeds soaked in tap water for 12 h were planted in trays filled with wet cocopeat and placed at 28 ± 1 °C to germinate. The 4 to 5 cm seedlings were transferred to plastic pots (two seedlings per pot) containing 1 kg of well-watered cocopeat-perlite (2:1) in a phytotron at 25 °C, a relative humidity of 60%, and a photoperiod of 16 h light/ 8 h dark. The seedlings were fed with 1/2 Hoagland’s nutrient solution at three-day intervals until their length reached 20 to 23 cm. Three days before salinity treatment, the seedlings were divided into two groups of 20 pots containing 40 seedlings. One group was treated with foliar application of 5 mM Arg, and the other group was left untreated and just received distilled water. Both groups were exposed to four levels of NaCl (0, 200, 400, 470 nm to measure Chl a, Chl b, and Car, respectively (Lichtenthaler and Buschmann 2001). Total Chl was obtained from the sum of Chl aand Chl b. Pigment contents were expressed in mg per g fresh weight.
Enzyme extraction and assay
Fresh shoot tissue (0.1 g) was ground in liquid nitrogen and homogenized with 1 ml of enzyme extraction buffer containing 100 mM cold potassium phosphate buffer (pH 7.4), 1 mM EDTA, 5 mM ascorbate, 50 mM 2-mercaptoethanol, and 1% (w/v) polyvinylpolypyrrolidone (PVPP). The homogenate was filtered through three layers of cheesecloth and centrifuged at 12,000 gfor 10 min at 4 ºC. Decoloration of the extract was done by adding 10 mg of charcoal before centrifugation. The 12,000 g supernatant was used for enzyme assay and soluble protein determination. Soluble protein content was determined by Bradford’s method (1976) using bovine serum albumin (BSA) as a standard.
Ascorbate peroxidase (APX) activity was determined spectrophotometrically by measuring the generation rate of dehydroascorbate in a reaction mixture (1 ml) containing 50 mM potassium phosphate buffer (pH 7.0), 1 mM H2O2, 0.5 mM ascorbic acid, and enzyme extract at 290 nm, assumingan extinction coefficient of 2.8 mM-1 cm-1 (Chen and Asada 1992).
Peroxidase (POX) activity was determined by evaluating the rate of guaiacol oxidation in an assay mixture (1 ml) containing 50 mM potassium phosphate buffer (pH 7.0), 5 mM guaiacol, 1 mM H O , and enzyme extract (at 470 nm and 600 mM) by the addition of salt to the1/2 Hoagland’s nutrient solution. Salt and Arg treatments were applied to seedlings at three-day and six-day intervals for up to 30 days, respectively. Seedlings were then harvested and evaluated for morphological and biochemical responses to salinity and Arg treatments. All studies were independently repeated using separate seedlings at least three times for each morphological and biochemical analysis.
Assessment of morphological traits and membrane stability index (MSI)
The measured weight characteristics included fresh weight (FW) and dry weight (DW) of both shoot and root, and longitudinal traits included shoot and root length.
MSI was determined according to Sairam and Saxena (2000). Two samples of fresh shoot tissue (0.1 g) were placed separately in a test tube containing 10 ml of distilled water. One sample was heated in a water bath at 40 °C for 30 min, and the other sample was boiled at 100 °C for 10 min. After determination of the electrical conductivity (EC) of the samples, MSI was calculated by the following formula:
MSI=1-(EC40/EC100) × 100
Determination of photosynthetic pigments
Chlorophyll (Chl) and carotenoids (Car) were extracted from 0.1 g of fresh shoot tissue with 2 ml of 95% (v/v) ethanol. The extracts were filtered through a Whatman No.1 filter paper, and their absorbance was read at 664, 648, and using an extinction coefficient of 26.6 mM-1 cm-1 (Srinivas et al. 1999).
The assay of polyphenol oxidase (PPO) was done according to the rate of purpurogallin production in 1 ml of a reaction mixture containing 50 mM potassium phosphate buffer (pH 7.0), 40 mM pyrogallol, and enzyme extract. The assay mixture was monitored at 430 nm, and the activity of the enzyme was calculated using the extinction coefficient of 2.47 mM-1 cm-1 (Nakano and Asada 1981).
Determination of total phenolic and flavonoid contents
Phenol compounds were determined by the method of Singletonet al. (1999) with some modifications (Einali et al. 2018) using gallic acid as the standard. Total flavonoids were measured according to Krizek et al. (1998). Freshshoot tissue (0.1 g) was extracted with 1 ml of acidified ethanol (ethanol: acetic acid, 99:1, v/v). The extract was centrifuged at 5,000 g for 10 min, and the absorbance of the resultant supernatant was recorded at 300 nm after heating at 80 °C for 10 min. Total flavonoid content was expressed as absorbance per mg of shoot tissue FW.
Other analytical methods
Proline content was estimated according to Bates et al. (1973) using proline as a standard. The level of lipid peroxidation was determined by measuring malondialdehyde (MDA) content as described by Heath and Packer (1968), assuming an extinction coefficient of 155 Mm-1cm-1.
Statistical analysis
All data were expressed as the mean ± standard error (SE) of triple analyses. Normality and equal variance were also tested. Statistically significant differences were determined in a factorial design by a two-way analysis of variance (ANOVA) at P < 0.05 using the Duncan method.
Results
Effect of exogenous Arg on plant growth and membrane stability during salt treatment. Salt and Arg treatments alone or in combination increased the apparent growth of S. europaea seedlings (Tab. 1, On-line Suppl. Fig. 1). Both shoot and root lengths were positively affected by salinity in a concentration-dependent manner. The highest increase in length was found in seedlings treated with a concentration of 400 mM NaCl. Arg treatment showed a significant effect on improving plant height either alone or in combination with salinity. However, shoot and root height remained statistically unchanged in Arg-treated seedlings grown at 600 mM NaCl compared to untreated controls (Tab. 1).
The fresh and dry weight of seedlings increased in response to salt treatment in shoot and root. Plant biomass and shoot fresh weight were positively changed by Arg treatment alone or in combination with salinity. However, fresh weight of root in Arg-treated seedlings remained unchanged or was drastically decreased compared to untreated controls (Tab. 1).
Salt treatment alone reduced MSI at all concentrations, especially in seedlings treated with 600 mM NaCl. Arg treatment alone or in combination with salinity up to 400 mM did not affect MSI but greatly improved this index in seedlings treated with 600 mM NaCl (Tab. 1).
Effect of exogenous Arg on plant pigments during salt treatment
Salt treatment at all concentrations significantly reduced the photosynthetic pigments of S. europaea seedlings (Fig. 1). Arg treatment alone did not change Chl a content, but its effect in combination with salinity depended on the salt concentration (Fig. 1A). In contrast, Arg treatment alone significantly reduced Chl b levels but had no effect when applied in combination with salinity (Fig. 1B). Total Chl content was positively affected by Arg treatment only in seedlings grown at 200 mM NaCl (Fig. 1C). The Chl a/b ratio increased in response to both salinity and Arg treatments (Fig. 1D). However, this ratio decreased in Arg-treated seedlings grown in 600 mM NaCl.
Arg treatment enhanced carotenoid content in 200 mM NaCl-grown seedlings but did not change it when applied alone or in combination with other salinities (Fig. 1E). The Chl / Car ratio was affected by 200 mM NaCl but not by other salt concentrations. Arg treatment alone or in combination with 200 mM NaCl reduced this ratio but was not effective at other salt doses (Fig. 1F).
Effect of exogenous Arg on plant phenolics during salt treatment
Salt treatment at all concentrations caused a significant reduction in the total phenol concentration of S. europaea seedlings (Fig. 2A). Arg treatment alone decreased phenol content, while it was effective in a salt-dependent manner when applied to seedlings grown at different salinity concentrations. In contrast, the flavonoid content of seedlings grown at 400 and 600 mM NaCl enhanced compared to the control (Fig. 2B). While Arg treatment reduced the level of seedling flavonoids exposed to the mentioned salt doses, it was ineffective when used alone or with 200 mM NaCl (Fig. 2B).
Effect of exogenous Arg on proline, soluble protein and lipid peroxidation during salt treatment
The proline content in seedlings grown at salinity up to 400 mM remained statistically unchanged but increased significantly in response to 600 mM NaCl (Fig. 3A). Arg treatment alone did not change proline levels, whereas this metabolite was highly accumulated when combined with salt. Arg-treated seedlings grown in 600 mM NaCl showed more than 4-fold proline accumulation than untreated controls (Fig. 3A).
Soluble protein concentration increased in 200 mm NaCl-grown seedlings but decreased in seedlings fed higher doses of salt. Protein content was not affected by Arg treatment alone or with salt up to 400 mM, whereas when combined with 600 mM NaCl, it changed positively (Fig. 3B).
Lipid peroxidation was increased by salinity in a dose-dependent manner. Arg treatment increased the MDA content in seedlings nourished with salt-free medium but decreased it in plants grown in combination with salt (Fig. 3C).
Changes in enzyme activities in Arg-treated seedlings during salt treatment
The activities of APX, POX and PPO in S. europaea seedlings were negatively changed by salt treatments (Fig. 4). Exogenous Arg treatment highly decreased the APX activity of salt-treated or salt-untreated seedlings (Fig. 4A). However, POX activity of seedlings treated with Arg alone or in combination with salinity up to 400 mM remained roughly unchanged but decreased abruptly in seedlings growing at 600 mM NaCl compared to untreated controls (Fig. 4B). In contrast, PPO activity was positively affected by Arg treatment alone or in combination with salt (Fig. 4C).
Discussion
In contrast to the destructive effects of salinity on plant growth and biomass in most plants (Qiu et al. 2014, Ahmad et al. 2018, Ghanem et al. 2021), our results showed that salinity could have a positive effect on height, fresh weight, and biomass of S. europaea seedlings. Similarly, the maximum growth of another Salicorniaspecies (S.dolichostachya) was observed at 300 mM NaCl (Katschnig et al. 2013), which is consistent with our results. Arg treatment alone or with salt has an additive effect on plant growth and biomass. This agrees with most studies showing that foliar spraying of mung bean (Qados 2010), wheat (Mostafa et al. 2010), and sunflower (Nejadalimoradiet al. 2014, Ramadan et al. 2019) with Arg increased the growth of these plants under salinity stress. The ameliorative effect of Arg on plant growth under salinity has been attributed to its role as a precursor of polyamines (Mostafa et al. 2010) or as a source of nitric oxide (NO) (Ramadan et al. 2019). However, the negative effect of Arg on root FW during salinity was noticeable. In contrast to the synergic effects of Arg and salinity on plant growth, root FW showed an antagonistic relationship between Arg and salinity treatments. Due to the enhancement effect of Arg and salt treatments on root biomass, this diminishing effect may refer to the potential for water maintenance in root tissues of salt-treated seedlings in the presence of Arg.
Reduction of photosyntheticpigments of S.europaea occurred under salt treatment, which is consistent with other studies (Zeid 2009, Ramadan et al. 2019, Ghanem et al. 2021). The decrease in pigment content under salinity can be attributed to their degradation by free radicals generated during stress (Ma et al. 2020) or activation of chlorophyll catabolic processes along with inhibition of biosynthetic enzymes (Rady et al. 2015). Unlike other studies (Zeid 2009, Ramadan et al. 2019), Arg treatment alone did not affect or decrease the pigment content of S. europaea seedlings. The positive effect of Arg on pigment content was observed only with 200 mM NaCl. This implies that Arg does not change photosynthetic pigments under high salinities. Thus, the improvement in the growth of Arg-treated seedlings under salinity is not directly related to photosynthetic pigments but may be due to the increased photosynthetic capacity through maintaining chloroplast structure. The reduction of lipid peroxidation during salinity due to Arg treatment associated with an increase in MSI could further support this suggestion.
One of the effects of salinity is lipid peroxidation, which occurs in plants both sensitive to and tolerant of salinity, due to oxidative damage, although its severity is higher in salt-sensitive plants (Kumar et al. 2020). Accumulation of MDA, as an indicator of lipid peroxidation, in S. europaea seedlings under salinity was associated with a decrease in MSI, which indicates the destructive effect of salt. However, these seedlings had a higher growth in the presence of salt, showing that they can tolerate these salinity concentrations well. Arg treatment in combination with salt but not alone, caused less lipid peroxidation and increased MSI. Similar results were reported in canola seedlings treated with Arg under salinity (Nasibi et al. 2014). This indicates that Arg enhances salt tolerance in S. europaea. To counteract the oxidative damage caused by abiotic stress, plants use defense mechanisms, including the accumulation of Osmo protectants and non-enzymatic compounds along with antioxidant enzymes (Ali et al. 2017, Polash et al. 2019). Proline is a compatible solute that acts as an Osmo protectant, stabilizer and protector of membranes, enzymes, and proteins,and scavenger of free radical (Ashraf and Foolad 2007, Kumar et al. 2020). As recently demonstrated (Ghanem et al. 2021), the increase in proline during salt treatments of S. europaea seedlings indicates the strategy of these plants in salt tolerance. Accumulation of proline in seedlings treated with Arg under salinity can be related to the positive effects of this amino acid on the production of proline, NO and polyamines (Liu et al. 2006). Thus, a more than fourfold increase in the proline content of Arg-treated seedlings exposed to 600 mM NaCl could explain a decrease in lipid peroxidation versus an increase in MSI in these plants.
Polyphenols and flavonoids, as antioxidant molecules, playan important role in scavenging free radicals by themselves and inducing antioxidant enzymes (Kofroňová et al. 2020). Our results showed that the content of total phenols decreases, but the flavonoids increase during salinity. However, increases in both polyphenols and flavonoids have been reported in plants under salt stress (Lim et al. 2012, Sarker et al. 2019). The phenolics changes during salinity were associated with a decrease in PPO activity, which can be related to salt tolerance. This is consistent with studies showing that decreased PPO activity is associated with stress tolerance (Thipyapong et al. 2004, Sofo et al. 2005). It indicates that among phenolic compounds, flavonoids may have a role in S. europaea tolerance to salinity. In contrast, Arg treatment combined with salinity had an additive effect on total phenols but decreased flavonoids content along with an increase in PPO activity. Considering the role of Arg in enhancing the growth of S.europaea seedlings under salinity, Arg treatment probably induces the production of phenolic compounds other than flavonoids to scavenge free radicals. It means that Arg with salt can change the pattern of phenolic production with PPO activity to induce high salt tolerance. This result in conflict with PPO activity points to the unclear role for the enzyme in stress tolerance, as reported previously (Boeckx et al. 2015).
The soluble protein content of S. europaea increased at200 mM NaCl but decreased under more salt concentrations (400 and 600 mM). This is consistent with previous reports of Pisum sativum (Velitcukova and Fedina 1998) and tomato (Manan et al. 2016) under salt stress, indicating a decrease in soluble protein levels due to inhibition of protein biosynthesis or increased protein degradation (Mersie andSingh 1993). Arg treatment increased the protein levels of seedlings fed with 600 mM NaCl. The positive effect of Arg on the increase of soluble proteins, which has also been reported in Lupinus termis under salinity (Akladious and Hanafy 2018), refers to the synthesis of specific proteins that are involved in the salinity tolerance of plants (Qados, 2010).
Incontrast to the findings of most studies (Ali et al. 2017, Polash et al. 2019, Kumar et al. 2020), in this research the activity of antioxidant enzymes, such as APX and POX as H O -decomposing enzymes in S. europaea seedlings decreased or remained unchanged in response to different salt concentrations. Similarly, Arg treatment decreased APX activity at all salinities but reduced POX at only 600 mM NaCl. Since Arg is involved in the production of proline, NO and polyamines (Liu et al. 2006), its protective effect, increasing salt tolerance in S.europaea seedlings, can be attributed to the subsequent metabolism of this amino acid. There are two lines of evidence that support this suggestion. First, the positive effect of Arg on proline accumulation, especially in 600 mM NaCl, confirms the role of Arg in inducing proline biosynthesis. The second line of evidence comes from a study showing that the protective effect of Arg on sunflower seedlings under salt stress may be due to the release of NO from Arg (Nejadalimoradi et al. 2014). However, in contrast to this report, our results show that Arg treatment decreased the activity of APX and POX in seedlings under salinity. Therefore, the role of Arg in enhancing the salt tolerance of this plant may be related to proline accumulation.
Inthe current study, salinity perse played a positive role in the growth and biomass of root and shoot in S.europaea seedlings, demonstrating that they tolerate these salt doses. Salt treatment was also associated with increased lipid peroxidation and decreased MSI, pigment content and phenolic compounds. Soluble protein content increased at low salinity but decreased under other salt concentrations. While the activity of antioxidant enzymes decreases, proline and flavonoids accumulate during salinity. Arg treatment alone exhibited a slight or no change in seedling growth and metabolite content, but in combination with salt showed a synergistic effect on enhancing growth parameters, MSI and accumulation of soluble protein, phenolic compounds and proline. It was associated with decreased lipid peroxidation, flavonoids and enzyme activity. It can be concluded that Arg treatment alone slightly impresses seedlings growth but when combined with salt, evidently plays an additive role in increasing salt tolerance of these plants by enhancing the accumulation of proline as an osmoprotectant and antioxidant molecules rather than enzymatic antioxidant.