5.1 Pre-treatment of biostimulants significantly impacted growth and photosynthetic pigments in P. distans seedling under salinity stress
To assist to our understanding of the effects of efficiency of biostimulants pre-tretament to neutralize the stress condition, we pre-treated P. distans seedling with foliar spray of two biostimulants (Algabon® and Bonamid®), and then exposed them to 300 mM NaCl and compared responses with those pre-treated with foliar spray of water.
Growth of NSC plants displayed a marked response towards biostimulants pre-treatment by both Algabon and Bonamid (Figure. 3B). Generally, there was a significant increase in the shoot and root biomass and water content of NSC plants pre-treated with Bonamid. Also, the application of Bonamid resulted in higher root/shoot biomass, which is the result of increased root growth and is consistent with root density (Figure 3. C). Similarly, the increase in dry biomass, root density and RWC of the plants sprayed with Bonamid compared to the Algabon treatment and control plants was most pronounced at 300 mM NaCl (Figure 3. C), and pre-treatment with Bonamid mitigated NaCl-induced growth prevention in P. distans (Figure 3. B).
In this study, we observed a composition-dependent effect of biostimulants in diminishing the effects of NaCl stress, so that Bonamid was more effective than Algabon. In most plants, salinity stress prevents growth and development. One of the primitive response of plants to salinity is inhibition of shoot and root growth. The effects of salinity on alteration in growth might be attributed to changes in water and ion absorption by the roots, production of hormonal signals that exchange messages to the shoot, and changes in gene expression patterns. Typically, when glycophytes are exposed to salinity stress, shoot growth is more affected than roots, leading to the enhance of root/shoot ratio (Kravchik and Bernstein 2013). However, the salinity-induced root growth response in halophyte plants may be different (Flowers and Colmer 2008).
Several studies have reported the affirmative effect of biostimulants on plant growth in a wide range of compounds such as seaweed extract, protein hydrolysis and humic acids under stress (Lucini et al. 2015; Latef et al. 2017; Saidimoradi et al. 2019). The application of seaweed extract- and protein hydrolysates-derived biostimulants might supply defence opposed to salinity stress in plants. Latef et al. (2017) suggested that foliar applications of two seaweed extracts improved plant growth and photosynthetic pigments of Cicer arietinum under saline soil condition. Also, exopolysaccharide extracts from Dunaliella salina diminished the salinity stress and alleviated the decrease in dry weight of the plant’s shoot and root systems in Solanum lycopersicum (Arroussi et al. 2018). Lucini et al. (2015) also demonstrated that foliar application of LISIVEG® (the plant-derived protein hydrolysate) biostimulant increased fresh yield, dry biomass and root dry weight of Lactuca sativa grown under salinity conditions. It is well known protein hydrolysates-derived biostimulant that play an important role in stimulation of growth, increase of yield and alleviation of the impact of salinity stress on crops, through the modulation of plant molecular and physiological processes. Protein hydrolysates-derived biostimulants directly influence on plant growth by the induction of carbon and nitrogen metabolisms, as well as regulation of N uptake and by interfering with hormonal activities, leading to the stimulation of root and shoot growth, and thus crop productivity (Colla et al. 2017).
Foliar and root applications of protein hydrolysates-derived biostimulants have been demonstrated to increase the uptake and use efficiency of both macro and micronutrients (Colla et al. 2015). Modifications of root density in protein hydrolysate-treated plants may be involved to improve the efficiency of water and nutrient uptake, thus promoting yield production (Colla et al. 2015). Subbarao et al. (2015) also indicated a positive effect of protein hydrolysate on enhancement of root and shoot growth, and found that soil application was more effective than foliar application. Similar results were obtained by Popko et al., (2018), who recommended foliar application of amino acids-based biostimulantsfor an efficient agricultural production of winter wheat. In our study, the Bonamid pre-treatment mediated-growth promotion effect on P. distans seedlings under salinity stress was simply explained by higher biomass and density of roots that led to the enhancement of the efficiency of water and nutrient (especially N) uptake.
Salinity stress decreased total chlorophyll content in control plants, whilst foliar spray of two biostimulants enhanced chlorophyll content and diminished detrimental impacts of salinity. Application of biostimulants plays an important role in enhancing chlorophyll content (Latef et al. 2017; Saidimoradi et al. 2019). Colla et al. (2014) also showed that a commercial plant-derived PH (Trainer) induced chlorophyll synthesis by the increasing of leaf nitrogen content. Generally, there was a significant increase in total chlorophyll in plants pre-treated with Bonamid under SC. Protein hydrolysate can also help plants to retain photosynthetic activity under stress conditions.
These results are in agreement with those of Lucini et al. (2015) and Di Mola et al., (2021) who stated that application of a plant-derived protein hydrolysate increased photochemical activity (Fv/Fm) in lettuce and hemp, respectively, grown under saline conditions. This factor ensures better photosynthetic metabolism and thus improves plant performance. Foliar spray of P. distans seedlings with two biostimulants enhanced carotenoid content in NSC. It has been shown that biostimulants are effective in increasing production of several classes of secondary metabolites such as carotenoids, which are involved in crop quality and stress response. Foliar application of seaweed extract from Ecklonia maxima increased chlorophylls and carotenoids concentrations in Brassica oleracea (Rengasamy et al. 2016). Application of an amino acid-derived biostimulant had positive effects on carotenoid content in two varieties of carrot (Grabowska et al. 2012). In general, under salinity conditions, the carotenoid content was unchanged in plants pre-treated with two biostimulants (Figure 4B), likely due to the great enhance in biomass produced by biostimulants, which in turn might dilute some of the compounds in the tissues.
5.2 Pre-treatment of biostimulants significantly impacted compatible solutes in P. distans seedling under salinity stress
The accumulation of compatible solutes, including proline, soluble sugar and glycine betaine was reported in halophytes plants likely for the modulation of osmotic signalling pathways in vacuoles, as well as stabilization of membranes and act as ROS scavengers (Sharma et al. 2019). At 300 mM of NaCl, proline, glycine betaine and soluble sugar content of roots and shoots enhanced in control plants of P. distans. Under salinity conditions, foliar spray of two biostimulants increased proline and soluble sugar concentrations of roots and shoots compared with their counterparts grown under NSC that diminished detrimental impacts of salinity in P. distans seedling. Previous studies also showed that foliar application of seaweed extract- and protein hydrolysates-derived biostimulants improved salt stress amelioration in plants by enhancing proline, soluble sugar and glycine betaine contents (Ertani et al. 2013; Arroussi et al. 2018; Latef et al. 2017). Osmotic regulation in stressed plants is highly dependent on soluble sugars, and in glycophytic plants, more than 50% of the maintenance of osmotic regulation under salinity stress is related to the production of soluble sugars. The important roles of carbohydrates in the alleviation of salinity-induced responses involves osmoprotection, scavenging of ROS and carbon storage. The enhancement of reducing sugars under salinity stress has been previously observed in various plants (Kerepesi and Galiba 2000). The upregulation genes associated with osmotic regulation has been also reported in halophytic plant of Spartina alterniflora under salinity stress (Baisakh et al. 2006).
Biostimulants produced based on protein hydrolysates and amino acids showed quality improvement in one cultivar of carrot by increasing soluble sugar content (Grabowska et al. 2012). Higher sugar level in plants treated with biostimulants have been detected in different species, together with higher carotenoid and chlorophyll contents, photosynthetic rates, stomatal conductance, total protein, phenols, ascorbic acid, as well as growth-promoting hormones (Abbas 2013; Abdalla 2013; Martinez Esteso et al. 2016; Arroussi et al. 2018). After the exposure of Bonamid-pre-treated plants under salinity conditions, we observed the reduction of glycine betaine in the leaves of P. distans. Similar to our results, some researchers revealed that by the application of biostimulants, the compatible solutes decreased in salinity-stressed plants (Aydin et al. 2012; Jarošová et al. 2016). One possible reason for this observation in plants pre-treated with Bonamid is the more participation of glycine betaine in the moderation of deleterious impacts of salinity in P. distans. Glycine-betaine is likely the major compatible solute other than proline. Application of a seaweed-based biostimulant had the effect of glycine betaine synthesis in spinach (Fan et al. 2011). The improvement in salinity tolerance in Bonamid-pre-treated plants may be attributed to the ingredients of the Bonamid (containing amino acid (85%)), which could induced the plant’s metabolism to biosynthesis of compatible solutes and these protective compounds increase the plant potential to exclude Cl− ions.
5.3 Pre-treatment of biostimulants significantly impacted antioxidants potential in P. distans seedling under salinity stress
Besides the accumulation of compatible solutes, to fight against salinity-induced oxidative stress, plants increase the biosynthesis and accumulation of non-enzymatic antioxidant compounds, particularly metabolic pathways associated with polyphenolic antioxidants biosynthesis (Cheynier et al. 2013). Under salinity stress, total phenolic compounds and radical scavenging activity increased in control plants of P. distans, also foliar spray of two biostimulants increased the antioxidant potential and total phenolic compounds compared with their counterparts grown under NSC and moderated detrimental impacts of salinity. Induction of oxidative damage under salinity stress through forming ROS has been reported in halophytes plants (Ellouzi et al. 2011; Bose et al. 2014). Hence, halophytes plants are able to synthesize non-enzymatic antioxidants such as total phenolic compounds under salinity conditions (He et al. 2020; He et al. 2021). Hsouna et al. 2020 revealed enhancement of polyphenol compounds in the leaves of the halophyte Lobularia maritima when exposed to saline conditions that might be related to the up-regulation of phenylalanine ammonia-lyase activity. Many quality characteristics of plants are linked to secondary metabolites, like polyphenols. These compounds are free radical scavengers, and may preserve plants against oxidative stress. Hence, interaction of biostimulants with the flavonoid metabolism can synergistically strengthen their effects. Enhancement in polyphenols contents of plant tissues by the biostimulant application has been shown in different glycophyte plants (Gurav and Jadhav 2013; Elansary et al. 2016). However, the research on the effects of biostimulants on the levels of phenolic compounds in halophyte plants has been very limited. Kaluzewicz et al. (2017) reported that the application of amino acid-based biostimulants and Ascophyllum nodosum filtrate increased the total phenolic content, sinapic acid content, as well as quercetin content in Brassica oleracea seedlings. According to the results of this study, Latef et al., (2017) and Arroussi et al. (2018), demonstrated that the foliar spray of seaweed extract-based biostimulant enhanced the levels of phenolic compounds in chickpea and tomato plants, respectively, under salinity stress, thus leading to salt stress alleviation. Amino acids-based biostimulants enhanced the activity of phenylalanine ammonia-lyase, which is the first step in phenylpropanoid pathway and starting point for secondary metabolics pathways and production of a wide range of phenolic compounds, such as flavonoids, anthocyanins, plant hormones, phytoalexins and lignins (du Jardin et al. 2020). In our study, biostimulants-mediated induction of phenylpropanoid compounds as well as the antioxidant capacity of plants under salinity stress would increase their health-related characteristics, leding to maintaining a high level of chemical defence capability and, in turn, better efficiency in respect of plant growth. Generally, a better status of the plants pre-treated with Bonamid, as well as potentially higher tolerance to salinity (Fig 3B) due to the presence of phenolic compounds, was also shown by the higher content of proline and proteins, indicative of the more magnitude of metabolic activity in plants treated with the biostimulant.
5.4 Pre-treatment of biostimulants significantly influenced protein and nitrogen percentage of shoots and roots in P. distans seedlings under salinity stress
Based on the results, root and shoot protein and nitrogen contents of P. distans can be affected by applying foliar spray of biostimulants under NSC. The highest content of proteins and nitrogen was found in roots and shoots of Bonamid pre-treated plants. Application of biostimulants have a major role in enhancing protein and nitrogen content (Ertani et al. 2013; Colla et al. 2014; Lucini et al. 2015). Abiotic stresses such as salinity increase ROS production and by upsetting redox balance, cause oxidative damage to organic molecules such as proteins, lipids, carbohydrates, and DNA. Under salinity stress, the protein content decreased in all pre-treated plants compared with their counterparts grown under NSCs, however the maximum content of proteins in roots and shoots was detected in Bonamid pre-treated P. distans. Salinity stress could also alter several metabolic processes in plants, in particular, photosynthesis, respiration, phytohormone regulation, protein biosynthesis and nitrate assimilation (Colla et al. 2010). It generally leads to a decrease of production and to the lower quality of the final product, due to an inhibition of leaves and roots growth (Bulgari et al. 2019). To confirm the effects eliciting from the applications of biostimulants, Lucini et al. (2015) showed that foliar application of a hydrolysate biostimulant increased yield and dry weight in lettuce plants, leading to resistance to salinity stress through the improvement of nitrogen metabolism and an increase of the Fv/Fm-ratio efficiency. It has been also reported that application of seaweed extract-based biostimulant mitigated salinity-mediated protein reduction in tomato plants grown under different NaCl levels, through the activation of various metabolic pathways (Arroussi et al. 2018). However, abiotic stresses such as salinity increase nitrogenous compounds such as proline and soluble proteins in the plant. These proteins are essential for all plant’s physiological processes including plant growth. Proteins produced under salinity stress in plants may be used as a source of nitrogen after stress. Since protein synthesis depends on the nitrate assimilation, it is obvious that the reduction of nitrogen assimilation under salinity stress can be the cause of the decrease in the protein content.
5.5 Pre-treatment of biostimulants significantly impacted Na+, K+, K+/Na+ and antiporters gene expression of shoots and roots in P. distans seedling under salinity stress
It has been shown that a disruption of Na+ and K+ homeostasis in cells followed by ion toxicity, adversely influences some major processes such as growth, photosynthesis, and development (Deinlein et al. 2014) and in both glycophyte and halophyte plants, sensitivity of cytosolic enzymes in front of salinity is similar. Hence, in all plants, maintaining cellular homeostasis of Na+ and K+ is crucial allowing plants to survive and grow under salt stress conditions (Cuin et al. 2011).
Our results showed that foliar spray of P. distans plants with two biostimulants reduced salinity-induced Na+ accumulations in roots and shoots. Salinity induced a reduction of K+ content of roots of P. distans; however, in the biostimulants pre-treated seedlings, it significantly compensated the negative impacts of salinity and enhanced K+ content in roots and shoots. Similarly, Latef et al. (2017) suggested that foliar applications of two seaweed extracts decreased the extent of Na+ accumulation and maintained higher K+ levels of Cicer arietinum under salinity stress. Furthermore, Wu et al., (2021) demonstrated that Na+ contents of roots in cucumber increased significantly under salinity stress; however, after foliar spray of 5-aminolevulinic acid (ALA), the Na+ content in the roots decreased significantly. Osmotic adjustments play important role in plant resistance under salt stress, which include the intake of inorganic solutes from the soil to help the maintenance of leaves’ turgor. Osmotic adjustments have been observed for the improvement of osmoregulation and resistance to salinity in halophyte plants (Jones and Gorham 2002; Hariadi et al. 2011). The effects of biostimulants in plant resistance against salinity can be attributed to the improvement of the osmotic adjustment by the accumulation of osmotic metabolites and the compartmentalization of salts in vacuoles. Our data indicated that application of biostimulants probably affected the mechanisms of uptake and translocation of ions in roots and shoots under both conditions. These biostimulant’s beneficial effects includes the inhibition of Na+ accumulations and improvement of K+ uptake in the leaves, leading to an increases K+/Na+ ratio of roots and shoots (Table. 3).
On the basis of phytoremediation potential (shoot and total) of Bonamid pre-treated P. distans seedlings (Figure. 6) that is the best criterion for comparison with the other pre-treatments, we decided to investigate this criterion in Bonamid pre-treated P. distans seedlings under both conditions at the molecular level, through determination of genes expression profile of SOS1: plasma membrane Na+/H+ and NHX1: tonoplast Na+/H+ antiporters. The mechanisms accepted by plants surviving in saline conditions include a balance between influx and efflux of Na+, either back into the apoplast across the plasma membrane or the tonoplast into the vacuole. In halophyte plants, the developed tolerance mechanisms like compartmentalization of ions in vacuoles are performed by Na+/H+ antiporter (NHX1) and salt extrusion through antiporter (SOS1) located in the plasma membrane (Wang et al. 2007; Hamed et al. 2013). The presence of Na+/H+ antiporter in the plasma membrane of plants is crucial for their growth under high salinity as it removes toxic Na+ from the cytoplasm. Salt stress increases the gene expression of SOS genes in Arabidopsis (Oh et al. 2009), Kochia scoparia (Fahmideh and Fooladvand 2018) and sugarcane (Brindha et al. 2021). Several reports indicate that NHX overexpression confers salinity tolerance in a wide range of plant species (Brini et al. 2007), tomato (Zhang and Blumwald 2001) and cotton (He et al. 2007).
Exposure of P. distans seedlings under salinity increased the expression of SOS1and NHX1 in roots and leaves. In halophytic plants as opposed to glycophytes, accumulation of Na+ in vacuoles and the regulation of activity of Na+/H+ exchanger (NHX1) have been seen under salinity conditions. For example, Wang et al. (2009) showed that the significant mechanism of halophytic species of P. tenuiflora to succeed in dealing with NaCl was excreting Na+. Also, Zhang et al., (2017) showed that SOS1, HKT1;5, and NHX1 synergistically regulate Na+ homeostasis by controlling Na+ transport systems at P. tenuiflora plant under both lower and higher salt conditions. In accordance with the results of the present study, the upregulation of NHX of Suaeda salsa (Qiu et al. 2007) and two antiporters of SOS1 and NHX in Kochia scoparia (Fahmideh and Fooladvand 2018) under salinity stress have also been reported.
It has been shown that biostimulants can elicit plant response to environmental stress, thus activating genes associated with signalling pathways related to stress response (Trevisan et al. 2017; Jithesh et al. 2019). In this regard, it is believed that the genes expression of Na+/H+ transporters generally increases by exogenously application of biostimulants. The foliar spray of 5-aminolevulinic acid (ALA) significantly upregulated the transcriptional level of SOS1 and NHX1 under normal and salinity conditions (Wu et al. 2021). This indicated that ALA application can improve the compartmentation of Na+ into vacuoles and enhance the salt resistance of cucumber seedlings. Similar to our results, stimulation of the expression of SOS1 in Arabidopsis with a commercially biostimulant (BC204) treatment (Loubser and Hills 2020) and the expression of vacuolar Na+/H+ exchanger (NHX1 gene) with silicon treatment have been reported in cucumber (Gou, 2020). On the contrary, Jithesh et al. (2018) found that the treatment of sos1 Arabidopsis mutant with Ascophyllum nodosum extract in media containing 75 mM NaCl did not reverse the lethality of salt. The results of our study showed that foliar application of Bonamid increased gene expression of SOS1 in roots, which coincided with a reduction of Na+ concentration in roots of Bonamid-pre-treated plants probably due to SOS1 proteins function that acts in loading of Na+ into the xylem of roots for regulating Na+ release to the leaves and contribution in osmotic adjustment. Furthermore, foliar application of Bonamid enhanced the gene expression of NHX1 under 300 mM NaCl, which suggested that the sequestration of Na+ to the vacuole increased to reduce Na+ toxicity in the cytoplasm.