1. Introduction
Salinity considers one of the major abiotic stressors causing severe damage to crops throughout the world. The surge in salinity of the aqueous component of soil will lead to a negative impact on the yield [
1]. It is predicted that the affected agricultural land will increase and the problem will get worse as a result of global climate change. All the important physiological and metabolic pathways of plants are affected by salinity [
2], besides its effects on nucleic acids (DNA and RNA) and mitosis [
3]. Various biological processes in plants are affected as a result of an imbalance in the nutrient content, as well as ionic and osmotic stress, and/or these factors combined as a result of salt stress [
4,
5]. To overcome the osmotic and ionic stress, plants were able to evolve their biochemical mechanisms such as modulating the osmotic and ionic pressure of cells as well as developing the enzymatic defense mechanism and synthesis of compatible solutes [
6]. Obvious oxidative stress markers resulting from high salinity stress are the formation of reactive oxygen species (ROS) such as superoxide ions, hydrogen peroxide, hydroxyl radicals and hydrogen peroxide (H
2O
2), which are proven to be highly detrimental to plants [
7]. In plants grown under salt stress, substantial elevations of ROS scavenging enzymes such as polyphenol oxidase (PPO), peroxidase (POD), superoxide dismutase (SOD) and catalase (CAT) have been documented [
8,
9]. In addition to physiological markers of salinity tolerance, both molecular and biochemical markers also show promise in helping tomato screening and breeding phenomena aimed at improving its salinity tolerance. Biochemical markers have provided great interest in recent years as the data more accurately reflect genetic variability since they are direct gene products.
Electrophoretic analysis of total soluble proteins by the sodium dodecyl sulfate (SDS) method and isozyme profiles are valuable in providing a basic need to assess some measures of genetic variability in and among cultivars [
10]. The electrophoretically separable variant of the isozymes system is widely used as a biochemical marker, and therefore their analysis can provide a precise tool to discriminate plants grown under saline stress conditions. The identification of isozymes patterns is very important to investigate each isoform activity. Isozyme markers are mostly co-dominant with a simple Mendelian inheritance in most loci and it can be resolved for most plant species regardless of habitat, size, or longevity. The use of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and isozymes were the simplest and best methods to provide clear information [
11,
12].
Chitosan is a natural-based linear polysaccharide derived from chitin, the second most abundant biopolymer in nature, and is present as a component in crustacean shells, insect exoskeletons, and fungal cell walls [
13]. After the suitable processing of raw chitin, a partial (at least 50%) or complete alkaline deacetylation process is carried out to prepare chitosan. The produced chitosan is composed of glucosamine and N-Acetylglucosamine units. The degree of deacetylation became higher if a larger amount of N-Acetylglucosamine is turned into glucosamine units, which determines its physical properties including solubility, adsorption capability, and biodegradability [
14]. Chitosan has two types of functional groups: hydroxyl groups and amino groups, while the functionality of chitosan increases with increasing amino groups [
15]. Chitosan induces various defensive responses related to salinity stress in plants [
16]. With the shift in climatic conditions and increased food demand leading to inefficient use of synthetic chemicals, the application of chitosan as an elicitor has a large prospect of resolving stress adaptation issues due to abiotic and biotic stresses. In plants, chitosan has been used to develop resistance to abiotic stressors [
17]. Their ability to scavenge ROS and eventually enhance stress efficiency has attracted researchers to deliver a more diverse application and continue exploring this new biopolymer. Chitosan at low concentrations could ameliorate the negative impact of salinity stress. The results of [
18] showed that the use of low concentration chitosan increased the resistance of safflower and sunflower plants to salt stress by reducing the enzyme activity in these plants. Besides, studies of [
19,
20,
21,
22] on
Trachyspermum ammi,
Plantago ovata,
Vigna radiata and
Zea mays, respectively showed that treatment with chitosan reduced the impacts of salinity on the previous plants by increasing the activity of antioxidant enzymes, which caused a decrease in the malondialdehyde (MDA) content. The intrinsic property of chitosan is that it is not dissolved in neutral aqueous solutions, but rather in acidic solutions of weak carboxylic acids, such as acetic, ascorbic, citric, lactic, and malic acids. Using acetic acid, as it is the associated organic acid to facilitate the dissolution of chitosan, is common in commercial formulations in the agricultural sector. Although acetic acid was reported to be the best associated organic acid in the case of coating fruits to prevent fungal growth [
23], the effect of the associated organic acid on plant biostimulant activity was not evaluated. Using other organic acids such as citric and ascorbic acids, which are known for their stimulant activities on plants [
24], could lead to synergetic effects, which might increase the performance of the biostimulant. Recently, several researchers demonstrated the stimulating effect of chitosan against abiotic stress on tomato plants [
25,
26,
27,
28]. Moreover, [
29] reported that foliar application of chitosan ameliorates the negative impact of salinity on tomato plants through enhancing growth aspects and photosynthetic pigments.
Tomato (
Solanum lycopersicum L.) is a short-lived perennial cropped and is annual. It is part of the Solanaceae (nightshade) family and is usually grown for its edible fruits. Tomatoes are considered among the most important crops grown over the world for their economic and nutritional value [
30,
31].
This research aims to evaluate and compare the anti-stress capabilities of the foliar application of chitosan, dissolved in four different organic acids (acetic acid, ascorbic acid, citric acid and malic acid) on tomato under salinity stress, suggesting the best solvent that could give synergetic effects with chitosan and incorporate within salinity anti-stress formulations in the commercial sector. It can be said that this is the first investigation into the application of isozymes and protein patterns in determining the impact of chitosan on salinized tomato plants at the molecular level.
3. Discussion
Salinity stress is considered one of the most critical challenges facing countries, especially Egypt. The growing knowledge of environmental problems, therefore, makes it important to seek alternatives that are easy to use and feasible to overcome the harmful impacts of salinity on plants [
33,
34,
35]. Morphological aspects (shoot length, root length, number of laterals branches per plant and number of leaves) were significantly decreased due to salt stress. In this regard, the reduction in growth may be correlated with different factors; among them are high osmotic stress and ion toxicity [
9,
36,
37]. The first standard to govern the occurrence of tolerance in tomato plants, foliar application with chitosan solutions was the enhancement of growth parameters. Reports have shown that the application of inducers such as chitosan improved morphological characteristics in the case of maize [
38,
39], rice [
40,
41], and common beans [
42] and stimulate tolerance of seedlings under stress conditions. Thus, the use of chitosan dissolved in some acids, which are low-molecular-weight organic acids such as citric acid, ascorbic acid and malic acids improves the plant’s ability to ameliorate abiotic stress [
16]. The chitosan solutions used in this study were not phytotoxic. Plants treated with chitosan dissolved in acetic acid have not shown phytotoxicity in different crops such as Japanese pear [
43], kiwifruit, or table grape [
44]. Concerning interaction effects, foliar application of chitosan enhanced morphological aspects of tomato plants. These stimulating effects of chitosan were clear due to the presence of ascorbic acid and citric acid as antioxidants.
Organic acids such as ascorbic acid, citric acid, acetic acid, and malic acid boosted the growth of different plants as they enhanced the photosynthetic process throughout increasing chlorophyll contents. Moreover, they play a major role in abolishing the adverse effects of abiotic stresses, protecting protein and lipid, increasing proline contents, and decrease lipid peroxidation [
45,
46,
47,
48,
49,
50].
Photosynthetic pigments were clear positive evidence as a result of the application of the chitosan solutions and became a visible piece of evidence of sufficient treatments. In the current study, the results clearly showed a lessening in the photosynthetic pigment levels in the leaves of tomato plants due to salt stress. The decrease in photosynthetic pigments may be due to a deficiency in the leaf area responsible for light capture and photosynthesis, or may also be due to the degradation of chlorophyll by increasing the activity of chlorophyll degrading enzymes and chlorophyllase under salt stress regimes [
34,
36]. On the contrary, data in the current study have shown that treatment of tomato plants grown under salinity stress conditions then treated with chitosan solutions significantly improved plant salt tolerance by increasing photosynthetic pigments. It was stated in [
51] that the application of chitosan enhanced photosynthetic rates of
Oryza sativa plants throughout enhancement photosynthetic pigments. This augmentation might be attributed to improved stomatal conductance, transpiration rate and/or cell size and number [
52]. This may also be since chitosan has been reported to cause plant defense reactions [
53], and it may trigger NADPH oxidase activity, thereby activating the production of H
2O
2. Thus, chitosan could activate ROS scavenging systems in plants [
54].
The accumulation of osmolytes serves as a common phenomenon that plays an important role in ROS scavenging, supply plant cells with energy as well as modulating cell redox homeostasis [
9,
55,
56]. In this work, there is a positive correlation between the reduction in osmolytes contents (soluble sugars and soluble proteins) and a reduction in photosynthetic pigments and the growth of tomato plants in response to salinity stress. However, the content of proline was increased due to its role in osmoregulation and ROS scavenging [
57,
58]. Foliar application of chitosan dissolved in different organic acids, especially ascorbic acid, enhanced osmolytes in the shoots of tomato plants. These results are in harmony with [
59]. A study by [
60] stated that a significant increase in osmolyte contents in chitosan-treated milk thistle (
Silybum marianum L.) plants. Chitosan caused an enhancement in the contents of soluble sugars, soluble proteins throughout its role in increasing the expression of enzymes involved in glycolysis [
61,
62]. Proline accumulation in tomato shoots prevents the photosynthetic process throughout, preventing damage of photosynthetic pigments caused by ROS [
57,
63].
ROS scavenging in plants occurs in two ways enzymatically and non-enzymatically to prevent plant cells from oxidative damage. Non-enzymatic pathways include phenolic compounds and ascorbic acid, which can overcome ROS production [
9,
64,
65]. In this study, salinity stress increased the content of total phenols and ascorbic acid in the shoots of tomato plants. Our results are in accordance with other investigators [
66,
67,
68]. Phenolic compounds and ascorbic acid support antioxidant roles by scavenging the free radicals, reducing their reactivity to the membrane components [
9,
48]. Moreover, Phenolic compounds are also able to stabilize cell membranes by lowering membrane fluidity, which results in reduced mobility of free radicals across membranes, thus limiting membrane peroxidation [
65]. Concerning the interaction effect of chitosan dissolved in different organic acids, especially the ascorbic acid foliar application of chitosan, enhanced total phenols and ascorbic acid contents over salinity-stressed plants. The aforementioned increases in ascorbic acid and total phenol contents are in correlation with the reduction in contents of MDA and H
2O
2. The accumulation of phenolic compounds and ascorbic acid serves as an adaptive strategy for salinity stress [
69,
70]. The obtained results are in line with [
71,
72]. Moreover, a study of [
73] indicated that treatment with chitosan increased significantly the content of phenolic compounds, which directly declined lipid oxidation throughout, transferring a phenolic hydrogen atom to a radicle. Moreover, [
74] reported the stimulatory role of chitosan on secondary metabolites as phenolic compounds through inducing certain genes involved in the biosynthesis of secondary metabolites.
Oxidative stress caused by salinity stress led to serious disruption to plant cells and increased the contents of MDA and H
2O
2 in the leaves of tomato plants. These findings are in harmony with [
34,
75,
76]. Application of chitosan lessened the production of MDA and H
2O
2 peroxide through increasing antioxidant compounds that scavenge ROS and prevent cellular membranes from oxidative stress [
38]. Moreover, [
73,
77] stated that chitosan application significantly reduced the contents of MDA in salinity-stressed wheat plants. The protective role of chitosan was more obvious due to the presence of different organic acids which help tomato plants diminish the harmful impact of salt stress [
47,
48,
78].
Salinity-induced growth deficits in crops are mainly associated with ion stress, which arises due to long exposure to salt stress [
6,
9]. Ionic stress occurs in response to the accumulation of sodium in plant cells, which caused plant toxicity and disrupt normal metabolism of salinity-stressed plants [
75,
79]. Our results showed an increase in sodium content in tomato shoots; however, potassium content was significantly decreased. These results explain the deleterious effect of sodium accumulation in the plant cell. Chitosan foliar application was reduced sodium accumulation and increase K level in tomato shoots, which caused homeostasis, which is generally observed in salt-tolerant varieties [
79,
80]. This change in Na
+ and K
+ level could be attributed to the ability of chitosan in improving the growth of tomato plants or throughout osmolytes content increases, which acquire a plant balance in facing salinity stress. These results are in harmony with the results of [
71,
81], who also reported that chitosan treatment significantly increased Na
+ and K
+ content in salinity stress wheat plants.
Antioxidant enzymes SOD, CAT, POD and POO provide a large number of defensive enzymes associated with salinity stress [
36,
82]. These enzymes act as initial steps in increasing plant resistance to various stresses as well as the formation of phenolic compounds [
83,
84]. The results showed that antioxidant enzyme activity increased in plants exposed to salt stress. The plants show different mechanisms to cope with salinity pressure as they increase the activity of certain antioxidant enzymes to keep ROS at the lower level in the cell. SOD helps in the conversion of O
2− to H
2O
2, which acts as the first line in facing oxidative stress, while CAT and POD help in the conversion of H
2O
2 to H
2O [
64]. SOD, CAT, POD and PPO activities were greater in the plants grown under NaCl stress and treated with chitosan solutions against salinized plants. Application of chitosan was reported to increase the activity of catalase and peroxidase in tomato [
85], eggplants [
86], and milk thistle [
60]. The chitosan chemical constitution includes uridine diphosphate N-acetyl-d-glucosamine (UDP-GlcNAc) as nucleotide sugars, which, when applied in plants recognized by cells throughout chitin synthase chitin deacetylase enzymes, caused the formation of chitosan oligomers that are involved in plant cell signals [
87,
88]. Chitosan oligomers enter the nucleus and act in cascade reactions as the production of hormones and the expression of antioxidant enzymes [
62,
87,
89,
90].
Multiple enzyme isoforms are considered a key control mechanism for cell metabolism in plants, and changes in isozyme profiles play an important role in cellular protection versus salt stress [
91,
92]. The induction of these isozymes is considered to constitute an important role in the cellular defense against oxidative stress [
93]. Activity staining of antioxidants after PAGE showed seven POD isozymes, four PPO isozymes and four SOD isozymes in the extract of leaf-soluble proteins (one Mn-SOD, two Fe-SODs, and one CuZn-SOD of tomato plants). In general, the antioxidant enzyme activities in salt-stressed plants treated with chitosan were always higher than those in control plants, because many new isozyme bands were induced by salt stress. Our results have shown that stressed tomato plants treated with chitosan either dissolved in ascorbic acid or citric acid appeared a maximum banding of POD isozyme compared to other treatments. These results reflecting the ameliorative role of chitosan treatments (Ch ASC and Ch CIT) in protecting tomato plants from salt stress. For the PPO isozyme pattern, the present analysis showed that there is no variation in the expression level of PPO isoforms among tomato sprayed with different chitosan treatments either alone or in combination with salinity stress. The only difference was observed for the PPO isozyme pattern in tomato treated with NaCl alone versus control plants. Indeed, the separation of SOD isozymes (after native PAGE) coupled with different specific inhibitors showed four SOD isozymes in the extract of leaf-soluble proteins: one Mn-SOD, two Fe-SODs (denominated Fe-SOD1 and Fe-SOD2), and one Cu/Zn-SOD. Quantification of the SOD band intensities revealed that Fe- SODs and Cu/Zn-SOD were the predominant isozymes. These results are in harmony with [
94] who reported enhanced Mn-SOD and Cu/Zn-SOD transcript abundances in maize and tomato plants. The intensity level of all SOD isozymes increased under chitosan treatments as compared to control untreated and salt-treated plants. The highest intensity level of SOD isozymes in tomato leaves was detected with Ch ASC alone. However, the lowest intensity level of SOD isozymes was observed in plants treated with NaCl alone and S + Ch MAL. The results are in line with [
95] who noticed that treating tomato plants with NaCl suppressed mRNA levels of SOD genes, whereas plants treated with zinc oxide nanoparticle inducers, in the presence of NaCl, showed an increase in mRNA expression levels, suggesting the beneficial impact of zinc oxide on plant metabolism under salt stress. Similarly, the present data indicate that chitosan, especially when dissolved in ascorbic acid had a positive response on tomato metabolism under salinity stress. The increments of Fe-SODs isozymes could be attributed to their abundance in chloroplasts of tomato plants under investigation [
96]. Furthermore, [
97] found that overexpression of Cu/Zn SOD in potato showed that transgenic plants exhibited increased tolerance to oxidative stress. The present data were found to agree with previous studies which reported that a variety of protein functions could act as scavengers for these ROS including CAT and POD [
93,
98]. Our results suggested that the differential responses of tomato plants to NaCl stress depended on the solvent type that chitosan was dissolved in. Thus, we could mention that chitosan dissolved in ascorbic acid (Ch ASC) and/or citric acid (Ch CIT), solvents appeared to have a higher tolerant level against salt stress against other treatments.
SDS-PAGE results illustrated differences in patterns of protein changes between chitosan treatments either alone or in combination with salinity stress and represented protein banding patterns with different molecular weight as an appositive marker and showed more changes in protein profile and a higher percentage of polymorphism in plants treated with chitosan dissolved in Ch ASC or chitosan dissolved in Ch CIT versus other chitosan treatments. The explanation for these findings is that tolerant cultivars are capable of adapting successfully to saline regimes by modifying their biochemical processes and, consequently, by accumulation or depletion of certain metabolite activities, which caused the suppression of a pre-existing protein synthesis and improved or de novo synthesis of proteins which induce resistance strategies. This explanation is also supported by previous results [
99,
100]. The protein band at molecular weight 72.115 kDa in control plants and salinity-stressed tomato plants treated with chitosan dissolved in acetic acid, ascorbic acid and citric acid can be considered as positive markers for stress, and it was noted that this band was diminished under salinity stress and induced again under the interaction between salinity and chitosan treatments except malic acid-treated plants.