One of the most common plant responses to salinity and drought stress is to reduce stomatal conductivity, thus minimizing water loss [
6]. This response limits the Calvin cycle’s access to CO
2 for carbon stabilization; consequently, the absorbed light exceeds the amount required for normal photosynthesis [
7]. Combined with the toxic impacts of sodium and chlorine accumulated in the cytosol of plants under salinity stress, this excess excitation light by depleting electron receptors (QA in PSII and NADP in PSI) affects electron transfer through photosystems and also increases ROS production (mainly O
2•− and
1O
2) by reducing O
2 [
8]. Limiting CO
2 availability increases the rate of photorespiration in C
3 plants, which in turn increases ROS production [
9]. Therefore, salt-stressed plants use mechanisms to reduce ROS production that can maintain an acceptable level of net photosynthesis under CO
2 limitation conditions and/or use alternative electron sinks that can prevent ROS formation from O
2 [
8].
2. Halophytes: Importance, Classification, and Salt Tolerance Mechanisms
Halophytes have the ability to naturally inhabit man-made areas like salt pans, roadside verges, and salt marshes [
26]. This makes them ideal for bioenergy production and saline agriculture because they do not compete with crops for arable land or freshwater resources [
27,
28]. However, we must be cautious about the impact of activities on wild halophyte populations as their preservation is at risk due to exploitation. To ensure their long-term survival it is crucial to develop management plans that prevent the gathering of these valuable halophytic species [
12,
26,
28].
Many studies have shown that at high salinity, the amount of halophyte production is much higher than the economic yield of commercial crops and trees [
6,
29]. However, it should be noted that the growth of the halophytes may be decreased at very high salinities. Therefore, is an inverse relationship between intensifying salinity and the proper halophyte number/abundance of halophytes [
29], so only a few halophytes, like some
Salicornia species, can grow with the salinity of seawater [
22].
In general, the reaction of plants to salinity depends on their tolerance to soil salinity. Therefore, plants are divided into four categories based on the amount of dry matter production under saline conditions [
30,
31]:
- (1)
-
Eu-halophytes: Growth of eu-halophytes is stimulated even in moderate salinities (such as Salicornia europaea and Suaeda maritima).
- (2)
-
Facultative halophytes: Growth of these halophytes is slightly stimulated at low salinity (such as Plantago maritima and Aster tripolium).
- (3)
-
Non-halophytes with low salinity tolerance: These plants are not halophytes and often include salt-tolerant crops and orchards. This group includes a wide range of economic plants such as barley (Hordeum vulgare), sorghum (Sorghum bicolor), cotton (Gossypium spp.), and pistachios (Pistacia vera).
- (4)
-
Halophobic: Plants that are sensitive to salinity and even at low salinity levels there is a significant reduction in their growth and yield, such as saffron (Crocus sativus), common bean (Phaseolus vulgaris), and most vegetables.
Halophytes are categorized according to the mechanism of salt extrusion [
32]:
-
Recretohalophytes: Include halophytes that excrete salts on the outer surface (Exo-recretohalophytes) or the inside (Endo-recretohalophytes) of plant tissue.
-
Euhalophytes: Or true halophytes with succulent leaves or stems.
-
Pseudo-halophytes: Unreal halophytes that store salts in the parenchymal organs of the root.
By all categories, halophytes are plants with special capabilities that are good choices for the current global situation where severely limited freshwater resources, poor soil quality, and climate change have restricted the production of conventional plants [
33]. Therefore, understanding the physiology of these species and elucidating their mechanisms for their high salinity tolerance is essential. Although good research has been carried out on the production of ROS, oxidative damage, and antioxidant enzymes in halophytes, more research is required to emphasize the role of ROS and antioxidants in the germination of halophytes.
Halophytes have various important mechanisms for salt tolerance, which work together to help them maintain ion balance and protect their cells from the effects of high salinity. The main important mechanisms of salt tolerance in halophytes include:
-
Halophytes have developed ways to reduce the uptake of sodium ions (Na
+) from the surrounding soil or water (reduction in Na
+ influx). By preventing the accumulation of sodium ions in their cells, they can avoid salt-related damage [
34,
35].
-
Halophytes possess compartments like vacuoles that can store and isolate excess sodium ions. This compartmentalization helps maintain sodium ion concentrations in the cytoplasm, which is crucial for cell health when dealing with high salinity conditions [
34,
35].
-
Some halophytes have evolved salt glands or bladders that actively excrete sodium ions from their tissues (excretion of sodium ions.) By removing these ions from their cells, they effectively regulate salt concentration and protect themselves against saline stress [
35].
These mechanisms collectively allow halophytes to tolerate and thrive in environments with levels of salt. By minimizing sodium influx compartmentalizing ions within structures and actively excreting them when necessary, halophytes ensure ion homeostasis and safeguard their cells from the damaging effects of elevated salinity [
36].
3. ROS and Antioxidants in Halophytes and Their Role in Salinity Tolerance
Plants primarily generate ROS in their chloroplasts during photosynthesis, resulting in the production of O
2•, H
2O
2, and O
21. Meanwhile, mitochondria produce O
2• and H
2O
2 as a byproduct of respiration, and peroxisomes generate H
2O
2 during the process of photorespiration [
4] as illustrated in
Figure 1. Plant antioxidant defense systems include non-enzymatic and enzymatic systems involving compounds and enzymes that are distributed in different cellular compartments. The synergic action of both systems is responsible for the enhancement of the antioxidative response under salinity stress for many halophytes [
37,
38]. Enzymatic components of antioxidant defense include superoxide dismutases (SOD), catalases (CAT), peroxidases (POX) (glutathione peroxidase—GPX, ascorbate peroxidase—APX), and reductases (dehydroascorbate reductase—DHAR and monodehydroascorbate reductase—MDHAR). Certain antioxidant enzymes, including SOD and CAT, are speculated to have emerged as early as 3.6–4.1 billion years ago, prior to the great oxidation event that enabled organisms to cope with reactive oxygen species (ROS). These ROS emerged on Earth around 2.5 billion years ago, along with atmospheric oxygen [
39]. The key non-enzymatic components are ascorbate, glutathione, tocopherol, phenolic, flavonoid, and carotenoid compounds [
40,
41].
Figure 1. Major compartments of plant cells responsible for ROS generation. Photosynthesis, photorespiration, and cellular respiration as crucial processes responsible for ROS production were highlighted in the plant cell. ROS are constantly synthesized in chloroplasts, mitochondria, and peroxisomes as part of normal plant cell metabolism. The leak out in the electron transport chain in chloroplasts and mitochondria leads to the formation of superoxide (O2−) or hydrogen peroxide (H2O2). Oxidative metabolism in peroxisome produces H2O2, O2−, and singlet oxygen (1O2). Created with BioRender.com.
By transforming O
2• into H
2O
2, superoxide dismutase acts as “the first line of defense against ROSs”. Three primary varieties of SOD, namely cytosolic Cu-Zn SOD, mitochondrial Mn-SOD, and chloroplastic Fe-SOD, have been identified in plants [
42]. Both glycophytes and halophytes exhibit a positive association between SOD activity and tolerance to salinity [
8]. Nevertheless, halophytes are recognized to have relatively higher levels of SOD activity than glycophytes. For instance, when exposed to salt,
Cakile maritima as a halophyte exhibits greater SOD activity than
Arabidopsis thaliana as a glycophyte [
43]. Yildiztugay et al. [
44] indicated high SOD activity under toxic salt concentrations for
Salsola crassa. However, after 30 days of salinity exposure, the activity of antioxidant enzymes in
S. crassa was decreased. Therefore, the fast and stronger enhancement in SOD activity in halophytes could play an important role in stress signaling in halophytes. For example, SOD activity for halophyte
Tripolium pannonicum can be dependent on salinity level and also on organs [
45]. However, in
Salicornia europaea, both ROS production and SOD activity are not growing at high NaCl concentrations i.e., 800 mM NaCl [
46].
Catalases are enzymes consisting of 4 haem-containing subunits that help convert H
2O
2 into oxygen and water (H
2O), effectively detoxifying it [
47]. The CAT performs a range of functions, such as participating in photorespiration, eliminating H
2O
2 during the β-oxidation of fatty acids in germinating seeds, and promoting stress tolerance. Multiple isoforms of CAT are frequently present in plants, primarily located in the mitochondria or peroxisomes [
48]. Catalases play a less significant role than SOD as was demonstrated for obligatory halophyte
S. europaea [
46]. Many other antioxidant enzymes such as glutathione peroxidase (GPX), glutathione S-transferases (GST), thiol peroxidase type II peroxiredoxin (Prx), and guaiacol peroxidase (GPOX) have also been reported from plants and contribute toward ROS homeostasis [
8].
Peroxidases (POX) are glycoproteins catalyzing the oxidation of substrates by degradation of H
2O
2 analogical to CAT activity. The increase in the activity of POX and SOD at 300 mM NaCl in
S. europaea was activated by the peroxidation of lipid membranes [
49]. The highest peak intensities of POX activity were observed for
S. europaea compared with
A. macrostachyum and
S. fruticosa from the same ecological habitat indicating the importance of POD activity in salinity tolerance strategy [
50]. Kumar et al. [
51] documented also that APX and POX are one of the main strategies for halophytes to control ion fluxes under high salinity for ten halophytic species.
Dehydroascorbate reductase (DHAR) and monodehydroascorbate reductase (MDHAR) are essential enzymes in the ascorbate-glutathione cycle, a key part of plant antioxidant defense mechanisms. DHAR and MDHAR regenerate strong antioxidant ascorbate utilizing dehydroascorbate (DHA) and monodehydroascorbate (MDHA) to maintain the pool of ascorbate in the cell [
52]. The activity of both APX and MDHAR are enhanced in salt-stressed plants compared with unstressed plants. The addition of external ascorbic acid and tocopherol to plants affected by salt further heightened the activities of these enzymes compared with untreated salt-stressed controls [
53]. DHAR and MDHAR activity was increased after long-term salinity stress for
S. crassa. Additionally, the expression of these enzymes was found to increase 2–3 fold with increasing salinity in the halophytes
Urochondra setulosa and
Dichanthium annulatum [
54].
To keep ROS levels within the tolerable range, plants also utilize low molecular weight non-enzymatic antioxidants such as ascorbate (AsA), glutathione (GSH), tocopherols, and flavonoids (
Figure 2). Studies Anjum et al. [
47] indicate a direct correlation between a plant’s tolerance to salinity and the levels of these antioxidants. AsA and GSH, two frequently occurring non-enzymatic antioxidants in plants, are present in all major compartments of plant cells, including the cytoplasm, apoplast, and chloroplast, with the ability to scavenge free radicals [
55]. Their significance in enhancing salinity tolerance has been well documented. Ascorbate is a multifunctional cellular compound with an antioxidant and cofactor function. AsA is thought to play a role in mitigating the effects of salt stress by maintaining the osmotic balance in cells [
56].
Sphaerophysa kotschyana as reported by Yildiztugay et al. [
57] and
Limonium stocksii as reported by Hameed et al. [
58], both halophytic species, demonstrated an increase in AsA and GSH levels in response to salinity. Tocopherols, also known as vitamin E, are lipid-soluble compounds found in four distinct forms and are known to serve as an effective antioxidant defense for biological membranes [
7].
Figure 2. Essential non-enzymatic antioxidants and their multifunctional role. Ascorbate, glutathione, tocopherols, and flavonoids function as protection molecules against oxidative stress in the cell were marked. As non-enzymatic antioxidants cooperate to neutralize ROS and their negative impact on cells and by additional function (listed in the frame) mitigate abiotic stress. Created with BioRender.com.
The α-tocopherol, as a dominant form of vitamin E in the green tissues of plants, plays a crucial role in reducing the production of reactive oxygen species in the chloroplast under environmental stress conditions [
59]. Ellouzi et al. [
43] demonstrated an impressive antioxidant capacity of
Cakile maritima correlated with the retention of a significant amount of α-tocopherol [
49]. However, the level of α-tocopherol did not change significantly under salt stress, which implies that
Crithmum maritima might neutralize the ROS production through direct quenching. The antioxidant role of phenolic compounds, including the subgroup flavonoids is particularly important in halophytes exposed to high salt conditions generating oxidative stress. The high content of flavonoids was noticed for
Salicornia europaea,
Crithmum maritimum L.,
Mesembryanthemum edule, and
Juncus acutus [
60].
The collaborative efforts of enzymatic and non-enzymatic antioxidants help maintain the levels of various ROS within a critical range necessary for regulating a variety of plant processes [
8]. For example, ROSs are involved in the regulation of seed germination and dormancy [
7], growth and development [
61], stress acclimation, and programmed cell death [
61]. However, these benefits are strictly dose-dependent. During periods of environmental stress, the production of ROS surpasses the cell’s ability to eliminate them, leading to elevated levels of ROS in the cell. This, in turn, results in oxidative damage to various cellular components such as nucleic acids, membrane lipids, and proteins [
8]. Therefore, an effective antioxidant system is crucial for plants to cope with salinity stress, maintaining a crucial balance between the production of harmful ROS and their removal protects plant cells from potential oxidative damage. Furthermore, in response to salinity stress, some plants may even boost the production or activity of specific antioxidants to establish their ability to effectively manage this challenging condition [
27,
41].