Towards a Sustainable Agriculture: Strategies Involving Phytoprotectants against Salt Stress

Abstract

Salinity is one of the main constraints for agriculture productivity worldwide. This important abiotic stress has worsened in the last 20 years due to the increase in water demands in arid and semi-arid areas. In this context, increasing tolerance of crop plants to salt stress is needed to guarantee future food supply to a growing population. This review compiles knowledge on the use of phytoprotectants of microbial origin (arbuscular mycorrhizal fungi and plant growth-promoting rhizobacteria), osmoprotectants, melatonin, phytohormones and antioxidant metabolism-related compounds as alleviators of salt stress in numerous plant species. Phytoprotectants are discussed in detail, including their nature, applicability, and role in the plant in terms of physiological and phenotype effects. As a result, increased crop yield and crop quality can be achieved, which in turn positively impact food security. Herein, efforts from academic and industrial sectors should focus on defining the treatment conditions and plant-phytoprotectant associations providing higher benefits.

1. Introduction

Plants are increasingly subjected to a variety of environmental stresses that adversely impacts plant growth and productivity [1]. In a recent report, the food and agricultural organization (FAO) highlighted the necessity of devising strategies to face the impacts of climate change on agriculture and food security worldwide [2]. Among environmental factors, salinity has a major impact, as most crop plants are salt stress-sensitive [3,4].

Soil salinity includes saline, sodic and alkaline soils, characterized by high salt content, high sodium cation (Na⁺) content, and high pH, respectively [5]. Soil salinity affects approximately 800 million hectares of farmlands on a world scale [5]. Saline soil is characterized by an electrical conductivity of 4 dS m⁻¹ generating an osmotic pressure of about 0.2 MPa and decreasing the yields of most crops significantly [4]. Increased water scarcity in semi-arid areas, as a consequence of both climate change (low rainfall and high temperatures) and population growth, will increase secondary salinization, i.e., salinization, due to human activity. In this sense, the use of unconventional water resources, such as municipal wastewater from wastewater treatment plants (WWTP) is one of the most important and current practices for sustainable agriculture. WWTP supplies an important amount of beneficial nutrients, which can improve soil fertility. However, its use may also result in serious problems associated with salinity and high boron concentrations [6,7,8,9,10,11,12,13].

Halophytes (1% of the world’s flora) are plants able to survive and reproduce in saline environments of 200 mM NaCl or more. Relevant examples include Salicornia sp, Spartina alterniflora Loisel, Anemopsis californica Nutt. and Atriplex sp., among others. Facultative halophytes occur on slightly saline soils, and include many species of Poaceae, Cyperaceae and Juncaceae families such as Glaux maritima L., Plantago maritima L., Aster tripolium L. and Chenopodium quinoa Kuntze. The major group of plants are glycophytes or halophobic, which represent 98–99% of the world’s flora. Within them, the vast majority are non-resistance to salt, and therefore, unable to successfully grow in saline soils. However, a small number of glycophytes (facultative glycophytes or indifferent to salt) may develop some resistance to salt and show higher levels of sodium in their tissues. As representative examples of this latter group, there are ecotypes of Festuca rubra L., Agrostis stolonifera L, Phragmites communis Cav., species or subspecies of Puccinellia, Lotus, Atriplex hastata L., Vigna unguiculata L. Walp. and sugar beet [14,15].

In the plant, salt stress first causes an ion-independent growth reduction driven by stomatal closure and inhibition of cell expansion, and in the long term, by ion toxicity, due to a cytosol over-accumulation of Na⁺ and Cl^-. This leads to a nutrient imbalance and the interference with many physiological processes as a lower energy production through photophosphorilization and phosphorylation in the respiratory chain, and an affected assimilation of nitrogen and protein synthesis, which in turn cause premature senescence and ultimately cell death [16]. In addition, salt stress is coupled with an oxidative stress, due to an over-accumulation of reactive oxygen species (ROS) at subcellular level [17,18,19,20]. As a consequence, under saline conditions, plants activate a variety of physiological and biochemical mechanisms to acquire resistance against saline stress, which include morphological changes, ion distribution, photosynthesis, and antioxidative metabolism activation [increased activity of reactive oxygen species (ROS)-scavenging enzymes and non-enzymatic antioxidant contents] [21,22,23,24].

To fulfill increasing food demands of the population, strategies towards enhanced crop resilience to environmental challenges are desirable. Over the past few decades, the use of phytoprotectants has become a hotspot in the amelioration of salt stress in crop plants. The present review compiles knowledge on the use of arbuscular mycorrhizal fungi (AMF), plant growth-promoting rhizobacteria (PGPR), osmoprotectants, melatonin, phytohormones, ROS and reactive nitrogen species (RNS) as natural phytoprotectants under salt stress.

2. Phytoprotectants of Microbial Origin

2.1. Arbuscular Mycorrhizal Fungi (AMF)

Arbuscular mycorrhizal fungi (AMF) are beneficial microorganisms which increase soil fertility of agricultural and natural ecosystems [25]. The term mycorrhiza applies to about 6000 different fungi, which develop symbiotic interactions with a huge range of plants. They build a direct physical interaction between plant roots and soil and represent an essential part of natural habitats [26]. Formation of AMF symbiosis brings numerous advantages to both symbionts, since the plant improves its uptake of water and mineral nutrients, while the fungus uses up to 20% of net photosynthate to support its growth and respiration processes [25]. Moreover, AMF may enhance plant resistance to biotic and abiotic stresses, among other functions [25,27].

AMF are able to live in saline ecosystems, since some of them have been isolated in these conditions [28], even though their growth and colonization of plants is usually reduced, due to specific ion toxicity and osmotic effects of salts [29,30,31]. AMF symbiosis has been found to enhance salinity tolerance in different host plants, such as cucumber, lettuce, tomato, maize, acacia and citrus [32,33,34,35,36,37]. AMF increase absorption of soil water through the mycelium and mobilization in the plant, enhance soil-root contact, reduce the deleterious effects of phytotoxic ions on the integrity of the plasmatic membrane and cell organelles, change phytohormone balance, increase antioxidant defenses (both enzymatic and non-enzymatic) and promote the expression of salt stress-related genes [37,38,39,40,41]. In addition, AMF may increase root hydraulic conductivity at low water potential [42], promote and change root system architecture, and enhance stomatal conductance by improving the gas exchange capacity [31,37,39,43]. Moreover, it has been found that in salt-stressed plants, chlorophyll synthesis is less interfered when plants are inoculated with AMF [31]. Thus, AMF can be implemented as phytoprotectants increasing growth potential and reducing fertilizer and water use. Herein, it is essential to make an accurate selection of AMF to ensure the effectiveness of the fungi species under salinity.

The mechanisms by which AMF are able to enhance salinity tolerance in plants are similar to those implied in drought conditions, since the first plant symptom under saline conditions is a water deficit in the plant, due to decreased water uptake. Inoculation with AMF results in a better soil structure under salt stress, due to intertwining of extraradical mycorrhizal hyphae and gluing of glomalin produced by AMF and roots [44], thereby providing relatively higher available water content [45]. Moreover, it improves nutritional status by the transportation of mineral nutrients across the root-soil interface, generating resistance in plants under osmotic stress conditions. Several studies show the enhanced nutritional status of AMF treated plants under osmotic stress [29,37,46]. AMF act as an initial defensive barrier, discriminating phytotoxic ions, and thus, favoring the absorption and selective transport of beneficial mineral nutrients. AMF reduces the mobilization of Na⁺ to plant tissues avoiding its concentration to reach harmful levels, because of their capacity to maintain this ion in structures, such as root cell vacuoles, vesicles and intraradical mycelium [27]. These fungal mechanisms help in the compartmentalization of toxic ions and preventing their accumulation from reaching plant tissues [47]. In this regard, lower concentrations of leaf Na⁺ were observed in mycorrhizal than in non-colonized plants subjected to saline stress [30,37,48]. Some authors claim that the decrease in leaf Na⁺ content could be partially due to a dilution effect, through an increase in dry matter accumulation [49]. AMF can reduce Cl^- and Na⁺ content and enhance beneficial nutrients, such as P, N, K⁺, Ca²⁺ and Mg²⁺ in different plants, such as citrus, wheat and ornamental plants [37,48,50], which consequently increase the K⁺/Na⁺, Mg²⁺/Na⁺, Ca²⁺/Na⁺ and K⁺/N ratios [30,51]. Improved plant nutrition by AMF symbiosis allows cells to regulate flowing ions more efficiently, enhancing nutrient uptake by transport or availability through mycorrhizal hyphae, conducive to improve salt resistance of mycorrhizal plants. Phosphorous (P) levels in the plant display significant increases by AMF colonization [25]. The better P nutrition of mycorrhizal plants has been related to the higher salinity tolerance of AMF plants in citrus and Jatropha curcas L. plants. [37,52]. In general, a better P nutrition in AMF-inoculated plants could improve their antioxidant enzyme activities [53], although the response of the individual enzymes varies in function of the fungal species and the hostplant interactions [54]. In this context, AMF symbiosis has been observed to enhance the antioxidative system in order to cope with the ROS generated by salinity in Olea europaea L. [55], and tomato [53]. Moreover, in citrus plants, it has been suggested that AMF decrease the antagonistic effect of Na⁺ on Mg²⁺ uptake, enhancing the chlorophyll content, and hence, improving photosynthetic efficiency and plant growth [37]. As a result of the mechanisms described above, AMF enhance plant nutrient absorption, which helps to withstand saline stress conditions [31,51]. In this sense, the higher water uptake is facilitated, since the external hyphae network allows host plants to uptake more water from soils, elevating leaf water potential [39,43,45].

On the other hand, some authors reported adverse effects of AMF when plants are fertilized [56,57]. Some recent studies have been focused on the feasibility of using reclaimed municipal wastewater from WWTP (as an alternative water resources) together with AMF inoculation [50,58,59]. In addition to high levels of Na⁺ and Cl^-, and depending on its source and treatment, reclaimed wastewater may contain high levels of beneficial elements, such as P, K⁺, Ca²⁺ and Mg²⁺, which can alter AMF function [58]. Gómez-Bellot et al. [59] found that the nutrient content of saline reclaimed water improved the nutritional status of euonymus plants, but no additive effect was observed when AMF was added to the same plants. In fact, the beneficial mycorrhizal effect was clearer in mycorrhizal plants irrigated with conventional water. These results are not in accordance with those found in lettuce [58], where irrigation with similar reclaimed wastewater (4 dS m⁻¹) and applying the same AMF colonization had a synergetic effect on promoting plant growth. Nevertheless, the inoculation of the same AMF using reclaimed wastewater with higher salinity (6 dS m⁻¹), improved the physiological behavior of laurustinus plants growing in soil, by ameliorating the negative effects of toxic ions [50]. Moreover, results suggested that plant mycorrhizal inoculation was more effective when the saline water for irrigation displayed a higher concentration of toxic ions, but lower content of beneficial nutrients [58,59].

At the molecular level, the elucidation of the mechanisms implicated in AMF symbiosis under salt stress has advanced over the past decade [60,61,62,63,64]. The stress perception is channelized inside the nucleus, resulting in the expression of multiple stress-responsive genes, related to cellular protection against the stress (e.g., antioxidants, late embryogenesis abundant proteins and chaperons) or implicated on the perception, transmission and transcription of the signal [38,60,61]. Recent studies revealed the molecular mechanisms of high K⁺/Na⁺ ratio in mycorrhizal plants, which are based on the induced expression of genes involved in K⁺ acquisition and Na⁺ extrusion to the soil [61,62,63,64]. For example, in Oryza sativa L., up-regulation of OsNHX3 (sodium/hydrogen exchanger) by AMF allows the compartmentalization of Na⁺ into the vacuole [63]. In black locust, AMF induced the expression of genes encoding for membrane transport proteins and aquaporins (SOS1, HKT1, NHX1, and SKOR), which contributes to maintaining ion homeostasis [64]

The current trend towards the use of biological products, such as mycorrhizal inoculation is helping to increase the efficiency of intensive agriculture [65,66]. Nowadays, the combination of AMF inoculation and controlled deficit irrigation in drought or salinity conditions are increasingly applied in arid and semi-arid areas [67,68,69,70,71] However, in order to be an effective and sustainable tool for agriculture, numerous factors have to be taken into consideration. Thereby, the fungal colonization in open-field experiments is more productive in terms of root biomass experiments, which is associated with the growth constrains of containerized roots in a greenhouse. Contrarily, shoot biomass, plant nutrition and yield seem to be independent on the location factor [72]. Another relevant factor determining the applicability of the AMF-plant association is the source of inoculant. In this sense, native AMF obtained from saline soils may cope with salt stress more efficiently compared to other AMF [73,74]. Therefore, the type of stress may determine the type AMF. Moreover, aspects, such as AMF evaluation under multiple environmental stressors, and the identification of specific AMF strain/plant species combinations, should be taken into account to obtain the most effective responses. Herein, it was observed that single species inoculation experiments use to confer higher benefits to plants in greenhouse trials, whereas, in open-field experiments where multiple stresses are acting, a more complex fungal community may provide a better output [75].

2.2. Plant Growth-Promoting Rhizobacteria (PGPR)

Plant growth-promoting rhizobacteria (PGPR) [76] referred to free-living soil bacteria that establish beneficial associations with plants. Around 1 to 2% of bacteria are classified as PGPR [77]. Among the diverse genera that compose the group, Pseudomonas and Bacillus spp are prevalent [78]. Independently on the strategy to promote plant growth, PGPR colonize either the root surface, the radicular tissues, or the rhizosphere [79]. Their beneficial action relies on indirect and direct mechanisms. The indirect stimulation of plant growth occurs by competing against plant pathogen, by means of antagonistic compounds produced by the PGPR or by induced resistance to pathogens in the plants [80,81,82,83,84,85]. This may include the production of antibiotics [86,87], lysing enzymes as proteases, cellulases, glucanases, chitinases, lactonases [88,89,90], siderophores to chelate iron [91,92] or other toxic compounds as cyanide [93,94], as well as volatiles compounds, such as dimethyl disulfide and 2-methylpentanoate [95,96]. On the other hand, the direct growth promotion occurs by increased nutrient availability (by nitrogen-fixing or solubilization of P and K⁺, among other nutrients), altered phytohormone and enzymatic production [97,98,99], and modulated osmotic balance [100,101]. PGPR has also been associated with modulation of ethylene level, increased nutrient solubilization and root absorption and production of phytohormones [102]. In addition, Mohamed and Gomaa [103] found that PGPR enhanced total chlorophyll pigments in radish plants grown under salinity, due to enhanced levels of proline and free total amino acids, which slowed down the chlorophyll degradation.

In agreement with the mechanisms described above, numerous studies have reported improved vegetative development and increased yield of different plant species by the utilization of PGPR [104,105,106,107]. Likewise, PGPR have also been applied to reduce the use of chemical fertilizers and pesticides, as a consequence of the more efficient nutrient uptake and the plant protection conferred by PGPR [108,109]. This results in improved economic viability, soil fertility, and environmental sustainability [110,111,112]. In this sense, several PGPR formulations are currently available as commercial products for agricultural production [113,114]. The ability to develop plant responses to certain PGPR may rely on the specificity of the interaction between the bacteria and the plant, and on the soil conditions [115,116]. Hence, efforts are focused worldwide on evaluating a wide range of rhizobacteria conferring resilience against environmental stressors, such as salt stress [117,118,119,120]. Overall, physiological and chemical responses in the modulation of abiotic stress in PGPR-inoculated plants have been termed as “induced systemic tolerance” (IST) [121]. Recently, a meta-analysis study has reported that PGPR inoculation induces more ostensible plant responses in non-optimal conditions [106]. In addition, PGPR effects mitigating salt stress have also been noticed in early plant stages of canola, lettuce, rice, chickpea and mung bean [122,123,124], wheat seedlings [125], and potato [126].

PGPR-inoculated plants grown under salinity conditions generally display an improved nutrient and water balance and enhanced rooting and plant growth [127]. Na⁺ reduction in leaves has been observed by PGPR inoculation of wheat [128]. Likewise, an altered selectivity of Na⁺, K⁺ and Ca²⁺ have been reported, resulting in an increased K⁺/Na⁺ ratio, and N, P, and K⁺ uptake in several crops, such wheat and maize [128,129]. In other cases, inoculated plants (soybean, lettuce and pepper) maintained their osmotic homeostasis by higher proline levels [130,131,132]. Moreover, an overall increase in the water use efficiency was observed in PGRP-inoculated plants under salt stress [117]. Furthermore, enhancement in the activities of antioxidant enzymes to cope with salinity-induced oxidative stress has been reported in lettuce [131], wheat plants [133], and Phaseolus vulgaris L. [134]. In addition, several studies showed that certain PGPR diminish the biosynthesis of salt-induced ethylene in several crops, such as tomato, canola and maize treated plants [117,135,136].

To date, our knowledge on beneficial microbes diversity, mechanism of action, application, and also physiological and biochemical inoculated plant responses, has facilitated the emergence of numerous PGPR commercial products. In the last decades, a large number of studies have demonstrated beneficial PGPR effects in both laboratory and greenhouse studies. However, fewer studies conducted field trials, and more comprehensive knowledge related to PGPR and plant interaction in different agro-ecosystems is required. The beneficial effects of PGPR are modulated by diverse growth habitats, native microbial communities and environmental conditions. For example, weather fluctuations influence the effectiveness of PGPR [137], which has to be considered in field experiments and in PGPR applications in agriculture. Regarding the molecular aspects of PGPR action, the expression of novel plant genes, such as SOS1, which codifies for a plasma membrane Na+/H+ antiporter [138], was found to be promoted in PGPR-applied wheat shoots and roots, resulting in improved salt stress resistance [139]. Moreover, the use of genes from halo-tolerant PGPR may confer salt tolerance to transgenic plants; for example, expression of Bacillus subtilis proline biosynthesis (ProBA) gene conferred salt tolerance in transgenic Arabidopsis through increased proline production [140]. Likewise, halotolerant properties of PGPR, such as Enterobacter ludwigii and other enterobacters rely on the role of Na+/H+ antiporters, such as NhaA. In conclusion, it is notable that PGPRs are postulated as an eco-friendly and versatile tool to stimulate crops to mitigate adverse conditions [89,127,141,142]. As our knowledge expands, more commercial formulations may be designed, accomplishing more sustainable agrarian systems.

3. Nitrogen-containing Phytoprotectants

3.1. Osmoprotectants: Proline, Glycine Betaine and Polyamines

The accumulation of osmolytes, i.e., low molecular weight water-soluble compounds, is a common strategy of many plants to cope with water deficit and salt stress as they confer stress tolerance without affecting cellular machinery [143]. Among them, the most common osmolytes are glycine betaine (GB), proline (Pro) and polyamines (PA) [143,144,145,146]. The accumulation of these molecules in the cytosol and organelles decreases plant internal osmotic potential, in response to the decrease in external soil osmotic potential, due to excessive accumulation of phytotoxic ions in the substrate. Consequently, the cell membrane and macromolecules integrity are maintained.

Pro biosynthesis from glutamate involves Δ1-pyrroline-5-carboxylate synthetase and glutamate dehydrogenase enzyme reactions. Pro accumulation depends on the balance between their biosynthesis and degradation rates, the latter catalyzed by the mitochondrial enzyme proline dehydrogenase (PDH) [147]. Studies on Pro mode of action have focused on its capacity to mediate osmotic adjustment, stabilize subcellular structures and ROS scavenging during salt stress. Likewise, increased Pro in response to stress contributes to maintaining the NAD(P)⁺/NAD(P)H ratio [148]. Yang et al. [147] reported Pro accumulation as a result of increased biosynthesis and inhibition of its degradation by H₂O₂.

Pro supplements were proven to confer salt tolerance and enhance plant growth by improving the antioxidative enzyme system, photosynthetic activity, water status, and consequently, plant performance in olive tree [149]. Moreover, Pro treated plants displayed lower soluble sugars content, which shows the role of Pro as osmoprotectant [149]. The application of 50-100 mM Pro in the irrigation water improved germination, seedling growth and chlorophyll contents of sorghum and wheat [150,151]. In another study, the application of seaweed extracts ameliorated the negative impacts of salt stress by increasing aminoacids, such as serine, threonine and Pro in roots of chickpea plant [152]. In soybean, 25 mM Pro applied as foliar spray increased nitrogen fixation and specific nodule activity [130]. Moreover, seed spraying of P. vulgaris at low Pro concentration (5 mM) alleviated oxidative stress and enhanced plant growth, increased carotenoids and ascorbate contents, and the endogenous levels of proline [153]. However, although the beneficial effects of Pro applied exogenously, toxic effects have also been reported. For example, Pro applied at high concentration in tomato resulted in ion imbalance and poor plant growth [154]. In rice, 30 mM Pro in the irrigation water ameliorated the negative effects of salt stress, whereas, higher concentrations (40-50 mM Pro) resulted in deleterious for plant growth [155]. Such toxic effect of exogenous Pro has been explained by the fact that high concentrations may mimic the effects of Cd, as reported in rice seedlings [156]

GB is a quaternary ammonium compound that is a highly effective osmoprotectant in many plant species in response to salt stress [143,144,145,146]. The metabolic pathway for GB biosynthesis in plants involves two enzymes, i.e., betaine aldehyde dehydrogenase (BADH) and choline monooxygenase (CMO), which are located in the chloroplasts [157]. This pathway takes place in some crops, like cotton and sugar beet, as well as in many drought-tolerant and highly salt-tolerant wild plants, including halophytes [158]. Evidence indicates that plants are able to translocate foliar-applied GB from their leaves to other organs mainly via phloem, where it may ameliorate stress tolerance by maintaining membrane integrity, adjusting cell osmolarity, and protecting the photosynthetic complex, especially photosystem II, among other mechanisms [159]. Furthermore, GB also acts in maintaining Rubisco activity and enhancing stomatal conductance [159]. GB accumulation has long been a target for engineering stress resistance. Remarkably, it has been found that transgenic carrot overexpressing BADH gene accumulated high amounts of GB, which in turn resulted in high salt tolerance at high salt concentrations (up to 400 mM) [160]. Likewise, the transgenic rice plants converted high levels of exogenously applied betaine aldehyde (up to 10 mol m⁻³) to glycinebetaine more efficiently than did wild-type plants acquiring greater resistance to salinity [161]. Conversely, transgenic rice plants overexpressing CMO gene were not effective for accumulation of GB and improvement of productivity, although displayed enhanced tolerance to salt stress in the seedling stage [162].

The use of GB applied exogenously to plants that accumulate little or none of this compound may alleviate the harmful effects of environmental stress [157]. There are several reports displaying the beneficial effects of exogenous application of GB on plant growth and final crop yield under salt stress or drought. Examples include those in rice, sunflower, maize, canola, eggplant, pepper, ryegrass, lettuce, safflower, pigeon pea, soybean, wheat and tomato [163,164,165,166,167,168,169,170,171,172,173,174,175]. Some reports have also shown that application of GB facilitated the accumulation of other osmotic substances, such as free Pro and soluble sugars in plant organs under salt stress conditions [168,176]. This has been attributed to a protective role of GB on key enzymes that catalyze the biosynthesis of soluble free proline [176].

PA are polybasic aliphatic amines extensively disseminated in plants and implicated in numerous cellular functions [177,178,179]. The most common PA present in halophytes are: cadaverine (Cad), putrescine (Put), spermidine (Spd) and spermine (Spm) [180,181]. These compounds can be located in all plant organs, being implicated in a plethora of metabolic processes, such as leaf senescence, morphogenesis, protection of photosynthetic tissues, growth and embryogenesis, serving as bioindicators of stress-tolerant genotypes [182,183,184,185,186,187,188]. Moreover, PA regulates the antioxidant machinery acting as free-radical scavengers [189], and contribute to abiotic stress signaling [190]. In these processes, PA have been proven to activate the expression of genes associated with stress response and establish the interaction with other metabolic pathways [190,191]. PA bind covalently to biomacromolecules to form bound PA, which usually exerts stronger physiological activity than free PA [192].

The use of exogenous PA and transgenic plants have demonstrated an important role of these compounds in a variety of adaptive responses to salt stress [185,193,194]. Exogenous application of Spm, Spd, Put and Cad resulted in improved plant growth by means of improved photosynthesis and activation of antioxidative metabolism, which ameliorate the negative effects of salt stress [195,196,197]. Ikbal et al. [184] reported that enhanced S-adenosylmethionine decarboxylase (SAMDC) activity would maintain high PA levels in salt-stressed micropropagated grapevine (Vitis vinifera L.) plants. The application of exogenous Spd (0.1 and 1 mM) enhanced salt tolerance (150 mM NaCl) of Panax ginseng C.A. Mey. plants by enhancing the antioxidant defenses and thus, alleviating salt-induced oxidative stress. As a result, Spd application increased plant growth, shoots and roots relative water content, and prevented chlorophyll degradation under salinity conditions [185]. More recently, Nahar et al. [187] observed that the pre-treatment of mung bean seedlings with PA—0.2 mM of either Put, Spd or Spm—improved the response to salt stress (200 mM NaCl) by reducing cellular Na⁺ accumulation, maintaining ion homeostasis, and by increasing the levels of antioxidant defenses, including ascorbic acid (AsA) and glutathione (GSH), as well as catalase (CAT), dehydroascorbate reductase (DHAR), glutathione peroxidase (GPX), and glutathione reductase (GR) activities [187]. Similarly, the up-regulation of SAMDC expression induced accumulation of Spd and Spm, resulting in an attenuation of salt stress in cucumber and grasses [198,199]. Under salt stress, the growth of transgenic rice seedlings overexpressing SAMDC was improved, in comparison to non-transformed rice plants, which was correlated with a three to four-fold increase in Spd and Spm [200].

In summary, exogenous application of osmoprotectants can be a suitable means to increase salt tolerance in different crop species by regulating osmotic potential, reducing the accumulation of phytotoxic ions and protecting essential macromolecules, such as proteins from the photosynthetic complex.

3.2. Melatonin

Melatonin (N-acetyl-5-methoxytryptamine) is a ubiquitous molecule derived from the aminoacid tryptophan which is present in almost all living organisms, including plants [201]. Melatonin was named because of its ability to pigment the skin of animals, such as fish, reptiles, and amphibians [202]. Numerous studies have demonstrated its role in seed germination, plant growth, organogenesis, and leaf and fruit senescence. All these functions might be mediated by the effect of melatonin up-regulated genes related to a multitude of cellular functions, including primary metabolism, redox processes, lipid and carbohydrate metabolism, cell cycle, DNA replication, photosynthesis, and senescence-associated genes, among others [203,204]. It also enhances the secondary metabolism and protecting the photosynthetic machinery. Moreover, it establishes crosstalk with plant hormones and polyamines, among other molecules, and regulates its own biosynthesis [205,206,207,208,209,210]. In addition, it is well established that melatonin helps to protect plants against different abiotic stresses, including salt stress [203,204,211,212].

Melatonin is synthesized by at least four different alternative biosynthetic routes, all implying serotonin as an intermediary, indicating that this molecule is a hub intermediate for this purpose in plants [213]. Each of the four known alternative routes for the biosynthesis of melatonin takes place in different organelles, where the production of the intermediates and melatonin occurred. Its chemical structure confers melatonin amphiphilic properties, which favors its movement through the plasma membranes and entry into different cellular compartments, especially chloroplasts, endoplasmic reticulum and cytoplasm [213].

In relation to salt stress, the functions of melatonin can be classified as follows: Activation of antioxidant defenses to protect plants against loss of water and physiological injuries associated with salinity, enhancement of photosynthesis, boost of ion homeostasis, regulation of plant hormone metabolism, and stimulation of polyamine metabolism pathway [212,214,215,216,217,218]. Under salt stress, the activities of ascorbate peroxidase (APX), peroxidase (POD), superoxide dismutase (SOD), CAT, GR, and GPX in maize seedlings treated with melatonin increased significantly compared to the levels on non-treated seedlings [215,219]. Similarly, the exogenous application of melatonin in Malus hupehensis Pamp. seedlings reduced hydrogen peroxide (H₂O₂) and superoxide radicals (O₂^.−) levels by activating the same antioxidant enzymes [220]. In addition, the contents of non-enzymatic antioxidants, such as GSH and AsA in cucumber subjected to salinity conditions were higher when ROS interacted with melatonin [216]. This evidence implies that exogenous application of melatonin could improve the antioxidant defenses (non-enzymatic and enzymatic) of plants mainly by removing the ROS generated by salt stress.

Globally, the application of exogenous melatonin helps plants to better perform photosynthesis by avoiding chlorophyll degradation and stomatal closure, due to salt stress [221,222,223]. The application of exogenous melatonin (50–150 μM) in cucumber significantly improved plant responses to salt stress by increasing maximum quantum efficiency of PSII, chlorophyll levels and leaf net photosynthetic rate [216]. Similarly, in watermelon, pretreatment with melatonin (50–500 μM) clearly favored salt tolerance by improving leaf net photosynthetic rate and stomata opening [224]. Melatonin significantly upregulated MdNHX1 and MdAKT1 transcript levels in M. hupehensis seedlings subjected to salt stress, providing a decrease in Na⁺ concentrations ⁺ and an increase in K⁺ that incremented K⁺/Na⁺ ratio [220]. In a similar way, melatonin provoked a remarkable increase in K⁺/Na⁺ ratio in shoots of maize seedlings when grew under salt stress [215]. Other genes, such as AKT, NHX, and SOS seem to be correlated with better ion homeostasis coherent with high K⁺ levels, and consequently, with a greater K⁺/Na⁺ ratio [225]. Melatonin also up- or down-regulates the expression of genes related to hormonal homeostasis, which encode for enzymes of the biosynthesis or degradation of plant hormones, such as indole-3-acetic acid, gibberellins (GA), abscisic acid (ABA) cytokinins, ethylene, salycilic acid (SA) and jasmonic acid (JA) [226,227,228,229].

In conclusion, melatonin participates in a variety of molecular stress mechanisms in plants related to the response to biotic and abiotic stresses. In general, increased melatonin levels—as a consequence of either a stressful situation or exogenous application—results in an improved stress tolerance mediated by changes in the hormone profile, antioxidative metabolism and expression of specific stress-responsive genes [208,210,227,230,231,232,233,234]. Recently, the novel characterization of a melatonin receptor in Arabidopsis opens up melatonin as a new phytohormone [235].

4. Phytoprotectants Related to Antioxidative Metabolism and Plant Hormones

4.1. Antioxidative Metabolism

Many studies have associated pre-treatments with different ROS, especially H₂O₂, reactive nitrogen species (RNS, especially nitric oxide (NO)), ascorbic acid (AsA), and plant hormones, such as gibberellins (GA) and salicylic acid (SA), with an improved resilience of plants against different abiotic stresses, such as salinity. Despite their harmful nature, H₂O₂ has a dual role in plant stress response; at high concentration, H₂O₂ is deleterious for the cell function, whereas, at low concentration it may act as a signal molecule in a variety of biotic and abiotic stresses [236,237]. Consequently, ROS production is tightly controlled in plant cells by enzymatic and non-enzymatic antioxidants. It has been reported that H₂O₂-priming seeds can induce salt stress tolerance by modulating the antioxidant defenses [238]. The pre-treatment of barley seedlings with low H₂O₂ concentrations (1 or 5 µM) for two days induced tolerance upon 150 mM NaCl treatment. This response was parallel with the induction of three SOD isoenzymes and the decrease in Na⁺ and Cl^- uptake [239]. The treatment of wheat seedlings with very low H₂O₂ levels (0.05 µM) improved its response to 150 mM NaCl in terms of plant growth (plant height, biomass and root length) coupled with a reduced O₂⁻ generation and lipid peroxidation levels and the increase of different antioxidant defenses [240]. More recently, [241] indicated that exogenous H₂O₂ could enhance salt tolerance in Vigna radiata L. plants by regulating the AsA and GSH metabolism.

AsA or vitamin C, as one of the most important antioxidants occurring in plants [242], plays an important role in ROS detoxification, providing protection and modulating other fundamental functions in plants under stress conditions [243,244,245]. AsA is also involved in key processes in plants, such as regulating photosynthetic capacity [246], flowering and senescence [247], and also as a co-factor for the biosynthesis and the signaling of several plant hormones (such as ethylene, JA, SA, ABA, and GA) under stressful environments [248]. AsA content and environmental conditions are linked in plants. As with other antioxidants, the variability of AsA content has been often considered relevant for explaining the different phenotype-dependent sensitivity against several stresses. It has been suggested that tolerance to stress is correlated with the capability of increasing AsA biosynthesis [243,249,250]. In this context, exogenous AsA is proposed to counteract the adverse effects of abiotic stress, when endogenous levels are not sufficient [251,252,253]. Thus, endogenous AsA can be increased by external treatments of AsA during different phases of development, applied on different organs (on roots, as a foliar spray or as seed priming), and in different types of crops, including vegetables, indeciduous fruits, oil-seeds, cereals and ornamental plants, among others. Exogenous application of AsA has been reported to enhance yield, growth and fruit quality [254,255,256,257], and more recently, it has been detected its potential effects on physiological responses of plants grown under abiotic stresses, such as salinity [258,259], and counteracting the adverse effects of NaCl when wheat grains were soaked with AsA [260]. Effectiveness of AsA in a wide concentration range (from 0.1 to 34 mM) has been studied. For instance, the addition of 0.5 mM AsA in tomato seedlings root medium attenuated the negative effects of salinity [261]. Particularly, 100 mg/L AsA applied on the root counteracted the adverse effects of salt stress on the growth of wheat under 150 mM NaCl [258], while 50–100 mg/l AsA applied as foliar spray protected the photosynthetic machinery from the damaging effects of salt stress in halophytes [262].

In addition to increased ROS production, salinity produces nitrosative stress in plants mediated by the overproduction of some RNS, such as NO and peroxynitrite (ONOO⁻) [263,264], to which peroxisomes are the main source [263]. However, the pre-treatment with a NO-donor (such as sodium nitroprusside, SNP) can improve the response of plants to NaCl-induced damage [265,266,267]. SNP is a small bioactive signaling molecule which plays multiple roles in plant growth and development under normal or stressful conditions [268,269]. SNP ameliorates salt stress acting as a ROS scavenger throughout the formation of ONOO^-, of less toxicity than NO [270] Moreover, SNP application stimulates proton-pump activity in the tonoplast, which in turn increases the K⁺/Na⁺ ratio [271]. Moreover, SNP regulates post-translational modifications of proteins involved in the cellular division [272]. The pre-treatment of Citrus aurantium L. plants with 100 µM SNP or 10 mM H₂O₂ for 48 h alleviated the physiological effects produced by 150 mM NaCl [265]. This response was linked to the regulation of protein carbonylation and S-nitrosylation of specific proteins [265]. Among the S-nitrosylated proteins there are involved in redox signaling and ROS scavenging, glycolysis, Calvin cycle, amino acids metabolism, photorespiration, and stress-related proteins. Some of the S-nitrosylated proteins were also subject of protein oxidation, including antioxidant proteins [(GST (glutathione S-transferase), SOD, peroxiredoxin), cytoskeleton proteins (tubulin, actin)], heat shock proteins (HSPs), translation and elongation factors, and metabolic proteins related to photosynthesis, glycolysis and the Krebs cycle [265]. The authors suggested the existence of a link between the different oxidative modifications events of proteins resulting from protein carbonylation or S-nitrosylation. They also pointed out that S-nitrosylation can prevent protein carbonylation events, thus, avoiding loss of function of proteins. In addition, S-nitrosylation of specific proteins can induce changes in protein function and/or structure. All these proteome changes, induced by H₂O₂ or NO treatments, can prepare the plants to cope with salt stress [265]. In soybean plants, the foliar application of 150 µM SNP increased relative water content, chlorophyll and ABA levels, and improved stomatal control under stress conditions (NaCl 200 mM) in soybean plants [266]. Moreover, at short-term (6 and 12 h), NO induced GST gene expression and GST enzyme activity under salt stress conditions. In addition, the pre-treatment of sunflower seeds with 250 µM SNP demonstrated that NO can modulate the GSH content and its redox state via GR activity in sunflower seedlings subjected to 120 mM NaCl [267]. Thus, NO can act as a regulator of GSH metabolism, modulating the activity of GSH-dependent enzymes [266,267,273]

4.2. Plant. Hormones (GA, SA)

SA is a phenolic plant hormone widely distributed in plants, playing an important role in the regulation of different physiological processes, such as seed germination, vegetative growth, photosynthesis, respiration, thermogenesis, flower formation, fruit yield, seed production and senescence [274]. It is widely documented that exogenous SA treatments improved plant growth and seed germination under saline stress in different plant species [275,276,277,278,279,280,281,282,283]. In general, SA-treated plants showed lower NaCl-induced symptoms and higher growth rate, chlorophyll contents, and photosynthetic capacity, as well as enhanced antioxidant capacity in relation to non-treated plants [275,276,277,278,279,280,281,282,283]. Hence, SA induces genes encoding antioxidants, chaperones and secondary metabolites, among others. However, evidence linking exogenous SA and resistance against salt stress remains controversial. In this sense, [284] observed that SA negatively affects the response of pea plants to NaCl stress, although low SA levels led to the induction of a pathogenesis-related protein (PR-1b) gene in leaves from NaCl-stressed pea plants. The induction of PR-1b gene could be an adaptive response in order to prevent an opportunistic fungal or bacterial infection in a weakness situation [284]. In this sense, the precise mechanisms of SA in the response of plants to salinity have to be unraveled, since SA effect seems to be dependent on the concentration, the plant species, the application mode, the physiological state of the plant during the application, as well as the level of salinity and the exposure time to NaCl. In Arabidopsis, SA has been reported to play a dual role: At low concentration, it induced antioxidant defenses, but excess application induced programmed cell death [285].

GA are a group of plant hormones involved in many developmental processes, such as seed germination, plant growth, flowering and fruit development [286]. Different studies showed that exogenous GA treatments can reverse the negative effects of salt stress. Kaur et al. [287] reported that low concentrations of exogenous GA₃ (6 µM) promoted seed germination and seedling growth of chickpeas under NaCl stress (75 mM). These authors also observed that GA₃ treatment increased α-amylase activity, which enhanced the starch mobilization, thus, leading to a better seedling growth [287]. Exogenous-applied GA₃ alleviated the effects of salt stress in tomato plants through a partial stomatal closure that reduced the upload of toxic ions to the shoots, and as a result, the appearance of toxicity symptoms [288]. Application of GA₃ also enabled the maintenance of photosynthetic capacity under salt stress by mitigating the salt-induced reduction of chlorophyll content [289]. Several studies have shown the positive effect of GA₃ in alleviating the toxic effects of NaCl on rice growth [290,291,292,293]. Exogenous application of 50 µM GA₃ improved the seed germination and early seedling growth of Arabidopsis in the presence of 100 mM NaCl. These positive effects of GA₃ was accompanied by an increase of endogenous SA levels, suggesting that the improved response of GA-treated plants under salinity conditions can be mediated by the modulation of SA levels [294].

5. Conclusions

Upon salt stress, typical effects in the plant are reduction of fresh weight, the appearance of chlorosis and necrosis and altered metabolic processes, such as photosynthesis, phytohormone regulation, respiration, nutrient balance, ion toxicity, and protein biosynthesis, which are generally mediated by oxidative stress. In recent years, the application of phytoprotectants, such as those of microbial origin (arbuscular mycorrhizal fungi and plant growth-promoting rhizobacteria), osmoprotectants, melatonin, phytohormones and antioxidant metabolism-related compounds has proven to be a sustainable agronomic practice to obtain plants more resistant to salinity avoiding losses in crop yields. Figure 1 shows an overview of factors involved in phytoprotectants application, their role in the plant and the output obtained from an agro-economical point of view. Beyond the phytoprotectant nature, other factors, such as plant or phenotype sensitivity towards a specific phytoprotectant, application form and targeted plant organ determine their applicability. Herein, efforts from academic and industrial sectors should focus on defining the conditions and associations providing higher benefits. Overall, phytoprotectants mode of action converge into the management of water and nutrient balance, increased photosynthesis efficiency and activation of enzymatic and non-enzymatic antioxidant defenses that leads to an alleviation of the oxidative stress produced in response to salinity. As a result, increased crop yield and crop quality are achieved, which in turn positively impact food security and consumer acceptance of the product. In this sense, improved tolerance of the plant to salt stress avoids growth abnormalities and provides greener leafy vegetables, which determine visual consumer preferences.

Table 1. Studies showing beneficial responses of plants of agronomic interest to phytoprotectants applications. The specific phytoprotectant, application mode and dose, plant species, and major effects in the plant in terms of physiological changes and phenotype are listed.

Type of Phytoprotectant	Agent/Treatment	Plant Species	Beneficial Effect
Microbial
AMF	Glomus mosseae	Zea mays	Higher dry weight of shoot and root, chlorophyll content, gas exchange capacity, and photosynthesis efficiency; improved water status [31]
	Glomus spp.	Cucumis sativus	Increased biomass and reduced wilting [32]
	G. mosseae	Solanum lycopersicum and Z. mays	Higher shoot and root dry matter yields, leaf area and P content [34,35]
	Rhizophagus irregularis or Funneliformis mosseae	Citrus spp.	Improved photosynthesis and nutrient acquisition [37]
	Glomus intraradices	Trigonella foenum-graecum	Improved nutrient acquisition and mitigation of ionic imbalance [38]
	Glomus spp.	Triticum aestivum	Higher plant growth, improved nutrient acquisition, and lower Na+ uptake [47]
	Glomus spp	T. aestivum	Enhanced growth, photosynthetic efficiency and protein synthesis; improved osmotic adjustment via compatible solutes accumulation. [48]
	Glomus iranicum	Viburnum tinus	Higher biomass, flower yield and water potential; decreased Na+ and Cl- contents [50]
	Gigaspora spp. or Glomus ssp.	Jatropha curcas	Higher dry weight of shoot and root, improved leaf water status, chlorophyll content and osmotic adjustment via compatible solutes accumulation; reduced leaf membrane damage [52]
	Glomus iranicum	Lactuca sativa	Improved plant growth, water-use efficiency, nutrient acquisition, net photosynthesis and stomatal conductance, and reduced Na+ plant uptake [58]
PGPR	Azospirillum brasilensis	L. sativa	Improved germination and vegetative growth [123]
	Aeromonas hydrophila, Bacillus spp.	T. aestivum	Increased dry matter yield of root, and restricted Na+ uptake [125]
	Bacillus pumilus or Bacillus. firmus	Solanum tuberosum	Increased photosynthetic efficiency, proline accumulation, and activity of ROS-scavenging enzymes [126]
	Bacillus subtilis	T. aestivum	Increased plant yield and N, P and Ca2+ leaf content, and reduced leaf Na+ content [128]
	Azospirillum spp	Z. mays	Restricted Na+ uptake and enhanced uptake of K+ and Ca2+; increased protein concentration [129]
	B. subtilis or Serratia sp	Glycine max	Increased antioxidant activity, the concentration of proline and specific nodule activity [130]
	Pseudomonas mendocina	L. sativa	Higher shoot biomass, leaf ratio of K+/Na+ and leaf proline content; enhanced antioxidant enzyme activities [131]
	Bacillus sp.	Capsicum annuum	Higher plant biomass, leaf proline content, and enhanced antioxidant enzyme activities [132]
	Serratia marcescens	T. aestivum	Enhanced growth, accumulation of osmoprotectants and antioxidant enzyme activities [133]
	Azospirillum brasilense and Rhizobium sp.	Phaseolus vulgaris	Promoted root branching [134]
	Pseudomonas putida	Brassica napus	Improved shoot and root fresh and dry weights [135]
	Pseudomonas syringae, Enterobacter aerogenes, or Pseudomonas fluorescens	Z. mays	Higher K+/Na+ ratio, relative water content and chlorophyll contents [136]
Nitrogen-containing phytoprotectants
Proline	25 or 50 mM in Hoagland’s solution	Olea europaea	Improved plant growth and ameliorated photosynthetic activity, antioxidative enzyme activities and leaf water status [149]
	50 mM or 100 mM in irrigation water	Sorghum bicolor	Improved germination, seedling growth and chlorophyll contents [150]
	50 or 100mM as foliar spray to the seedlings	T. aestivum	Improved germination, plant growth and chlorophyll contents [151]
	5 mM as seed spray	P. vulgaris	Alleviated oxidative stress and enhanced plant growth, increased antioxidant enzyme activities, carotenoids concentration, ascorbate content, and endogenous proline [153]
	30 mM/L in irrigation water	O. sativa	Higher K+/Na+ ratio and growth in seedlings [155]
	25 mM as foliar spray	G. max	Increased nitrogen fixation and specific nodule activity [173]
Glycine betaine	15 mM in nutrient solution	O. sativa	Increased relative water content, antioxidant enzyme activities, and reduced lipid peroxidation [163]
	50 mM as foliar spray	Helianthus annuus	Increased plant growth and improved photosynthetic rate [164]
	50 or 100 mM as foliar spray	Z. mays	Improved growth and water status, especially when applied at vegetative stage [165]
	1 or 5 mM during imbibition and seedling growth	B. napus	Improved fresh and dry weight of plant [166]
	50 mM as foliar spray	Solanum melongena	Improved growth, photosynthetic rate, transpiration, stomatal conductance, and yield of plant and fruit [167]
	Imbibition in 10 mM	Piper nigrum	Improved germination and radicle emergence, reduced lipid peroxidation and elevated superoxide dismutase enzyme activity [168]
	20 mM in Hoagland’s nutrient solution	Lolium perenne	Reduced electrolyte leakage and lipid peroxidation, suppressed Na+ accumulation and increased shoot K+/Na+ ratio [169]
	25 mM as foliar spray	L. sativa	Increased dry matter, antioxidant enzyme activities and total phenolic contents, and altered contents of organic acids and amino acids [170]
	Imbibition in 30 or 60 mM	Carthamus tinctorius	Increased germination rate and decreased lipid peroxidation [171]
	25 mM as foliar spray	G. max	Increased nitrogen fixation and specific nodule activity [173]
Polyamines	0.5 mM Put and 0.5 mM Spd	Hordeum vulgare	Restored root tonoplast function in seedlings by increasing phospholipids, endogenous polyamines and activities of H+-ATPase, H+-PPase and vacuolar Na+/H+ antiport [182]
	1mM Spd	O. sativa	Partial restore of H+-ATPase activity and increased K+/Na+ ratio [183]
	0.01, 0.1, or 1mM Spd in nutrient solution	Panax ginseng	Improved seedling growth by preventing chlorophyll degradation, increasing endogenous polyamines levels, and antioxidant enzyme activities [185]
	0.2 mM Put, Spm or Spd	Vigna radiata	Improved plant growth and nutrient homeostasis, decreased ROS accumulation and increased enzymatic and non-enzymatic antioxidant defenses [187]
	8 mM Put	Cucumis sativus	Improved plant growth and stimulated glycolysis and Krebs cycle pathways of leaves [188]
	100 mg/L Spd, 150 mg/L Spm or 150 mg/L Put as foliar spray	G. max	Enhanced seedling length and root growth, and peroxidase and catalase activities [195]
	Combination of 1 mM Put, 1 mM Spd and 1 mM Spm as foliar spray	Arachis hypogaea	Improved plant height, branching, dry weight, chlorophyll contents, and antioxidant enzyme activities, and reduced relative electrolytic leakage and lipid peroxidation [196]
	1 mM Spd as foliar spray	Cucumis sativus	Enhanced tolerance to salinity by regulation of the metabolic status of polyamines [198]
Melatonin	Seed coating in 50 or 100 µM	G. max	Promoted soybean growth (leaf size and plant height), production and fatty acid content [203]
	10 or 20 µM in solid medium at seedling stage	O. sativa	Reduced chlorophyll degradation, suppressed the transcripts of senescence-associated genes, delayed leaf senescence and cell death; enhanced antioxidant protection by melatonin role as a potent free radical scavenger [204]
	1 µM in Hoagland’s nutrient solution	Citrus aurantium	Reduced toxicity symptoms in leaves (chlorosis, dehydration, necrosis, twisted leaves) lipid peroxidation, and electrolyte leakage; increased photosynthetic pigments, Cl- accumulation in leaves and roots, glutathione levels and superoxide dismutase activity in roots [211]
	1 µM in Hoagland’s nutrient solution at the seedling stage	Z. mays	Increased plant dry matter, K+/Na+ ratio, net photosynthetic rate, chlorophyll contents, antioxidant enzyme activities, and decreased electrolyte leakage and lipid peroxidation [215]
	50 or 150 µM in the Hoagland’s solution	C. sativus	Increased net photosynthetic rate, maximum quantum efficiency of photosystem II, total chlorophyll content, and enhanced enzymatic and non-enzymatic antioxidant system [216]
	1 µM at the seedling stage	T. aestivum	Improved plant growth, shoot dry weight, leaf photosynthesis rate, maximum photochemistry efficiency of photosystem II, and chlorophyll content; reduced accumulation of H2O2 [217]
	Factorial combinations of melatonin in nutrient solution (0.1, 0.5, and 1 μM) and foliar spray (50, 100, and 200 μM) before salt stress	Ipomoea batatas	Reduced K+ content in tissues and Na+ content in the shoot; enhanced plasma membrane H+–ATPase activity, triacylglycerol breakdown and fatty acids β-oxidation [218]
	100 µM in the Hoagland’s solution	Z. mays	Alleviated ROS burst and protected photosynthetic activity in maize seedlings via increased antioxidant enzyme activities [219]
	0.1 µM pre-treatment	Malus hupehensis	Ameliorated growth inhibition and photosynthetic capacity; reduced oxidative damage [220]
	Seed soaking in 100 or 500 µM	T. aestivum	Enhanced growth, photosynthetic pigments and indol-acetic acid content of plants; in yielded grains, increased production, phenolic content, nutrient balance, protein content and antioxidant activity [221]
	1, 50, 100, 150, or 200 μM seedling pre-treatment	S. lycopersicum	Ameliorated growth and maintained net photosynthetic rate; decreased ROS accumulation [222]
	50, 150, or 500 μM in the nutrient medium at the seedling stage	Citrullus lanatus	Improved growth, photosynthesis and redox state in a dose-dependent manner [224]
	1 μM seedling pre-treatment	B. napus	Ameliorated seedling growth, reestablished redox and ion homeostasis (decreased ROS production and lipid peroxidation; increased K+/Na+ ratio [225]
	1 μM seed pre-treatment	C. sativus	Enhanced germination rate mainly via up-regulation of ABA catabolism genes and down-regulation of ABA biosynthesis genes [226]
Antioxidative metabolism-related
H2O2	1 or 5 μM seedling pre-treatment	H. vulgare	Decreased lipid peroxidation, endogenous H2O2 and proline contents in seedlings [239]
	0.05 µM in the Hoagland’s solution	T. aestivum	Decreased lipid peroxidation, superoxide anion production; enhanced antioxidant enzyme activities, glutathione and carotenoids contents [240]
	0.25 or 1 mM as foliar spray	Vigna radiata	Increased ascorbate and glutathione contents, reduced lipid peroxidation and electrolyte leakage [241]
	10 mM in Hoagland’s solution	C. aurantium	Recovered ascorbate redox state and inhibited protein carbonylation [265]
Ascorbate	150 ppm as foliar spray	Pennisetum glaucum	In combination with zinc sulfate increased the plant height and total plant biomass [257]
	50 or 150 mg/L in nutrient solution	T. aestivum	Enhanced growth, endogenous ascorbate level, catalase activity, photosynthetic capacity and leaf K+ and Ca2+ contents [258]
	2000 and 3000 rpm as foliar spray	O. europaea	Enhanced leaf characters, fruit set, yield and fruit quality traits [259]
	Seed soaking in 0.6 mM	T. aestivum	Decreased Na+ and increased K+, Mg2+ and Ca2+ in the different organs, increased shoot and root starch [260]
	0.5 mM in the nutrient medium	S. lycopersicum	Reduced lipid peroxidation and facilitated seedling recovery and survival after stress [261]
	20, 40 or 60 mM seed soaking for 3 h	Atriplex stocksii and Suaeda fruticosa	Ameliorate germination rate and seedling growth [262]
Sodium nitroprusside	10 µM in Hoagland’s solution	C. aurantium	Increased glutathione redox state, inhibited protein carbonylation, enhanced antioxidant enzyme activities, ameliorated DNA oxidation and leaf chlorotic symptoms [265]
	150 μM sprayed to seedlings	G. max	Reduced leaf chlorotic damage, improved stomatal control, increased chlorophyll content, ABA content, GST activity and root water content [266]
	250 μM in Hoagland’s solution to seedlings	H. annuus	Decreased ROS accumulation and stimulated melatonin accumulation [267]
	50 µM in Hoagland’s solution	S. lycopersicum	Up-regulated antioxidant metabolism, osmolyte synthesis, and metabolite accumulation [269]
	0.03 or 0.06 g/L as foliar spray	P. vulgaris	Increased plant dry weight, carotenoids and ABA contents, pod diameter and fiber percentage [270]
	0.2 or 1 mM SNP as foliar spray	C. arietinum	Decreased lipid peroxidation, H2O2 content and root water content; increased glutathione and ascorbate redox states [272]
Salycilic acid	1 mM as foliar spray	S. lycopersicum	Improved plant growth, increased K+ and Mg2+ contents in shoots and roots, glutathione, ascorbate and antioxidant enzyme activities; decreased lipid peroxidation and Na+ contents [276]
	100 µM pre-treatment in hydroponic medium	S. lycopersicum	Reduced leaf K+ content and leaf water potential; triggered ABA accumulation and free putrescine and spermine levels [277]
	0.5 or 1 mM as foliar spray	Gerbera jamesonii	Reduced lipid peroxidation and electrolyte leakage; increased superoxide dismutase and peroxidase activities [278]
	50 µM during imbibition	A. thaliana	Ameliorated germination by reducing oxidative damage [279]
	100 μM sprayed on seedlings	B. napus	Increased ascorbate and glutathione contents, glutathione redox status and antioxidant enzymes activities [280]
	100 μM as foliar spray	Gossypium hirsutum	Increased growth rate, net photosynthetic rate, transpiration rate and reactive oxygen species-scavenging enzymes activities; reduced H2O2 accumulation in seedlings, leaf electrolyte leakage; alleviated inhibition of plasma membrane H+-ATPase [281]
	0.5 mM as foliar spray	Brassica juncea	Improved growth, photosynthesis, activity of enzymes of ascorbate-glutathione cycle and glutathione content [282]
	0.1 or 0.5mM pre-treatment in the growth medium	Medicago sativa	Alleviated plant growth and photosynthetic capacity, increased H2O2-scavenging enzyme activities [283]
Gibberellic acid	3, 6, 9 or 12 µM in the solid medium	C. arietinum	Reduced activities of amylase 1 and amylase 2 and sucrose uptake in cotyledons, increased starch mobilization [287]
	100 mg/L in the nutrient solution	S. lycopersicum	Reduced stomatal resistance; enhanced plant water use; increased fruit number [288]
	100 mg/L as foliar spray	T. aestivum	Ameliorated decrease of dry weights of shoot and root, plant height, leaf area and net photosynthesis rate; increased grain size, but not grain yield [289]
	10 ppm pre-treatment as foliar spray	O. sativa	Increased growth; reduced net accumulation of Na+ and Cl- maintained high level leaf K+ accumulation [291]
	100 µM during germination	O. sativa	Ameliorated germination and growth inhibition [292]
	10 µM pre-treatment in hydroponic medium	O. sativa	Up-regulation of lipid biosynthesis and increased chlorophyll concentration [293]
	50 μm during imbibition	A. thaliana	Improved germination and seedling establishment; increased salicylic acid levels [294]

AMF, arbuscular mychorrhizal fungi; PGPR, plant growth-promoting rhizobacteria; Put, putrescine; Spd, spermidine; Spm, spermine.

shows the literature collected in this review concerning the exogenous application of phytoprotectants, which resulted in beneficial effects in the plant. The type of phytoprotectant, application mode and dose, plant species, and major effects in the plant in terms of physiological changes and phenotype are listed.

Further investigation should be conducted for understanding the physiological and molecular mechanisms involved during the action of phytoprotectants on plants and their effectiveness. In addition, aspects, such as phytoprotectant evaluation under multiple environmental stressors, or innovative approaches, such as the combination of phytoprotectants and intercropping systems should be taken into account. As our knowledge expands, more commercial formulations may be designed, accomplishing more sustainable agrarian systems.