Role of Exogenous Nitric Oxide in Protecting Plants against Abiotic Stresses

Abstract

The continuously changing environment has led to devastating effects on the normal growth and development of plants. This necessitates the understanding of different components that can be involved in alleviating these effects. In the last two decades, nitric oxide (NO) has been largely focused on as a molecule whose endogenous production and exogenous supply lead to several molecular and physiological changes in plants under stressed conditions. Although its role as a signaling molecule in endogenous production has been largely discussed, its function in dealing with contemporary abiotic stress conditions on exogenous supply remains comparatively less explored. There is growing evidence that NO plays a critical role in many physiological processes; however, there is debate about the exact mechanism(s) through which NO lessens abiotic stress on external supply. In this review, we discuss the studies that were focused on observing the effect of exogenous NO on different abiotic stresses including heavy metal stress, element deficiency or toxicity stress, salt stress, drought stress, ultraviolet radiation stress, waterlogging stress, and chilling stress. Though the positive effects of endogenous NO have been discussed in brief in different sections, the focus of the review is to discuss the effects of exogenous NO on plant grown under abiotic stresses. Deciphering the underlying mechanism of exogenous NO treatment may open up new ideas that can suggest the successful application of NO in agricultural regions to reduce the damaging influences of different abiotic stresses.

1. Introduction

Plants continuously deal with several biotic and abiotic stresses that largely impede their growth, consequently, enhancing global food insecurity [1]. Thus, it is necessary to decode the mechanism via which plants respond to different stress signals. The production of reactive oxygen species (ROS) is one of the major responses of plants to any abiotic stress condition [2,3]. However, the generated ROS can have beneficial or harmful effects reliant on their concentration and type [4]. Plants deal with ROS production via different signaling pathways and molecules such as nitric oxide (NO) [5].

Plant cells witness the endogenous production of the reactive free radical NO that is involved in the defense strategy against stress conditions. Several properties such as effortless dispersion through the cell membrane, existence as a free radical, and neutral charge make NO an appropriate molecule as a signaling messenger [6]. NO participates in developing different physiological responses in plants such as senescence, flowering, development, and germination [7]. The interaction of NO with different molecules changes the gene expression and functioning of proteins, thus influencing the phenotypic responses of plants. NO regulates the toxicity level of oxidative stress induced by ROS and its disastrous effects [8]. It leads to the enhanced formation of antioxidants and scavenges the produced ROS in respiratory pathways in mitochondria [9]. Both endogenous and exogenous NO are involved in different mechanisms related to plant development. While endogenous NO production regulates the production of hormones, facilitates stomatal movement, and impedes apoptosis, exogenous supply controls the production of superoxide anions [10,11]. These processes regulate responses toward abiotic stresses confirming the involvement of NO in increasing crop yield under a stressed environment [12]. In this review, we mainly discuss different studies that examined the effects of exogenous NO on the tolerance level of plants towards different abiotic stresses (Figure 1). A detailed conversation of its role in plant growth and underlying mechanisms may help in its successful utilization in developing stress tolerance in different crops.

2. NO Production in Plants

The mechanism of NO generation in plants is still up for debate despite the identification of important oxidative mechanisms by researchers [13]. Different oxidative and reductive routes are involved in the production of NO, with the former depending on the oxidation of aminated molecules and the latter on the reduction of nitrites to NO. The oxidative pathways involve the oxidation of aminated molecules such as L-arginine, polyamines (PAs), and hydroxylamine, while reductive pathways involve: (a) cytochrome-c oxidase; (b) nitrate reductase (NR); (c) plasma membrane-bound nitrite—NO reductase; (d) xanthine oxidoreductase (XOR), and/or reductase enzymes for the reduction of nitrites to NO and nonenzymatic reduction of nitrite in acidic conditions [14].

While NO synthase (NOS) has been identified as the enzymatic source of NO synthesis in animals, plant science is currently looking for the enzymes that produce NO in plants [15], and numerous potential sites have been proposed for its synthesis. Even though there is no evidence of homology between the NOSs and plant genome sequences, different studies including fungal elicitor xylanase treatment-based NO accumulation in tomato cell cultures and Arginase-dependent NO accumulation in Arabidopsis roots suggest the existence of some other forms in advanced plants [16]. Several studies reported the presence of NOS-like enzymes in plants with NOS-like activity in proteins extracted from purified organelles, cultured cells, and plant tissues [15,17,18,19]. Meanwhile, others revealed their absence in land plants exploring the homologies between the transcriptomes of land plants and the human neuronal NOS gene [20]. Such studies not only rejected the requirement of NOS-like protein for the production of NO in plants but also proposed that if a NOS-like enzyme is present in plants its structure will be different from the conventional mammalian NOSs [20].

NR which plays an important role in nitrogen assimilation in plants, is considered a crucial enzymatic source of NO [21]. It uses nitrate as its main substrate that is extensively dispersed in plants. NR passes electrons from NAD(P)H to molybdoenzyme NO-forming nitrite reductase (NOFNiR) which is also involved in the synthesis of NO from nitrite [22]. Different plant organelles, such as the chloroplasts, peroxisomes, mitochondria, and plasma membrane, have been recognized as probable sources of NO production under different abiotic stress conditions [23]. The generation of NO in chloroplasts due to abiotic stress has been first reported in tobacco leaves [24]. The addition of different substrates leads to the production of NO in chloroplasts in in vitro experiments [25]. Herbicide administration decreases plant photosynthetic activity, which significantly decreases the amount of NO in cotyledons and supports the idea that chloroplasts have an important role in NO synthesis [26]. An increment in Ferritin 1 expression due to NO synthesis in the chloroplasts of Arabidopsis under iron treatment suggested the Arginase-dependent production of NO [27]. While the Atnoa 1 mutant of Arabidopsis showed the absence of endogenous NO in chloroplasts, Brassica napus and Arabidopsis plants showed the chloroplastic production of endogenous NO and peroxynitrite (ONOO-) [28]. The drought stress condition reportedly increases NO synthesis in the mesophyll cells of pineapple plants [29]. The plasma membrane regulates the exchange of substances and information in and out of the cell and, thus, the molecules or enzymes situated in the plasma membrane are involved in the movement of stress signals in plants. NO production in the plasma membrane of tobacco roots occurs via nitrite:NO oxidoreductase (Ni:NOR) that reduces nitrite in the apoplast [30]. The H⁺-ATPase enzyme in the plasma membrane generates an electrochemical gradient that is responsible for ion transport across the membrane [30].

The concentration of NO in mitochondria is affected by the levels of ROS under different abiotic stress conditions [31]. Plant mitochondria are involved in endogenous NO production under both anoxic (lack of oxygen) and hypoxic (less oxygen) conditions via the participation of different electron transport chain components and cytochrome c oxidase [32]. Under hypoxic stress, mitochondria produce more NO in the reductive pathways as compared to the other compartments [23]. Oxygen production during photosynthesis facilitates greater tolerance to leaves under hypoxia which largely increases the NO production in mitochondria [32]. In addition, nitrite-dependent NO production in tobacco leaves is facilitated by mitochondrial electron transport [33]. NOS activity and the presence of NO were observed in the leaf peroxisomes of Pisum sativum [34,35]. Peroxisomes produce NO through oxidative pathways [36]. Peroxisomal enzymes such as hydroxypyruvate reductase, glycolate oxidase, malate dehydrogenase, and catalase are involved in S-nitrosylation under abiotic stresses [37]. NO production in peroxisomes is also facilitated by the oxidoreductase enzyme, XOR, which uses molybdenum as a cofactor [38]. The transformation of nitrite to NO under growth conditions void of free oxygen and nitrates (anaerobic) is catalyzed by XOR [39]. In addition, the peroxisomes of Arabidopsis take part in the nitrosative stress development under salt stress conditions and are required for the accumulation of NO in the cytosol [40]. The peroxisomes of Arabidopsis take part in developing the nitro-oxidative stress response against cadmium (Cd) stress via the overproduction of the peroxisomal superoxide anion (O₂^.−), NO, and peroxynitrite [41].

3. NO Signaling and Functioning in Plants under Abiotic Stress Conditions

The discovery of sources of NO production coincided with the recognition of its critical function in plant signaling [42]. NO-mediated signals are coregulated with numerous signaling pathways such as protein kinases, calcium ions (Ca²⁺), and cyclic nucleotides. Ca²⁺ mobilization facilitates the regulation of the signaling cascade via NO. Several studies on different plants showed that cytosolic Ca²⁺ homeostasis in plant cells is regulated by NO [24,43,44,45]. NO stimulates intracellular Ca²⁺-permeable and plasma membrane channels and, consequently, expresses the Ca²⁺ reporter apoaequorin in drought-stressed Tex–Mex tobacco cells [43]. MAP kinases (MAPKs) were also reportedly induced by NO in Arabidopsis [46]. Khan, Siddiqui [47] reported that H₂S and NO interact with each other in the presence of exogenous Ca to reduce the Cd stress in fava beans. In Arabidopsis, the AtNOGC1 protein (Arabidopsis flavin monooxygenase) has guanylate cyclase activity bound with NO and produces cGMP [48]. Likewise, the downstream signaling of NO under plant stress involves different molecules including Ca21 and cyclic ADP-ribose (cADPR) [49]. NO, as a signaling component, also plays a role in increasing stress-based kinase expression and its association with phytohormones [50]. The biological importance of NO can be understood by exploring its in vivo synthesis. Omics approaches are actively being used to identify the genes having a role in posttranslational modifications (PTMs) and signaling of NO [51]. PTMs such as carbonylation, nitration, and nitrosylation have been reported to affect ascorbate peroxidase (APX) activity in drought stress conditions [52]. Some plant species produce NO-based S-nitrosylated proteins after the enzymatic modification of proteins following translation in response to stress [5]. Nitrosylation has become a model redox-based PTM in various phylogenetic kingdoms [53]. In S-Nitrosylation, NO attaches to cysteine thiol via a covalent bond to form an S-nitrosothiol. Bai, Yang [54] reported that NO-mediated S-nitrosylation in Antiaris toxicaria reduced the H₂O₂ synthesis by triggering the antioxidant activity and, thus, increased the desiccation tolerance of seeds.

NO-mediated PTMs engage several target signaling molecules, such as the K1 channel at the plasma membrane of the guard cell, salicylic acid-binding protein 3, AtRhobD, and the auxin signaling proteins TIR1, TGA1, NPR1 [55,56]. Salicylic-acid-dependent plant immunity is also regulated via denitrosylation by TRXh5 [57]. In addition to S-nitrosylation, several other PTMs such as acetylation, persulfidation, phosphorylation, and SUMOylation are also regulated by NO and related molecules [58]. The association of NO with PAs is found to have suppressing effects on different abiotic stresses [59]. The increment in NO production by PA application in Arabidopsis thaliana seedlings also directs towards the association of NO and PA [60]. PA signaling increases NO production which stimulates ABA synthesis and enhances tolerance toward abiotic stresses [61]. NO application under salinity stress reportedly decreases the activity of polyamine oxidase (PAO), which is a catabolic enzyme of PA, while increasing the expression of PA biosynthetic genes [62].

NO also crosstalks with phytohormones to deal with different abiotic stress conditions (Figure 1). Cytokinins (CKs), salicylic acid (SA), jasmonic acid (JA), and abscisic acid (ABA) are the phytohormones that interact with NO to alleviate the suppressive effects of drought stress [63]. CKs share a complex interaction with NO to modify the plants’ responses towards drought stress. While CKs reduces the level of NO in guard cells of broad beans causing the opening of closed stomata [64], it increases the NO levels in drought-stressed maize plants, thus increasing their photosynthetic rate [65]. Similar to CKs, SA acts both synergistically and antagonistically with NO to regulate the responses of plants toward drought stress [61,66]. JA induces the synthesis of NO in the guard cell and works synergistically with NO to regulate the closing of stomata during water deficits [67]. ABA needs NO as a signaling molecule for stomatal closure to reduce water loss through transpiration under drought-stress conditions [63]. ABA is generated in plants in response to ultraviolet-B (UV-B) stress that leads to an increase in the production of NO. This NO regulates the opening and closing of the stomatal aperture controlling the photosynthetic activity of plants in response to UV-B [68,69]. For salinity stress, auxin is reported to work synergistically with NO to alleviate salt stress damage in B. juncea [70]. However, in Arabidopsis and cucumber, NO works as an upstream and downstream signal in auxin-induced tolerance against salt stress [71,72]. NO also regulates salinity stress responses by integrating the signaling of gibberellic acid (GA) and auxin. Positive effects of NO on CK content and, thus, the alleviation of salt stress damage by decreasing the sodium ions accumulation and delaying salinity-induced leaf senescence have also been revealed in several studies [73,74,75]. NO reportedly enhances ABA accumulation in salinity-stressed maize, rice, and wheat plants [76,77,78]. Ethylene works as a downstream signaling molecule for NO functioning in Arabidopsis and sunflower plants under salinity stress conditions [79,80]. However, there are other studies reporting the synergistic action of NO and ethylene on alleviating salinity stress responses in plants [81,82]. NO also has a signaling interaction with phytohormones to regulate heavy metals (HMs) stress [83]. While HMs stress decreases the auxin levels to inhibit plant growth, the external application of SNP regulates auxin signaling, consequently promoting plant growth. HMs stress enhances ethylene levels to suppress plant growth; however, the external NO donor decreases its levels to alleviate HM toxicity. Similarly, ABA levels are decreased by NO generated by HMs stress, thus, reducing the oxidative damage and increasing the stress tolerance [83]. The phytohormones regulate the stress responses via the involvement of signaling cascades and different pathways. Despite several studies on NO-based signaling in plants under different abiotic stresses, its interaction with other signaling molecules should be explored in detail in different plant species.

4. Role of NO in Heavy Metal/Element Stress Tolerance in Plants

Industrial and agricultural wastes, mining, sewage sludge, and fertilizer application lead to the accumulation of different elements including heavy metals and metalloids in soil that is harmful to plant growth at higher concentrations [84,85]. After entering the cells via the plasma membrane, these metals bind and interrelate with different components including nucleic acids and proteins, and negatively influence the normal functioning of plants [86]. The most frequently observed heavy metals hazardous to plants are arsenic (Ar), aluminum (Al), and copper (Cu) [87]. NO is reported to have a controversial interaction with heavy metals sometimes alleviating their adverse effects and occasionally contributing to their toxicity [88,89,90] (Table 1). For stress alleviation, NO inhibits the over-accumulation of heavy metals in the cells and hinders the entry of heavy metals into the roots [84] (Figure 1). Moreover, it increases the activity of antioxidant molecules and the expression of defensive genes to protect against metal stress [37].

Aluminum stress reduces mineral and nutrient uptake in plants, which in turn inhibits crop development and production in acidic soils. The root tip is known to be targeted by Al toxicity, where excess Al inhibits the elongation and division of cells causing reduced growth and decreased uptake of nutrients and water [91,92]. These changes lead to programmed cell death and the yellowing of leaves causing the early senescence of plants [93,94]. Al toxicity enhances ROS developing oxidative stress in plants and causing membrane damage via lipid peroxidation [95,96,97,98]. Al toxicity has variable effects on endogenous NO concentration. Toxic Al reduces the endogenous NO concentration in the transition zone of Arabidopsis via depolarization of the plasma membrane [99] and in the root apex via hindering the activity of NOS in Hibiscus moscheutos [100], while an increase in endogenous NO in roots of common bean and rice was reported under Al toxicity [101,102].

Plants’ tolerance to Al can be induced via the scavenging of ROS. Exogenous NO serves as an antioxidant scavenging the ROS and delaying the programmed cell death in stressed plant tissues. It regulates hormonal balance and modifies cell wall polysaccharides to reduce oxidative stress generated by Al toxicity ([101,103,104]. The negative effects of excess Al on root elongation in Hibiscus moscheutos were reduced by an external supply of SNP [100]. Similarly, in Artemisia annua, SNP application enriched the root secretion of citric acid and malic acid, consequently restricting the movement of Al in roots and its accretion in cells [105]. SNP controls the hormonal balance in root apexes of wheat and rye plants to increase their tolerance against Al stress [104]. Moreover, SNP application has a positive effect on the antioxidant activity of Al-stressed wheat plants irrespective of the tolerance level of the genotypes [106]. SNP treatment not only has a positive effect on the root elongation of rice plants under Al stress, but it also decreases the accumulation of hemicellulose in root tissues, decreases lipid peroxidation, and enhances antioxidant activity. The diminished accumulation of Al in the cell wall also reduces the structural damage of cells [107].

Table 1. Previous studies conducted to observe the effects of nitric oxide (NO) on different plants grown under heavy metals/nutrient stress condition.

Metal Type	Plant Species	Tissue Exposed	Source of NO	Exogenous NO	Observed Effect on Plant	References
Pb and Cd	Lupinus luteus	Root	SNP	10 x	Augmented antioxidant enzymes activity, scavenging of ROS, and improved root structure	[108]
Zn	Triticum aestivum and Phaseolus vulgaris	Seedlings	SNP	100 x	Augmented antioxidant enzymes activity and reduced toxicity	[109]
Cd	M. truncatula	Seedlings	SNP	100 x	Reduced oxidative damage	[110]
As	Vigna radiata	Germinating seeds	SNP	75 x	Augmented antioxidant enzymes activity	[111]
As	Pistia stratiotes	Plants	SNP	0.1 y	Increased photosynthetic activity, augmented antioxidant enzymes activity	[112]
As	Triticum aestivum L.	Seedlings	SNP	250 x	Augmented enzymatic and nonenzymatic antioxidant activity; enhanced proline, chlorophyll content, and relative water content	[113]
Cu	Arabidopsis thaliana L.	Plants	SNP	10 x	Increased cell viability	[114]
Cd	Brassica juncea	Plants	SNP	10–2000 x	Augmented antioxidant enzymes activity, improved root structure, enhanced leaf water content and photosynthetic activity	[115]
Cd	Cucumis sativus	Plants	SNP	100 x	Decreased yellowing of leaves and enhanced scavenging of ROS.	[116]
Pb	Lolium perenne L.	Seedlings	SNP	50, 100, and 200 x	Augmented antioxidant enzymes activity and photosynthetic rate	[117]
Cu	Oryza sativa L.	Seedlings	SNP	200 x	Augmented antioxidant enzymes activity	[118]
Pb	Triticum aestivum	Roots	SNP	100 x	Reduced oxidative damage	[119]
As	Eichhornia crassipes	Plants	SNP	100 x	Reduced oxidative damage	[120]
Cd	Arachis hypogaea L.	Leaves	SNP	250 x	Augmented antioxidant enzymes activity and photosynthetic rate	[121]
Cu and Cd	Nicotiana tabacum	Plants	SNP	50 x	Augmented antioxidant enzymes activity and photosynthetic rate, reduced oxidative damage	[122]
Cu	Catharanthus roseus	Plants	SNP	50 x	Reduced oxidative damage	[123]
As	Vicia faba L.	Plants	SNP	100 x	Enhanced production of metabolite, phytohormone, photosynthetic pigments, yield, and growth	[124]
As	Oryza sativa	Seedlings	SNP	100 x	Reduced oxidative damage	[125]
Cd	Oryza sativa ssp.	Seedlings	SNAP	30 x	Augmented antioxidant enzymes activity and dry weight	[126]
Cd	Typha angustifolia	Seedlings	SNP	100 x	Augmented antioxidant enzymes activity, reduced oxidative damage	[127]
As	Pistia stratiotes	Plants	SNP	0.1 x	Reduced oxidative damage	[128]
Ag	Pisum sativum	Seedlings	SNP	100 x	Reduced oxidative damage	[129]
Cd	S. lycopersicum	Seedlings	SNP	100 x	Reduced oxidative damage	[12]
As	Spirodela intermedia	Plants	SNP	50 x	Reduced oxidative damage	[130]
As	O. sativa	Seedlings	SNP	100 x	Reduced oxidative damage	[131]
Ni	O. sativa	Seedlings	SNP	200 x	Reduced oxidative damage	[132]
As	O. sativa	Seedlings	SNP	100 x	Reduced oxidative damage	[133]
Cr	Solanum lycopersicum L.	Seeds and Seedlings	NO	100 x	Increased action of scavenging enzymes and seed germination	[134]

^x μM; ^y Molar; SNP—sodium nitroprusside; SNAP—S-nitroso-N-acetylpenicillamine.

Table 1. Previous studies conducted to observe the effects of nitric oxide (NO) on different plants grown under heavy metals/nutrient stress condition.

Metal Type	Plant Species	Tissue Exposed	Source of NO	Exogenous NO	Observed Effect on Plant	References
Pb and Cd	Lupinus luteus	Root	SNP	10 x	Augmented antioxidant enzymes activity, scavenging of ROS, and improved root structure	[108]
Zn	Triticum aestivum and Phaseolus vulgaris	Seedlings	SNP	100 x	Augmented antioxidant enzymes activity and reduced toxicity	[109]
Cd	M. truncatula	Seedlings	SNP	100 x	Reduced oxidative damage	[110]
As	Vigna radiata	Germinating seeds	SNP	75 x	Augmented antioxidant enzymes activity	[111]
As	Pistia stratiotes	Plants	SNP	0.1 y	Increased photosynthetic activity, augmented antioxidant enzymes activity	[112]
As	Triticum aestivum L.	Seedlings	SNP	250 x	Augmented enzymatic and nonenzymatic antioxidant activity; enhanced proline, chlorophyll content, and relative water content	[113]
Cu	Arabidopsis thaliana L.	Plants	SNP	10 x	Increased cell viability	[114]
Cd	Brassica juncea	Plants	SNP	10–2000 x	Augmented antioxidant enzymes activity, improved root structure, enhanced leaf water content and photosynthetic activity	[115]
Cd	Cucumis sativus	Plants	SNP	100 x	Decreased yellowing of leaves and enhanced scavenging of ROS.	[116]
Pb	Lolium perenne L.	Seedlings	SNP	50, 100, and 200 x	Augmented antioxidant enzymes activity and photosynthetic rate	[117]
Cu	Oryza sativa L.	Seedlings	SNP	200 x	Augmented antioxidant enzymes activity	[118]
Pb	Triticum aestivum	Roots	SNP	100 x	Reduced oxidative damage	[119]
As	Eichhornia crassipes	Plants	SNP	100 x	Reduced oxidative damage	[120]
Cd	Arachis hypogaea L.	Leaves	SNP	250 x	Augmented antioxidant enzymes activity and photosynthetic rate	[121]
Cu and Cd	Nicotiana tabacum	Plants	SNP	50 x	Augmented antioxidant enzymes activity and photosynthetic rate, reduced oxidative damage	[122]
Cu	Catharanthus roseus	Plants	SNP	50 x	Reduced oxidative damage	[123]
As	Vicia faba L.	Plants	SNP	100 x	Enhanced production of metabolite, phytohormone, photosynthetic pigments, yield, and growth	[124]
As	Oryza sativa	Seedlings	SNP	100 x	Reduced oxidative damage	[125]
Cd	Oryza sativa ssp.	Seedlings	SNAP	30 x	Augmented antioxidant enzymes activity and dry weight	[126]
Cd	Typha angustifolia	Seedlings	SNP	100 x	Augmented antioxidant enzymes activity, reduced oxidative damage	[127]
As	Pistia stratiotes	Plants	SNP	0.1 x	Reduced oxidative damage	[128]
Ag	Pisum sativum	Seedlings	SNP	100 x	Reduced oxidative damage	[129]
Cd	S. lycopersicum	Seedlings	SNP	100 x	Reduced oxidative damage	[12]
As	Spirodela intermedia	Plants	SNP	50 x	Reduced oxidative damage	[130]
As	O. sativa	Seedlings	SNP	100 x	Reduced oxidative damage	[131]
Ni	O. sativa	Seedlings	SNP	200 x	Reduced oxidative damage	[132]
As	O. sativa	Seedlings	SNP	100 x	Reduced oxidative damage	[133]
Cr	Solanum lycopersicum L.	Seeds and Seedlings	NO	100 x	Increased action of scavenging enzymes and seed germination	[134]

^x μM; ^y Molar; SNP—sodium nitroprusside; SNAP—S-nitroso-N-acetylpenicillamine.

High concentrations of heavy metal Cu produce more ROS causing oxidative stress in plants and limiting plant growth and development. Cu toxicity reduces photosynthetic efficiency, photosynthetic pigments, GSH levels, and antioxidant activity in plants [135]. It not only increases lipid peroxidation in plants but also disturbs their nutrient balance [136]. Several studies reported that endogenous NO production is enhanced in plants in response to Cu toxicity. Cu stress produces endogenous NO which activates key proline-synthesizing enzymes such as pyrroline-5-carboxylate synthase modulating the cellular proline [137]. Cu treatment significantly enhances endogenous NO via the involvement of NOS-like enzymes in vascular bundles of fava bean roots. This Cu-induced NO played a crucial role in reducing the suppressing effect of Cu on root growth [138].

Several studies have documented the role of externally applied NO in reducing Cu stress responses in plants [139] (Table 1). NO supply reduces oxidative damage and ameliorates the levels of maximum quantum yield of PS II and oxygen fixation in Chlorella affected by Cu toxicity [140]. The imbibition of wheat seeds with SNP reduces lipoxygenase activity, enhances the CAT and SOD activity, and reduces lipid peroxidation and hydrogen peroxide accumulation to reduce the negative effects of Cu on seed germination [141]. Exogenous NO efficiently reduces Cu-induced toxicity in Panax ginseng roots via maintaining the cellular redox, modification of antioxidant enzymes activities, and detoxification of H₂O₂ radicals [142]. NO application increases the antioxidant activity and stimulates H1-ATPase activity in the plasma membrane of tomato plants to reduce the growth damage caused by CuCl₂ [143].

GSH is involved in regulating cellular redox potential. According to Mostofa, Seraj [118], the interaction between NO and GSH significantly reduced Cu-induced toxicity in Oryza sativa seedlings by decreasing oxidative damage, increasing the GSH content, and strengthening the antioxidant mechanism. SNP application decreases oxidative burst, modifies the antioxidant defense system, enhances photosynthesis, enhances Cu accumulation in roots, and decreases its content in the leaves of Lolium perenne grown in Cu-toxic growth condition [136]). The regulation of nutrient uptake, increase in the amount of total phenolics and amino acids in roots, and decrease in ROS are some of the strategies involved in the reduction of Cu toxicity response via SNP application in Catharanthus roseus [123]. SNP supplementation increases the RUBISCO, rubisco activase, and total chlorophyll contents in in-vitro-grown tobacco (Nicotiana tabacum) plants to deal with Cu stress [122]. Exogenous NO suppresses Cu toxicity in barley seedlings by enhancing GSH and AsA, increasing antioxidant activity, decreasing lipid peroxidation, and inhibiting Cu uptake [135].

Cd is a heavy metal with disastrous effects on plant growth. Excess Cd inhibits growth, increases the yellowing of leaves, enhances metabolic disorder, inactivates antioxidant enzymes, and causes the accretion of different ROS [144]. The replacement of Ca ions in plant photosystem II by Cd ions reduces the photosynthetic capacity via hindering the light response activity of PSII, consequently decreasing the biomass [145]. To reduce stress, plants either make complexes between Cd and metal ions or synthesis oxidants to scavenge ROS produced by excess Cd [146]. Several studies reported the synthesis of internal NO on exposure to Cd stress. A major increase in NO was observed within 3 and 7 hrs of Cd treatment in wheat and Arabidopsis roots [147]. The short-term and long-term release and accumulation of NO was reported in the roots of Triticum aestivum, Pisum sativum, and Brassica juncea [148]. On the contrary, a few studies showed a decrease in NO accumulation in the roots of peas and rice upon Cd exposure [149,150]. In all, Cd stress creates an imbalance in endogenous NO production in plants.

Exogenous NO acts as both a prooxidant and antioxidant to reduce the effects of Cd stress in plants. SNP application enhances the activities of antioxidant enzymes such as CAT and SOD, protects chlorophyll, and decreases lipid peroxidation and biomass reduction due to Cd stress in sunflower seedlings [151]. NO inhibits the entry of Cd by increasing the accumulation of pectin and hemicellulose in the roots’ cell wall and phosphoric acid in the plasma membrane, thus, reducing the response towards Cd stress. Cd tolerance in Lolium perenne seedlings was also induced by SNP application that increases antioxidant production, consequently, decreasing the malondialdehyde (MDA) and ROS levels [152]. An escalated photosynthetic activity along with restricted translocation of Cd in roots and shoots had been reported in Arachis hypogeae [121]. The effect of exogenous Ca supply on the synthesis and coordination of two signaling molecules, hydrogen sulfide and NO, thus, alleviating the Cd stress symptoms was also observed in fava bean [47]. Ca not only improved hydrogen sulfide (H₂S) and NO content but also enhanced cysteine levels in seedlings that, finally, reduced ROS and improved antioxidant enzymes to reduce the symptoms of Cd stress. Boron (B) is a micronutrient whose deficiency and toxicity both are known to be affecting crop yields [153]. B toxicity enhances lipid peroxidation leading to membrane injury and an increase in oxidative stress in plants [1]. It causes the yellowing of leaves with patches on different regions depending on the plant species [154]. High B decreases the leaf area and photosynthetic rate of plants [155,156]. It also hinders root growth and leads to reduced biomass with enhanced B accumulation in tissues [157]. Though there are limited reports on the endogenous production of NO under high B, thiourea application on wheat increases the endogenous NO content that further improves the antioxidant activities under B toxic conditions, thus improving the tolerance of plants towards stress [158]. The exogenous NO application as SNP not only increased the growth of maize plants under B toxicity but also increased the antioxidant activity including peroxidase (POX), CAT, and SOD reducing lipid peroxidation and electrolyte leakage [159]. Exogenously applied NO protected the watermelon seedlings from both B deficiency and toxicity via the inhibition of lipid peroxidation and ROS and upregulation of antioxidant enzymes [160]. Thiourea-mediated endogenous NO reduced the oxidative stress and increased the B toxicity in both durum and bread wheat [158].

Positive effects of exogenous NO have been reported to reduce the toxicity effects of several other elements (Table 1). The external supply of SNP had been reported to decrease the accumulation of Arsenic (As) in mung beans and enhance the antioxidant activity in wheat plants [111,113]. The treatment of NO donors on rice leaves reduced the effects of manganese (Mn) toxicity [161]. Exogenous NO also reduced the oxidative stress caused by Nickel (Ni) in beans and tomatoes [162,163]. NO not only alleviates the toxicity of elements but also reduces their deficiency in plants. The foliar application of NO reduced sulfur (S) deficiency in tomato seedlings via the reduction in lipid peroxidation and ROS in roots and leaves. NO also increased the activity of S-assimilating enzymes, thus, improving the S utilization efficiency of S-deficient plants [164]. Thus, the activation of antioxidant enzymes and reduction of oxidative stress are found to have an important role in the regulation of heavy metal/element stress tolerance by NO. Although the role of exogenous NO in plants’ responses to heavy metal stress has been thoroughly explored, studies are still needed to understand the mechanisms underlying the interaction between exogenous NO and other molecules including hormones and TFs. Moreover, continuous environmental changes increase the co-occurrence of combined heavy metals stress in plants [165]; thus, the role of NO in developing tolerance towards different combined heavy metals stresses should be separately studied.

5. Role of NO in High-Temperature Stress Tolerance in Plants

Understanding how plants adapt to climate change is now crucial due to rising global temperatures. Heat stress disintegrates membrane lipids and increases the permeability of cell membrane that increases the loss of electrolytes, increases the accumulation of ROS, degrades proteins, inactivates enzymes, and decreases the photosynthetic efficiency of plants, mostly affecting PSII [166,167]. Heat stress increased the nitrosothiol content and triggered the main constituents of reactive nitrogen species (RNS) metabolism in Brassica and peas [168,169]. An increase in endogenous NO production has emerged as an important adaptive response of plants under heat stress [24] (Figure 1). NO production in plants under heat stress may vary according to the temperature with higher release at a comparatively higher temperature [170]. In addition, short heat exposure may cause higher NO bursts in plant leaves [24]. Heat induces the NO fluorescence in the plastids, nuclei, and cytosol of tobacco leaves [24]. Several studies reported that the level of NO/SNO might be associated with heat stress tolerance [171,172,173]. The NO/RNS equilibrium is crucial for heat acclimation and the disturbance in this equilibrium hinders the detoxification of reactive nitrogen/oxygen species causing nitrosative/oxidative damage during heat stress. The activation of antioxidant enzymes such as APX, catalase (CAT), and superoxide dismutase (SOD) by NO during heat stress lowering the ROS levels confirms the role of NO in heat stress management in plants [174,175]. In fava beans, heat stress enhanced the biosynthesis of proline and NO that stimulated the antioxidant defense system [176].

External NO application reportedly decreases the cellular damage caused by heat stress [175,177] (Figure 1). NO treatment enhanced the survival rate of wheat (Triticum aestivum) leaves and maize (Zea mays) seedlings and reduced heat-induced damage in rice (Oryza sativa) seedlings [178,179]. Exogenous NO activates scavenging enzymes to develop heat tolerance in mung beans [180]. In some cases, the antioxidant activity stimulated during heat stress via SNP application continues even after the end of heat stress [181]. SNP increases glyoxalase I and II, dehydroascorbate reductase, and monodehydroascorbate reductase activities and glutathione and ascorbate content in wheat seedlings to deal with the heat stress [177]. Exogenous NO has a more suppressing and positive effect, respectively, on the lipid peroxidation and membrane thermostability of the shoot as compared to the roots of wheat plants grown under heat stress [182]. In addition, the survival of maize seedlings under heat stress increased with a decrease in MDA content and electrolyte leakage upon SNP application [183]. Similarly, recovery of electrolyte leakage, chlorophyll content, and relative water content via SNP application was reported in heat-stressed Gingiber officinale leaves [167]. Higher levels of Chl a and b were achieved in Solanum lycopersicum as a result of reduced Chl degradation and increased RuBisCo, carbonic anhydrase, and NR activities when SNP and calcium were applied together [184]. Likewise, ABA and NO when applied together decrease the hydrogen peroxide content and enhance the production of osmolytes and antioxidant activity in wheat grown under heat stress [185]. Most studies that have examined the possible benefits of exogenous NO during heat stress to date have relied on physiological and biochemical data. Other than focusing on molecular responses, more studies should be conducted in field growth conditions to observe the effects of NO on heat stress alleviation in plants.

6. Role of NO in Chilling Stress Tolerance in Plants

Cold stress develops metabolite imbalance, impaired functioning of cells, hindrance in enzyme activity, and membrane damage. Though low-temperature stress is known to have damaging effects on plant growth, different adaptation mechanisms have evolved to alleviate the responses. By scavenging ROS and lowering levels of phytohormones such ABA and jasmonates as well as sugars, flavonoids, anthocyanins, and PAs, NO regulates the cold-responsive network [186]. Cold acclimation in plants is activated by a brief prior exposure to a low temperature and enhances freezing tolerance to plants on further subsequent low-temperature exposure. Lipid signaling is reprogrammed under cold acclimation and tolerance. The cold-acclimated and tolerant plant species showed different compositions and contents of sphingolipids as compared to the nonacclimated and susceptible species [187]. The transitory phosphorylation of sphingolipids occurs due to cold stress and NO regulates this transient phosphorylation confirming a potential association between lipid metabolism and NO under cold stress [188]. A few studies have informed the synthesis of NO under low-temperature stress in several plant species confirming its role in cold acclimation. NOS-like enzymes and NR lead to endogenous NO production under cold stress [188]. NO increases cold acclimation via proline synthesis that positively influences membrane integrity and enzymes under cold stress [189].

Endogenous and exogenous NO have been reported to enhance cold acclimation and freezing tolerance in plants [188,189,190] (Figure 1). Exogenously applied SNP reduces the effect of chilling stress in cucumber seedlings by reducing the MDA content and increasing the chlorophyll and soluble sugar content and antioxidant enzymes including SOD, GR, POX, and CAT [191]. SNP decreases the generation of peroxide radicals and lipid peroxidation and enhances the activity of antioxidant enzymes in wheat plants suffering from chilling stress either alone or combined with SA [192]. NO alleviates the response of Bermuda grass to cold stress by enhancing SOD, POX, and chlorophyll content and lowering electrolyte leakage and MDA content [193]. SNP when combined with melatonin regulates cold damage in cucumber seedlings via activating the photosynthetic process and antioxidant enzymes and reducing lipid peroxidation, ROS accumulation, and electrolyte leakage [194]. Although different studies have been conducted to observe the effects of exogenous NO on cold tolerance in cultivated plants, wild relatives of crops have not been explored at all in this direction. It will be interesting to explore whether NO has similar effects on the chilling stress tolerance of wild plants or not.

7. Role of NO in UV-B Stress Tolerance in Plants

Sunlight radiations present in the UV spectrum are segmented into UV-A (315–400 nm), UV-B (280–315 nm), and UV-C (200–280 nm). Among these three forms, UV-B possesses the highest energy and can have damaging effects on plants [10,195]. UV-B radiations increase the oxidation in the thylakoid membrane enhancing the hydrogen peroxide content and ion leakage leading to cell death [196,197]. It also damages DNA affecting the replication and transcription process and inhibiting normal plant development [198,199,200]. The oxidation of DNA, RNA, lipids, and proteins of plants due to the production of ROS is a major effect of UV-B stress [201,202,203]. UV-B decreases chlorophyll content and Rubisco activity and damages the proteins D1 and D2 of PSII disturbing the photosynthetic activities in plants [204,205,206,207]. On UV-B exposure, while a few studies suggested the production of NO in guard cells by NR activity [208], others proposed that the generation was due to NOS-like activity [209]. NOS-like activity in maize hypocotyls was induced under UV-B radiations that activated the antioxidant activity confirming the role of NO as a second messenger [210]. In addition, NOS-like activity produced NO that facilitated the ROS action to enhance ethyl synthesis so that leaves can be protected from UV-B exposure [211]. Though chloroplasts have the capacity to produce NO upon treatment with UV-B radiations to reduce radiation-induced oxidative damage, their capacity for NO production decreases if the extent of radiation exposure is more than the protective capacity of NO [10].

Different studies reported the chances of increasing UV-B stress tolerance through the exogenous application of SNP (Figure 1). SNP reduces chlorophyll loss and electrolyte leakage lessening the damage caused by UV-B radiations [196]. A decrease in proline and hydrogen peroxide content and lipid peroxidation along with an increase in antioxidant enzymes has been reported in wheat seedlings upon SNP application to deal with the UV-B stress [212]. Likewise, SNP treatment induces protein synthesis and glucosidase activity to protect the maize plants from UV-B radiation. The apocynin treatment and simultaneous NO accumulation restrict the oxidative and cellular damage caused by UV-B radiation in the mesophyll and chloroplast cells of maize seedlings. Moreover, while hydrogen peroxide production and ABA concentration increase due to UVB radiation, simultaneous NOS-like activity enhanced the NO production to sustain the cell equilibrium and reduced cellular damage caused by UV-B radiation [69].

In soybeans, SNP regulates the UV-B stress response by reducing the accumulation of superoxide anion and hydrogen peroxide and inhibiting the membrane damage and leakage of ions [213]. Additionally, APX, CAT, and SOD activity increase upon NO supply with an increase in two APX, one CAT, and three SOD isoforms, respectively [213]. External SNP increases indole-3-acetic acid (IAA), gibberellic acid (GA), chlorophyll and carotenoid content, and total phenolic concentrations and reduces MDA, peroxide radicals, SA, and ABA content to reduce the effect of UV-B radiation [214]. Different studies revealed that NO helps plants to cope with increased levels of UV-B via modifying the hormonal responses, anthocyanin and flavonoid synthesis, and antioxidant enzymes activity overall decreasing the oxidative stress in plants.

8. Role of NO in Salt Stress Tolerance in Plants

Soil salinity is a major problem that influences plant growth and agricultural yield affecting more than 20% of agricultural land and almost 50% of irrigated land [215,216]. Salt stress leads to membrane damage, metabolic function impairment, ion leakage, DNA dissolution, and consequent cell death [217]. It not only disturbs the osmotic balance and ion homeostasis but also hinders the physiological and biochemical processes of plants [218]. Decreased growth, antioxidant enzymes activity, and chlorophyll content and increased electrolyte leakage, generation of ROS, and lipid peroxidation are the common symptoms of salinity stress in plants [219].

The production of signaling molecules such as NO is among the different defensive mechanisms that plants have developed to cope with salinity stress (

Table 2. Previous studies conducted to observe the effects of nitric oxide (NO) on different plants grown under salt stress condition.

Plant Species	NaCl Conc. (mM)	SNP Conc. (μM)	Observed Effects	Reference
Rice (Oryza sativa L.)	0–100	<10 x	Augmented antioxidant enzymes activity	[179]
Lupin (Lupinus luteus L.)	200	100 x	Augmented antioxidant enzymes activity and root structure	[108]
Maize (Zea mays L.)	100	100 x	Augmented antioxidant enzymes activity and seedling development	[220]
Red kidney bean (Phaseolus vulgaris)	100	50 x	Augmented antioxidant enzymes activity	[221]
Cucumber (Cucumis sativus L.)	100	5000 x	Reduced oxidative damage and increased scavenging of ROS	[11]
Alfalfa (Medicago sativa)	100	100 x	Enhanced plant growth and germination of seeds	[222]
Barley (Hordeum vulgare L.)	50	50 x	Augmented antioxidant enzymes activity	[223]
Wheat (Triticum aestivum L.)	300	0 or 100 x	Increased dry weight and coleoptile and radicle length	[224]
Wheat (Triticum aestivum)	150 and 300	1 x	Augmented antioxidant enzymes activity, reduced oxidative damage	[225]
Mustard (Brassica juncea L.)	150	200 x	Augmented antioxidant enzymes activity, reduced oxidative damage	[226]
Cucumber (Cucumis sativus L.)	100	100 x	Augmented growth of radicles and hypocotyls	[227]
Soybean (Glycine max L.)	80	10 x	Augmented antioxidant enzymes activity	[228]
Tomato (Solanum lycopersicum L.)	120	100, and 300 x	Decreased peroxide level, increased ascorbate and proline	[229]
Spinach (Spinacia oleracea L.)	200	200 z	Augmented secondary metabolites and antioxidant enzymes activity	[230]
Wheat (T. aestivum L.)	100	100 x	Augmented photosynthetic activity and antioxidant enzymes activity	[219]
Chickpea (Cicer arietinum L.)	50 and 100	50 and 100 y	Augmented osmolytes production, antioxidant enzymes activity, and growth	[231]
Sunflower (Helianthus annuus)	120	250 x	Augmented seedling growth and antioxidant enzymes activity	[232]
Sunflower (Helianthus annuus seeds)	120	100, 250, 500, and 1000 x	Augmented antioxidant enzymes activity and decreased reactive oxygen species	[233]
Jatropha (Jatropha curca)	100	75 y	Decreased accumulation of toxic ions and alleviated oxidative damage along with enhanced seedling growth	[234]
Brassica juncea L.	100	100 x	Improved photosynthetic activity	[235]

^x μM; ^y Molar; ^z nl/L.

) (Figure 1).

Though endogenous NO is known to be regulating the responses to salt stress, the regulation is dependent on the type of plant species, and level of salt treatment [54,229]. Plant defenses against salt stress involve PAs, and it is recognized that their induction is strongly related to NO generation [236]. Thus, NO-polyamine interaction can be an important molecular mechanism for plant stress tolerance. The plasma membrane H⁺-ATPase which maintains a constant K⁺:Na⁺ ratio is induced via NO and protects plants from salinity stress [237]. Atnoa1 mutants of Arabidopsis grown in vivo showed more susceptibility, a higher shoot Na⁺/K⁺ ratio, and less survival than wild plants under salt stress due to reduced endogenous NO levels [238,239]. Moreover, nitric reductase catalyzes NO production with the involvement of some dehydrogenase enzymes alleviating the salinity stress response [221]. Signal transduction through the G-protein enhances NO synthesis that induces the production of ROS scavengers and antioxidants protecting the plants against salt stress.

External SNP applications have shown the enhanced growth of seedlings in different plant species grown under salinity stress alleviating the effect of osmotic stress [240,241,242] (Table 2). NO application decreases sodium ions and increases potassium ion accumulation in maize seedlings suffering from salt stress. It increases the chlorophyll content and dry weight of plants along with a decrease in membrane leakage [243]. It is involved in increasing Na⁺/H⁺ antiport in the tonoplast and proton pump activities, thus, improving salt tolerance. In salt-stressed tomatoes and spinach, external SNP increases the production of flavonoids, proline, total phenolic content, glutathione, and ascorbate to deal with the stress [230,244] (Table 2). In addition to SNP, other sources of NO such as NO 2,2′(hydroxynitrosohydrazono) bis-ethanimine (DETA/NO) were found to be helpful in alleviating the effects of long-term salinity stress. DETA/NO increases the root, shoot, and nodule dry biomass of soybean plants grown under salinity stress. The NO donor increases APX activity in salt-stressed soybean plants which ultimately decreases the salt-induced hydrogen peroxide levels to a minimum [228]. SNP either alone or in combination with another signaling molecule, Ca, suppresses the effects of salinity stress by increasing soluble sugar and chlorophyll content, activating different antioxidant enzymes, decreasing lipid peroxidation, and increasing the mineral concentrations in wheat seedlings [219]. NO, when applied together with N or S, efficiently mitigates the damaging effects of salt stress on mustard plants via promoted photosynthetic and antioxidant enzyme activity and enhanced N and S assimilation [235]. Different studies have provided evidence that exogenous NO maintains cell permeability, decreases ROS production, lessens the osmotic injury, and enhances secondary defense to protect plants from salinity stress (Table 2). However, more detailed research is needed to understand the strategy by which NO enhances the tolerance level and allows better growth of plants under salt stress.

9. Role of NO in Drought Stress Tolerance in Plants

Global warming has significantly increased drought stress in different agricultural regions continuously decreasing crop productivity. Plants show both short-term and long-term responses to drought stress [245]. While short-term responses include changes in turgor pressure, osmolyte concentrations, organ growth, and hydraulic and stomatal conductance, long-term responses include changes in root–shoot phenological cycles, root architecture, nutrient accumulation, and slowed-down senescence [246]. Curling of leaves, stunted growth, chlorosis of leaves, and permanent wilting are the main morphological symptoms of water deficit in plants [247]. Drought stress negatively affects water use efficiency in plants, stomatal conductance, stem elongation, leaf growth, the oxidative mechanism, membrane stability, and photosynthetic activity in plants [248,249]. Water deficit causes early and greater production of ROS in plants as compared to their scavenging capacity developing oxidative stress in them.

The free radical signal molecule, NO, is reported to be involved in different physiological processes to regulate the effects of drought stress (Figure 1). Several plant species have shown the synthesis of NO under drought stress [63]. The synthesis of NO under drought stress and alleviation of oxidative damage is dependent on species, ecotypes, level of drought stress, duration of exposure, and stages of plant growth. For example, drought stress induces NO production in dune reeds but not in swamp reeds leading to reduced levels of free radicals and enhanced activity of antioxidant enzymes developing tolerance against drought stress [250]. The drought interval is seen to affect the production of NO in the cucumber root cells [251]. The application of NOS and NR inhibitors reduce the NO synthesis confirming the NOS activity and NO production in mesophyll cells and cytosolic fractions of maize leaves under drought stress. However, the hampered antioxidant activity was activated and hydrogen peroxide accumulation was decreased by the external supply of NOS [252]. NOS-like activity has a crucial role in the production of NO under drought stress as the NO scavenger enhanced the NOS-like activity and the NOS inhibitor NG-nitro-L-arginine methyl ester (L-NAME) hindered the accumulation of NO in drought-stressed maize seedlings [253]. The application of CK on maize plants grown under drought stress induced the production of endogenous NO [65]. The produced NO signal decreases the ROS generated due to drought stress and enhances the plant’s photosynthetic performance by regulating the electron donation capacity of PS II [65]. Ammonium supplementation induces NO production that helps to alleviate water stress in rice [254]. Brassinosteroid (24-epibrassinolide) application on pepper plants grown under drought stress reduced oxidative stress via the enhanced production of endogenous NO that, consequently, improves the antioxidant mechanism of plants [255]. The indirect production of NO from the additional nitrate supply reduces the damaging effects of drought stress by activating the photosynthetic mechanism and plant growth and reducing oxidative damage [256] (Table 3). An application of nTiO₂ to drought-stressed fava beans induced NO synthesis that, consequently, decreased the production of oxide radicals and enhanced the activity of antioxidant enzymes [257].

Studies have shown that exogenous NO is involved in coping with drought stress. SNP application enhances the plant’s tolerance to drought stress by closing the stomata and reducing the transpiration rate and leakage of ions [258]. Zhao, Chen [259] observed a decrease in ABA accumulation upon NOS inhibitor (L-NNA) application in wheat seedlings suggesting that NO activates ABA synthesis and alleviates water loss under drought stress. An external SNP supply decreased oxidative damage, increased RWC, and enhanced growth in wheat seedlings suffering from PEG-induced drought stress [260]. SNP application reduces oxidative damage, increases photosynthetic and antioxidant activities, enhances the synthesis of proteins, and decreases the evaporation of water from wheat leaves under drought stress.

Seed priming with SNP alleviates the drought stress responses of tomato plants via increase in antioxidant enzymes and photosynthetic activities and proline biosynthesis [261]. A foliar spray of SNP enhances drought stress tolerance responses irrespective of the tolerance level of Vigna unguiculata (tolerant) and Phaseolus vulgaris (sensitive) via increasing the electron transport rate [262]. External NO activates the production of hydrogen sulfide that reduces the osmotic stress symptoms by increasing the accumulation of osmolytes, antioxidant enzyme activity, and maintaining cysteine homeostasis in wheat seedlings [263]. Even in in-vitro-cultured Allium hirtifolium grown under drought stress, exogenous SNP not only protects plants from drought stress via increasing RWC and photosynthetic pigments but also facilitates the propagation and allicin production through an increased regeneration rate [264]. NO protects watermelon plants from drought stress by increasing the APX antioxidant enzyme activity [265]. However, the extent of protection is dependent on the genotypes. NO treatment promotes osmotic homeostasis through the upregulated accumulation of compatible solutes in stressed plants and by activating the antioxidant defense system [266]. Exogenous SNP along with melatonin reduces lipid peroxidation and the accumulation of ROS, and enhances water content, photosynthetic activity, and plant biomass to alleviate the effects of drought stress in soybean plants [267]. Although there are several studies on the effects of NO on drought stress in plants, it is important to investigate more and more plants suffering from water deficit under field growth conditions. Moreover, detailed studies should be encouraged to explore the interaction between exogenous and endogenous NO.

Table 3. Previous studies conducted to observe the effects of nitric oxide (NO) on different plants grown under drought stress condition.

Plant Species	Source of NO	Applied Amount	Observed Effects	Reference
Tradescantia	SNP	150 μM	Reduction of electrolyte leakage, cellular damage, and closing of stomata	[258]
Populus przewalskii	SNP	>500 μM	Increased amino acid content, proline synthesis, antioxidant activity, and enhanced photosynthesis	[268]
Ginkgo biloba	SNP	250 μM	Increase in flavonoid, soluble sugar, and proline content	[269]
Solanum lycopersicum	SNP	100 μM	Increased photosynthesis, carbonic anhydrase activity, and antioxidant activity	[261]
Malus hupehensis	SNP	300 μM	Increased photosynthesis	[254]
Poncirus trifoliate	SNP	100 μM	Closing of stomata and enhanced antioxidant activity	[270]
Dendrobium huoshanense	SNP	50 μM	Decreased DNA methylation, MDA content, enhanced relative water content	[271]
Tagetes erecta	SNP	10 μM	Decreased starch content; enhanced chlorophyll fluorescence parameters; increased protein, carbohydrate, and leaf chlorophyll content	[272]
Zea mays	SNP	100 μM	Increased glycine-betaine accretion and antioxidant activity	[273]
Agrostis stolonifera, Lolium arundinaceum	SNP	150 μM	Increased antioxidant activity, proline, and chlorophyll content; decreased electrolyte leakage	[274]
Medicago truncatula	DEA-NONOate a	500 μM	Decreased germination of seeds, closing of stomata	[275]
Phaseolus vulgaris	SNP	100 μM	Increased antioxidant activity, decreased cellular damage and electrolyte leakage	[262]
Vigna unguiculata	SNP	100 μM	Increased antioxidant activity, decreased cellular damage and electrolyte leakage	[262]
European searocket (Cakile maritima Scop.)	SNP	100	Reduced lipid peroxidation; ion leakage; increased growth, photosynthetic pigment, antioxidant activity, and proline content	[276]
Sugarcane (Saccharum spp.)	GSNO	10, 100, 500, and 1000	Enhanced leaf and root dry matter, photochemical activity	[277]
Apple rootstocks (Malus spp.)	SNP	50, 100, 200, 300, and 400	Increased photosynthetic and antioxidant enzyme activities	[278]
Wheat (Triticum aestivum)	SNP	50, 100, and 150	Reduced lipid peroxidation; enhanced yield, proline, and chlorophyll content	[279]
Rapeseed (Brassica napus)	SNP	500	Enhanced enzymatic antioxidant activity and nonenzymatic antioxidant content	[280]
Tomato (Lycopersicon esculentum Mill.)	SNP	50 and 100	Reduced hydrogen peroxide and enhanced SOD activity	[281]
Cucumber (Cucumis sativus L.)	SNP	1, 10, 50, and 100	Increased root length and number	[282]
Canola (Brassica napus L.)	SNP	20	Increased antioxidant activity, accumulation of osmoprotectant, and decreased lipid peroxidation	[283]
Crambe (Crambe abyssinica)	SNP	75, and 150	Reduced hydrogen peroxide level; lipid peroxidation; increased antioxidant enzyme activities, photosynthetic activity, and chlorophyll content	[284]
Persian shallot (Allium hirtifolium)	SNP	10, 40, and 70	Decrease in lipid peroxidation; hydrogen peroxide; increase in leaf relative water content (LRWC), proline content, photosynthetic pigments, and antioxidant enzyme activity	[264]
Wheat (Triticum aestivum L.)	SNP	0.5	Increase in proline and endogenous NO content and activation of antioxidant defense system	[285]
Milk thistle (Silybum marianum)	SNP	100 and 200	Increase in photosynthetic pigments and activity along with an increment in seed yield	[286]
Rice (Oryza sativa L.)	SNP	20	Decrease in root lipid peroxidation, carbonyl, and oxide radicals content; increase in antioxidant activity	[287]
Physalis angulata	SNP	25, 50, 75, and 100	Lower supply increased the photosynthetic activity and enhanced growth	[288]
Broccoli (Brassica oleracea L.)	SNP	20	Increase in total soluble proteins, SOD, POX enzymes, growth parameters, and photosynthetic pigment	[289]
Thyme (Thymus serpyllum Serpolet and T. Vulgaris L.).	SNP	50, 100, 150, and 200	Decrease in antioxidant activity and increase in proline accumulation	[290]
Perennial ryegrass (Lolium perenne L.)	GSNO	100	Decrease in oxide radicals, lipid peroxidation; increase in fructan content and photosynthetic pigments	[291]
Indian mustard (Brassica juncea)	SNP	100	Activation of antioxidant defense system	[292]
Wheat (Triticum aestivum)	SNP	5	Elongation of primary and lateral root length	[293]
Safflower (Carthamus tinctorius L.)	SNP	25	Reduced phenol, flavonoid and anthocyanin content, and root growth. Increased chlorophyll content and shoot growth.	[294]
Soybean (Glycine max)	SNP	100	Enhanced biomass and photosynthetic rate	[295]
Wheat (Triticum aestivum L.)	SNP	100	Reduced lipid peroxidation and hydrogen peroxide; increased intercellular carbon dioxide, photosynthetic rate, total soluble protein, and seedling growth	[296]
Marjoram (Origanum majorana L.)	SNP	30 and 60	Decreased hydrogen peroxide, lipid peroxidation, and electrolyte leakage; enhanced antioxidant defense mechanism, essential oil production, and plant dry weight	[297]
Watermelon (Citrullus lanatus)	SNP	100	Decreased electrolyte leakage, osmotic potential, and lipid peroxidation; increased antioxidant activity and root length	[265]
Stevia rebaudiana Bertoni	SNP	50, 100, 250, and 500	Increase in number of leaves and shoots and shoot length	[298]
Sugarcane (Saccharum spp.)	NO3−:NH4+ ratios 100:0 and 70:30	5	Decreased accumulation of reactive oxygen species; increased root growth and photosynthesis	[240]
Soybean (Glycine max)	SNP	100	Decreased lipid peroxidation, electrolyte leakage, and hydrogen peroxide content; enhanced total tocopherol, flavonol and phenol content, and antioxidant activity	[266]
Soybean (Glycine max)	SNP	50, 100, 200, 400, and 600	Increased expression of G6PD7, G6PD6, and GPD5 genes encoding Cytosolic glucose-6-phosphate dehydrogenase	[299]
Alfalfa (Medicago sativa L.)	SNP	100	Differential expression of genes participating in different mechanisms; decrease in lipid peroxidation and root length; increase in antioxidant activities, proline content, chlorophyll content, and leaf relative water content	[300]
Banana (Musa acuminata)	SNP	5	Increase in antioxidant enzyme activities, root number, and biomass	[301]

^a DEA-NONOate—NO-donor diethylamine NONOate sodium.

Table 3. Previous studies conducted to observe the effects of nitric oxide (NO) on different plants grown under drought stress condition.

Plant Species	Source of NO	Applied Amount	Observed Effects	Reference
Tradescantia	SNP	150 μM	Reduction of electrolyte leakage, cellular damage, and closing of stomata	[258]
Populus przewalskii	SNP	>500 μM	Increased amino acid content, proline synthesis, antioxidant activity, and enhanced photosynthesis	[268]
Ginkgo biloba	SNP	250 μM	Increase in flavonoid, soluble sugar, and proline content	[269]
Solanum lycopersicum	SNP	100 μM	Increased photosynthesis, carbonic anhydrase activity, and antioxidant activity	[261]
Malus hupehensis	SNP	300 μM	Increased photosynthesis	[254]
Poncirus trifoliate	SNP	100 μM	Closing of stomata and enhanced antioxidant activity	[270]
Dendrobium huoshanense	SNP	50 μM	Decreased DNA methylation, MDA content, enhanced relative water content	[271]
Tagetes erecta	SNP	10 μM	Decreased starch content; enhanced chlorophyll fluorescence parameters; increased protein, carbohydrate, and leaf chlorophyll content	[272]
Zea mays	SNP	100 μM	Increased glycine-betaine accretion and antioxidant activity	[273]
Agrostis stolonifera, Lolium arundinaceum	SNP	150 μM	Increased antioxidant activity, proline, and chlorophyll content; decreased electrolyte leakage	[274]
Medicago truncatula	DEA-NONOate a	500 μM	Decreased germination of seeds, closing of stomata	[275]
Phaseolus vulgaris	SNP	100 μM	Increased antioxidant activity, decreased cellular damage and electrolyte leakage	[262]
Vigna unguiculata	SNP	100 μM	Increased antioxidant activity, decreased cellular damage and electrolyte leakage	[262]
European searocket (Cakile maritima Scop.)	SNP	100	Reduced lipid peroxidation; ion leakage; increased growth, photosynthetic pigment, antioxidant activity, and proline content	[276]
Sugarcane (Saccharum spp.)	GSNO	10, 100, 500, and 1000	Enhanced leaf and root dry matter, photochemical activity	[277]
Apple rootstocks (Malus spp.)	SNP	50, 100, 200, 300, and 400	Increased photosynthetic and antioxidant enzyme activities	[278]
Wheat (Triticum aestivum)	SNP	50, 100, and 150	Reduced lipid peroxidation; enhanced yield, proline, and chlorophyll content	[279]
Rapeseed (Brassica napus)	SNP	500	Enhanced enzymatic antioxidant activity and nonenzymatic antioxidant content	[280]
Tomato (Lycopersicon esculentum Mill.)	SNP	50 and 100	Reduced hydrogen peroxide and enhanced SOD activity	[281]
Cucumber (Cucumis sativus L.)	SNP	1, 10, 50, and 100	Increased root length and number	[282]
Canola (Brassica napus L.)	SNP	20	Increased antioxidant activity, accumulation of osmoprotectant, and decreased lipid peroxidation	[283]
Crambe (Crambe abyssinica)	SNP	75, and 150	Reduced hydrogen peroxide level; lipid peroxidation; increased antioxidant enzyme activities, photosynthetic activity, and chlorophyll content	[284]
Persian shallot (Allium hirtifolium)	SNP	10, 40, and 70	Decrease in lipid peroxidation; hydrogen peroxide; increase in leaf relative water content (LRWC), proline content, photosynthetic pigments, and antioxidant enzyme activity	[264]
Wheat (Triticum aestivum L.)	SNP	0.5	Increase in proline and endogenous NO content and activation of antioxidant defense system	[285]
Milk thistle (Silybum marianum)	SNP	100 and 200	Increase in photosynthetic pigments and activity along with an increment in seed yield	[286]
Rice (Oryza sativa L.)	SNP	20	Decrease in root lipid peroxidation, carbonyl, and oxide radicals content; increase in antioxidant activity	[287]
Physalis angulata	SNP	25, 50, 75, and 100	Lower supply increased the photosynthetic activity and enhanced growth	[288]
Broccoli (Brassica oleracea L.)	SNP	20	Increase in total soluble proteins, SOD, POX enzymes, growth parameters, and photosynthetic pigment	[289]
Thyme (Thymus serpyllum Serpolet and T. Vulgaris L.).	SNP	50, 100, 150, and 200	Decrease in antioxidant activity and increase in proline accumulation	[290]
Perennial ryegrass (Lolium perenne L.)	GSNO	100	Decrease in oxide radicals, lipid peroxidation; increase in fructan content and photosynthetic pigments	[291]
Indian mustard (Brassica juncea)	SNP	100	Activation of antioxidant defense system	[292]
Wheat (Triticum aestivum)	SNP	5	Elongation of primary and lateral root length	[293]
Safflower (Carthamus tinctorius L.)	SNP	25	Reduced phenol, flavonoid and anthocyanin content, and root growth. Increased chlorophyll content and shoot growth.	[294]
Soybean (Glycine max)	SNP	100	Enhanced biomass and photosynthetic rate	[295]
Wheat (Triticum aestivum L.)	SNP	100	Reduced lipid peroxidation and hydrogen peroxide; increased intercellular carbon dioxide, photosynthetic rate, total soluble protein, and seedling growth	[296]
Marjoram (Origanum majorana L.)	SNP	30 and 60	Decreased hydrogen peroxide, lipid peroxidation, and electrolyte leakage; enhanced antioxidant defense mechanism, essential oil production, and plant dry weight	[297]
Watermelon (Citrullus lanatus)	SNP	100	Decreased electrolyte leakage, osmotic potential, and lipid peroxidation; increased antioxidant activity and root length	[265]
Stevia rebaudiana Bertoni	SNP	50, 100, 250, and 500	Increase in number of leaves and shoots and shoot length	[298]
Sugarcane (Saccharum spp.)	NO3−:NH4+ ratios 100:0 and 70:30	5	Decreased accumulation of reactive oxygen species; increased root growth and photosynthesis	[240]
Soybean (Glycine max)	SNP	100	Decreased lipid peroxidation, electrolyte leakage, and hydrogen peroxide content; enhanced total tocopherol, flavonol and phenol content, and antioxidant activity	[266]
Soybean (Glycine max)	SNP	50, 100, 200, 400, and 600	Increased expression of G6PD7, G6PD6, and GPD5 genes encoding Cytosolic glucose-6-phosphate dehydrogenase	[299]
Alfalfa (Medicago sativa L.)	SNP	100	Differential expression of genes participating in different mechanisms; decrease in lipid peroxidation and root length; increase in antioxidant activities, proline content, chlorophyll content, and leaf relative water content	[300]
Banana (Musa acuminata)	SNP	5	Increase in antioxidant enzyme activities, root number, and biomass	[301]

^a DEA-NONOate—NO-donor diethylamine NONOate sodium.

10. Role of NO in Waterlogging Stress Tolerance in Plants

Saturation of soil around roots with water causes the inhibition of aerobic respiration (hypoxia) leading to waterlogging stress in plants [302]. With negative effects on both vegetative and reproductive growth, waterlogging increases the reduction in crop yield finally inducing plant death. Recurrent unexpected rainfalls and flood disasters due to environmental change worsen the situation of waterlogging stress in plants [303]. Waterlogging stress imbalances the nutrient content in wheat cultivars by decreasing the K⁺, Ca²⁺, and Mg²⁺ and increasing the Fe²⁺ and Mn²⁺ ions [304]. The photosynthetic rate decreases in waterlogged plants with the closure of stomata, decline in chlorophyll content, and increment in leaf senescence [305]. Waterlogging induces the formation of adventitious roots with a greater number of aerenchyma along with a change in root architecture [306,307]. Several studies reported an increase in osmolyte accumulation and ROS production in plants under waterlogged conditions consequently activating their antioxidant defense system [308]. Both short- and long-term waterlogging aggravates lipid peroxidation and oxidative stress in leaves [309]. Hypoxic conditions due to waterlogging or flooding stress induce levels of endogenous NO in plants via the reduction of nitrite [310,311]. These changes in NO levels in plants suffering from hypoxia alter the levels of hormones such as ethylene, JA, and IAA [312].

An exogenous application of NO has been reported to help different plant species cope with flooding and waterlogging stress. SNP induces waterlogging tolerance in cucumber seedlings by reducing the MDA content and regulating the chlorophyll content and activities of antioxidant enzymes such as SOD, POD, CAT, and APX. The regulation of different mechanisms reduces oxidative damage in plants alleviating membrane injury [313]. However, the alleviating effects of SNP on waterlogging are dependent on the amount of SNP and the tolerance level of the genotypes [314]. SNP increases the endogenous NO levels and formation of adventitious roots along with a reduction in growth inhibition in waterlogged Suaeda salsa. The exogenous NO donor S-nitroso L Cysteine (CySNO) and SNP mitigate the suppressive effects of flooding stress in soybean cultivars by decreasing the superoxide anions and increasing glutathione activity [315]. External SNP not only improves short-term but also long-term flooding stress tolerance in soybean by enhancing the activity of antioxidant enzymes such as POD, CAT, and SOD, reducing the H₂O₂ accumulation and MDA content, improving the chlorophyll and proline content, and modifying the photosynthetic rate [316]. A foliar spray of SNP on waterlogged cotton plants enhances the NO content that, consequently, decreases the ABA and ethylene content and increases the GA and IAA enhancing the photosynthetic rate and decreasing yield loss [317]. Although studies have been conducted in different directions to explore the effects of NO on waterlogging stress tolerance, it is encouraged to investigate the involvement of hormones in the process in detail.

11. Role of NO in Abiotic Stress Tolerance in Plants with Mycorrhizal and Rhizobial Symbiosis

NO has been reported to regulate nitrogen-fixing symbiosis by several strategies [318]. Coagnate symbionts produce NO in the roots of Medicago sativa and Lotus japonicas [319]. NO makes a complex with the major hemoprotein of N2-fixing legume nodules called leghemoglobin [320,321,322]. NO synthesis involves the participation of both bacterial and plant partners that leads to the establishment of a proper symbiotic interaction [318]. NO is not only found to be involved in bacterial symbiosis but is also involved in the colonization process of Arbuscular mycorrhizal (AM) fungi. NO gets accumulated in the roots of Medicago truncatula plants treated with AM fungal exudates [323]. An inoculation of soybean and clover plants with Rhizophagus irregularis and Glomus mosseae, respectively, increases the NO synthesis in plants [324,325].

An exogenous SNP supply reportedly has controversial effects on plants with mycorrhizal and rhizobial symbiosis when grown under different abiotic stress conditions. SNP treatment does not have any significant effects on SDW or RDW, while it increases the stomatal conductance of AM-lettuce plants grown under drought stress conditions [326]. AM symbiosis and SNP treatment also improve root water consumption in drought-stressed lettuce plants. However, conclusively, Sánchez-Romera et al. [326] suggested that NO accumulation in leaves under drought conditions is reduced by AM symbiosis. The external supply of NO enhances the proline concentration in low-temperature stressed mycorrhizal rice [327]. Similarly, exogenous SNP increases the denitrifying bacteria and colonization rate of Tuber indicum in the roots of Carya illinoinensis seedlings under low P stress along with an increase in root activity as compared to nonstressed condition [328]. The increased production of NO in fava bean plants nodulated with a Rhizobium laguerreae strain contributes towards plant growth through Pgb–NO respiration and protects them from severe hypoxia caused due to drought stress [329]. Although a few studies have been conducted to understand the role of NO in developing abiotic stress tolerance in plants with mycorrhizal and rhizobial symbioses, the efforts have been too limited to draw any conclusions. Thus, more experiments should be conducted to properly understand the mechanism behind the process.

12. Effects of Exogenous NO on the Transcriptomic Response of Plants Suffering from Different Abiotic Stresses

Transcriptome profiling via RNA sequencing is commonly used to investigate and discover differentially expressed genes (DEGs) involved in plant growth and development under different stressed conditions [330]. This is because modifications of chromatin and transcription factors under stressed environments can regulate gene transcription. Several studies have demonstrated the effect of NO on alleviating abiotic stress responses via involvement in transcriptomic pathways and mechanisms. NO increases the expression of Metallothionein (MTs)-based genes with a simultaneous increase in the activity of antioxidant enzymes such as APX, SOD, POX, and CAT in Solanum lycopersicum to increase the tolerance to Cu toxicity [331]. The salt stress in chickpeas is mitigated by the exogenous application of NO via the upregulation of SOD, CAT, and APX genes [231]. The upregulation of genes supports a decline in MDA content, electrolyte leakage, and H₂O₂ and an increase in levels of osmolytes, photosynthetic pigment production, antioxidant activities, and growth parameters on NO supply. NO alleviates heavy metal stress by the induced expression of HM-associated-domain-containing genes. These genes act as metallochaperones that facilitate the movement of metal ions in the cells [332]. CySNO application differentially regulates fourteen HM-associated-domain-containing genes in the RNA sequencing of Arabidopsis thaliana [333]. Wu, Hu [334] reported the role of NO production in alleviating drought stress in wheat plants by facilitating the Mo-induced antioxidant defense system at least partially through the regulation of NR. The TaAPX, TaCAT, and TaSOD genes encoding SOD, CAT, and APX are upregulated upon SNP treatment in line with the increase in the activities of APX, CAT, and SOD enzymes. The transferase, reductase, dehydrogenase, oxidoreductase genes, and transcription factors including enzyme activator, GTP binding, ATP binding, transcription factor complex, and GTPase activator activity are upregulated by external NO supply and reduce the effects of Cd stress on fungus Pleurotus eryngii [335]. SNP mitigates salinity stress in susceptible rice genotypes with an increased expression of OsHIPP38, OsGR1, and OsP5CS2 genes [336].

Exogenous SNP application improves drought tolerance in alfalfa with the differential expression of 24 known and 31 new miRNA [300]. Metabolic pathways related to protein transport, ribosome, amino acid synthesis, starch and sucrose metabolism, tyrosine metabolism, and ascorbic acid are regulated by NO supply under drought stress. Among different processes, NO has more effect on carbohydrate metabolism including carbohydrate transmembrane transport, the amylopectin biosynthetic process, the galactolipid biosynthetic process, the starch biosynthetic process, and the cellulose biosynthetic process. Irrespective of the type, whether SNP and CySNO, both the NO sources cause a range of biochemical and transcriptional changes that can alleviate the detrimental effects of short-term flooding stress in soybeans. While the transcripts of GSNOR1, NOX1, and NR show a significant increase in accumulation, the transcript accumulation of ABAR and TOC1 shows a decrease in response to early or later phases of flooding stress [315]. The external supply of SNP in tomato plants grown under hypoxia shows differential regulation of 395 genes with the up- and down-regulation of 97 and 154 genes, respectively. Cell-wall-related genes, genes encoding proteins involved in the regulation of transcription (TFs) and PTM, redox and cellular transport-associated genes, genes encoding enzymes of glycolysis, carbon metabolism, starch degradation, photosynthesis, amino acid synthesis, protein synthesis, protein degradation, ROS metabolism-related genes, salt and heat stress-related genes, phytohormones-related genes, and aquaporin encoding genes are transcriptionally regulated upon exogenous NO application to alleviate low oxygen stress [337].

Other than the genes involved in signal transduction pathways, amino-acid metabolism, lipid metabolism, saccharide metabolism, biosynthesis of other secondary metabolites, and the sugar metabolism-related genes encoding fructose-bisphosphate aldolase and glucan-endo-1,3-β-glucosidase are transcriptionally regulated by exogenously applied NO in melon seedlings suffering with chilling stress [338]. The significant enrichment of biological processes such as photosynthesis, phenylpropane metabolism, and nitrogen metabolism upon an exogenous supply of NO increases the capacity of watermelon plants to endure aluminum stress [339]. The increased expression of genes encoding CAT and POD upon NO application activates the antioxidant enzymes defense system to protect plants against Al stress. It also significantly increases the expression of the sucrose phosphate synthase (CmSPS) gene along with an increase in the antioxidant enzyme activities, chlorophyll, and soluble solutes [340]. Though a few transcriptomics-based studies have been done to explore the effects of NO on different abiotic stress conditions, there is a long way to explore and completely understand the involvement of NO in diverse transcriptomic pathways of different crops under different abiotic stresses.

13. Future Prospects for NO Research for Abiotic Stresses

NO which was entitled the ‘Molecule of the Year’ in 1992 [341] was reported to be released from plants in the 1970s [342,343]. However, the research on its role in plant development gained speed in the late 1990s [174]. Till then, several experiments have been conducted to document different aspects of NO including its production, mode of action in plants, signaling, crosstalk with other components, and its functioning during different abiotic stress conditions. In this article, we summarized a few studies that focused on the effects of NO on different abiotic stress conditions. It can be seen from the given literature that although the effects of the exogenous supply and endogenous production of NO on the alleviation of abiotic stresses have been studied in different crops, the studies are mostly conducted to understand the underlying mechanisms. Unfortunately, further advanced studies are required to be conducted to get an agricultural or economic benefit from the understood mechanisms. For example, numerous studies directly evidenced the stimulation of antioxidant enzymes on NO application to ameliorate the abiotic stress responses. Thus, efforts should be made that can bring the understood mechanism to the agricultural fields in the form of an application, either as a fertilizer or a chemical solution for exogenous NO supply or a factor that can stimulate endogenous NO production. Nowadays, nanomaterials are successfully being used via different strategies to increase crop yields [344,345]. The employment of NO-releasing nanomaterials such as S-nitrosothiol-containing chitosan nanoparticles that can increase higher NO levels in tissues as compared to nonencapsulated NO donors can be promoted to improve crop production under stressed conditions [346]. Wild and close relatives of crops are considered to be a hub of tolerant genes to different abiotic stress conditions. However, the effect of NO on wild crop relatives grown under different abiotic stresses has not been explored at all. It will be interesting to see the effect of exogenous and endogenous NO on the growth of wild genotypes as compared to modern cultivars grown under different abiotic stress conditions and to understand the interaction mechanism. In addition, in the literature, most of the experiments have been conducted in hydroponic environments or pots. More experiments should be repeated in fields to unfold/underline the unexpected/upcoming challenges that may come under field growth conditions. In molecular studies, although several genes were found to be upregulated or downregulated upon NO application to alleviate abiotic stress responses, their functional characterization has been less explored and should be studied in detail for different plant species.