*5.1. Regulation of Gene Expression*

The functional analogy of ROS, RNS, and RSS has been extensively studied by applying them exogenously to analyze their potential in modulating growth and defense-related processes in plants exposed to environmental cues [18]. Recent studies have shown that both NO and H2S are potentially involved in regulating seed dormancy and germination via modulating ABA signaling [51]. Molecular investigation revealed that NO exerts this effect by activating PYR/PYL/RCAR (pyrabactin resistance 1/PYR, such as the regulatory component of ABA receptor), SnRK2 (SNF1-related protein kinase), and ABI5 (abscisic acid insensitive 5) protein, whereas H2S signaling stimulated seed germination in Arabidopsis by modulating the expression of L-DES (L-cysteine desulfhydrases) protein [66,67]] Recent investigations in Arabidopsis indicated that H2S signaling results in the accumulation of constitutive photomorphogenesis 1 (COP1) in the nucleus that stimulates the degradation of elong hypocotyl 5 (HY5). The increased degradation of HY5 resulted in decreased expression of the ABI5 gene, thereby enhancing seed germination under heat stress [68]. In another study, the combinatorial effect of NO and H2S signaling regulated the adventitious rooting in tomato plants by activating the expression of auxin-related genes (ARF4 and ARF16) and cell cycle-related genes (CYCD3, CYCA3, and CDKA1) [69]. Exogenous application of NaHS leads to the activation of transcription and activity of L-DES1 that stimulates the H2S biosynthesis, which in turn activates the germination of Arabidopsis seeds [70]. In mutant (lcd/des1 defective) plants, exogenous application of NaHS does not induce the germination of the Arabidopsis seeds. In wild plants (lcd/des1 induced), NaHS-mediated increase expression of L-DES1 protein activated the H2S biosynthesis have successfully maintained the GSH/GSSG ratio within the cells that may have triggered alternative oxidase (AOX) mediated cyanide resistant respiration to regulate seed germination [70]. Similar observations have also been made where the exogenous application of NO and H2S positively modulated the seed germination and lateral root formation in cucumber and maize plants by triggering the expression of the heme oxygenase-1 (HO-1) gene and by reorganizing the arrangement of F-actin bundles [71–73].

Another complex process that is sophistically regulated via signaling molecules (ABA, ethylene, and K+/Ca2+) is the opening/closing of stomata, and recent investigations have confirmed the role of NO and H2S signaling in controlling guard cell function [67,74]. Previous investigations have concluded that NO signaling modulates stomatal movement by regulating the expression of SNF-1 (sucrose nonfermenting 1) and SnRK2.6 genes [75]. In contrast, H2S signaling negatively regulated the opening of stomata via inducing NOmediated activation of 8-mercapto-cGMP, which restricts the inward flow of K+ in guard cells [76,77]. Recent investigations have reported H2S signaling downstream of mitogenactivated protein kinase 4 (MAPK4), thereby inducing ABA-mediated stomatal closure in response to drought stress [78]. A study concluded that the exogenous application of NaHS significantly altered the drought-tolerant capacity of mapk4 and slac1 mutant plants compared to wild-type Arabidopsis plants. H2S signaling could not effectively regulate the lcd/des1 gene expression, which has affected the ABA-mediated stomatal movement in mutant plants [78]. Similarly, the exogenous application of H2O2 induced H2S signaling by activating the expression of the L-DES1 gene significantly regulated the salt stress tolerance in *Vicia faba* plants by altering the movement of guard cells [79]. Recently, researchers have confirmed that the NO can modulate ABA-induced stomatal closure by activating the GSNO reductase gene, which in turn causes differential regulation of nitrate reductase genes (NIA1 and NIA2) and CLE 9 peptide (Clavato3/Embryo Surrounding region) [52]. The differential accumulation of NIA genes alters the ABA-induced activity of K<sup>+</sup> inward and activates the outward flow of K+ in wild-type Arabidopsis plants. In contrast, the down-regulation of NIA genes abolishes the inward movement of K<sup>+</sup> [52].

Several studies have demarcated NO and H2S signaling in leaf senescence and fruit ripening. Previous works have reported a substantial role of exogenous NO in reducing ethylene production and ABA-induced senescence in rice and strawberry plants [80,81]. NO regulated ethylene production and senescence by modulating the expression of NOS-like enzymes, leading to differential accumulation of endogenous NO [82]. Later, researchers indicated that exogenous application NO evidently interacts and activates SA and JA signaling, which induces the expression of NOS-like enzymes, thereby delaying leaf senescence upon upregulation of the antioxidant defense system [83]. Recently, H2S signaling in regulating leaf senescence was analyzed by inducing mutation in an H2S-producing enzyme encoding L-cysteine desulfhydrase 1 (L-DES1). They concluded that mutant (Des1) Arabidopsis plants showed enhanced susceptibility to drought stress and accelerated leaf senescence [84].

In contrast, over-expressed Arabidopsis plants (OE-DES1) exhibited more tolerance to drought and delayed leaf senescence [84]. The effect of LCD1 on the regulation of leaf senescence was explored in wild-type, mutant, and overexpressed tomato plants. The mutant LCD1 plants showed early development of leaf senescence, whereas overexpressed LCD1 plants exhibited delayed leaf senescence compared to wild-type tomato plants. Further investigations revealed that the delayed leaf senescence in overexpressed tomato plants was due to the downregulation of various chlorophyll degradation genes, such as NYC1, PAO, PPH, and SGR1, and senescence-associated genes (SAGs), along with active scavenging of ROS [85].

Fruit ripening is governed by a complex physiological process fine-tuned via various signaling molecules, plant growth hormones, and environmental stimuli [86]. In recent years, extensive studies have been conducted to unravel the mechanism of regulation of fruit ripening mediated via reactive species (ROS/RNS/RSS). Exogenous application of NO and H2S differentially regulated the endogenous level of NO and H2S, accentuating fruit ripening in *Capsicum annum* fruits. The differential regulation of endogenous NO and H2S levels altered the activity of the NADP isocitrate dehydrogenase enzyme, thus promoting fruit ripening in pepper plants [87]. A year later, they conducted a different study to reveal that an increase in the endogenous NO and H2S levels differentially regulated the fruit ripening process in *Capsicum annum* fruits by downregulating the expression of the NADP malic enzyme and the 6-phsphogluconate dehydrogenase enzyme [88].

Furthermore, Zhu et al. [89] reported the synergistic effect of NO and H2S on the inhibition of peach fruit ripening during storage. They concluded that the combined application of NO and H2S caused a significant reduction of ethylene production, i.e.,

it decreased the expression of ACC synthase and ACC oxidase enzyme and cell wall metabolism to delay fruit ripening in peach plants. Interestingly, the exogenous application of strigolactones regulated the endogenous level of NO and H2S by upregulating NOSlike enzymes and L-cysteine desulfurase, which concomitantly delayed the ripening of strawberries [90]. They further concluded that the delayed development of strawberries was due to differential regulation of the antioxidant defense system mediated via increased NO and H2S during storage.

#### *5.2. Regulation of Post-Translational Modification*

Recent research has confirmed that ROS/RNS/RSS regulates the expression of various stress-responsive genes and modulates specific proteins' activity via post-translational modifications (PTMs) [6]. PTMs such as Persulfidation, tyrosine nitration, S-nitrosylation, and carbonylation are activated upon exogenous application of RSS/RNS/ROS, thereby influencing plant growth and developmental processes under stress conditions [37]. Cysteine thiols are a highly nucleophilic group that regulate a plethora of protein regulatory functions, such as their catalytic efficiency, stability, and interaction with ligands upon reacting with ROS/RNS/RSS [91]. Persulfidation, previously known as *S*-sulfhydration, is a reversible oxidative PTM process where the thiol group (-SH) is converted into persulfide form (-SSH) in a complex reaction, in which protein thiol reacts with an anionic and protonated form of H2S or reactive species-mediated interaction of protein thiols with inorganic polysulfides [91]. The advent of ultra-throughput proteomic approaches and advancement in mass spectrophotometry has led to the identification of several proteins undergoing persulfidation [91]. Exogenous application of H2S (NaHS) coupled with proteomic analysis identified 1000 differentially regulated proteins in Spinacia oleracea plants, of which few were persulfidated [92]. Similarly, researchers employed the biotin switch method with liquid chromatography–tandem mass spectrometry (LC-MS). They identified 106 to 2015 (using the modified switch method) persulfidated proteins in Arabidopsis leaves [12]. In addition, exogenous H2S persulfidated the mitogen-activated protein kinase 4 (MAPK4), which causes up to a 10-fold expression of MAPK4, leading to the enhanced tolerance of Arabidopsis plant's chilling stress [93]. Concomitantly, the exogenous application of NaHS reverses the adverse effect of heavy metals inducing oxidative damage in tomato plants by inducing the persulfidation of enzymatic antioxidants such as CAT, APX, GR, and SOD [94]. H2S-mediated persulfidation significantly regulated the expression of the L-DES1 gene, which modulated the ABA signaling in guard cells, leading to the opening and closing of stomata [68]. They further concluded that H2S mediated persulfidation of Open Stomata 1/SNF1-related protein kinase 2.6 (SnRK2.6) increases its ability to interact with another transcription factor, such as DREB and ABA-responsive factor 2 (ABF2), to promote stomatal closure [68]. Interestingly, H2S-mediated persulfidation of ABI4, RBOHD, L-DES1, ATG4, and ATG18a have improved plant cellular and physiological processes under stressful conditions by preventing reversible protein oxidation and DNA damage [95–97].

*S*-nitrosation is a PTM process essentially regulating discrete signaling functions in plants ranging from seed germination to fruit maturation and is also mechanistically involved in regulating abiotic/biotic stress tolerance [51,98,99]. Much of the NO-derived PTMs in plants are accentuated by tyrosine nitration followed by *S*-nitrosation, which involves adding nitric oxide radical to protein thiols to generate *S*-nitrosoglutathione (GSNO) [13,64]. Extensive research on NO-mediated PTMs has been conducted in Arabidopsis plants, where the researchers have identified several *S*-nitrosated proteins after treating them with a NO donor (GSNO) [100]. Similar reports have also been published [101–103] where they used a combination of BSM and affinity tags to identify targets of *S*-nitrosation proteins in Arabidopsis plants exposed to biotic and abiotic stresses. *S*-nitrosation and tyrosine nitration was involved in heavy metal-stressed mutant and wildtype Arabidopsis plants [104]. In their study, the researcher identified that *S*-nitosothiol signaling was responsible for enhancing NADPH-dependent thioredoxin reductase activity, which is involved in the regulation of hydrogen peroxide, and further application of

LC-MS/MS analysis indicated that heavy metal-induced *S*-nitrosation of APX resulted in enhanced tolerance to stressed plants [104]. Ripening of *Capsicum annum* fruits is also differentially regulated by NO-mediated PTMs [105]. They exploited BSM, nano-liquid chromatography, and mass spectrometry to identify that Cys377 of peroxisomal catalase potentially undergoes *S*-nitrosation, thereby modulating ROS/RNS levels in fruits.

Further, Silva et al. [106] identified potential targets and substrates for *S*-nitrosation in plants. Their study determined that cytosolic and plastid glutamine synthetase is differentially regulated by *S*-nitrosation, where Cys369 is a possible site. Later, [107] postulated that the exogenous application of NO-mediated nitration of tyrosine residues and *S*-nitrosation of cysteine residues differentially affected vital amino acid residues involved in the binding of FAD and molybdenum cofactors in Arabidopsis plants. Conversely, *S*-nitrosation of protein thiols has modulated tomato plants' stress defense response against *Phytophthora infestans* [108]. Their study identified decreased activity of the *S*-nitrosoglutathione reductase enzyme in both tolerant and susceptible genotypes.

In contrast, *S*-nitrosation protein was increased in the tolerant genotypes as the infection progressed. Correspondingly, proteomic approaches identified tolerant plants exhibiting increased protein-*S*-nitrosation and showed enhanced accumulation of osmolytes, metabolites, and enzymatic and non-enzymatic antioxidants [108]. Recently, time-course gene expression analysis and non-denaturing PAGE have confirmed that PTMs such as tyrosine nitration, *S*-nitrosation, and glutathionylation heterologously regulated the expression of APX enzyme in the fruits of *Capsicum annum* plants [109].

Carbonylation is an irreversible PTM mediated by various ROS such as hydroxyl radical (HO˙), superoxide anion (O2˙ −), and hydrogen peroxide (H2O2) that potentially add aldehyde/ketone group into side chains of specific amino acids [110]. Carbonylation is a complex chemical process driven in conjunction with lipid/sugar derivatives and is basically of two types, viz. primary/secondary carbonylations [111]. Primary protein carbonylation is often modulated via metal-associated oxidation of Lys, Pro, and Thr residues or through α-amidation of glutamyl residue to form aldehyde and ketone derivatives [110]. Conversely, Secondary protein carbonylation is driven by adding reactive carbonyl species (RCS) to Cys, His, and Lys residues of specific proteins [112]. A large body of literature has confirmed that the protein carbonylation is exaggerated upon an increase in cellular ROS levels that differentially regulate plant cell growth and physiology under stress conditions [111,112]. The effect of protein carbonylation has been profoundly studied in Arabidopsis plants, where researchers have reported that the protein carbonylation first increases and then decreases with the age of the plants, thereby drastically affecting the reproductive and senescence phase [113]. Researchers used liquid chromatography–tandem mass spectrometry to identify carbonylation sits in *Phaseolus vulgaris* plants [114]. They further concluded that approx. 238 proteins were carbonylated, of which maximum effect was observed in two essential nodule proteins, i.e., glutamine synthetase and glutamate synthase, that conversely regulated cell metabolism and nodule senescence in legume plants [114].

Furthermore, a proteomics approach was employed to identify carbonylated proteins in premature, mature, and desiccated seeds of *Medicago truncatula* [115]. The PM34 protein, a protein responsible for seed growth and also known to possess cellulase activity, was carbonylated more readily than other proteins. Effect of As accumulation on protein carbonylation was analyzed in *Oryza sativa* root and shoots [116]. Researchers concluded that increased As accumulation drastically enhanced lipid peroxidation and protein carbonylation, affecting rice plants' yield. Likewise, ROS-mediated protein carbonylation of enzymatic and non-enzymatic antioxidants invariably decreases seed vigor and other physiological activity of the seed of wheat plants [117]. Similarly, [118] identified 180 carbonylated proteins in ABAtreated *Arabidopsis thaliana* leaves using affinity chromatography that differentially regulated stomatal closure upon ABA supplementation.

*S*-acylation, previously known as *S*-palmitoylation, is a reversible PTM that instigates the palmitate group's addition to the specific Cys residues of soluble/membrane proteins [119]. *S*-acyl transferases are the main catalyzing this reaction, having conserved

the Asp-His-His-Cys motif and formed a thioester bond in the target proteins [120]. Numerous studies have identified and quantified *S*-acylated proteins in Arabidopsis and poplar plants using a proteomics approach coupled with BSM labeling [119,121]. Researchers have corroborated that the proteins which are most commonly *S*-acylated in plants growing under climate extremes are calcium-dependent protein kinases (CDPK), mitogen-activated protein kinases (MAPK), ATPases, calcineurin-B-like proteins, and several integral proteins [122,123]. *S*-acylation of several receptors, such as kinases proteins adjacent to the transmembrane domain, differentially regulated the flagellin perceiving receptor kinases 2 (FLS2) and associated signaling function [124]. Interestingly, another study concluded that *S*-acylation of plant immune receptor proteins such as P2K1, FLS2, and CERK1 activated the defense response of Arabidopsis plants more prominently compared to their corresponding non-mutant plants [125]. Recently, *S*-acylation of Alpha/Beta Hydrolase domain-containing protein 17-like acyl protein thioesterases invariably affected cell death/apoptosis in Arabidopsis and *Nicotiana tabaccum* plants [126]. They further concluded that protein-*S*-acyltransferases and de-*S*-acylation enzymes are progressively involved in regulating subcellular localization, stability, and activity of enzyme proteins. Likewise, the *S*-acylation of FLS2 controlled and stabilized the ligand-induced receptor kinase complex, thereby triggering plant innate immune response against pathogen attack [127]. *S*-acylation-mediated regulation of plant immune receptors (DORN1, LecRK-1.9, and ATP receptors) inhibited bacterial invasion in Arabidopsis plants via P2K1-mediated autophosphorylation and protein degradation [128].

#### *5.3. Regulation of Epigenetic Modification*

Epigenetic modifications, such as DNA methylation and histone modifications, are potentially involved in the modulation of growth, development, and defense process of plants exposed to climate extremes [129]. The epigenetic modifications exert these stimulatory/inhibitory functions by regulating gene expression or performing chromatin remodeling dynamics strongly influenced by ROS/RNS/RSS signaling [129,130]. Accumulating shreds of evidence have indicated that reactive species can potentially affect the post-replication modification of DNA molecules by methylating cytosine at the five positions or adenine at the N4 or N6 positions [131]. Several excellent review papers have comprehensively described plant methylation and histone modifications [129–131]. Therefore, this section is mainly confined to ROS/RNS/RSS mediated regulation of epigenetic modifications influencing plant growth and development under stress conditions. These reactive species accentuate epigenetic mechanisms of gene regulation by modulating the functioning of various proteins/enzymes such as histone deacetylases, DNA methylases, Demeter1, a repressor of silencing 1 and Demeter-like 2 and 3 [132,133].

Researchers have corroborated that these reactive species directly interact with the Fe-S cluster of Demeter proteins, resulting in the generation of oxidative stress in plants, thus revealing the linkage between redox signaling and epigenetic modification [134]. Some of the pioneering studies in redox regulation of epigenetic modification in transgenic rice plants indicated that plants over-expressing OsSRT1 (silent information regulator 2 (SIR2) related HDAC gene) exhibited less DNA methylation and oxidative damage compared to wild-type plants [135]. A decade later, Zhang et al. [136] confirmed the above notion and showed that OsSRT1 enhanced the tolerance to oxidative of transgenic plants by differentially regulating the expression of glyceraldehyde-3-phosphate dehydrogenase that had prevented the excessive generation of reactive species, thereby inhibiting DNA damage. Since then, a large body of literature has confirmed that differential regulation of ROS/RNS/RSS signaling can modulate epigenetic modification, such as acylation and nitrosation in Arabidopsis and rice plants via reactive species-mediated development of oxidative stress and decreased expression of HDA19 and HDA9 [33].

Likewise, the exogenous application of salicylic acid and nitric oxide accentuated ROS scavenging, thereby inducing DNA de-methylation and enhancing high-temperature tolerance in *Lablab purpureus* L. plants [41,42]. Reactive species-mediated PTMs, such as acylation

and nitrosation, have also been implicated in regulating secondary metabolite synthesis and the formation of crown roots in rice plants [33]. In another study, the exogenous application of nitric oxide efficiently modulated the ROS/RNS signaling, thereby improving the tolerance of lettuce plants exposed to metal stress by decreasing DNA methylation and activating the expression of stress-responsive genes [137]. Furthermore, the knockdown of argonaute 2 (Os-AGO2) protein in rice anthers exaggerated ROS biosynthesis leading to increased methylation levels, abnormal growth, and apoptosis [138]; whereas an overexpression of hexokinase 1 (Os-HXK1) differentially regulated the ROS level and tapetal apoptosis, leading to a significant decrease in the methylation level of the concerned promoter.

An increasing body of literature has demonstrated DNA methylation's involvement in fruit ripening, which was analyzed recently by [139]. They concluded that the treatment of grape plants with azacitidine significantly lowered the methylation rate in grapefruit compared to non-treated plants and enhanced the expression of the superoxide dismutase enzyme, thus establishing a link between ROS metabolism and epigenetic regulation. Recently, non-thermal plasma (NTP) treatment of seedlings of *Glycine max* displayed alteration in methylation patterns [140]. Researchers employed two doses of NTP that differentially regulated the ROS/RNS mediated methylation pattern that accentuated seed vigor and germination, resulting in improved seedling growth. In addition, genome-wide methylome status under the influence of H2O2 production was analyzed in transgenic tobacco plants [141]. They identified that overexpressing the CchGLP gene of transgenic plants stimulated endogenous ROS/RNS signaling, thereby enhancing its biotic and abiotic stress tolerance. Several recent findings have argued and confirmed the multifaceted role of ROS/RNS/RSS in epigenetically regulating gene expression and stress tolerance in plants. Still, this regulation's molecular mechanism is far-fetched. Therefore, future efforts must be diverted toward unraveling the functional mechanism by which ROS/RNS/RSS strengthens the plant's innate immunity against biotic/abiotic stresses.

#### **6. Potential Biotechnological Application in Agriculture**

In the recent decade, the plant science community has diverted its attention toward un-earthing the functional mechanisms by which ROS/RNS/RSS play a substantial role in regulating plant growth, development, and stress amelioration (Figure 3) [37]. Researchers have confirmed that these ROS/RNS/RSS regulate important cellular, physiological and biological processes in plants after interacting with other phytohormones [4,7]. A large body of literature has contemplated the role of NO during seed germination, fruit development, maintaining ion/osmotic homeostasis, and coping with biotic/abiotic stresses [6,11,13]. Furthermore, NO has also been known to differentially regulate the virulence of bacterial and fungal pathogens by acting as an antimicrobial or antifungal agent [142]. In addition, studies have also delineated that the perception of NO gas by microorganisms, such as bacteria and fungi, can induce transcriptional reprogramming of genes involved in metabolic and virulence systems, thereby regulating their adaptation [143]. Correspondingly, the genes involved in NO perception and signaling have been identified in both gram-positive and gram-negative bacteria [142]. The potential biotechnological application of NO in agricultural or horticultural crops depends on the donor molecules, application, tissue type, environment, and possible interaction with other secondary metabolites [142]. The most frequently used NO donor is sodium nitroprusside, and the application of foliar, seed soaking, or irrigation has tremendously improved plant growth and development under stress conditions [41]. Increasing evidence has contemplated that exogenous application of NO (Figure 3) can explicitly break seed dormancy and improve seed vigor, an essential prerequisite for attaining better growth and increasing crop yield [144,145]. Several studies have found that applying NO donors can effectively modulate seed dormancy even at low concentrations compared to the mechanical and chemical methods of breaking seed dormancy, such as H2SO4 and NaOCl [146,147].

**Figure 3.** Crosstalk of ROS, RNS, and RSS in cell signaling, gene regulation and their role in ameliorating abiotic/biotic stress tolerance in plants. Exogenous application of SNP (NO donor), NaHS (H2S donor), and H2O2 results in the activation of a plethora of signaling pathways, thus regulating cell signaling and activation of several downstream stress-responsive genes/transcription factors leading to improved stress tolerance.

Recent investigations have shed light on the functional mechanisms by which NO mediates regulation of seed dormancy/germination by activating PTMs, such as tyrosine nitration of abscisic acid receptors, such as *S*-nitrosylation SnRK2s and ABI5, followed by degradation of ethylene-responsive factors [66]. In another study, Nagel et al. [146] identified novel loci in barley plants capable of regulating NO signaling, thereby regulating seed dormancy and preharvest sprouting. They concluded that the HvGA20ox1 gene is upregulated by NO signaling, alleviating dormancy and initiating flowering in barley plants [146]. Similarly, the significance of H2S in helping seed dormancy has been reported by various researchers where exogenous H2S upregulated the expression of COP1/HY5 stimulated seed germination in heat-stressed Arabidopsis plants [68]. The implication of exogenous application explicitly improves seed germination and seedling growth in heat-stressed *Zea mays* by regulating the expression of antioxidants and osmolyte biosynthesis [148].

Interestingly, the exogenous application of H2S resulted (Figure 3) in the persulfidation of ethylene biosynthetic enzymes, thereby decreasing the sensitivity of ethylene receptors and leading to enhanced seed germination due to the expression of antioxidative enzymes under stress conditions [149]. Recently, the antagonistic effect of the exogenous application of H2S has also been observed by [18]. The authors confirmed that H2S-mediated persulfidation has also improved seed dormancy/germination by downregulating the expression of DES1 and ABI4 genes.

NO is also known to regulate fruit ripening and post-harvest shelf life by antagonizing ethylene signaling and contributing to shelf life extension [150]. Exogenous application of NO in post-harvest stages of *Magnifera indica* showed decreased membrane damage, ethylene production, and polygalacturonase activities, prolonging the fruits' shelf life without losing quality [151]. Likewise, the exogenous application of NO positively regulated the physiological weight loss, antioxidants, lipooxygenase, and pectin methylesterase activities, improving the quality/shelf life of *Prunus persica* fruits [152]. In addition, exogenous application of H2O2 and NO to guava and tomato fruits exhibited delayed ripening and minimum loss in TSS and ascorbic content, thus improving the storage life of these fruits under storage conditions [153]. Similarly, the exogenous application of H2S has been demarcated to improve the shelf life and quality of tomato post-harvest fruits by inhibiting the protease activity and decreasing the ERF and beta-amylase gene expression with higher nutritional contents [154]. In another study, exogenous application of H2S differentially regulated the expression of cell wall modifying related genes and critical genes involved in pectinase enzyme, thereby preventing rapid fruit softening in the fruits of *Fragaria chiloensis* [155]. Recently, exogenous application of H2S on the activity of various biochemical markers linked to fruit ripening in the fruits of banana and pointed gourd in the post-harvest stage was analyzed to detect their quality and shelf life. They unraveled that exogenous application of H2S significantly lowered membrane damage, lignification, and chlorophyll loss and correspondingly maintained higher PAL and PPO activity, thereby increasing the shelf life of the fruits without compromising their quality and extending their marketing duration [156,157].

NO and H2S have been known to modulate legume rhizobia symbiosis, a critical process in agriculture, as it accounts for the availability of biological nitrogen in the soil [158]. NO is believed to regulate two crucial aspects of nitrogen fixation in legume plants (i) by controlling the rhizobial infection and (ii) by activating nodule organogenesis, both of which are significantly regulated upon the expression of nodulation factors (nod genes) [158]. Increasing evidence has confirmed the regulatory role of NO as a signaling molecule for promoting legume–bacteria interaction and nodule development [159–162]. Exogenous application of NO to *Phaseolus vulgaris* and *Glycine max* stimulated the nodule formation, as indicated by the over-accumulation of nod factors [163]. In addition, studies have suggested that NO and ROS positively regulate legume–rhizobia interaction and nodule formation via ROS/RNS-mediated differential expression of ROS/RNS-producing enzymes [163].

Furthermore, the exogenous application of NO and H2S compellingly enhanced the heavy metal stress tolerance of legume–rhizobia symbiosis by increasing the soil enzyme activity and bacterial diversity, thereby improving their growth and productivity [158]. Likewise, the exogenous application of NO regulated the expression of phytoglobin 1.1 in *Medicago truncatula*, accentuating the symbiosis by controlling nodule development and nitrogen fixation [158]. ROS/RNS/RSS amplifier effect in stimulating legume–rhizobia symbiosis is mainly due to the various PTMs catalyzed by these reactive species that activate essential enzymes, such as thiol peroxidase and phytoglobin genes, that efficiently form nodules in legume crops [6]. Future application of these reactive species in a precise and controlled way can potentially regulate crop growth and productivity by regulating many distinct physiological and biological processes.

### **7. Conclusions**

Earlier, ROS/RNS/RSS has been described as toxic molecules affecting various metabolic and regulatory functions in plants exposed to several biotic and abiotic stress conditions. With the advent of technological breakthroughs, different redox biologists have implicated their role in regulating critical cellular and metabolic processes in plants exposed to climate extremes. When these redox regulators are maintained below a threshold level, they control many signal transduction pathways by mediating PTMs and epigenetic modification in plants. PTMs, such as carbonylation, *S*-nitrosation, tyrosine nitration, and persulfidation, instigate dynamic signaling of different components of enzymatic and non-enzymatic defense

systems at gene and protein levels. The precise regulation induced by these redox regulators at the biochemical and molecular level strengthens plant innate immune response, thereby maintaining the growth and productivity of plants under adverse growing conditions.

Moreover, researchers have also reported that these redox molecules essentially regulate DNA methylation and histone modification in plants, thus significantly impacting the transcription and activities of various genes and enzymes. Nevertheless, the precise mechanisms by which ROS/RNS/RSS regulate these epigenetic marks and other biochemical and molecular processes under stress conditions need further investigation. Furthermore, in-depth explorations of how these redox regulators interact with other signaling molecules and plant growth regulators must be unraveled. The unraveling of molecular mechanisms underlying the regulatory functions of ROS/RNS/RSS will open a new realm for formulating new biotechnological strategies for their possible application in agriculture for fostering new/improved cultivars in the era of climate change.

**Author Contributions:** K.K.R. conceived the idea, prepared all the figures and tables, and wrote the final draft of the manuscript; P.K. critically reviewed and edited the manuscript. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Acknowledgments:** The authors are also thankful to the anonymous reviewers for their careful reading.

**Conflicts of Interest:** The authors declare that no conflicts of interest exist.

#### **References**


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