**1. Introduction**

In the 21st century agriculture and various climatic stresses, such as high temperature, drought, and salinity, have redundantly affected crop growth and productivity, prompting severe threats to global food security for ever-growing global populations [1]. In the Asian continent, a rainfed agriculture system is usually standard and is followed by most farmers. These climatic stresses have become a daunting challenge that has imposed severe repercussions on crop health, thereby ultimately affecting its productivity to a certain extent and leading to livestock death [2]. In addition to sessile, plants are constantly exposed to these climate extremes that instigate various cellular, physiological, biochemical, and molecular responses. Reactive oxygen species (ROS) and reactive nitrogen species (RNS) act as integral components of signal transduction processes regulating vital functions in plants exposed to climate extremes [3]. Initially, ROS and RNS are considered toxic molecules where elevated levels provoke oxidative stress in plants leading to cellular damage and death [4]. However, several recent reports have highlighted that ROS and RNS also function as signaling molecules (when their generation is critically maintained below a threshold level by antioxidative systems), catalyzing several oxidation reactions, thereby modulating vital signaling cascades [5].

**Citation:** Rai, K.K.; Kaushik, P. Free Radicals Mediated Redox Signaling in Plant Stress Tolerance. *Life* **2023**, *13*, 204. https://doi.org/10.3390/ life13010204

Academic Editors: Wajid Zaman and Hakim Manghwar

Received: 24 November 2022 Revised: 5 January 2023 Accepted: 9 January 2023 Published: 10 January 2023

**Copyright:** © 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

Redox chemistry is inextricably engaged in the generation, regulation, and sustainment of life on earth by exhilarating reduction–oxidation (redox) reactions essential for driving crucial cellular and metabolic processes such as photosynthesis, respiration, and other biochemical reactions in diverse life forms [6]. Interestingly, recent reports have restricted the involvement of ROS and RNS in stimulating thermodynamically favorable reactions that are essential for sustaining life, including (i) their ability to enhance metabolic reactions, (ii) regulate enzymatic and non-enzymatic antioxidants, (iii) cross barriers (membranes) to activate signaling cascades, and (iv) provide a source of energy (electrons) to defend against oxidative stress [7]. In the recent decade, a plethora of research has been conducted to assess the positive side of ROS/RNS signaling in plants' growth, development, and defense response. A concomitantly large body of literature has pinpointed their exemplary role [8].

Reactive sulfur species (RSS) is a term still to be entered into the general scientific vocabulary due to its low expression and lack of consideration of its role in signal transduction [9,10]. Early prebiotic life forms, i.e., before photosynthesis, likely thrived under a sulfur-rich environment, and several reports claimed that when life originated, approx. 3.8 billion years ago, RSS was the first reactive molecule that influenced expansion [9]. Before photosynthesis, RSS, mainly hydrogen sulfide (H2S) released from volcanic eruptions and other geothermal activity, served as building blocks for nucleic acid biosynthesis and protein synthesis for early life forms, such as Beggiatoa, pupfish, giant tubeworms, and mollusks [10]. Researchers have also confirmed the involvement of RSS in providing reducing powers for fixing CO2 via the Calvin cycle in various green and purple sulfur bacteria [6,11]. Furthermore, a large body of literature has implicated the significant role of RSS in initiating oxidation reactions, thereby controlling redox homeostasis, cell signaling, and defense response in plants [12].

Recent studies have corroborated that ROS, RNS, and RSS have similar chemical structures, yet RSS is more versatile and reactive. In addition, ROS/RNS-mediated biosynthesis of RSS under an oxidative environment is its unprecedented source [9]. Due to its similar chemical nature, RSS triggers oxidation reactions by modifying cysteine sulfur and produces an identical effector response as ROS and RNS. Still, the reaction of RSS is more prominent and stable for a much longer duration [13]. Recent discoveries have spectated the growth stimulatory effect of RSS on plant growth and development under stress conditions [6,11,12]. The functional mechanism by which RSS exerts a change stimulatory effect in plants has been unearthed by a few researchers who identified that RSS signaling stimulates post-translational modification of cysteine residues (Cys-SSH) that regulate the expression of stress-responsive genes/proteins [13].

Further, RSS-induced sulfidation, or sulfhydration of proteins, accentuates critical metabolic pathways that stimulate the biosynthesis of secondary metabolites and enzymatic antioxidants, thereby improving the physiology and morphology of plants exposed to climate extremes [12]. Despite their versatility and stability, RSS chemistry, biosynthesis, and plant functions remain anonymous to date. Several excellent review articles have been published on ROS and RNS signaling in plants [9,10]. Discussing them in detail will result in the lengthening of the context. Therefore, readers are requested to consider the articles mentioned above. This review provides an in-depth understanding of RSS biosynthesis in plants and its chemical and biological properties in regulating the functions of proteins/genes and signaling pathways under stress conditions. In addition, this review also examines how RSS interact with other reactive oxidants, such as ROS and RNS, and their combinatorial effect in regulating the growth and metabolism of plants.

#### **2. Chemical Biology of RSS in Plants**

In recent years, RSS has been proclaimed to be inexorably interlinked with all life forms from its inception to the present day [14]. Several studies have confirmed that ROS and RSS are chemically similar and are often grouped under the category of chalcogens, i.e., both belong to group 16 of the periodic table [15]. Concomitantly, ROS and RNS mediate the production of RSS under oxidative environments, which is a potential route

for RSS biosynthesis [9]. RSS, like ROS, is also categorized as free radicals and non-radicals. Non-radicals are comprised of thiol (RSH), disulfide (RSSR), sulfenic acid (RSOH), and thiosulfate (RSOSR), whereas free radicals are composed of thiyl-radical (RS˙) [14]. Various organisms such as plants, bacteria, fungi yeasts, nematodes, and humans employ different chemical reactions at intracellular and molecular levels, thereby affecting physiological and molecular processes in the concerned organisms [15].

During the prebiotic era, hydrogen sulfide (H2S) was the primary energy source catalyzing significant steps for incepting life that drove the evolution [16]. In a study, Wachtershauser's group tried to mimic the conditions before photosynthesis, where organic carbon and nitrogen molecules in the presence of H2S catalyze the formation of thiosulfoxide, which is then converted to persulfide via one-electron oxidation reactions [17]. Correspondingly, upon its subsequent exposure to additional thiosulfoxide, persulfide produces polysulfides (H2Sn) capable of catalyzing oxidative metabolism in purple and green sulfur bacteria [17]. Thiosulfoxide, persulfide, and polysulfide can be stored and recycled/reduced back to H2S to accentuate future oxidation reactions, thus confirming their capability to induce oxidation/reduction reactions for stimulating various signaling pathways [18]. In contrast, ROS and RNS do not possess such ability; they cannot be stored or reused in signaling or related pathways because they function as "one and done".

However, despite dissimilarity, RSS exhibits higher similarity with ROS than RNS because both are chalcogens with six electrons in their valence shell [19]. Nonetheless, RSS is considered the most versatile, promiscuous, and stable reactive oxidant of its counterparts, i.e., ROS/RNS. Due to higher electronegativity, outer shell electrons in oxygen are near the nucleus, as in sulfur, where electrons are farther from the nucleus [9]. Furthermore, the most stable oxidation state for oxygen is −2 and 0; however, it may also exist in a less stable form of −1, +1, and +2; on the other hand, the most stable oxidation state for sulfur ranges from −2 to +6 [14]. Additionally, sulfur contains more than 30 allotropes compared to oxygen, which has fewer than 10, further attesting to the flexibility of RSS under extreme environments [9]. Strikingly, ROS and RSS exist in various forms with similar chemical and functional properties, i.e., ROS is produced from the one-electron reduction of oxygen (Table 1). In contrast, RSS is produced from one-electron oxidation of H2S [10]. ROS, such as hydrogen peroxide (H2O2), superoxide anion (O2˙ −), hydroxyl radical (OH˙), perhydroxy radical (HO2), and singlet oxygen (1O2), are exclusively involved in signaling under stress conditions [10]. Below, Equation (1) depicts the generation of ROS via a single oxygen reduction process:

$$\text{O}\_2 \overset{+/-\text{E}\_-}{\longleftrightarrow} \text{O}\_2\text{-}^- \overset{+/-\text{E}\_-}{\longleftrightarrow} \text{H}\_2\text{O}\_2 \overset{+/-\text{E}\_-}{\longleftrightarrow} \text{OH}\text{-}^\cdot \overset{+/-\text{E}\_-}{\longleftrightarrow} \text{H}\_2\text{O} \tag{1}$$

While perhydroxy radical (HO2) and singlet oxygen (1O2) are less intense, signaling molecules often scavenged into peroxide and oxygen are relatively impermeable across membranes. They have a short half-life, are unstable, and are less reactive.

In contrast, 'RSS, which are produced by single-electron oxidation of H2S, are often composed of thiyl radical (HS˙), hydrogen persulfide (H2S2), and persulfide radical (S2˙ −) (Table 1). This biosynthetic reaction ends with the formation of elemental sulfur; the steps of which are depicted in Equation (2):

$$\text{S}\_2 \overset{+/\_{-}\text{e}}{\longleftrightarrow} \text{S}\_2\text{-}^- \overset{+/\_{-}\text{e}}{\longleftrightarrow} \text{H}\_2\text{S}\_2 \overset{+/\_{-}\text{e}}{\longleftrightarrow} \text{HS} \overset{+/\_{-}\text{e}}{\longleftrightarrow} \text{H}\_2\text{S} \tag{2}$$



Abbreviations: O2: molecular oxygen, O2˙−: superoxide anion, Fe3+: ferric ion, 1O2: singlet oxygen, Fe2+: ferrous ion, H2O2: hydrogen peroxide, OH−: hydroxyl anion, OH˙˙: hydroxyl radical, NO2˙: nitric dioxide, NADPH: nicotinamide adenine dinucleotide phosphate dehydrogenase, NO˙: nitric oxide radical, L-Arg: L-arginine, FAD: flavin adenine dinucleotide, MOCO: molybdenum cofactor, Cyt C: cytochrome C, L-Cys: L-cysteine, H2S: hydrogen sulfide, NH3: ammonia, SO−<sup>2</sup> <sup>3</sup> : sulfite ion.

#### **3. RSS Biosynthesis in Plants**

In plants, the genus Allium provides an intriguing insight into the biosynthetic pathway of RSS from natural sulfur agents [22]. The first step in RSS production is the biosynthetic transformation of sulfoxides to thiosulfinates, i.e., allin to allicin catalyzed by the alliinase enzyme C-S-lyase type enzyme of 103 kDa with two similar subunits of 448 amino acids [22,23]. The alliinase enzyme contains pyridoxal 5- -phosphate (PLP) at the active site, enhancing the enzyme's efficiency when the substrate is present at a low level, i.e., allin. In the presence of allin, alliinase catalyzes the formation of highly reactive aldimine, resulting in the generation of sulfenic acid (RSOH) and a PLP amino acylates complex [22]. The complex becomes detached from the RSOH and combines with ammonia and pyruvate to facilitate the formation of thiosulfinate (RSOSR), such as allicin/water [9].

Thiyl radicals, a free radical RSS, are synthesized via single-electron oxidation of the thiol group occurring under oxidative stress conditions, where a proton from the S-atom is removed before or after electron dissemination leading to the formation of disulfide [22]. A notable example of thiyl free radical formation is seen during the glutathionylation of several proteins in plants, where thiyl radical intermediates are provoked by ROS/RNS and other enzymatic antioxidants to generate RSS radicals [23]. Furthermore, researchers have indicated that thiol/disulfide exchange between RSSR and RSH can potentially shift the redox potentials of RSH in a powerful way that can oxidize RS (O) SR from four thiol radicals (R'SH) which are later converted to thiol and R'SSR [24].

Thiosulfinates are highly reactive non-radical RSS that can react with any molecule containing the thiol (R'SH) group to form sulfenic acid and disulfide (RSSR) [24]. Additionally, disulfides can be oxidized by themselves under stress conditions or can be corrupted by ROS/RNS to thiosulfinates and later into thiosulfonates (RD (O)2SR), thus validating the involvement of ROS/RNS in RSS biosynthesis [23]. Concomitantly, ROS/RNS-mediated oxidation of the S-atom of sulfenic acids results in sulfinic acid formation. The S-atom of the latter has a higher oxidation state than the former, which can cause further oxidation of sulfinic acid to sulfonyl radicals, which are considered the progenitors of RSS biosynthesis in plants [23].

Interestingly, sulfenic acids also have the potential to oxidize thiols to disulfides and, at the same time, can initiate the reduction of cumene and linoleate and, therefore, are also called ultimate reductants [24]. Like superoxide anions, sulfenic acids also possess electrophilic and neutrophilic properties, thus acting as powerful oxidizing and reducing agents leading to the formation of RSS even by reducing sulfur-containing molecules or by oxidizing sulfur atoms from cell proteins, such as cysteine and methionine [13]. A large body of literature has indicated that sulfenic acid-mediated oxidation of protein-thiols could result in the generation of RSOH, RS (O)2H), RS (O)3H, and RSSR [6]. Sulfenic acids are pivotal RSS molecules that can readily react with thiols to form disulfides, exponentially increasing thiol-containing proteins' catalytic efficiency. In addition, sulfenic acids are included upon hydrolysis of nitrosothiols (nitric oxide donors), modulating critical biological processes and signal transduction.

#### **4. RSS Signaling in Plants**

The functional mechanism by which RSS works as a signaling molecule is relatively analogous to that of ROS/RNS signaling; nonetheless, the former is more intricate and perplexing at the biochemical and molecular levels [22]. Several researchers have established that ROS and RSS exploit cysteine residues as the "junction" to mediate redox signaling, sensing, and catalysis of critical metabolic functions [23]. Recent studies have corroborated that RSS function as a signaling molecule after interacting with thiol (-SH) containing proteins (cysteine and methionine), thus regulating persulfidation through post-translational modification (PTMs). Furthermore, RSS can modulate various PTMs such as *S*-glutathionylation, *S*-nitrosation, *S*-cyanylation, and *S*-acylation upon interaction with RNS, glutathione, cyanide, and fatty acids [13]. The profound biochemical implication of RSS in plant cells has delineated its intricate interaction with various antioxidant enzymes, such as glutathione reductase, peroxidase, and sulfite oxidase, thus mediating sulfur metabolism in plant peroxisomes [25]. Correspondingly, this sulfite oxidase, in conjunction with the catalase enzyme, catalyzes the conversion of sulfite to sulfate, thus resulting in the generation of ROS [26]. The existence and inception of RSS have been recently mapped in peroxisomes of root tips and guard cells of Arabidopsis using confocal laser scanning microscopy (CLSM) and proteomic techniques [13,27].

Furthermore, researchers have also mapped the position of corresponding protein genes involved in RSS biosynthesis and observed that the majority of them were present on chromosomes 1, 2, 3, and 5 (Figure 1A,B) along with the protein/genes of ROS and RNS biosynthesis in Arabidopsis [28]. In addition, conserved motif analysis and gene sequence alignments demonstrated that all the reactive species producing enzymes/proteins have a highly conserved arrangement of motifs with varying sequences (Figures 1C and 2A) [29]. The phylogenetic and gene co-expression analysis (Figure 2B,C) of the major reactive species producing enzymes/proteins diverge from one another as they evolve from a common ancestor, and co-expression of the corresponding genes is essential for activating the transcription of stress-responsive genes and transcription factors [28,29].

**Figure 1.** Chromosomal localization, synteny analysis, and motif elucidation of important ROS-, RNS-, and RSS-producing genes in model plant *Arabidopsis thaliana*. (**A**) Chromosomal localization, (**B**) synteny analysis, and (**C**) conserved motifs are represented in different color boxes. AT5G07390/RBOHA: respiratory burst oxidase homolog protein A, AT1G77760/NIA1: nitrate reductase 1, AT1G52340/ABA2: ABA xanthoxin dehydrogenase, AT2G27150/AAO: abscisic-aldehyde oxidase, AT3G01910/SOX: sulfite oxidase, AT1G05240/POX: peroxidase 1, AT3G62130/LDES: L-cysteine desulfhydrase, AT3G26115/DDES: D-cysteine desulfhydrase 2, AT5G04590/SIR: assimilatory sulfite reductase, AT3G59760/CYSC1: cysteine synthase, AT3G61440/QASC: nifunctional L-3-cyanoalanine synthase/cysteine synthase C1.

Scientists have only recently uncovered that the peroxisomal enzymes are the primary targets of the persulfidation of thiol-containing proteins leading to the generation of RSS under stress conditions [30]. Later, in vitro analysis in Arabidopsis and *Capsicum annum* unraveled the mechanistic insight into the enzymatic pathway of RSS biosynthesis, where catalase functions both as oxidase and reductase to catalyze the synthesis of H2S [30]. Apart from catalase and peroxidase, superoxide dismutase can generate persulfide by stimulating the reaction between O2 and H2S in mitochondria and peroxisome, thus validating that apart from ROS/RNS, these organelles also serve as the site of RSS biosynthesis [31]. Still, scientists are trying to delve deeper and further to identify the precise biochemical and

metabolic pathway involved in the biosynthesis of RSS in plants, as well as to unravel the mechanism by which they interact with other signaling molecules [13]. Few studies have identified possible enzymatic routes of RSS biosynthesis in the different subcellular compartments of plants, such as cytosol, chloroplast, mitochondria, and peroxisomes. They postulated that the enzymes such as D-cysteine desulfhyfrase, cyano alanine synthase, sulfite reductase, cysteine synthase, and cysteine synthase-like modulate RSS signaling in plants exposed to climate extremes [13,25].

**Figure 2.** Basic sequence analysis, phylogenetic relationship, and co-expression prediction of important ROS-, RNS-, and RSS-producing genes in the model plant *Arabidopsis thaliana*. (**A**) Gene sequence, (**B**) phylogenetic analysis, and (**C**) co-expression of putative genes using TBtools and iTOL software. AT5G07390/RBOHA: respiratory burst oxidase homolog protein A, AT1G77760/NIA1: nitrate reductase 1, AT1G52340/ABA2: ABA xanthoxin dehydrogenase, AT2G27150/AAO: abscisic-aldehyde oxidase, AT3G01910/SOX: sulfite oxidase, AT1G05240/POX: peroxidase 1, AT3G62130/LDES: L-cysteine desulfhydrase, AT3G26115/DDES: D-cysteine desulfhydrase 2, AT5G04590/SIR: assimilatory sulfite reductase, AT3G59760/CYSC1: cysteine synthase, AT3G61440/QASC: bifunctional L-3-cyanoalanine synthase /cysteine synthase C1.

Plant peroxisome is the house of various signaling molecules such as glutathione, *S*-nitrosoglutathione, hydrogen peroxide, and sulfite oxidase that are explicitly involved in the RSS biosynthesis and metabolism [32]. Recent discoveries have indicated that RSS signaling profoundly mediated the peroxidation of cysteine sulfur resulting in the formation of sulfenyl via a process known as sulfenylation. Sulfenylation is analogous

to persulfidation, a method capable of accentuating RSS-mediated redox sensing and signaling, thus modulating protein folding, histone modification, DNA–protein interaction, and other regulatory functions [33]. One of the mechanisms by which RSS signaling works is that both sulfenylation and persulfidation can reduce hydrogen peroxide to water, which in turn induces the restoration of protein thiols, thus provoking their interaction with other low molecular weight thiols (thioredoxins and reduced glutathione) [34]. The interaction between persulfidated proteins and low molecular weight thiols results in the generation of polysulfide, which is actively involved in the storage and transportation of sulfur molecules capable of initiating RSS signaling [33]. RSS signaling is also incepted upon the NADPH-mediated reduction of persulfidated proteins, which upon interaction with peroxisomal ROS, results in the generation of RSS [35]. Some researchers have also advocated accentuating RSS signaling by incorporating cysteine persulfides into proteins via ROS-mediated PTMs [30]. Furthermore, peroxide signaling is often associated with unresolved conundrums regarding RSS signaling, yet it is hypothesized peroxide signaling acts as a relay between peroxidase and thiol proteins. Correspondingly, the resulting reaction product (oxidants) establishes a complex with the peroxidase proteins, thus categorizing the oxidant signals involved in the modulation of RSS signaling [36].

#### **5. Crosstalk between ROS/RNS/RSS in Plant Defense**

Plant stress signaling comprehends an intricate network of various signaling molecules that function synergistically or antagonistically to regulate multiple physiological and metabolic processes under specific environmental conditions [37]. Several plant growth regulators, such as salicylic acid, auxin, jasmonic acid, ethylene, abscisic acid, polyamines, and melatonin, are involved in modulating a broad range of plant functions [6]. Apart from these, the gasotransmitters, such as H2S and NO, have also been recognized as an indispensable combination for regulating plants' physiological and stress-related processes (Table 2) with high efficacy and stability compared to their natural counterparts [6]. A molecule can act as a signal only when its level reaches beyond the optimum threshold, driven by complex processes occurring inside the cell. Despite the complex network, all the reactive species, whether ROS, RNS, or RSS, diverge at some metabolic checkpoints where they encounter identical targets responsible for their production, accumulation, and scavenging [18]. When these signaling molecules (they may contain reactive species or plant growth regulators) are exogenously applied, it results in the transient accumulation of compounds that exaggerate the endogenous level of ROS, RNS, or RSS [37]. This transient accumulation of reactive species could have synergistic or antagonistic effects before or after stressful events.

Recent studies have corroborated that the exogenous application of these signaling molecules is integrally involved in regulating complex networks (Table 2) such as redox status, stomatal movement, root development, and apoptosis in plants exposed to climate extremes [38,39]. However, the effects of these signaling molecules are primarily dependent upon their dosage, subcellular compartment, duration of the application, and their relative flux in the treated plant cell/tissue [40]. A large body of literature exists on the stimulatory role of H2S and NO in plants exposed to biotic/abiotic stresses, which has been summarized in recent review articles [6,41–43]. Therefore, this crosstalk will provide current updates on the involvement of H2S and NO in modulating plant functioning at the molecular level, which is imperative for understanding the role of these gasotransmitters in stress alleviation.


**Table 2.** Major regulatory effects of H2O2, NO, and H2S on the physiological and metabolic process in plants exposed to climate extremes.


**Table 2.** *Cont.*

Abbreviation: H2O2: hydrogen peroxide, NO: nitric oxide, H2S: hydrogen sulfide, GA: gibberellic acids, ABA: abscisic acid, ROS: reactive oxygen species, NADPH: nicotinamide adenine dinucleotide phosphate dehydrogenase, MAPK: mitogen-activated protein kinase, BHLH: basic helix loop helix, MYB: myeloblastosis transcription factor, IAA: indole acetic acid, Des1: l-cysteine desulfhydrase, Ash: ascorbate, Gsh: glutathione, ACC: 1-Aminocyclopropane 1-carboxylic acid.
