**Regulation of Ascorbate-Glutathione Pathway in Mitigating Oxidative Damage in Plants under Abiotic Stress**

#### **Mirza Hasanuzzaman 1,\*, M. H. M. Borhannuddin Bhuyan 2,3, Taufika Islam Anee 1, Khursheda Parvin 2,4, Kamrun Nahar 5, Jubayer Al Mahmud <sup>6</sup> and Masayuki Fujita 2,\***


Received: 30 June 2019; Accepted: 5 September 2019; Published: 9 September 2019

**Abstract:** Reactive oxygen species (ROS) generation is a usual phenomenon in a plant both under a normal and stressed condition. However, under unfavorable or adverse conditions, ROS production exceeds the capacity of the antioxidant defense system. Both non-enzymatic and enzymatic components of the antioxidant defense system either detoxify or scavenge ROS and mitigate their deleterious effects. The Ascorbate-Glutathione (AsA-GSH) pathway, also known as Asada–Halliwell pathway comprises of AsA, GSH, and four enzymes viz. ascorbate peroxidase, monodehydroascorbate reductase, dehydroascorbate reductase, and glutathione reductase, play a vital role in detoxifying ROS. Apart from ROS detoxification, they also interact with other defense systems in plants and protect the plants from various abiotic stress-induced damages. Several plant studies revealed that the upregulation or overexpression of AsA-GSH pathway enzymes and the enhancement of the AsA and GSH levels conferred plants better tolerance to abiotic stresses by reducing the ROS. In this review, we summarize the recent progress of the research on AsA-GSH pathway in terms of oxidative stress tolerance in plants. We also focus on the defense mechanisms as well as molecular interactions.

**Keywords:** antioxidant defense; free radicals; glyoxalase system; hydrogen peroxide; plant abiotic stress; reactive oxygen species; redox biology; stress signaling

#### **1. Introduction**

With the advancement of lifestyle the natural resources are being exploited and the interruption of natural environment is increasing the extremity of various kinds of abiotic stress, including salt stress, drought stress, waterlogging, temperature extremes, including high and low, excess and low light intensity, radiation stress, ozone, metal and metalloid toxicity, and other organic or inorganic pollutants. Environmental extremity narrows ways to increase plant productivity. Ever-increasing population demands newly cultivable areas, including the adverse land areas, even the higher crop production in per unit area.

Any abiotic stress impaired stomatal function, photosystem activity, Calvin cycle, or photosynthetic enzyme activities, as well as altered electron transport chain reactions. Moreover, unfavorable peroxisomal or cytosolic atmosphere led to overwhelm of electron absorption and generate ROS as a common outcome and subsequently causes oxidative damage [1,2]. If the challenges of plant scientists are increasing productivity against the abiotic stresses, their concentrations are moving to the depth for breaking the obstacles at the cellular or organelles levels, where abiotic stresses impose common types of barrier to hinder their function. Reactive oxygen species is an inescapable outcome of aerobic reactions, which are partly reduced or activated by the appearance of oxygen. Reactive oxygen species is a combined name that indicates different, highly active components. Superoxide (O2 −), hydroxyl (OH•), and peroxyl (ROO•) are some examples of oxygen radicals. Hydrogen peroxide (H2O2), singlet oxygen (1O2), and ozone (O3) are the non-radical types of ROS [3]. Reactive oxygen species are important for plants. They have dual role in plants: a small amount of those acts as a signal for inducing abiotic stress responses towards adaptation process, while the excess generation of those causes oxidative damage. However, in severe cases, oxidative damages to membranes (lipid peroxidation), proteins, nucleic acid, including RNA and DNA, and even directs to the oxidative obliteration of the cell [4]. Chloroplast, mitochondrion, membranes of the cell or its ultrastructural organelles, apoplast, and nucleolus are the locations of ROS production. Nonetheless, peroxisome is also considered as a powerful source of ROS since the electron transport chain (ETC) and photochemical reactions are the majority of the processes generating ROS [5–7].

Plants have an antioxidant defense system having non-enzymatic and enzymatic antioxidants in cellular organelles, which scavenges different ROS up to a certain level. If the ROS generation is higher than the scavenging ability of the antioxidant system, then oxidative damage occurs. Antioxidant defense system comprises ascorbate (AsA), glutathione (GSH), carotenoids, tocopherols, flavonoids, etc., which are some commonly known non-enzymatic antioxidants [5]. Ascorbate peroxidase (APX), monodehydroascorbate reductase (MDHAR), dehydroascorbate reductase (DHAR), glutathione reductase (GR), superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPX), glutathione *S*-transferase (GST), and peroxiredoxin (PRX) are well known enzymatic antioxidant components [8,9]. Among all of these, AsA, GSH, APX, MDHAR, DHAR, and GR comprise the AsA-GSH cycle.

Ascorbate is one of the most powerful substrates for scavenging H2O2. Ascorbate maintains the reduced state of α-tocopherol. Ascorbate is supposed to be concerned in zeaxanthin biosynthesis dissipating excess light energy in the thylakoid membranes of chloroplast and prevents oxidative stress. Ascorbate sustains reduce the state of prosthetic metal ions and maintain the activity of antioxidant enzymes [6]. Glutathione regulates various metabolic functions; it acts as an antioxidant. Glutathione peroxidase and GST utilize GSH as substrate; GPX is responsible for ROS detoxification, whereas GST is liable for xenobiotic detoxification [1]. The glyoxalase system consisting of glyoxalase I (Gly I) and glyoxalase II (Gly II) enzymes detoxifies cytotoxic and oxidative stress creator methylglyoxal (MG), where Gly I uses GSH and after finishing MG detoxification, GSH is recycled [2]. The positive role of AsA-GSH cycle components has been documented in many plants that are affected by abiotic stresses [1,2]. Participation of the GSH/glutathione disulfide (GSSG, the oxidized form of GSH) redox in maintaining a favorable cellular environment and in stress signal and adaptation were discussed in some previous findings. Glutathione participates in signal transduction, the proper pathway, of which remains unrevealed. The presence of AsA and GSH has been reported to improve osmoregulation, plant water status and nutrient status, water use efficiency, photosynthetic performance, and the overall productivity of plants. Exogenous AsA and GSH applications have been reported to enhance the antioxidant defense as well as the overall tolerance of plants against abiotic stresses. Accordingly, the enzymatic antioxidants of AsA-GSH cycle participates in scavenging ROS, whereas AsA and GSH not only directly scavenge a range of ROS but also perform many other functions to maintain a favorable state in cytosol and other cellular organelles to enhance antioxidant capacity and to reduce oxidative stress, which is induced by different abiotic stresses; AsA and GSH also improve the physiological performance of plants. Since the discovery of the AsA-GSH cycle, its most discussed

topics are related to antioxidative protection. However, in this aspect, various other factors should be revealed like physiological factors/processes involved in generating oxidative stress, role of AsA-GSH cycle components in regulating those physiological processes and ultimately the oxidative stress. Considering the multiple vital roles of AsA-GSH cycle in mitigating oxidative stress, this review accommodates presently available and updates of research findings and perspectives.

#### **2. Ascorbate-Glutathione Pathway—An Overview**

Ascorbate-Glutathione pathway (also called as Asada–Halliwell pathway) is the major pathway of antioxidant defense, which mainly detoxify the H2O2 in a plant cell. Apart from AsA and GSH, its enzymes—APX, MDHAR, DHAR, and GR [6]—have significant roles. Both AsA and GSH are found in the cytosol, nucleus, chloroplast, mitochondria, and peroxisome, where they operate the functions assisted by four enzymes and, therefore, each enzyme has several isoforms that are based on the cellular localization [10]. Both AsA and GSH are present in cellular organelles in a millimolar range, for instance, in *Arabidopsis thaliana*, AsA concentration is the highest (22.8 mM) in the peroxisome, where GSH is highest (14.9 mM) in mitochondria [11,12]. AsA and GSH both have high redox potentials and, therefore, interact with many components and pathways towards the maintenance of a generally reduced state. There are few steps, by which AsA and GSH work coordinately to detoxify H2O2, and at the same time, both AsA and GSH are regenerated. First, the enzyme APX converts H2O2 into the water with the help of AsA as an electron donor, which is also converted into monodehydroascorbate (MDHA). This MDHA again regenerates AsA by the activity of MDHAR and a part of this is spontaneously converted into dehydroascorbate (DHA). Later, DHA is reduced to AsA again by using GSH, which results in its oxidation to produce GSSG. Finally, this GSSG regenerates GSH by the activity of GR using NADPH as the electron donor (Figure 1). Both AsA and GSH are strong antioxidants, but the maintenance of their redox state is important in conferring stress tolerance in plants, which largely depends on the activities of the four enzymes that are associated with the AsA-GSH cycle [6,13]. In the next sections, we have described all of the components of the AsA-GSH pathway.

**Figure 1.** Ascorbate-Glutathione (AsA-GSH) (Ascorbate-Glutathione) pathway [ascorbate, AsA; ascorbate peroxidase, APX; monodehydroascorbate, MDHA; monodehydroascorbate reductase, MDHAR; dehydroascorbate, DHA; dehydroascorbate reductase, DHAR; glutathione, GSH; oxidized glutathione, GSSG; glutathione reductase, GR; Nicotinamide adenine dinucleotide phosphate (reduced form), NAD(P)H; Nicotinamide adenine dinucleotide phosphate (oxidized form), NAD(P)+].

#### **3. Components of AsA-GSH Pathway**

#### *3.1. Ascorbate*

All living organisms either make AsA (also known as Vitamin C) or get it in their foodstuffs. Naturally abundant l-AsA is of a simplest chemical structure and is related to C6 sugars. It is a hexonic acid aldono-1,4-lactone (either l-galacturonic or l-gulonic acid), having an enediol group at C2 and C3 [14]. The enediol group enables l-AsA for donating one or two electrons to form an initial oxidized intermediate (MDHA) and further to an oxidized (DHA) form. The C5 and C6–OH group serves to provide alcoholic nature. They can react with produced acetals, ketals by reacting with aldehydes and ketones, respectively. Having two asymmetric C, l-AsA illustratesa positive optical rotation, which is unaltered by the acidicpH of solution but greatly affected by alkaline pH, which increases over +160◦ in 2N NaOH solution [14].

In solid-state, l-AsA is stable but oxidizes readily in solution, in particular in the presence of Cu, Fe, or alkali to form DHA. Afterward, two MDHA can undergo spontaneous reaction to rejuvenate one molecule of l-AsA and one molecule of DHA [15].

Ascorbate biosynthesis system is one of the ancient pathways and formed in very primitive life process on this planet. In plant tissue, AsA can be synthesized from several biochemical pathways. d-glucose is the primary substrate for producing AsA, and in this pathway, a set of ten reactions occurred (Figure 2). Ascorbate can be formed via four pathways viz. l-galactose, l-gulose, d-galacturonic acid, and myo-inositol pathway [16–18]. The biosynthesis of AsA is lineal with the cell wall formation. After the initial reactions, the d-galacturonic acid and L-galactose pathways both yielded l-galactono-1,4-lactone. Besides, in l-gulose and myo-inositol pathway, l-gulonic acid is produced, which is further hydrolyzes to form l-gulono-1,4-lactone, are catalyzed for the synthesizing of AsA in the mitochondria (Figure 2) [19].

In an organism, AsA metabolism comprises of biosynthesis (catabolism) and degradation (anabolism), and the balance between catabolism and anabolism determines the intracellular concentration of AsA. In the previous paragraph, we briefly discussed the biosynthesis of AsA. Hence, we will discuss the degradation and turnover of AsA in this paragraph. In some plants, the AsA turnover rate is relatively very high [20]. On the other hand, AsA is neither stable nor is restricted to oxido–reduction, which changes the equilibrium of AsA and DHA in plant tissue. The AsA pool undergoes turnover in plants. As AsA has prominent responsibility in the redox function metabolism, therefore the recycling of AsA from MDHA (catalyzed by MDHAR using NADPH) and DHA (DHAR using GSH) is the necessity to keep the redox balance as well as the higher total AsA pool [21]; and the functioning of the water-water cycle to optimize photosynthesis [22]. If the oxidized forms are not recovered, they will undergo further degradation to form oxalic or l-tartaric acids (Figure 2) [23]. In a plant cell, AsA act as a multifunctional biosynthetic precursor. While using radioactive 14C AsA, some studies tried to understand the degradation of AsA and DHA, but the mechanism is still not fully understood (Figure 2) [24]. However, it is well understood that the cleavage between C2 and C3 results in oxalate formation, whereas the cleavage between C3 to C6 produces l-threonate, via l-idonate [25,26]. Furthermore, DHA can be hydrolyzed into 2,3-l-diketogulonate, being further oxidized to unknown intermediate (Figure 2) and catalyzed by ascorbate oxidase (AO). Sometimes, this intermediate produces toxic H2O2 non-enzymatically and it may inhibit peroxidase [24,27].

**Figure 2.** Ascorbate biosynthesis and metabolism is a complex set of reactions, some involving unidentified enzymes; some of the products are reactive and potentially damaging carbonyl compounds, (**A**) biosynthetic pathway; and, (**B**) regeneration and degradation pathways in plants. The metabolites in the violate box represent the name of each biosynthetic pathway. The elaborated name of enzymes are as follows (HK: Hexokinase; PGI: glucose-6-phosphate isomerase; PMI: mannose-6-phosphate isomeras; PMM: phosphomannomutase; TC1 or GMP: GDP-d-mannose pyrophosphorylase/mannose-1-phosphate guanylyltransferase; VTC2 or GGP: GDP-d-mannose 3 ,5 -epimerase, GME: GDP-l-galactose phosphorylase;VTC4 or GPP:l-galactose-1-phosphate phosphatase; GalDH: l-galactose dehydrogenase; l-GalLDH: l-galactono-1,4-lactone dehydrogenase; ?: nucleotide pyrophosphatase or sugar-1-phosphate guanyltransferase; ??: sugar phosphatase; l-GulDH: l-gulose dehydrogenase; l-GulL: l-gulonolactonase; l-GulLOX: l-gulono-1,4-lactone oxidase; PPGT: polygalacturonate 4-alpha-galacturonosyltransferase; d-GalPUT: d-galacturonate-1-phosphate uridyltransferase; d-GalUR: d-galacturonate reductase; AL: aldonolactonase; PGM: phosphoglucomutase; UDPGluPP: UDP-glucose-pyrophosphorylase; UDP-GluDH: UDP-glucose dehydrogenase; UDP-GluPUT: glucuronate-1-phosphate uridylyltransferase; d-GluPP: d-glucurono-1-phosphate phosphatase; MIOX: myo-inositol oxygenase;d-GluR: d-glucuronate reductase; MDHAR: monodehydroascorbate reductase; DHAR: dehydroascorbate reductase;l-IDH: l-Idodonate dehydrogenase).

#### *3.2. Glutathione*

Glutathione is an omnipresent low molecular weight tripeptide (γ-l-glutamyl-l-cysteinylglycine; γ-Glu-Cys-Gly), which is a strong antioxidant and an essential metabolite with a multifarious role in plants [28,29]. It was first discovered from yeast cells subsequently in many plants and animal tissue. Later on, in 1936, it was found as the reducing agent present in the plant tissue [30].

Although GSH is composed of glutamine (Glu), cysteine (Cys), and glycine (Gly), three essential amino acids; but some plant may contain homologues of GSH, where Gly is replaced by other amino acids [31]. In plants, reduced GSH accounts for >98% of total GSH [10]. Generally, cells possess three major reservoirs of GSH cytosol (80–85%), mitochondria (10–15%), and endoplasmic reticulum [32]. The thiol group is specific to γ-glutamyltranspeptidase (GGT) and it allows GSH a higher degree of stability [32,33]. Nevertheless, GSH plays a vital role, including antioxidant defense, xenobiotics detoxification, cell cycle regulation, and apoptosis, reserving cysteine, maintaining redox balance as well as immunity modulation and fibrogenesis [10,29].

In plants, GSH biosynthesis involves two enzymatic steps, which require ATP and the constituent amino acids (Figure 3). In the earliest stratum, γ-glutamylcysteine (γ-EC) is produced by γ-glutamylcyteine synthetase (γ-ECS, EC 6.3.2.2) catalysis and participation from Glu and Cys. In the next stratum, GSH is synthesized from γ-EC and Gly via bonding from the Cys residue of γ-EC with α-amino group of Gly, catalyzed by GSH synthetase (GSH-S, EC 6.3.2.3, also known as GSH synthase). After synthesis in the cytoplasm, GSH is transported to other cellular organelles [34].

**Figure 3.** Glutathione biosynthesis, metabolism, and degradation in plants. (**A**) Biosynthesis the first step occurred in plastid: Glu and Cys form γ-glutamylcysteine (γ-EC) catalyzed by γ-EC synthetase (γ-ECS). The second step occurred in the cytosol or in plastid: γ-EC and Gly bond together to form GSH catalyzed by GSH-S (glutathione synthase). Further, GSH participates in ROS scavenging and is converted into GSH/glutathione disulfide (GSSG) by the enzyme glutathione peroxidase (GPX), glutathione *S*-transferase (GST), and DHAR. Further GSSG can be recycled to GSH by the activity of glutathione reductase (GR). (**B**) In the degradation pathway, GSH and S-conjugated compound (GS-X) can be degraded to γ-EC and γ-EC-X by phytochelatin synthase (PCS). While, carboxypeptidase (Cpep) and γ-glutamyl transpeptidase (GGT) both could degrade GS-X to form γ-Glu-aa (aa, amino acid) and γ-EC-X, respectively. Similarly, GSSG is degraded by GGT and Cpep to form γ-Glu-aa and γ-EC, respectively. Further, the produced γ-Glu-aa is converted to 5-oxoproline (5-OP) by γ-glutamyl cyclotransferase (GGC). Besides, GSH is also converted to 5-OP. Although it is thought that this reaction is catalyzed by GGC, still it is unclear. 5-OP is converted to Glu in the next step by the action of 5-oxoprolinase (OPase).

Glutathione is very important for various physiological processes, especially during abiotic stress; it coordinates with AsA turnover and is oxidized to GSSG [35]. Moreover, some other thiol-dependent enzymes, GPX and GST use GSH as co-factor, hence converted to GSSG, which is further reduced back to GSH, with GR catalysis. In higher plants, there are two genes that are reported to encode GRs (*GR1* and *GR2*), where *GR2* is essential for plant development [31,34].

The degradation of GSH is another important phenomenon of GSH metabolism (Figure 3). Up to now, as many as four types of GSH degrading enzymes have been described, which either use GSH or act on GSSG or other GSH-conjugates. Among them, carboxypeptidase activity could degrade GSH itself or GSH-conjugates. Cytosolic PCS is another enzyme that is responsible for the breakdown of GSH-conjugates that are mainly activated during metal/metalloid(s) stress. Another enzyme γ-glutamyl transpeptidase (GGT) acts in GSH transpeptidation or hydrolysis, which is further converted to free Glu by the action of GGC (γ-glutamyl cyclotransferases) and 5-oxoprolinase (5-OPase) (Figure 3) [31,36]. Moreover, another vacuolar GGTs have been reported in *Arabidopsis*, which breaks GSH-conjugates [37]; hence, along with PCS, GGTs is important for metabolizing GSH-conjugates that is formed during secondary metabolites synthesis [37,38].

#### *3.3. Ascorbate Peroxidase*

The class I heme-peroxidases; APX (EC 1.11.1.11) occurred in several isoforms in plant cell, viz. cytosolic APX isoform (cAPX), mitochondrial APX isoform (mitAPX), peroxisomal and glyoxysomal APX isoform (mAPX), and chloroplastic APX isoform (chAPX) differed in their substrate specificity, molecular weight, optimal pH ranges between 7 and 8 for maximum activity and stability [39–41]. More importantly, isoforms activity is not stable when AsA is absent. For example, AsA concentrations those are lower than 20 μM greatlyreduced chAPX activity. All of the APX isoforms are heme peroxidase they are inhibited by cyanide and azide. Iron plays a vital role in the APX catalytic site; hence, despite the presence of high AsA concentration, Fe deficiency reduced the activity of cAPX [42]. If the single Cys32 residue near arginine (Arg) 172 residue is altered, APX loses the ability to oxidize AsA to DHA, but it can oxidize other small aromatic molecules. Therefore, the APX properties differ with the guaiacol peroxidases, but they are 33% identical with cytochrome c peroxidase (CCP) [43].

During ROS (H2O2) detoxification, APX binds H2O2 producing intermediate (I), and heme iron [Fe(V)] is oxidized forming oxyferryl species (Fe4<sup>+</sup> = O). Afterward, APX is regenerated from I, in a two-step reaction withAsA, where the AsA donate an electron and become oxidized. Detailed reactions are shown below (HS = Substrate, S = One electron oxidized form of the substrate).

> APX + H2O2 → Intermediate (I) + H2O Intermediate I + HS → Intermediate II + S Intermediate II + HS → APX + S +H2O

In plant cell, APX scavenges H2O2, which mainly participates in AsA-GSH cycle catalyzes the reactions produces MDHA (Figure 4) and, subsequently, MDHA yields DHA [6,10,44].

**Figure 4.** The function of Ascorbate peroxidase (APX) for the abolition of excess reactive oxygen species (ROS) generation in various cellular compartments. Additional details are in the text.

#### *3.4. Monodehydroascorbate Reductase*

The MDHAR (EC 1.6.5.4) helps in the revival of AsA [35], having several isoforms that are found in different organelles. Reports suggested that there are three MDHAR genes in tomato, five genes and six isoforms in *Arabidopsis* and rice, and as many as nineteen genes in wheat. In the plant cell, MDHAR activity was detected in different cell organelles, for instance, cytosol, mitochondria, chloroplasts, peroxisomes, and glyoxysomes (Figure 5) [45].

**Figure 5.** The antioxidant of MDHAR in regenerating AsA to support the removal of reactive oxygen species (ROS) (lower left) contrasts the pro-oxidant role of MDHAR creating 2,4,6-trinitrotoluene (TNT) toxicity.

Although the enzyme is purified from several sources, the detailed structure of this enzyme was published in recent past. Begara-morales et al. [46] coined three-dimensional structure after conducting silico analysis of pea peroxisomal MDHAR. More recently, Park et al. [47] described details MDHAR composition from japonica rice. Those indicated that the structure of MDHAR consists of flavin adenine dinucleotide (FAD) and pyridine binding domain. The structure resembles Fe–S protein reductase [47]. The elucidated structure also indicates that rice MDHAR contains a typical α/β fold, and Arg320 and tyrosine (Tyr) 349 residues are vital for its activity. On the fad-binding domain, the fad is bounded by hydrogen and van der waals bond, where, Gly13, 15 and 297, alanine (Ala) 122 and 319, and threonine (Thr) 123 are involved and highly conservedin the bottom of the crevice. Moreover, Lys53 and Glu178 bridged together, which further bonded with proline (Pro) 49, where both Glu178 and Pro49, are highly conserved, as well as lysine (Lys) 53, Glu178, and FAD bonded each other. Among others, Arg48 compensate FAD phosphate group's negative charge.

In rice, MDHAR α-helices surround β-sheets in the nicotinamide adenine dineucleotide (NAD)-binding domain, where the Tyr174, histidine (His) 315, and phenylalanine (Phe) 348 residues are shifted. Sandwiching between the FAD isoalloxazine ring and Tyr174, as well as steric hindrance moved Tyr174 away. The Phe348 residue shifts outward while His315 comes towards NAD binding site. In addition, the hydrogen bond is formed between Glu178 and nicotinamide ring; Arg202 and ribose and phosphate group, Glu314 and ribose; Glu196 and adenosine ring. Moreover, Glu196 offers rice MDHAR selectivity to NAD; preferring NADH over NADPH [47].

Interestingly, MDHAR can bind substrates other than MDHA, such as isoascorbic acid Evidence showed that phenoxyl radicals, like ferulic acid, quercetin, chlorogenic acid, and coniferyl alcohol, might be reduced by MDHAR [48]. In *Arabidopsis*, MDHAR activity reduces 2,4,6-trinitrotoluene (TNT) and creates its toxicity (Figure 5), but the MDHAR6 mutants are more tolerant, as TNT could not reduce and thus autooxidizes to creates O2 − [49].

Reports imply that MDHAR response to abiotic stress conditions by reducing MDHA that produces by the excess ROS scavenging, which was observed in many test species (Figure 5) [50,51]. The chlMDHAR is involved in photosynthetic activity during lack of peroxiredoxin [52]. In addition, chlMDAHR activity increased by three- to six-fold during pepper fruit ripening [53].

#### *3.5. Dehydroascorbate Reductase*

A major enzyme for GSH assisted DHA recycling is DHAR (EC 1.8.5.1), which is also known by GSH:DHA oxidoreductase or GSH dehydrogenase (AsA) [51,54]. This regeneration process is accomplished at alkaline pH and it is a well known biochemical reaction in plants.

The plant GSH-dependent DHAR is a monomeric enzyme, which is a member of the GSH*S*-transferase superfamily [55]. *Arabidopsis* possesses three functional DHAR encoding genes, DHAR1 (*At1g19570*), DHAR2 (*At1g75270*), and DHAR3 (*At5g16710*). In recent decades, the attention of researchers towards the DHAR activity in plants for regenerating DHA increased, and a number of investigations were carried out to elucidate the structure and molecular mechanism of DHAR.

The overall three-dimensional structure of DHAR from different plant origin is almost identical, except with some additional short-chain before the α1-helix. The enzyme has several binding sites. The G site is responsible for binding the GSH in the enzyme. The GSH cysteinyl sulfur bonded Cys20 and occupied disulfide bond. The GSH γ-glutamyl is stabilized, via H-bonds H2O molecule, and then forms the backbone with serine (Ser) 73 and aspartic acid (Asp) 72. The Phe22 is engaged with the γ-glutamyl group by the van der Waals bond, in addition to hydrogen bonds with Lys59. The Val60 stabilizes the central cysteinyl region. The glycinyl group of GSH is loosely bound, forming a salt bridge with Lys47 [54].

The substrate-binding site or DHA binding site or H-site of DHAR enzyme typically exhibits more structural plasticity, but not simultaneously. From the structure of *Pennisetum glaucum* DHAR gene (*PgDHAR1)*, it was observed that Lys8 and Asp19 are responsible for DHA binding [56].

The DHAR catalyzing accomplished by the following three reactions (Figure 6):

DHAR-S + DHA →DHAR-SOH + AsA (Reaction 1)

```
DHAR-SOH + GSH → DHAR-S-SG (Reaction 2)
```
DHAR-S-SG + GSH → DHAR-S + GSSG (Reaction 3)

**Figure 6.** The mechanistic scheme, the ping-pong mechanism for the enzymatic reduction of dehydroascorbate (DHA).

Therefore, the process can be summarized by the following reaction:

2GSH + DHA → GSSG + AsA (Reaction 4)

As stated earlier, the integral function of DHAR is to reduce DHA to regenerate AsA. During this process, the active site of Cys is oxidized by DHA and further converted to the sulfenic acid. The reaction requires one molecule of H2O. Knockout mutants of *Arabidopsis DHAR1*, *DHAR2*, and *DHAR3*, did not show any significant differences in total AsA content until facing the abiotic stress condition, which confirmed the necessity of DHAR in reducing the DHA during stress [55].

#### *3.6. Glutathione Reductase*

The flavoprotein oxidoreductase GR (glutathione reductase, EC 1.8.1.7) and reduced GSSG to GSH, also known by the term GSR or NADP<sup>+</sup> oxidoreductase, as it employs NADPH for its cellular activity. Although GR is stated as a dimer, the monomeric, heterodimeric, and heterotetrameric forms have also been illustrated [29].

No less than two genes that encoded GR were reported, viz. GR1 and GR2 from higher plants. Where GR1 is cytosolic or peroxisomal and shorter, contrary, GR2 comprises a long N-terminal sequence and mitochondrial or chloroplastic [57]. Up to date, several researchers reported GR isoforms in many plants, for instance, tobacco, spinach, etc. [10]. Although being found in above-stated organelles of the

cell, the chloroplastic isoforms are accounted for 80% of GR [58]. The enzyme possesses a different quaternary structure that is based on the source from which it was purified [29].

Resembles with flavin-containing enzymes, GR exhibits the Rossmann folds, which is very much conserved and serves as the FAD and NADPH binding domains [59]. There is a controversy regarding the number of domain present in GR. Some reports suggested three, while some suggested four. Some researchers suggested an interface domain in GR protein; therefore, the enzyme has four domains, viz FAD-binding domain, NADPH-binding domain, GSSG-binding domain, and an interface domain [60]. Two Arg residues Arg287 and Arg293 are exclusively necessary for the enzymatic activity of GR [61]. Two Cys residue formed a disulfide bridge, which is redox-active and highly conserved. Serl64 replaces Cys residue in higher plants [62].

The enzyme shows high specificity to substrate binding, although the enzyme reduces GSH conjugates as well as mixed GSSG. Although Plant GR can employ NADP<sup>+</sup> <sup>−</sup>, its affinity towards NADPH is high [29]. The catalytic mechanism of GR accomplishes in two steps. The first step involves NADPH dependent reduction of the flavin moiety, which is further oxidized, meanwhile the disulfide bridge in active site reduced to form an anion–thiolate and release Cys. In the next step, GSSG molecule binds in the active site forming a disulfide bond together with a Cys and histidine (His) separately of the active site. Afterward, one GSH leaves the His, while another followed it and then leaves the Cys residue leaving the disulfide the bridge in the enzyme active site [63].

The brief reaction catalyzing by GR is as follows (Figure 7):

GSSG + NADPH + H<sup>+</sup> = GSH + NADP<sup>+</sup>

**Figure 7.** Mechanistic scheme for the enzymatic reduction of glutathione/oxidized glutathione (GSSG) in a plant cell.

During catalysis, pH, and NADPH, and GSSG concentration modulate GR activity. It was reported that low NADPH concentration reduces the GR activity, while below pH 5.5 and over 7.0 is not suitable for GR actions. On the other hand, NADPH-induced GR inhibition was prevented by GSSG [64].

#### **4. Ascorbate and glutathione Redox and its Role in Plant Metabolism**

Balanced metabolism is the prerequisite for better productivity in plants, which is always disturbed due to biotic and abiotic stresses. Thus, redox balance is one of the key features of life, by which oxidized products are reduced for further oxidization and energy supply. Moreover, plant cells should counter the oxidation of vital cellular component that occurs continuously due to the presence of 21% atmospheric molecular O2, which is further complicated due to light-induced overproduction of ROS during photosynthesis. In addition, to keep the electron transport cascades active, simultaneous conversion of electron carriers between reduced and oxidized forms are required. Furthermore, photosynthetic and respiration needs regular electron flux to the electron transport chains from a different site. Therefore, the primary consequence is the generation of O2 −and, subsequently, other ROS, from different enzyme catalysis [10,65]. Although playing a signaling role, over generation of ROS is harmful to cells; thus should be regulated to govern the redox homeostasis [66,67]. For example, AsA, GSH, tocopherols, thioredoxin, glutaredoxin, and peroxiredoxin, and energy metabolism mediators and electron carriers, for example, AsA/DHA, GSH/GSSG, FADH/FAD+, NADPH/NADP+, and NAD+/NADH play vital roles in plant cell for maintaining the redox balance and are termed as redox managers [68]. Among the redox managers, there are significant contributions drawn by AsA and GSH, hence in this section; we will discuss their role to keep redox balance as well as maintaining smooth cellular metabolism.

Reports suggested that, under control condition, the AsA/DHA ratio remains >9. Ascorbate becomes oxidized during ROS scavenging, electron donation to photosystem II (PSII), violaxanthin de-epoxidation, and α-tocopherol reductive quenching [69,70]. While the direct reduction of MDHA by ferredoxin at photosystem I (PSI) and by MDHAR, as well as DHA reduction by GSH dependent DHAR activity, maintains a highly reduced state of AsA pool [2,71]. The biosynthesis and metabolism of AsA are discussed earlier in this article (Section 3.1). In the apoplast and vacuoles, AsA pool is an important redox buffer for ROS detoxification, where AsA recycling is mainly accomplished in the cytosol, and AsA/DHA acts as an oxidative stress sensor [72].

The GSH redox potential depends on the GSH concentration and the ratio of GSH/GSSG. In the GSH pool [GSH/GSSG], if the GSSG remains constant, but total GSH decreases, the equilibrium position dropped and redox balance is disrupted. Thus, proper judgment of the GSH/GSSG could give the idea of the redox ratio [73]. Glutathione serves in a multiplicity of metabolic functions; for instance, it participates in the regeneration of AsA from DHA in the chloroplast by DHAR [74]. Moreover, GSH plays a role in reducing glutaredoxins, functions as a precursor of phytochelatin (PC) synthesis for chelating heavy metal, signal transduction, sulfur metabolism, xenobiotics detoxification, and protects protein thiols against irreversible oxidation with disulfide formation or glutathionylation, which inhibited enzymes, like enolase and 6-phosphogluconolactonase [75]. Plant cells have distinct compartmentation of GSH. Although all other cellular compartments, except vacuole, contain GSH/GSSG redox buffer, the only vacuole is the storehouse of GSH where the GSH-conjugates are degraded [76].

As discussed in earlier (Section 2), both AsA and GSH are connected to the reactions network, the AsA-GSH pathway, and the cellular redox homeostasis depends on the pathway components [76]. In this cycle, AsA performs electron donation for APX that works for H2O2 detoxification. Due to its high attraction for H2O2, APX is capable of efficient ROS scavenging, even in a low concentration, which gives rise to DHA. The produced DHA is further recycled back, which maintains a high ratio of AsA/DHA. If DHA cannot reduce, it might further be irreversibly hydrolyzed, which decreases the ability of AsA redox pool [77]. In the catalysis process GSSG produced, which is further recycled back, which maintains not only a high GSH/GSSG ratio, but also the balance between GSH and AsA pools [10].

In addition, the redox couples of AsA/DHA and GSH/GSSG can also function in another way for accomplishing redox signaling [78]. As discussed earlier, the AsA/DHA couples create redox balance inside cells. Moreover, AO converts AsA to DHA in the apoplast [79], and it creates a redox

gradient to connect intra- and extra-cellular atmosphere transverse the plasma membrane. Hence, AsA/DHA redox pair functions in apoplastic and cytoplasmic signals [80]. In contrast, the GSH/GSSG couple plays their functions in balancing intracellular redox potential, which in the intracellular redox signaling [81]. In this regard, the GSH distribution in different cellular organelle is very important for understanding cellular redox situation, for which signaling, as well as cellular metabolism, are smoothly going on [12].

#### **5. Overview of Oxidative Stress and Antioxidant Defense in Plants**

The production of ROS in living organisms is a usual cellular metabolism, and it is found in a large number in the internal constituents of the cell-like chloroplast, mitochondria, cytosol, peroxisomes, etc. [82–84].

Each plant cell maintains a dynamic balance between ROS and ROS-scavenging antioxidants. Abiotic stress destroys such cellular balance in favor of oxidative reactions by producing a huge amount of ROS [85]. Insufficient energy indulgence in the photosynthetic process during abiotic stresses reduces molecular oxygen and then produces a large amount of ROS, including H2O2, O2 −, 1O2, OH•, and so on (Figure 8) [10,86]. Reactive oxygen species are extremely reactive molecules and they can damage a large variety of cellular biomolecules, including carbohydrates, nucleic acids, lipids, proteins, etc., and alter their functions [85,87]. In addition, MG, a cytotoxic compound and reactive oxidizer, spontaneously produced in a cell in little amount but under abiotic stresses, its production increased and participated in developing oxidative stress (Figure 8). Similar to ROS, MG production is increased under abiotic stress, which can damage the ultra-structural constituents of cell and cause mutation, and ultimately provokes programmed cell death (PCD) [10].

**Figure 8.** Abiotic stress-induced oxidative stress through the generation of ROS. Additional details are in the text.

Besides causing oxidative stress, ROS and MG play signaling roles for stress tolerance, which controls acclimation and defense responses by modulating some antioxidants and their respective genes [10,86]. The excess generation of ROS and MG is also able to activate interruption in redox homeostasis, which can give the signal for cellular death or shortening plant life cycle [10,82].

Plant cells have well established antioxidant defense and glyoxalase system for scavenging toxic ROS and MG, respectively. The antioxidant defense system consists of some non-enzymatic components (AsA, GSH, alkaloids, α-tocopherol, non-protein amino acids, and phenolic compounds) and enzymatic components (SOD, CAT, APX, MDHAR, DHAR, GR, GST, and GPX [5,28]). Within the antioxidant defense system, the AsA-GSH pool performs the direct and significant role for minimizing stress effect through scavenging of ROS by using key four enzymes, e.g. APX, MDHAR, DHAR, and GR [28,88,89]. In our previous section (Section 3.3, 3.4, 3.5, and 3.6), we elaborately discussed the function of these four enzymes in ROS detoxification. Usually, in the antioxidant defense system, SOD gives frontline protection against ROS by converting O2 <sup>−</sup> to H2O2. Subsequently, CAT and APX scavenge H2O2 to H2O. Glutathione peroxidase and GST also scavenge H2O2 to H2O with the help of GSH (Figure 9) [28].

**Figure 9.** AsA-GSH pathway of the antioxidant defense system and its relation with the glyoxalase system. Additional details are in the text.

Toxic MG is detoxified in the cell by glyoxalase system. Glutathione is not only the major element of AsA-GSH cycle, but it also plays a significant function in the MG detoxification system. Glyoxalase system is composed of two vital enzymes, Gly I and Gly II. In glyoxalase system, MG is detoxified to non-toxic compound in two steps reactions; in the initial step, MG is transformed to *S*-D-lactoyl-glutathione through the utilization of GSH and in the final step *S*-d-lactoyl-glutathione transformed in to d-Lactate, where GSH is recycled back [11]. Moreover, GSH contributes to metal chelation. It enhances the amount of PC under stress condition, which makes a complex with metal and drives into the cell vacuole as inert form [90].

#### **6. Role of AsA-GSH in Regulating Oxidative Stress under Abiotic Stresses**

Abiotic stress-induced excess ROS causes oxidative stress in plants followed by cellular damage, even death. Hence, the plant itself defends against this higher ROS accumulation by their defense mechanism. Plant significantly activates the AsA-GSH pathway for ROS detoxification. In this section, we will discuss the involvement of AsA-GSH cycle for alleviating oxidative stress upon various abiotic stresses reviewing recently published articles (Tables 1–3).

#### *6.1. Salinity*

One of the most devastating abiotic stress factors—salinity by which cultivable land is becoming barren thus reduces total crop production day by day. Oxidative stress is the most dangerous event under salt inundation is imposed by salinity-induced ionic and osmotic stress [10]. Hence, these ionic and osmotic stress both disturb the photosystem, and thus cause excess ROS, such as 1O2, O2 <sup>−</sup>, H2O2, and OH. Salinity-persuaded acute ROS accumulations, then bother cellular redox followed by cellular damage counting membrane dysfunction, DNA damage, collapse the enzymatic action, along with distraction of the antioxidant defense system [91,92]. At this point, the plant synthesizes cellular AsA and GSH, which act as non-enzymatic antioxidants by involving their enzymatic components to detoxify ROS up to tolerable levels (Table 1).

However, the enzymes of AsA-GSH pathway showed their differential responses intolerant and sensitive varieties due to saline toxicity. Among salt-tolerant (Pokkali) and sensitive (BRRI dhan29) rice cultivars. Pokkali responded by enhancing the enzymatic activities of the AsA-GSH cycle, where, lowered APX and higher DHAR activity along with unchanged MDHAR and GR activities were found from BRRI dhan29. Rahman et al. [91,93] reported about the well involvement of AsA-GSH cycle in salt-stressed *O. sativa* where ROS generation was extreme. Here, salt exposed rice enhanced the reduced and oxidized GSH content with a lesser amount of AsA by the higher APX, MDHAR, DHAR, and GR activities against overproduced ROS. *Vigna radiata* was grown under the saline condition [94] and where salt-induced oxidative stress was marked with extreme O2 <sup>−</sup> and H2O2 overgeneration. Salt-stressed *V. radiata* augmented GSH and GSSG contents along with lowered AsA, whereas up-regulated the activity of all enzymatic antioxidants of AsA-GSH cycle and thus responded with elevated ROS [95]. Salt exposed *Lens culinaris* up-stimulated both MDHAR and DHAR activities, which resulted in a lesser amount of AsA and indicated the overproduced H2O2 detoxification [96]. Recently, Singh et al. [97] disclosed the incremental activity of enzymatic antioxidants, including APX, DHAR, and GR, with lower AsA, GSH, and GSSG contents, because of salt-induced higher ROS accumulation in *Solanum lycopersicum*. Similarly, 150 mM salt-treated *S. lycopersicum* also decreased AsA content, which might be used in H2O2 detoxification, while better GSH showed its role in lowering H2O2. Ahmad et al. [98] also observed higher APX, and GR activities, while MDHAR and DHAR activities again reduced as well as supported AsA-GSH mediated ROS regulation. Ahanger et al. [99] reported the same response of *S. lycopersicum* upon saline toxicity. Both activities of APX and GR were enhanced in salt-treated *Triticum aestivum* besides elevated H2O2 generation and resulted in higher GSH accumulation [100]. The activity of APX, MDHAR, DHAR, and GR enhanced in salt-stressed *S. lycopersicum* to check the excessive H2O2 generation, which resulted in lowered AsA and GSH contents [92].

The changes in AsA-GSH pathway were investigated in salt-stressed *Nitraria tangutorum* by applying a varied level of NaCl (100, 200, 300, and 400 mM) [101]. They noticed a gradual enhancement of AsA, DHA, GSH, and GSSG contents by keeping pace with sequential increment of salt-induced H2O2. Here, increased MDHAR and DHAR activities in stressed seedlings also contributed to increasing AsA, and higher DHAR and GR were responsible for better GSH and GSSG contents [92,102]. Talaat et al. corroborated these results with salt-exposed *Phaseolus vulgaris* [103]. Thus, as a part of plant antioxidant defense under salinity, AsA-GSH pathway is very efficient to regulate extra ROS for being tolerant.

#### *6.2. Drought*

Drought is another most important abiotic stress, which generates excess ROS accumulation and thus causes variation in the enzymatic activities of AsA-GSH pathway for ROS detoxification. The enzymatic responses of AsA-GSH pathways varied, depending upon plant species, plant age, drought intensity, and duration [10]. Commonly, drought up-regulated the enzymatic antioxidant activities of AsA-GSH pool [10,104]. Plant tolerance to drought stress is categorized based on stress-induced endogenous antioxidants contents along with enzymatic activities (Table 2). *Dendranthema grandiflorum* responded differentially according to their tolerant and sensitive varieties, where tolerant one comparatively displayed better enzyme activity of antioxidants than sensitive [105]. Lou et al. [106] demonstrated how *T. aestivum* responded upon drought exposure. Hence, they noticed that the AsA-GSH cycle responded considerably with excess ROS generation by significant variation of GSH/GSSG and AsA/DHA redox along with the steady increment of H2O2. Their team also observed the enzymatic up-stimulation of AsA-GSH pathway to alleviate stress by scavenging excess ROS in *T. aestivum* spike. Thus, *T. aestivum* showed higher participation of AsA with higher APX activity in drought exposure for scavenging extra H2O2, as well as higher enzymatic activity to run the AsA-GSH pathway systematically [107].


**Table 1.** Role of AsA-GSH in regulating oxidative stress under salinity and drought.





Drought-stressed *A. thaliana* enhanced GSH and GSSG content along with the higher GR activity [108]. Hence, *Arabidopsis* showed the GSH dependent H2O2 detoxification to attain tolerance. Higher total AsA was accumulated in *Cajanus cajan* upon complete water restriction conditions for up to nine days to defend against excess H2O2 toxicity [109]. Hence, drought enhanced the enzymatic activity of APX, DHAR, and GR for decreasing GSH/GSSG, as well as controlling ROS level.

Similarly, tolerant genotype VA13 of *Amaranthus tricolor* showed comparatively better tolerance under drought stress than sensitive one (VA15) by expressing differential responses of the enzymatic and non-enzymatic ROS detoxification pathways [110]. Hence, VA13 expressed remarkable increment in AsA-GSH redox by accelerating the enzymatic antioxidative actions by which increased non-enzymatic antioxidants (AsA and GSH) accumulation, which are vital for ROS detoxification.

*Vigna radiata* responded differently regarding different drought intensity [111] to control diverse levels of ROS. Moderate drought imposed by 10% polyethylene glycol (PEG) induced comparatively lowered ROS than severe drought (by 20% PEG). Therefore, severe drought-stressed *Brassica* showed a larger use of AsA-GSH pathways against higher H2O2 generation than moderate stress. Here, higher stress caused a higher increase of APX activity along with lowest MDHAR and DHAR activity, while GR activity reduced differently than lower stress exposure to rapeseeds seedlings. Additionally, Hasanuzzaman et al. [52] also observed AsA and GSH both antioxidants contents reduced under severe drought condition, but increased under moderate stress. Bhuiyan et al. [104] found increased AsA content in *B. rapa* under drought (20% PEG). They also observed increased APX activity in drought-stressed seedlings, which assisted in efficiently scavenging the H2O2. Another two enzymes related to AsA regeneration MDHAR and DHAR also upregulated, as a result the AsA level was increased and strongly maintained its redox balance during oxidative stress situation. Nahar et al. [111] narrated the function of AsA as ROS detoxifier under drought stress where AsA content reduced in *V. radiata* with the increasing of ROS generation. Here, drought-induced higher APX activity enhanced the oxidation of AsA by scavenging H2O2 and improved GR activity increased the supply of GSH for involving ROS detoxification. *Anacardium occidentale* also showed the active participation of AsA-GSH cycle by integrative responses of both non-enzymatic and enzymatic antioxidants for drought-induced excess ROS regulation, where the higher accumulation of AsA and GSH, along with APX activity, coordinately reduced the overproduced H2O2 [112]. Thus, the AsA-GSH pathways involve in ROS detoxification as well as ROS homeostasis by eliminating excess ROS for keeping them up to the requirement of functioning cell signals.

#### *6.3. Toxic Metals*/*Metalloids*

Due to fast industrialization of the modern world and unrestrained anthropogenic activities, toxic metals/metalloids stresses have become a gargantuan problem for the plant growth and

development [121]. Plants experience toxic metals/metalloids stress try to survive to some extent by using their well established antioxidant defense system. But, the activity and performance of defense system differ with stress concentration, stress duration, plant type, and age of the plant.

The enzymes of AsA-GSH pathway confirmed their differential responses to different toxic metals/metalloids stress (Table 2). Mahmud et al. [122] confirmed that due to Cr stress, the few components of AsA-GSH pathway increased their amount or activity in *B. juncea* L. cv. BARI Sharisha-11. They found five days duration of 0.15 mM and 0.3 mM K2CrO4 treatment decreased the content of AsA, but did not change the GSH content. Moreover, activities of APX and GR were enhanced; however, the activities of MDHAR and DHAR were diminished. The higher APX and GR activity might play a function in scavenging excess ROS. A similar upregulation of APX and GR was also recorded in *B. napus* L. cv. BINA sharisha 3 due to Cd treatment [123]. From two separate experiments, they also found Cd stress (0.5 mM and 1.0 mM CdCl2) for 48 h decreased the AsA content, but increased GSH content only under 0.5 mM CdCl2 treatment. Exposure of *Gossypium* to 50 and 100 μM Pb(NO3)2 for six weeks increased the H2O2 content and APX activity [124]. The addition of 150 μM NiCl2·6H2O in growing media of *B. juncea* L. for one week increased the H2O2 content. Moreover, Ni stress decreased the AsA level but augmented the content of GSH and GSSG. Nickel also diminished the function of DHAR and MDHAR, however enhanced APX and GR activity [125]. Similar differential responses of AsA-GSH pathway components were also observed under As [126] and Al [50] toxicity. It can be stated that overproduced ROS plays the signaling role to some extent and inaugurate the higher activity of AsA-GSH enzymes under metals/metalloids toxicity. The upregulation of enzymes plays a significant role in maintaining the redox balance of AsA-GSH pathway under stress condition.


**Table 2.** Status of AsA-GSH in regulating oxidative stress under metal/metalloid stress.




#### *6.4. Extreme Temperature*

Along with the rise in average global temperature, HT stress has been turned into a topic to be concerned about among environmentalists and researchers worldwide. In general, a 5 ◦C temperature rise above the optimum temperature of growth is considered to be extreme temperature stress or HT stress or heat shock to any plant species [142,143]. Heat stress causes denaturation of protein and membrane lipids, enzyme inactivation, inhibited protein synthesis, and loss of membrane integrity [144], which results from the disruption of cellular homeostasis through the ROS formed in a mass amount under heat stress [143,145]. Focusing on the role of AsA-GSH pathway to scavenge these ROS, different crop species under different levels of extreme or HT stress have been studied (Table 3).

Khanna-Chopra and Chauhan [146] selected a warmer season to induce HT stress to two different cultivars of wheat (*T. aestivum*), which are Hindi62 (heat-tolerant) and PBW343 (heat-sensitive). They sowed the wheat seeds in mid-January and considered it as heat stress environment, while the control plants were sown in mid-November and considered as the non-stress environment. Data were collected at seven days interval up to 35 days after anthesis (DAA), and the results showed a sharp increase in H2O2 content up to 14 days, but then declined. Whereas, MDHAR and DHAR enzymes' activity only increased in Hindi62, but APX and GR activities showed a fluctuating pattern of alteration in both cultivars [146]. Another cereal *Z. mays* when experimented similarly with two different cultivars; LM-11 (heat-sensitive) and CML-32 (heat-tolerant), exposed to 40 ◦C for 72 h, resulted in higher APX and GR activities in CML-32 roots, while a reduction occurred in the shoot. In LM-11, none of the enzyme activity or AsA content was affected [147]. Higher levels of O2 <sup>−</sup> production rate and H2O2 content were observed in *Ficus concinna* seedlings under 48 h of HT (35 ◦C and 40 ◦C) stress condition, where AsA and GSH contents were unaffected at 35 ◦C, while declining AsA at 40 ◦C temperature [148]. The activity of APX, MDHAR, DHAR, and GR enzymes increased at 35 ◦C, but then again reduced at 40 ◦C to the level of control plants [148]. Under similar heat stress condition (40 ◦C, 48 h), *V. radiata* seedlings resulted in decreased GSH content and MDHAR-DHAR activities, but higher APX-GR activities [50]. Kiwi fruit (*Actinidia deliciosa*) seedlings, when exposed to 45 ◦C in an incubator for 8 h, resulted in higher AsA content and enhanced activity of all the AsA-GSH cycle enzymes [143]. Tomato seedlings were studied in two different aspects: short-term heat shock (40 ◦C, 9 h) [149] and long-term heat stress (38/28 ◦C day/night, seven days) [150]. In both experiments, the enhancement of O2 − generation rate and H2O2 content were recorded, but enzyme (APX and GR) activity was only increased at short-term stress condition [149], while the long-term heat exposure reduced all four enzymes activities and GSH content [150]. Similar enzymatic activity was observed in *Nicotiana tabacum* seedlings after seven days of heat (35 ◦C) stress [151]. From the above discussion, it can be stated that heat stress prevailing for longer duration is less likely to have the capability to modulate AsA-GSH pathway as compared to short-term heat stress.


**Table 3.** Role of AsA-GSH in regulating oxidative stress under extreme temperature, flooding, and atmospheric pollutant.



#### *6.5. Flooding*

Changes in global climate result in the frequent or unexpected occurrence of heavy rainfall in different regions of the globe, which causes a sudden flood and disrupts the normal ecosystem [2]. Such changes in the ecosystem may cause the extinction of plants species and imbalance in the natural environment [2]. Flooding induced production of ROS and subsequent cellular damage has been authenticated in many studies so far [152–154]. Following are the discussion regarding crop species facing flooding stresses and modulation of their AsA-GSH pathway by flooding stress (Table 3).

Pigeon pea (*C. cajan*) seedlings that are exposed to waterlogged condition for six days revealed that tolerant cultivar could increase APX and GR activities, but a susceptible one cannot [155]. They also observed that, unlike other cases, waterlogging caused a lower accumulation of H2O2 and O2 − [155]. In another experiment with *V. radiata*, Sairam et al. [156] showed that waterlogging similarly reduced the H2O2 and O2 −production rate in susceptible cultivar, while the tolerant ones remained unaffected. However, both APX and GR enzymes' activity increased in tolerant genotypes, while the susceptible one got reduced [156]. The enhanced production rate of O2 <sup>−</sup>and H2O2 content under flooding stress have been reported in cotton [154], Welsh onion [157], and clover [158] plants. Cotton (*G. hirsutum* cv. Siza) plants after three and six days of flood exposure raised the AsA content, but reduced the activity of APX, MDHAR, and GR [154]. A similar reduction in APX and GR enzymes activities was also recorded in Welsh onion (*Allium fistulosum* L.) after 10 days of waterlogging stress [157]. When *Z. mays* seedlings were waterlogged for 21 h at their root portions, they resulted in reduced AsA content and increased APX activity [159]. On the other hand, under long duration (14 days) flooding stress, *Glycine max* L. plants showed a reduction of GSH activity in roots and GR activity in the shoot, but the GSH in shoot and GR in root were not affected [153]. In case of complete submergence of *O. sativa* L. plants for two, four, or eight days, elevated levels GR enzyme activity was recorded, while APX enzyme activity increased only in tolerant cultivar [152]. Accordingly, the discussion reveals that the impact of flooding stress on AsA-GSH pathway varies depending upon the plant species and duration.

#### *6.6. Atmospheric Pollutants*

Atmospheric pollutants are the substances that are assembled in the air to a level or magnitude that is dangerous for living beings. Plants that are grown under different levels of atmospheric pollution have shown their oxidative stress responses and AsA-GSH pathway regulation in different manners (Table 3).

*Erythrina orientalis* plants were grown in three different locations of Philippines: La Mesa (a non-polluted area); and, Makati and Quezon (highly air-polluted cities). The results revealed that plants grown in the non-polluted area had lower activities of APX and GR as compared to the ones grown in highly polluted areas [160]. A similar increase in APX activity along with higher AsA content was recorded in *Prosopis juliflora* plants grown under polluted industrial region [161]. In a recent experiment, Lucas et al. [162] studied *Lolium perenne* plants that were grown under two different areas of Spain, Madrid, and Ciudad Real, where Madrid was considered to be more polluted than Ciudad Real. The findings indicated that the pollens of *L. perenne* accumulated higher concentration of H2O2 and in shoots APX and DHAR activity declined, but the activity of MDHAR and GR increased in the shoot of *L. perenne* plants that were grown in Madrid [162]. When rice seedlings were exposed to continuous O3 treatment, the results showed a remarkable increase in both O2 − generation rate and H2O2 content. In addition, contents of AsA and GSH reduced, while APX, MDHAR, DHAR, and GR activity increased upto 70 days of O3 exposure in SY63 cultivar and upto 79 days of O3 exposure in WXJ14 cultivar [163]. Ascorbate and GSH contents were not affected by O3 exposure in the *Populus* seedlings, but DHAR activity was lower, while the activity of GR and MDHAR was higher after 17 days of O3 treatment [164]. Young strawberry (*Fragaria* x *anansa*) seedlings were exposed to three different levels of CO, NOx, and SO2, which are as follows: CO @ 133, 267, and 533 ppm, NOx and SO2 @ 25, 50, and 199 ppm corresponding to low, medium, and high dose, respectively. As a result of exposure to these atmospheric pollutants, H2O2 content as well as O2 − generation rate increased. However, at low and medium doses of their exposure APX and GR activity increased, while at a high dose that decreased [165]. All sorts of atmospheric pollutants have a remarkable effect on AsA-GSH pathway, but further studies are required to demonstrate that those pollutants completely induced the modification of the AsA-GSH pathway.

#### *6.7. Other Stress*

Conklin et al. confirmed the positive role of AsA in protecting plants from ultraviolet (UV) radiation [159], where they found that Vit-C deficient mutant of *A. thaliana* was suffered by stress-induced damages than that of wild type. AsA-deficient mutants also showed sensitivity to O3 stress due to a lower biosynthesis of AsA [171]. Gao and Zhang [172] reported that vitc1 mutants of *A. thaliana* showed physiological disorders and greater oxidative damages than the wild type, which was due to lower activities of antioxidant enzymes. Mutant plants also showed lower GSH/GSSG and higher DHA/(AsA+DHA) ratio than the wild type. Singh et al. [173] observed a decrease in AsA-GSH cycle enzymes in UV-exposed plants, which in turn affected the plants with oxidative stress. Similar to higher plants, marine macroalga *Ulva fasciata* also showed a positive correlation between enhanced the functions of AsA-GSH cycle and better tolerance of plants to UV radiation [174]. In their study, scavenging of H2O2 was regulated by AsA-GSH cycle components, especially APX and GR. Noshi et al. [175] reported that AsA-GSH redox pool provided better protection of *Arabidopsis* from high-light mediated oxidative stress, which was mainly attained due to the higher activities of DHAR. However, both AsA and GSH were found to be responsible for conferring high light (HL) stress [175]. Later, Zheng et al. [176] that susceptibility of *Arabidopsis* mutant was to HL stress were related to the deficiency of AsA and GSH. When AsA deficient *A. thaliana* mutant (vtc2-1) exposed to HL, they generated a high level of H2O2 (an oxidative stress marker) than the wild type, which was highly and negatively correlated with the total AsA content. The lack of AsA also resulted in lower chlorophyll (chl) content, chl fluorescence parameters, and PSII photochemistry [176]. Recently, Choudhury et al. [177] studied the metabolomics of *A. thaliana* grown under HL and found that the increased biosynthesis of GSH supports the photochemistry that supports *Arabidopsis* better survival under HL stress.

The pivotal role of the AsA-GSH cycle was also observed in low pH stress also. Bhuyan et al. [170] tested five spring wheat cultivars at different levels of low pH stress. Their observation exhibited that low-pH stress resulted in elevated O2 <sup>−</sup>and H2O2 generation. A decrease in AsA content with increased DHA content was observed, although the APX activity decreased. Increased MDHAR activity was observed, but the ratio of AsA/DHA was not increased. Decreased GSH content and increased GSSG content were found where DHAR and GR activity decreased, resulting in a drop of the GSH/GSSG ratio.

#### **7. Exogenous Use of AsA and GSH in Conferring Abiotic Stress Tolerance**

While considering the vital role of both AsA and GSH and their redox researches have been trying to explore the possibilities of using exogenous AsA and GSH in protective plants from abiotic stress. However, the effects are not straightforward due to their species and dose dependency. In the next sections, we provided a summary of the recent results on plant abiotic stress tolerance while applying exogenous AsA and GSH.

#### *7.1. Exogenous AsA*

As a non-enzymatic antioxidant, AsA is vital for plant defense mechanism by involving in stress perception and subsequent signaling, and therefore plant responses [178]. Besides its regenerative nature, AsA is also able to donate electrons with and/or without the help of enzymes, and thus significantly detoxifies ROS [179]. Thus, exogenous AsA application is the most prominent for enhancing plant tolerance due to its efficient protection against lipids and proteins oxidation under abiotic stresses [180].

Ascorbate can be exogenously applied as a foliage application, seed treatment, and co-treatment for the alleviation of stress-induced damages [181]. Many researchers reported about the supplemental AsA-mediated antioxidant defense regulation in various plant species under different stressors, such as salt stress [182], drought [183], extreme temperature [184], ozone [185], and heavy metal stress [186].

Supplemental AsA application effectively lowered the oxidative stress in salt-stressed *Phaseolus vulgaris*, as indicated by the reduction of malondialdehyde (MDA) and ROS accumulation through activating their immune systems related with up-regulation of SOD, CAT and GR activities [187]. The AsA recovered salinity-induced oxidative damage in *Caralluma tuberculata* by lowering the activity of APX, POD, CAT, and GR, which were increased upon saline toxicity [188]. Exogenous AsA-induced plant tolerance, especially on the AsA-GSH pathway, is cultivar dependent [189]. Hence, they used both salt-tolerant *O. sativa* cv. Pokkali and salt-sensitive *O. sativa* cv. Peta to exogenously apply AsA as co-treatment with salinity and found a reduction of H2O2 generation in both cultivars. Here, AsA enhanced endogenous AsA and GSH, along with higher SOD, APX, and GR activities in salt-stressed both cultivars in line with lowered ROS and MDA production. However, Pokkali showed more prominent responses of salt tolerance than Peta. Finally, Wang et al. [189] suggested that exogenous AsA differentially increased the salt tolerance mechanism, and thus lessened salt-induced ROS in two rice cultivars. Exogenous AsA enhanced the salinity tolerance of *Z. mays* through protecting oxidative stress with stimulation of plant antioxidant defense [190]. In this study, AsA was used as seed priming against 100 mM NaCl, and AsA restored the salt-induced membrane damage. Hence, external AsA improved the non-enzymatic antioxidants, including Pro, AsA, and GSH accumulation, where SOD and GPX activities increased. Rady and Hemida [190] found lowered CAT activity in AsA treated seedlings under salt stress, which pointed out the AsA-induced decline of H2O2 generation.

Plants get relief from drought stress by exogenous AsA application, which was reported by previous researchers [114,191]. Alam et al. [114] studied the AsA induced attenuation of oxidative stress in *B. napus*, *B. campestris*, and *B. juncea* under 15% PEG, indicated by decreasing lipoxygenase (LOX) activity, H2O2, and MDA contents. This AsA mediated oxidative stress mitigation was described by AsA caused the strengthening of plant antioxidant defense mechanisms. Hence, exogenous AsA not only responsible for modulating AsA-GSH cycle, but also increased other enzymatic antioxidants activities, such as CAT, GPX, Gly I, and Gly II in all plant species, except GST, which was only increased in *B. napus*. Exogenous AsA mitigated the PEG-induced oxidative stress in *Z. mays* where AsA used as co-treatment, later endogenous AsA content increased, followed by scavenging surplus H2O2 generation and a reduction of lipid peroxidation [191].The higher transcript levels of SOD, CAT, APX, GR, MDHAR, and DHAR were induced in tall fescue by AsA application under PEG-induced water crisis, in respect with the only stressed condition [192]. Subsequently, Xu et al. [192] recommended AsA as a phytoprotectant to improved plants tolerances upon drought stress.

Exogenously applied 50 μM AsA decreased high temperature (HT, 45/35◦C)-induced elevated H2O2 and MDA contents with lowered electrolyte leakage (EL) in *V. radiata* [184]. Hence, supplemental AsA altered the heat-induced lowered SOD, CAT, APX, and GR activities with increasing endogenous AsA and GSH contents.The AsA also enhanced the antioxidant capacity of tomato to cope with low-temperature stress [193]. The foliar application of AsA decreased the EL and MDA content in *T. aestivum* seedlings when exposed to the combined stress of herbicide and low temperature (−2 ◦C) [194]. This AsA-induced lowered oxidative damage might be because of ROS scavenging under stress indicated by AsA mediated lowered O2 <sup>−</sup> and H2O2, which were attributed by increasing POD, APX, and GR activities.

Seed priming with AsA also increased plant tolerance to metal stress. Hence seed priming with AsA of *A. esculentus* showed the alleviation of Pb-induced oxidative stress that was confirmed by lowered H2O2 and MDA contents [135]. This AsA-induced alleviation of oxidative stress supported by exogenous AsA mediated increment of endogenous AsA contents, as well as upregulation of SOD, POD, and CAT activities in Pb-stressed *A. esculentus*. The AsA priming also increased the anthocyanins content in Pb-exposed seedlings, which again enhanced the metal tolerance by checking ROS production. Previously, exogenous foliar application of AsA on rice seedlings increased AsA, and

GSH contents, while enhanced both AsA/DHA and GSH/GSSG redox status, along with higher APX and GR activities under Cd stress [195].

Alamri et al. [196] investigated the potentiality of exogenous AsA to remove the metal-induced oxidative stress in *T. aestivum*. They observed that AsA suppressed the higher content of MDA and H2O2 in Pb exposed seedlings by improving the antioxidant enzymes activities, including SOD, CAT, and GR. Thus, AsA-mediated higher activity of enzymatic antioxidants could be responsible for the lowered membrane damage indicated by EL as well as Pb tolerance.

The AsA supplementation also showed its effective role in the mitigation of Cd-induced oxidative stress. Zhang et al. [141] reported that AsA application, as a foliar spray, could become a potent tool to alleviate Cd toxicity in *Z. mays*. They used 0.1, 0.3, and 0.5 mM of AsA in against 3.36 mM Cd contamination, while they observed a remarkable gradual reduction of both H2O2 and MDA contents under stressed conditions with increasing AsA levels. Foliar AsA application improved endogenous GSH along with the augmentation of SOD, POD, CAT, and GR activities, which are in line with AsA-induced lessening of oxidative stress in Cd-exposed *Z. mays*.

Thus, exogenous AsA application scavenges ROS in the plant under abiotic stresses, and then protects cell membrane stability. Therefore, reduced MDA content and EL were reported with AsA application as a sign of AsA-induced alleviation of oxidative damages. Accordingly, such exogenous AsA-induced strengthening of plant antioxidant defense, along with lessening oxidative stress, explained the potential of AsA for conferring abiotic stresses.

#### *7.2. Exogenous GSH*

At endogenous level, being an active participant of AsA-GSH cycle, GSH scavenges H2O2 in enzyme-dependent pathways; GSH is a substrate for GPX; GSH detoxifies lipid hydroperoxides together with GSTs; and, GSH/GSSG induces signals for abiotic stress adaptation [6]. Moreover, several research studies reported that the exogenous application of GSH proved to confirm the additional beneficial effects for enhanced the antioxidant defense system and abiotic stress tolerance.

After exogenous GSH pretreatment, mung bean plants were imposed with HT (42 ◦C), and beneficial effects were noticed. It enhanced chl and leaf RWC; increased cellular GSH content and GSH/GSSG lowering GSSG content; amplified APX, MDHAR, DHAR, GR, GPX, GST, and CAT activities; exogenous GSH pretreatment upheld the activity of Gly I and Gly II of MG detoxification system. The upregulation of both antioxidant and glyoxalase system ensured the HT tolerance. Meanwhile, GSH supplementation with HT decreased H2O2, O2 −, MDA level, the activity of LOX, and MG content [95]. Increased temperature (35 ◦C) in root-zone variably affected physiological processes, growth, and Calvin cycle, which mediated inconsistency in antioxidant components; HT also affected antioxidant enzymes' gene expression of *Cucumis sativus* L. seedlings. HT-induced reduction of GSH content, the ratio of GSH/GSSG, photosynthetic pigments level, photosynthesis, and changes of linked gene expression were evident. HT also augmented soluble protein, proline (Pro), O2 − generation and MDA level, expression of genes, and antioxidant enzymes functioning. The application of supplemental GSH with HT upheld soluble protein, Pro, antioxidant enzymes activity, and its linked gene expression, as well as inhibited O2 − generation and lipid peroxidation, than to HT treatment without GSH [197].

Exogenous GSH improved AsA, and GSH contents, GSH/GSSG, APX, MDHAR, DHAR, and GPX activity of antioxidant system in drought exaggerated mung bean seedlings, which helped to relieve the adverse effect reducing the ROS including H2O2 and O2 −, both in content and visually in the leaf spots of which were visualized through histochemical detection. Exogenous GSH also decreased the LOX activity, which caused the oxidation of lipid. Exogenous GSH also up-regulated the activity of Gly I and Gly II, therefore, reduce the toxic consequence of MG, and MG-induced oxidative damage [111].

Exogenous GSH (1.0 mM) positively regulated an antioxidant system in wheat plants facing lead (Pb) stress. The imposition of Pb diminished growth, the relative water content of leaf, and chl *a* and *b* content; amplified Pro level, H2O2, and O2 − generation, and lipid peroxidation. Glutathione supplementation with Pb stress improved the AsA and GSH contents, GSH/GSSG, activities of MDHAR, DHAR, GR, SOD, CAT, and GPX, and decreased oxidative damage. The decline of H2O2 and O2 − generation and membrane lipid peroxidation was clear evidence, together with an increased level of Pro and chl, which contributed overall tolerance to Pb toxicity [35]. Pretreatment with 100 μM GSH with 50 μM Cd reversed growth reduction and concealed Cd-provoked MDA buildup. In contrast to Cd compelled plants, GSH pretreatment reversed photosynthetic pigment destruction, downregulated Cd accumulation in root and shoot. Exogenous GSH considerably increased the functioning of POD and SOD. In contrast to the Cd affected plants, exogenous GSH pretreated plants extensively reassured decrease in Cu or augmented in Fe levels, which were due to Cd [198]. Exogenous GSH and Cys were applied on lead (100 and 500 mg L−1) affected *Iris lactea* var. chinensis, growth, accumulation of Pb, and nonprotein thiol (NPT) accumulation pattern were observed. The addition of GSH improved GSH biosynthesis in root and shoot. Endogenous shoot level Cys was recorded for exogenous Cys addition. Exogenous GSH application, together with buthionine sulfoximine (BSO) addition, regulated enzymes involved in GSH biosynthesis. This GSH played an imperative function in Pb accumulation and adaptation to this stress [199]. Exogenous GSH application improved the germination and growth of *Arabidopsis*, tobacco, and pepper under mercury (Hg) stress. Exogenous GSH also conferred Cd, Cu, and Zn stress tolerance. Exogenous GSH downregulated H2O2 and O2 − generation and MDA content, whereas upregulated chl level under Hg. Outstandingly, exogenous GSH reduced Hg accumulation in *Arabidopsis*. GSH showed high binding empathy to Hg, as compared to Cd, Cu, or Zn [200].

Salt-tolerant Pokkali and sensitive cultivar Peta of rice were scrutinized for the role of exogenous GSH on them. Exogenous GSH increased the activity of SOD, APX, and GR, the amount of AsA and GSH, and reversed chloroplasts' H2O2 and MDA accumulation in either cultivar affected by salinity (200 mM NaCl). However, tolerance was prominent in cv. Pokkali [163]. The supplementation of GSH inverted the pessimistic properties of salinity stressed (NaCl 100 mM) tomato plants improving the transcript levels and activities of enzymes that are linked to GSH biosynthesis and metabolism. The biosynthesis-related enzymes were gamma-glutamylcysteine synthetase (γ-ECS), glutathione synthetase (GS), whereas, others were GST, GPX, and GR. Exogenous supplemental GSH helped to upregulate the activity of SOD, peroxidase (POD), CAT, APX, MDHAR, DHAR and GR, GSH level, and GSH/GSSG in salt-stressed plants [201]. Externally applied GSH lessened the oxidative damage in different soybean genotypes via reducing H2O2 and MDA level, which were produced due to salinity. Glutathione supplementation minimizing the oxidative damage further contributed in yield attributes, and yield performance, which was seeds plant−<sup>1</sup> and pods plant−1, 100-seed weight and yield [202]. Defensive function of supplemental GSH (1 mM GSH) was examined for salt (200 mM NaCl) stressed mung bean. Mung bean plants when imposed with exogenous GSH and NaCl elevated AsA and GSH levels, GSH/GSSG, enhanced APX, MDHAR, DHAR, GR, SOD, CAT, GPX, and GST activities were recoded. Exogenous supplemental GSH also augmented the activity of Gly I and Gly II under salinity. Enhanced antioxidant and glyoxalase system components that resulted from the effect of exogenous GSH application had several beneficial effects. MDA, H2O2, and MG, O2 −production turned down, and leaf RWC and chl level raise; all of which made mung bean seedlings capable to perform better under saline growing media [95]. Glutathione was exogenously applied on tomato plants affected by salinity. Additional of GSH decreased oxidative stress. The reason behind this was revealed as redistribution of light energy in PSII, higher cellular GSH, GSH/GSSH ratio and activities of SOD, CAT and APX, MDHAR, DHAR, GR, and GRx. Glutathione supplementation revolutionized growth inhibition, Na+and Cl−ions balance, and Na+/K+. Choloplast, as well as stomatal function related to photosynthetic performance, were documented to improve after the application of GSH with salinity [203].

#### **8. Interaction of Other Pathways with AsA-GSH Pathways in Regulating ROS Metabolism**

Beside oxidative stress mitigation, the AsA-GSH cycle also interacts other pathways to reduce ROS and oxidative stress. Therefore, in this section, we will discuss the potentiality of AsA-GSH pathway components and their interaction with other pathways to modulate the ROS metabolism in plants.

#### *8.1. Interaction of AsA-GSH Cycle with NO Metabolic Pathway*

Although AsA-GSH cycle protects cellular components from oxidative damage, its components, especially the proteins (APX, MDHAR, DHAR, and GR), are also vulnerable to the oxidative damage, which can modify their activity, hence breaking down the antioxidant defense. In plants, nitric oxide can be produced from several biochemical pathways, both oxidative and reductive [35]. In the GSH pool, the reduced form of GSH can interact with NO and produce GSNO, which is further catalyzed by the action of GSNO reductase (GSNOR) and release NO and GSSG, and maintains the equilibrium of NO and nitrosothiols, as well as balance the redox state in the cell [204]. Moreover, *S*-nitrosylation could modify protein interactions, thus tinkering the antioxidant response [205]. Reports suggest that all of the proteins of the AsA-GSH cycle are influenced through *S*-nitrosylation and/or nitration, which are accomplished from the interaction with NO. Among the AsA-GSH cycle, enzymes APX is the most studied, which is directly influenced by NO metabolism. For example, the inactivation of APX1 is caused due to the oxidation of Cys32 [205]. Contrary, nitrosylation of APX1 active-site Cys32 increases its activity and this post translation modification (PTM) is performed during salinity stress, which increases oxidative stress as well as *S*-nitrosothiols [206]. Among the other enzymes, MDHAR is negatively modulated by nitration, which cuses enzymatic inactivity by altering the position of the cofactor binding site [206], and hence disturb the AsA recycling process. Although information is available on the nitration and activity modulation of DHAR proteins [207,208], but the involvement of Try in this process, as well as the structural alteration impact of the enzyme, is still unclear. Moreover, GR is also targeted for nitration, which is reported to inhibit its activity in a mammalian cell, but the chloroplasic and cytosolic GR of pea is not affected by nitration [206].

#### *8.2. Signaling Role of AsA-GSH Cycle Components and Interaction with Other Pathways*

Ascorbate serves as the co-factor for redox enzymes, as well as a precursor for several biosynthetic pathways. In addition, AsA is an important reducing agent for Fe, Cu, and Mn, thus act as a pro-oxidant controlling toxic OH• production from the Haber–Weiss and Fenton reaction [209]. Besides, its role as an antioxidant is the most important part of detoxifying ROS. As a pro-oxidant AsA regenerate α-tocopherol. Moreover, AsA also works in the photo-protection that is mediated by the xanthophylls cycle, where violaxanthin de-epoxidase use AsA as a co-factor [210]. Moreover, AsA is employed as the substrate for organic acid (oxalate and tartrate) biosynthesis (Figure 2). Rapid cell expansion is correlated with AO activity, which oxidized AsA [211]. During cell expansion, Pro residues present in the glycoproteins of cell wall undergo hydroxylation where prolyl hydroxylase use AsA as a cofactor [210]. Furthermore, AsA can potentially upregulate cytosolic free Ca2<sup>+</sup> via anion channels and play a signaling role [212]. More than this breakdown of AsA to DHA by APX or AO creates an electrochemical gradient over the plasma membrane, which also has a signaling role.

Under abiotic stress conditions, GSH triggers adaptation or PCD by intercellular signaling [213]. Glutathionylation of protein Cys residues suggests its redox signaling role, which alters the transcription of proteins [214]. In *Arabidopsis*, stomatal movement induced from methyl jasmonate (MeJA) is regulated by intracellular GSH [215]. In tobacco, both GSH and GSSG application induce Ca2<sup>+</sup> signaling as well as the expression of a specific gene, which supports the involvement of GSH with signal pathways that connect the Ca2+-dependent protein kinase [216]. The protein family peroxiredoxins (Prxs) are also GSH dependent and catalyze the reduction of H2O2 [217]. The GSSG can be exchanged with sulfhydryl groups of proteins and produce protein–GSH disulfide conjugates, which has a long half-life and plays a vital role in cellular signaling [218]. Moreover, GSH influences translation, and PTM of proteins, modulation of metabolism, and gene expression [219]. Hence, the mechanistic process of GSH signaling role should be focused on in future studies.

#### *8.3. AsA-GSH Cycle Interaction with Phytohormone Biosynthesis Pathways*

Ascorbate regulates phytohormone biosynthesis; hence, modulating plant development [220]. Ascorbate shows activity, where cell developments are affected by hormonal signaling and modulate effective signaling processes [221]. The abscisic acid (ABA) involvement in stagnating growth and metabolism suggests the crucial role of AsA sensing for plant survival [222]. In addition, a number of dioxygenases that are directly related to hormonal biosynthesis require AsA is a cofactor [223]. Moreover, a low AsA induces PR proteins, but do not alter antioxidative enzymes. Thus, AsA acts as a "crosstalking" signal, where ABA acts as an important intermediary signal induces PR1 proteins in many plants. Hence, phytohormone signaling arises the AsA-dependent PR genes regulation [224]. On the other hand, 1-aminocyclopropane-1-carboxylate (ACC) synthase (*ACS*) and ACC oxidase (*ACO*) genesencoding ethylene biosynthetic enzymes is induced by GSH. Further, GSH increases serine acetyl transferase (SAT) level and confers Ni toxicity tolerance [225]. In rice, the overexpression of SA metabolism genes gave raise to both SA and GSH content under oxidative stress [226]. Therefore, GSH triggers phytohormones, and vice versa, along with other signaling genes [227].

#### *8.4. Interaction of AsA-GSH Pathway with Glyoxalase Pathway*

There is an intimate relationship between AsA-GSH cycle and the glyoxalase pathway through GSH, where it plays a vital role in the detoxification of MG. Methylglyoxal is a respiratory byproduct and produced usually in plants and detoxifies by the glyoxalase system. However, MG is overproduced under stress, which causes toxicity [51]. Moreover, MG can disfunctionate antioxidant enzymes [1]. In the MG detoxification process, Gly I (EC 4.4.1.5) and Gly II (EC 3.1.2.6) work simultaneously to detoxify MG (Discussed in Section 6). In this pathway, Gly I uses GSH as a cofactor and conjugates MG with GSH to form *S*-D-lactoylglutathione (SLG), Gly II, and then produce d-lactate breaking SLG, and regenerate GSH (Figure 9) [1], thus playing important interaction with glyoxalase system.

#### *8.5. Interaction AsA-GSH Pathway with Xenobiotics Detoxification Pathways*

Xenobiotic detoxification involves the conjugation of toxic xenobiotics with GSH, which are further transferred to the vacuole by using ATP driven tonoplast transporter. This detoxification enables secondary metabolites biosynthesis as well as storing in the vacuole, such as anthocyanin. Plants are having GSH-dependent enzyme, GST, which detoxify herbicides by conjugating it with GSH. Therefore, the glutathionylated metabolites are imported to vacuolar by ABC (ATP-binding cassette) transporters. However, the GST mainly functions in catalyzing natural products that were observed with xenobiotics and, similar to those, catalyzes alternative GSH-dependent biotransformation reactions and binds and carries phytochemicals between cellular compartments [228].

#### *8.6. AsA-GSH Cycle Interaction with Metal Chelation Process*

Maintaining lower metal/metalloid(s) level inside the cell involves metal sequestration by low molecular weight thiols, for instance, metallothioneins (MTs) and phytochelatins (PCs). The two important enzymes involved in this process, glutaredoxin (GRx) and thioredoxin (TRx), are GSH dependent and neutralize H2O2 or controls of protein thiols [229]. On the other hand, PCs are another important chelating agent containing that thiol group that are upregulated by different metal/metalloid(s) [32]. The basic component for this PC is GSH. The biosynthesis of PCs is accomplished by the enzyme PC synthase (PCS), which requires GSH as a substrate. The enzyme is crucial for metal detoxification, metal homeostasis, and stress tolerance [137].

#### **9. Genetic Manipulation of AsA-GSH Pathway and Its Role in Abiotic Stress Tolerance**

The regulation of AsA and GSH pool plays an important role in mitigating oxidative stress in plants. To attain this, the regulation of the enzymes that are related to the AsA-GSH pathway is vital. There are many plant studies that considered the genetic manipulation of AsA-GSH pathway. These

studied revealed that the overexpression of AsA-GSH pathway enzymes provided the plants better protection against oxidative stress under various environmental adversities (Table 4). Transgenic tobacco plants overexpressing *PcAPX* showed enhanced tolerance to salt and drought [230]. Transgenic plants exhibited a 347% increase in APX activities under drought stress, as compared to control, which resulted in a remarkable decrease in H2O2 content (136%) than that of wild type (309%). The ascorbate content was also higher 63%) when compared to wild type (42%). Similar results were also observed in the case of salt stress [230]. Chin et al. [231] found that transgenic *Arabidopsis* overexpressing *OgCytAPX1* scavenged ROS effectively and showed enhanced tolerance to salt and heat. The overexpression of *Malpighia glabraMDHAR* gene resulted in a higher biosynthesis of AsA, which provided tobacco plants tolerance to salt stress [232]. Shin et al. [233] observed that the coexpression of *B. rapaBrMDHAR* and *BrDHAR* genes provided a remarkable improvement of oxidative stress in *A. thaliana*. The overexpression of *BrMDHAR* and *BrDHAR* showed enhanced MDHAR and DHAR activities and higher AsA/DHA ration. These plants also provided better radical scavenging capacity, which resulted in lower H2O2 content. Yin et al. [234] found that *Arabidopsis* plants overexpressing the gene *AtGR1* conferred Al stress tolerance by reducing reactive carnoyl species, which was mainly due to higher GSH level and GR activity. The plant that overexpressed *AtGR1* also maintained the activity of H2O2-scavenging enzymes. For instance, GPX and APX activities in Al-treated plants were decreased by 21 and 46%, respectively, but the wild-type plants only showed 8 and 30% decreases in such activities [234].

Modulating several *NAC* genes [*NAC* domain consists of three different genes; *NAM* (no apical meristem)-*ATAF* (*Arabidopsis* transcription activation factor)-*CUC* (cup-shaped cotyledon)] are also an efficient way to transform the AsA-GSH cycle, consequently enhancing stress tolerance. The overexpression of wheat *TaNAC2* in *Arabidopsis* lines showed tolerance against freezing, salt, and drought stress by modulating the AsA-GSH cycle [235]. Moreover, ectopic expression of *SlNAC2* conferred both salt (200 mM) and drought (20% PEG) tolerance up to 10 days in transgenic *Arabidopsis* lines, which is correlated with the lower accumulation of ROS. In addition, the transcriptomic abundance GSH metabolizing genes was also observed in transgenic lines, leading to increased GSH synthesis and lesser oxidative damage [236].


**Table 4.** Overexpression of genes related to AsA-GSH pathway and their role in ROS scavenging.


**Table 4.** *Cont.*

#### **10. Conclusions and Outlook**

Ascorbate and GSH have roles in decreasing oxidative stress, and it has been reported in numbers of research findings. Most of the research findings reported about their roles in antioxidant defense system for scavenging ROS. However, exogenous GSH related research on antioxidant defense system needs further confirmation at the genetic and molecular level. Moreover, without the commonly known ROS, like H2O2, OH, O2 −, etc. some other oxidative stress-inducing agents, like reactive nitrogen species, MG, etc., should be brought under consideration for research. How GSH can affect the generation of other kinds of oxidative stress-inducing damage. Research that is related to exogenous AsA or GSH-induced GST activity concerning xenobiotic detoxification is rare. The regulation of tocopherol by AsA or GSH can be an interesting area of research. For the reduction of metal-induced oxidative stress protection, GSH plays a vital role by producing PCs and inducing vacuolar sequestration. The credible function of AsA-GSH cycle in this area is so far to explicate. The GSH/GSSG redox is a well-reported term when discussing stress-induced oxidative damage and signal transduction process towards adaptation though the process is not well revealed. Interaction between and among the AsA-GSH cycle components and the hormones, other signaling molecules or any other molecules in oxidative stress, redox regulation, or plant adaptation process is not well understood. It is well known that chloroplast, its photosystem, and Calvin cycle activity or photosynthesis process is the maximum contributor of most of the ROS and oxidative stress under any abiotic stress condition. Several research findings reported about the role of AsA and GSH in improving the chl or carotenoid levels. However, very few of them reported regarding the roles of AsA and GSH in regulating stomatal conductance, Calvin cycle, RuBP activity/regeneration, or photosystem efficiency, which directly generates ROS and results in oxidative stress [203]. Some of the research findings show the positive roles of GSH improving/regulating Pro, which is cellular ROS scavenger or cytosol stabilizer to reduce ROS generation. These are the promising area of future research, which not only will alleviate the oxidative stress, but also improve the photosynthetic efficiency of plants for increasing plant production for the constantly growing population of the planet.

Although, in this review article, we focused on abiotic stress-induced oxidative damage and the role of AsA-GSH cycle to mitigate such adversities, biotic stress (fungi, bacteria, virus, nematodes, and parasitic organisms, etc.) might also alter the essential plant processes as well as cellular metabolism.For example, the production of ROS and oxidative stress, disruption of membranes, hampering photosynthesis, changing enzyme activities, cell death, and yield loss might also be attributed to biotic stress, which is in line with abiotic stress.Biotic stress also disrupts signal transduction, as well as transfigures signal pathways that are associated with stress acclimation. Over the past decade, AsA-GSH cycle has also emerged as an important component for the plant biotic stress response. Similar to abiotic stresses, biotic stresses also alters the metabolism and changes in antioxidant activity. Therefore, AsA-GSH cycle also directly impacts the important metabolomic processes, thus providing an important link between metabolism, signal transduction, and acclimation to plants during biotic stress.

**Author Contributions:** M.H. constructed the main conceptual ideas and proof outline. All authors participated in the drafting of this paper as individual subject matter experts in their fields. M.H., M.H.M.B.B., and J.A.M. prepared the figures. M.H., T.I.A., K.P. and K.N. prepared the tables. M.H. and M.F. has contributed critically to the improvement and editing of the manuscript. All the authors contributed to collecting the literature, improving the paper, and approved the final manuscript.

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

**Acknowledgments:** We thank Khadeja Sultana Sathi, Tonusree Saha, Mira Rahman and Naznin Ahmed for the formatting and proof checking of the manuscript.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


© 2019 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 (http://creativecommons.org/licenses/by/4.0/).

## *Review* **The Role of the Plant Antioxidant System in Drought Tolerance**

#### **Miriam Laxa \*, Michael Liebthal, Wilena Telman, Kamel Chibani and Karl-Josef Dietz**

Department of Biochemistry and Physiology of Plants, Faculty of Biology, University of Bielefeld, Universitätsstr. 25, 33615 Bielefeld, North Rhine Westphalia, Germany; mliebthal@uni-bielefeld.de (M.L.); wtelman@uni-bielefeld.de (W.T.); kamel.chibani@uni-bielefeld.de (K.C.); karl-josef.dietz@uni-bielefeld.de (K.-J.D.)

**\*** Correspondence: miriam.laxa@uni-bielefeld.de; Tel.: +49-521-106-5590

Received: 14 March 2019; Accepted: 2 April 2019; Published: 8 April 2019

**Abstract:** Water deficiency compromises plant performance and yield in many habitats and in agriculture. In addition to survival of the acute drought stress period which depends on plant-genotype-specific characteristics, stress intensity and duration, also the speed and efficiency of recovery determine plant performance. Drought-induced deregulation of metabolism enhances generation of reactive oxygen species (ROS) and reactive nitrogen species (RNS) which in turn affect the redox regulatory state of the cell. Strong correlative and analytical evidence assigns a major role in drought tolerance to the redox regulatory and antioxidant system. This review compiles current knowledge on the response and function of superoxide, hydrogen peroxide and nitric oxide under drought stress in various species and drought stress regimes. The meta-analysis of reported changes in transcript and protein amounts, and activities of components of the antioxidant and redox network support the tentative conclusion that drought tolerance is more tightly linked to up-regulated ascorbate-dependent antioxidant activity than to the response of the thiol-redox regulatory network. The significance of the antioxidant system in surviving severe phases of dehydration is further supported by the strong antioxidant system usually encountered in resurrection plants.

**Keywords:** antioxidant; drought; ROS; RNS; stress; acclimation

#### **1. Introduction**

During their ontogenesis, plants face a dynamically changing environment defined by abiotic factors (e.g., light/dark, temperature, nutrient and water availability, and toxic compounds such as heavy metals) and biotic interactions (e.g., beneficial and pathogenic microbes, fungi, insects, other herbivores) [1]. Environmental perturbations which significantly disturb metabolism, development and yield, are considered as stress situations and cause stress responses in biological system. Such imposed stress is commonly accompanied by an increase in the production of reactive oxygen species (ROS) and reactive nitrogen species (RNS) that lead to an imbalance between their production and scavenging. Despite their reactive and thus toxic nature, ROS and RNS are also key components of signal transduction pathways that trigger stress responses. Furthermore, ROS and RNS are involved in plant developmental processes [2–4] and plant-microbe interactions [5,6]. However, excessive ROS and RNS production must be counteracted by the antioxidant system to prevent damage development and cell death.

Drought stress severely impacts plant development, growth and fertility. Drought triggers water loss and a decrease in water potential, which concomitantly leads to a reduction in cell turgor (Figure 1). Among the fastest processes induced by drought is the abscisic acid (ABA)-mediated closure of stomata [7]. Prolonged drought stress and increased stress intensity lead to further acclimation reactions. These responses include osmotic adjustment [8,9], decreased shoot-root ratio [10], cell wall modifications [11,12], reprogramming of metabolism [13], and activation of the antioxidant system [14,15]. Many of these modifications are measurable and are used to characterize the severity of drought stress. Measurable traits are, for example, the stomatal and mesophyll conductance, net photosynthesis, photorespiration, abundance of osmoprotectants, tissue water potential, ABA content and membrane integrity. Drought avoidance includes morphological adaptations, like leaf curling and increased wax deposition on the leaf surface [16] (Figure 1).

**Figure 1.** Physiological and biochemical processes triggered by drought.

During evolution, plants developed mechanisms to acclimate to drought or even to withstand dry periods. Extensive research has unraveled the molecular mechanisms of drought and desiccation tolerance. Figure 2 summarizes characteristic features of drought-sensitive, drought-tolerant and desiccation-tolerant plants. Tolerant plants are equipped with higher levels of both osmolytes and non-protein antioxidants, reprogram their metabolism and enhance their antioxidant capacity. Interestingly, sensitive species also activate their antioxidant system. Nevertheless, despite this apparent contradiction, drought tolerance seems to be a function of the antioxidant capacity realized in response to drought. Furthermore, the antioxidant activity not only is important during acute drought stress, but also interferes with recovery from water limitation and resurrection from dehydration.

**Figure 2.** Characteristic features of drought-sensitive, drought-tolerant and desiccation-tolerant plants. The figure summarizes properties related to metabolism, antioxidant defense, and recovery which often are associated with the physiological traits. Red arrow: reactive oxygen species (ROS)/reactive nitrogen species (RNS) gain prevalence; green arrow: status is preserved following drought. Fond size correlates with the strength of stress responses measured. ROS, reactive oxygen species; RNS, reactive nitrogen species; HSP, heat shock protein; LEA, late embryogenesis abundant protein; ELIP, early light-inducible protein; Suc/Fru, sucrose to fructose ratio; CAT, catalase; APX, ascorbate peroxidase; SOD, superoxide dismutase; ASC, ascorbate; GSH, glutathione.

In the beginning of the review we will recall the classification of drought and how drought stress conditions are experimentally induced. This is important information to relate the production of ROS and RNS to the applied stress later in this review. Our review centers on the sites of production and roles of ROS and RNS during dehydration and their detoxification by the antioxidant system. Where possible we will correlate the activation of the antioxidative system to drought tolerance. Furthermore, we will evaluate which antioxidants are involved in drought response in particular. The last section describes the role of the antioxidative system in resurrection plants as an intriguing case of exceptional drought tolerance.

#### **2. Classification and Application of Drought Stress**

Drought is classified in mild, moderate and severe stages of stress (Table 1). The transition between the different stages occurs steadily and reflects the progression of drought stress severity both in duration and dehydration strength. Hence, an absolute value of dehydration cannot be assigned to the individual stages of drought stress. The stages are rather categorized in certain ranges. Various units have been used to describe water limitations (Table 1). The overall consensus is that the relative water content (RWC) in mild drought stress ranges between 60–70% compared to the control of ≥90%, in moderate stress between 40–60% and in severe stress between 0–40% (Table 1, Figure 1). Interestingly, these classifications are quite consistent between different species, even though the length of the applied stress to reach these states differs considerably (Table 1). Severe drought

stress conditions can be reached rapidly within a week in soils with low water holding capacity. Mild stress conditions, corresponding to a soil field capacity (SFC) of 70%, are already reached after two days, severe (SFC < 50%) and very severe wilting (SFC < 30%) after five and eight days, respectively, as determined for 25 day-old soybeans grown in a sand-vermiculite mixture [17]. A time period of 1–2 weeks without watering was shown to be the most suitable condition for testing both drought tolerance and recovery of various mesophytic species grown on soil (Table 2). Drought stress can be induced either by withholding water in the case of soil-grown plants or by polyethylene glycol (PEG) in both agar-plates and liquid cultures [18]. The use of PEG-infused agar systems allows generating a defined water potential in the substrate [19]. However, the majority of these systems were only applicable for seedlings for a long time. Recently, Frolov and colleagues [20] established an agar-based polyethylene glycol infusion drought model for six-to-eight-week-old *Arabidopsis* plants. This system is extremely valuable as it allows analyzing the response of adult plants and thus a more appropriate developmental stage in terms of agricultural application.

The occurrence and severity of drought-induced injury varies between different developmental stages of the plant and also depends on duration and strength of the applied stress.


**Table 2.** Exemplary experimental design for testing drought tolerance in different plant species.


day(s); h, hour(s); MS medium, Murashige–Skoog medium; PEG, polyethylene glycol.

#### **3. ROS and RNS Generation during Dehydration and Its Combination with Other Stresses**

Stress-induced production of ROS and RNS occurs in different cell compartments [45]. They are used to transmit signals to the nucleus and other compartments to reprogram cell performance including gene expression [46,47]. The underlying mechanisms are known as retrograde and anterograde signaling pathways [1,48]. This paragraph focuses on the sources of ROS and RNS, and their accumulation in response to drought stress.

#### *3.1. ROS during Drought*

The first response of plants to drought is the closure of stomata in order to minimize water loss due to transpiration. Because of ongoing photosynthesis in the light, the increased gas diffusion barrier facilitates depletion of the intercellular carbon dioxide (CO2) concentration. Decreased availability of CO2 stimulates ribulose–1,5–bisphosphate oxygenation and, thus, photorespiratory hydrogen peroxide (H2O2) production in the peroxisomes. This effect has been studied in detail and was frequently summarized, e.g., with respect to drought and H2O2 production in wheat and potato as C3 field crops [49]. Insufficient availability of the electron acceptor CO2 slows down the oxidation of nicotinamide adenine dinucleotide phosphate (NADPH) in the Calvin–Benson cycle. Lack of NADP<sup>+</sup> causes a backlog of electrons and over-reduction of the photosynthetic electron transport which in turn increases the reduction rate of oxygen as alternative electron acceptor in the Mehler reaction at photosystem I (PSI) and enhanced release of superoxide anion (O2•<sup>−</sup>) and hydrogen peroxide (H2O2). Hence, chloroplasts are primary targets of excess light and CO2 starvation in drought. In addition, photorespiration produces NADH in the mitochondrion.

A highly reduced chloroplast NADPH-pool via thioredoxin (TRX) reduction activates the NADPH-dependent malate dehydrogenase and, thereby, the malate valve for export of reducing equivalents to the cytosol and mitochondrion. The disequilibrium between electron supply and consumption in photosynthesis is efficiently transmitted to the respiratory electron transport chain (ETC) in the mitochondrion. Activation of alternative oxidase (AOX) and induction of *aox* gene expression are hallmarks of drought response [50–52]. Even under normal conditions, 1–2% of oxygen is consumed to produce ROS due to an over-reduction at complex I and III in the oxidative phosphorylation [53]. Under drought, the capacities of AOX, plant uncoupling proteins (PUCPs) and ATP-sensitive potassium channels are stimulated to dissipate excess electron flow in ETC [54]. Respiratory functions are inhibited by about two-thirds in drought-stressed plants as reviewed by Atkin and Macherel [55]. These studies included dehydration regimes of various intensities and on different time scales. The authors commented that the missing response in tolerant species might be due to enhanced antioxidant defense. Additionally, ROS are produced at the apoplast. Interestingly, the production of apoplastic ROS is coupled to calcium signaling [56]. Respiratory burst oxidase homolog (RBOH) proteins in the plasma membrane are calcium and phosphorylation-sensitive enzymes generating superoxide anions in the apoplast in response to drought, but also many other stresses [57,58]. Cell wall-associated kinases (WAKs) are members of the receptor-like kinase (RLK) family and participate in the perception of turgor pressure changes during drought probably linking ROS bursts with phosphorylation of RBOHs [59]. Apoplastic ROS also induce lipid peroxidation giving rise to malondialdehyde (MDA) as an indicator for membrane damage especially during drought. After dismutation of superoxide to H2O2 in the apoplast, transfer of H2O2 from the apoplast to the cytosol may also contribute to the intracellular ROS signature.

Table 3 summarizes changes of ROS and RNS amounts in response to drought stress. Maize growing in soil at 20% water saturation deficit accumulated twice the H2O2 amount of well-watered control plants [60]. Likewise, H2O2 reached thrice the contents of control rice if exposed to 200 mmol/L mannitol for two days [61,62] and in *Ailanthus altissima* plants that were kept unirrigated for 14 days [63], respectively. Thus, accumulation of ROS under drought is a prototypic case of stress-induced responses. **Table 3.** Changes in reactive oxygen species (ROS) and nitric oxide (NO) amounts upon drought or osmotic stress treatment in various plant species. Data originate from green leaf tissue if not indicated otherwise. Increase in percent was chosen due to different detection methods with different units. Effects were estimated from graphs, figures and tables if not directly given in the text or supplements.


MWHC, maximum water holding capacity; RWC, relative water content; SWC, soil water content; SFC, soil field capacity; d, day(s); h, hour(s.).

#### *3.2. ROS, Oxidative Post-Translational Modifications and Redox Signalling*

Within proteins, the thiol groups of both cysteine (Cys) and methionine (Met) are the major sites of oxidative post-translational modifications (PTMs) [90]. Thiols are prone to successive oxidation to sulfenic (R-SOH), sulfinic (R-SO2H), and sulfonic (R-SO3H) acids [91]. Cys oxidation and reduction efficiently regulates enzyme activities. A well-established system is the redox system of chloroplasts in which the redox input is provided by ferredoxin (Fd), NADPH and glutathione (GSH), redox signals are transmitted on target proteins by TRX, NADPH-thioredoxin reductase (NTRC) and glutaredoxins (GRX) [92]. Peroxiredoxins (PRX) are thought to sense the redox state of the cell and act in signaling instead of ROS detoxification [92]. Oxidative PTMs and the role of PRX in plant redox signaling are subjects of recent reviews and, thus, are not discussed in detail here [92,93].

#### *3.3. RNS during Drought*

Reactive nitrogen species are less diverse than ROS. Nitric oxide (NO) is a gaseous signaling molecule involved in germination, development, hormone regulation, and stress management. While homologues of animal NO synthase are absent from plants [94], the described mechanisms for NO production include (i) nitrate reductase (enzymatic, cytosol/plasma membrane), (ii) xanthine oxidoreductase (enzymatic, peroxisome), (iii) NO-associated proteins (enzymatic, mitochondria/plastids), (iv) nitrite: NO reductase (enzymatic, plasma membrane), (v) electron transport chain (non-enzymatic, mitochondria/chloroplast), and (vi) a poorly understood mechanism using arginine, polyamine or hydroxylamine [95–97]. The bioactive NO concentration is influenced by the nitrogen nutrient supply, the concentration of the storage compound nitrosoglutathione (GSNO), the activity of the GSNO reductase, and turnover mechanisms including the interaction with hemoglobins [98–100].

Osmotic stress, established by exposing rice roots to 200 mmol/L mannitol, increased the NO amount threefold within 24 h in rice leaves [61]. The same increase in NO was observed in rice after withholding irrigation for nine days, while a significant increase was undetected after three days [86]. Since both studies focused on leaves, the large time scale difference is striking and may reflect the time span needed to establish similar stress levels. This interpretation is supported by the fact that an osmotic shock treatment with 210 mmol/L mannitol corresponds to an applied osmotic potential of approximately −1.1 MPa [101], while an equivalent osmotic potential after withholding water was reached only at days 4 and 5 [86]. The data also point to changes in drought sensitivity during development. Most plants respond more sensitive to dehydration in early developmental stages. Therefore, one explanation for the discrepancies between the above mentioned studies might be attributed to differences in the plant growth stages of 16 [61] versus 42 days [86], leaving juvenile leaves more sensitive to drought. In this context, it should be mentioned that the ratio of developing to mature cell in the leaf lamina changes significantly during the early phase of development. Furthermore, the antioxidant response to paraquat was compromised in young *Arabidopsis* leaves [102]. Mature leaves were able to compensate ROS accumulation much more efficiently due to an increase in APX activity. The authors suggested different photoprotective regulatory mechanisms in the two leaf types. Furthermore, it was concluded that the redox-state of plastoquione A (QA) is the determinant of tolerance to paraquat-induced oxidative stress [102]. A similar observation was made in *Fagus sylvatica* L. Here, resistance to paraquat-induced oxidative stress was mediated by an increase in SOD activity in mature leaves [103]. In the tea plant (*Camellia sinensis*), cold-sensitivity of young leaves is correlated with inhibited expression of genes related to cell membranes, carotenoid metabolism, photosynthesis and the antioxidative system [104]. In contrast, transcripts belonging to the gene ontology groups of chloroplasts, cell membranes, redox processes, glutathione metabolism and photosynthesis were increased in mature leaves in response to cold. Hence, the antioxidative system plays an important role in establishing acclimation and hardening to stress.

In tree species like *Ailanthus altissima,* NO amounts increased three-fold after withholding water for 14 days [63]. NO is reported as an important positive regulator for Crassulacean acid metabolism (CAM) in pineapple leaves as described by Freschi et al. [79]. Emission of NO gradually increased from 40 to 140 pmol. h<sup>−</sup>1g−<sup>1</sup> dry weight upon treatment with 30% PEG 6000 for 5 days. Of PEG, 30% corresponds to a water potential of −1.03 MPa [105] and, thus, is similar to osmotic stress induced by 200 mmol/L mannitol [87]. NO quantification was mostly achieved by using fluorescence probes like diaminofluorescein (DAF) or diaminorhodamine (DAR) derivates. To overcome drawbacks related to limited specificity, new probes are presently engineered to improve sensitivity and specificity [106]. Nevertheless, cell- and tissue-imaging with DAF-2 diacetate in dehydrating pineapples localized NO in chlorenchyma, trichoma and epithelial cells but did not resolve subcellular compartmentation.

NO also plays a significant role in regulating germination during drought in grasses like wheat and rice [87,107]. Endogenous NO counteracts programmed cell death and vacuolization induced by gibberellic acid. The NO amount in aleurone layers drops by 75% after 24 h of osmotic stress compared to controls (20% PEG-6000). Exogenous application of NO donors alleviates the effect and delays germination. Thus, a synergistic effect of NO is seen with ABA allowing postponing germination until growth conditions improve. Under such conditions, germination is inhibited and resumed only after growth conditions have improved. Expression of rat neuronal NO synthase (nNOS) in plants constitutively increases NO levels twofold in *A. thaliana* [80] and 1.5-fold in *O. sativa* [61]. These nNOS-plants accumulate more biomass and less H2O2 after withholding water for 14 d (*A. thaliana*) or upon treating rice with 200 mmol/L mannitol. These results assign a significant role to NO in shaping the acclimation to drought. They also show that the NO effect partly antagonizes the effects of ROS in this process.

In general, information on plant specific endogenous RNS signaling is still scarce. The production of NO occurs in similar subcellular compartments as ROS but our knowledge on its induction, regulation of enzyme activities, and substrates emerges only slowly. Hence, many groups use NO donors to artificially expose plants to RNS. Currently, research focuses on synergistic versus antagonistic effects of RNS and ROS, especially in the field of abiotic stress, and promises a more integrative concept. Experiments on genetic model systems are needed which link the dynamics of specific markers for RNS signaling with proteomic and transcriptomic analyses.

#### *3.4. Nitrosylation by ONOO*− *and GSNO*

Antagonistic and synergistic effects relate to reaction products of RNS and ROS and antioxidants, respectively. Thus, GSNO forms by reaction of NO with reduced glutathione, while peroxynitrite (ONOO<sup>−</sup>) forms at sites of simultaneous formation of O2•<sup>−</sup> and NO. GSNO triggers S-nitrosylation, while ONOO− causes tyrosine nitration. Several targets of these reactions are part of the antioxidant defense system like PRX, ascorbate peroxidase (APX), monodehydroascorbate reductase (MDHAR), dehydroascorbate reductase (DHAR) and catalase (CAT) [108,109]. Especially during drought in *Lotus japonicus* NO amounts doubled in roots, but interestingly not in leaves [84]. S-nitrosylation of proteins is promoted in roots. The authors hypothesized that roots are prone to nitrosative stress, and leaves to oxidative stress.

Higher NO concentrations in roots compared to leaves were also reported in sugarcane [89] and bluegrass [110] and support this rule of thumb. One function of NO in roots concerns root patterning as described for pea, tomato, tobacco, and cucumber facing drought conditions [82,111–113]. Such differential effects have also been reported for pollen development and stigma function which respond preferentially to either RNS or ROS, respectively. Apparently, ROS and RNS play unique roles in developmental signaling which should be explored further [114]. Furthermore, GSNO serves as a mobile carrier of NO allowing for long distance signaling. In contrast, ONOO− is highly reactive and characterized by a short half-life of 10 to 20 ms, and thus is discussed as a linker between ROS and RNS signaling [115]. Moreover, specific analyses are needed to clarify the NO-related effects on metabolism and to see whether RNS signaling is exclusively transmitted by ONOO− and GSNO.

#### *3.5. ROS*/*RNS in Stress Combinations with Drought*

Responses to drought are accentuated if dehydration is combined with other abiotic stresses. Exceptions from this rule concern drought combined with ozone and high CO2. The antagonising effect is traced back to stomata closure triggered by ozone [116] or high CO2 [117]. Iyer and collegues [116]

described this phenomenon in *Medicago truncatula*. Here, ROS levels increase in response to drought and ozone by 2-fold and 2.8-fold, respectively, compared to the well-watered condition. However, ROS levels in response to combined drought and ozone stress are indistinguishable from the control (well-watered plants). In contrast, NO levels are elevated only in response to drought by approximately 2-fold, while ozone has no effect. Simultaneous application of the two stresses again did not lead to significant changes. Interestingly, jasmonic acid and salicylic acid synthesis are induced after application of NO-donors in *A. thaliana* which might explain the mitigating effect of ozone in combination with drought [118]. Again, both reports vary in species and treatment, but indicate that RNS signaling is directly involved in stress response and alters the ROS effects.

In the natural environment, dry periods often coincide with high temperature and high light. Malondialdehyde (MDA) is an indicator for lipid peroxidation and oxidative damage and significantly increases in green tissue of citrus cultivars exposed to a combination of drought and heat (10 d, 40 ◦C). The increase is absent in single stress applications [119]. The stronger effect of a drought/heat combination is also seen in maize. Here, MDA levels increase by 225%, while the single applications elevated MDA levels by only 45% (−0.7 MPa PEG for 8 h) or 92% (2 ◦C/h increase from 28 to 42 ◦C for 8 h), respectively [120]. In cotton cultivars, no significant differences in H2O2 levels are observed for drought and combined drought/heat stress [121].

Combining heat (42 ◦C) and drought in succulent purslane for seven days doubles MDA content, while single stress treatments increase the MDA amount only by 20%. Interestingly, O2•<sup>−</sup> amount raises 2.5-fold under heat and combined stress, but not in plants exposed to drought [122]. Surprisingly, the leaf H2O2 level decreases in grapevine upon deprivation from water for four days followed by treatment with heat (1 h, 42 ◦C) or high light (1 h, 2000 μmol quanta. s<sup>−</sup>1m−2) [123]. None of the double or triple stress treatments including drought alters the H2O2 amounts above the levels measured during control treatments. Significant variations between cultivars are only seen in single treatments and a heat/high light treatment.

These examples support the theory by Suzuki and colleagues [1] that the response to a combined stress is unique and cannot be simply extrapolated from the responses to single stresses. For instance, the response to stress combinations on signaling pathways and responses can be synergistic, antagonistic or independent. Antagonistic and, thus, positive interactions are observed for the combination of drought and high CO2 [124]. However, combined stress often leads to negative interactions, and the consequences are synergistic rather than additive [1]. This is also true for high light and drought [125]. Both, high light and drought realize an over-reduced state of photosynthetic ETC. With respect to high light the over-reduction is caused by an excess of light energy, while the over-reduction following drought is caused by a limited CO2 availability after stomatal closure and the concomitant inhibition of the Calvin–Benson cycle. Consequently, in both cases ROS and RNS are generated, but the ROS/RNS signatures differ in both cases [126].

The described examples demonstrate the importance to investigate plant responses and signaling pathways in combined stress. However, most laboratory studies on plant stress responses consider one stress at a time, whereas plants in the field usually are exposed to different stresses simultaneously. For example, drought stress is often accompanied by heat and high light intensities [117,127]. Therefore, it has to be kept in mind that any treatment applied under controlled growth chamber conditions fails to reflect field conditions. Ecotypes of the same plant species adopt distinct adaptive responses to acclimate to their local habitats. Such naturally occurring biodiversity in terms of sensitivity vs. tolerance of closely related species, the extreme adaptability of specialists and the special case of crop plant monocultures cannot be treated in this review focusing on ROS and RNS-dependent signaling.

#### **4. Response of the Redox Network under Drought**

The activation of the antioxidant system via retrograde signaling is a key process in plant acclimation to oxidative stress. Thus, the upregulation of antioxidant enzymes represents an important marker for drought stress. In the cell, the production and scavenging of ROS and RNS is strictly controlled and the equilibrium can be perturbed by several biotic and abiotic stresses [128]. Plants have evolved complex redox signaling networks in which ROS and RNS are used as signals to regulate normal and stress-related physiological processes including antioxidant mechanisms to combat the toxic effects of ROS and RNS [129,130]. Plants keep ROS under control by an efficient and versatile scavenging system. The antioxidant defense comprises low molecular weight compounds such as GSH, ascorbate (ASC), α-tocopherol, carotenoids, and enzymes including CAT, SOD, and the thiol peroxidases of the PRX and glutathione peroxidase (GPX) type [131].

Thiol peroxidases are linked to the NADPH-thioredoxin reductase (NTR), ferredoxin-dependent TRX reductase (FTR) and GSH/GRX systems [132,133]. Mechanism of ROS production and their scavenging by high antioxidant capacity has been associated with tolerance of plants to abiotic stresses [128]. Recently, a new function was assigned to thiol peroxidases in redox regulation, namely as TRX oxidases [134]. This mechanism allows for reading out the balance between reductive electron input and oxidative electron drainage and tunes the redox and activity state of target proteins.

#### *4.1. E*ff*ect of Drought Stress on the Antioxidant System and Redox Homeostasis*

During drought stress, up-regulation of antioxidant systems occurs at both the transcriptional and post-transcriptional level. Table 4 gives examples for quantitative drought responses of antioxidative enzymes and enzymes involved in regeneration of non-protein antioxidants. APX, catalase (CAT) and GPX represent the principal ROS scavengers in plants. Among these three, APX appears to be induced most strongly on post-transcriptional level (Table 4). In contrast to CAT and GPX, APX is also regulated on transcriptional level based on the data summarized in Table 4. Cytosolic, chloroplastic and peroxisomal APX activities are commonly enhanced in all species of the plant kingdom. The activity of cytosolic APX is increased during drought in pea [135]. The *alx8* mutant (altered expression of APX2) of *Arabidopsis* reveals improved drought tolerance [136,137]. Over-expression of peroxisomal or cytosolic APX from poplar in transgenic tobacco increases plant performance under drought [138,139]. CAT is a tetrameric, heme-containing enzyme that catalyzes the dismutation of H2O2 into H2O and O2 in the peroxisome. CAT2 plays a crucial role when the plant is exposed to a severe drought stress [140]. Compared to APX activation, stimulation of CAT is moderate (Table 4). Even though CAT activation seems predominantly taking place on post-transcriptional level, there are examples for complex regulation of CAT activity under severe drought which involves gene expression, translation and protein turnover [141].


 2.29-fold (rel. expression)

Wheat

 35%

 [157]

Antioxidantenzymesregulatedinplantsunderdrought.




**Table4.***Cont*.

#### *Antioxidants* **2019**, *8*, 94



APX, ascorbate peroxidase; CAT, catalase; DHAR, dehydroascorbate reductase; GPX, glutathione peroxidase; GR, glutathione reductase; GST, glutathione-S transferase; MDHAR, monodehydroascorbate reductase; PDI, protein disulfide isomerase; PRX, peroxiredoxin; SOD, superoxide dismutase; TRX, thioredoxin. Black color, up-regulation; red color, down-regulation; ns, not significantly changed.

Besides APX, other components of the ASC-GSH cycle, namely MDHAR, DHAR, glutathione-S-transferase (GST) and glutathione reductase (GR), work synergistically in different cell compartments. MDHAR, DHAR, GST and GR transcripts and activity are predominantly induced under drought stress (Table 4). Among these four enzymes, GR is activated strongest. GR activation can be compared to the one observed for CAT. In general, upregulation of the ASC-GSH metabolism and associated enzymes efficiently scavenge H2O2 under drought stress as observed in wheat [167].

Moreover, PRXs are also up-regulated and accumulated in cotton [150], date palm [151] and wheat [161] upon drought (Table 4). This indicates that plants activate compensatory mechanisms to counteract enhanced H2O2 production in response to drought stress. In addition to their reductive function in detoxifying H2O2, alkyl hydroperoxide and ONOO<sup>−</sup>, PRX play a role in redox signaling and transmit information on the cell ROS state to target proteins [134,168].

SODs are a class of metalloenzymes that catalyze the dismutation of two molecules of O2•<sup>−</sup> into molecular oxygen and H2O2. The activation of SOD isoforms (Mn-SOD, Fe-SOD, Cu,Zn-SOD) is interpreted as a measure to counteract O2•− accumulation in diverse cell compartments under drought in e.g., *Arabidopsis* [158], blue grass [160], citrus [147], *Co*ff*ea canephora* [148], date palm [151], fescue [160], pea [135], poplar [153], tepary bean [145] and wheat [159]. Apparently, SOD is a critical component of the ROS-scavenging system likely by minimizing the reaction of O2•− with, e.g., NO to form ONOO−, unsaturated fatty acids for peroxidation or with proteins. In line with this assumption transgenic plants overexpressing Cu,Zn-SOD are more tolerant to drought stress [168].

A set of other important proteins belonging to the TRX superfamily is usually highly activated under drought stress. In general, TRXs are induced under different environmental stresses including dehydration, salinity, heat or cold [169]. Under several stresses, atypical and canonical TRX have the capacity to reduce oxidized antioxidant enzymes in the chloroplast, cytosol and mitochondria [170,171]. TRXs are localized in cytosol, chloroplast, mitochondrion, endoplasmic reticulum and nucleus [132]. Strongly responding oxidoreductases are represented by atypical chloroplastic TRX (CDSP32 and CDSP34), cytosolic or mitochondrial NADPH-TRX reductase (NTRA or B), endoplasmic reticulum-associated protein disulfide isomerase (PDI) and canonical cytosolic TRX (TRX h). NTRA-overexpressing plants exhibit extreme drought tolerance with high survival rates, low water loss and reduced ROS accumulation compared to wildtype and *ntra*-knock out plants [144]. However, TRX transcripts and activity measurements in date palm [151] and wheat [161] also indicate a down-regulation of some TRX members in response to drought stress.

#### *4.2. Distinct Patterns of Antioxidative Sytem Activation in Sensitive and Tolerant Species*

As summarized in Figure 2, drought-sensitive species also activate their antioxidative system. The data given in Table 4 confirm this assumption. However, they point out that not only the magnitude of activation might be decisive but also which enzymes are activated. For instance, the activation of the major scavenger APX and CAT is stronger in tolerant species compared to their sensitive counterparts. In contrast, sensitive species activate GPX more than tolerant species. Changes in the activation of the antioxidant system between sensitive and tolerant species are visualized in Figure 3. Obviously, sensitive plants predominantly activate the glutathione-dependent scavenging system, while the ascorbate-dependent system is only induced moderately or are even down-regulated (Figure 3). On the other hand, tolerant species showed a stronger activation of ascorbate-dependent scavenging system compared to the glutathione-dependent system. Moreover, inactivation is only apparent for the TRX-dependent scavenging system in tolerant species. Because drought stress leads to an over-reduction of the electron transport chain, down-regulation of TRX may counteract excessive reduction of target proteins. On the other hand, TRX-dependent reduction of PRX is compromised under this condition. However, PRX can be regenerated by other enzymes like GRX and NTRC [92]. Moreover, drought conditions necessitate a high capacity of detoxifying enzymes such as APX and CAT to suppress ROS accumulation. Furthermore, PRX are involved in redox-signaling [92] which might be their predominant function under drought stress.

**Figure 3.** Changes in the activation of the antioxidative system in sensitive and tolerant species. Orange, downregulation, blue, upregulation, grey, no significant changes, no color, no data. APX, ascorbate peroxidase; CAT, catalase; DHAR, dehydroascorbate reductase; Fd, ferredoxin; GPX, glutathione peroxidase; GR, glutathione reductase; MDHAR, monodehydroascorbate reductase; PRX, peroxiredoxin; SOD, superoxide dismutase; TRX, thioredoxin.

There is not much information on drought tolerance and NO signaling. However, a recent study investigated root extracellular and leaf intracellular NO contents in drought-tolerant and –sensitive sugarcane genotypes. Here, drought tolerance was correlated with an increased extracellular NO concentration due to an increased nitrate reductase (NR) activity [89]. Furthermore, the simultaneous decrease in S-nitrosoglutathione reductase (GSNOR) implicates that tolerant plants possess a higher GSNO reservoir. As mentioned before, GSNO is a mobile carrier of NO allowing long distance transport. As observed for roots, likewise, the leaf intracellular NO content was elevated in the tolerant species when compared to the sensitive [89].

When evaluating the role of the ascorbate- and glutathione-dependent pathways in drought tolerance, it must be taken into consideration that the basal levels of the different antioxidants in sensitive and tolerant species were not compared. However, *Arabidopsis* plants lacking the cytosolic APX1 show a collapse in the entire chloroplast-located H2O2-scavenging system, which is accompanied with increased H2O2 levels and protein oxidation, respectively [172]. In a direct comparison with TRX-dependent peroxidase activity, APX activity was 7-fold and 2-fold higher in leaf extracts and chloroplasts, respectively [173]. Thus, a predominant role of the ascorbate-dependent antioxidative system should be assumed. At this point, a deeper screen through the literature may not be helpful to test the hypothesis since most studies only present data on changes of selected antioxidant enzymes in a few tolerant and sensitive species. Future investigations should explicitly address the hypothesized role of the ascorbate-dependent ROS defense in drought tolerance in tolerant and sensitive genotypes within plant families. If the hypothesis can be confirmed, the ascorbate-dependent scavenging system can be a target for improving plant tolerance towards drought in biotechnological application.

#### **5. The Role of the Antioxidative System in Desiccation Tolerance**

Drought stress induces major transcriptional reprogramming in plants via ABA-dependent and ABA-independent pathways regardless whether a plant is sensitive or tolerant to drought. This is also true for resurrection plants. Research has shown that resurrection plants use similar mechanisms and strategies to respond and adapt to drought as sensitive species. However, if processes like perception, signaling and responses are as similar as assumed, which specific features provoke the tolerance to desiccation of vegetative tissues? The major difference to drought-sensitive plants is that the protective machinery of resurrection plants is held in an activated, 'primed' state. To achieve this, the basal

levels of osmolytes like sugars and polyamines, non-enzymatic and enzymatic antioxidants are often increased in desiccation tolerant plants. High levels of sugars like trehalose, sucrose and raffinose prevent protein denaturation, stabilize membranes and act as ROS scavengers [174,175]. In addition, unique sugars such as the C8-sugar octulose also accumulate to up to 90% of the soluble sugars in photosynthetically active leaves [176]. Despite this, Djilianov and colleagues [177] found that the initial Suc/Fru ratio is increased in the desiccation-tolerant plant *H. rhodopensis* compared to the sensitive species *C. eberhardtii*. The differences and similarities between drought sensitivity, and drought and desiccation tolerance are compiled in Figure 3.

Significant evidence indicates that the strong antioxidant status is a prerequisite of desiccation tolerance in resurrection plants. Thus, glutathione is suggested to be an important player in the dehydration response [178]. The non-enzymatic antioxidants ascorbate and glutathione turn more oxidized during dehydration [177,179], while the total glutathione content increases. The increase in GSSG remains elevated during desiccation of the tolerant species *H. rhodopensis*. In addition, activities of antioxidant enzymes like SOD, peroxidase POD), CAT and GR increase in response to drought in the fern *Selaginella tamariscina* [180]. Resurrection plants are well equipped with genes encoding antioxidant enzymes. For instance, *H. rhodopensis* contains more genes encoding SOD, CAT, MDHAR and GR than the model plant *A. thaliana* [181]. The *H. rhodopensis* genome encodes eight catalase genes and, thus, five more than the *Arabidopsis* genome [181]. Expression of specific *Cat* genes is upregulated following drought/desiccation. The importance of CAT activity during desiccation is shown by an experiment in which leaves were sprayed with the catalase inhibitor 3-aminotriazole (0.1 mmol/L 3-AT). Plants that were treated by 3-AT never recover completely from desiccation and die within a month after the treatment [181]. The increased sensitivity of dehydrating plants to CAT inhibitors is interpreted as indication of enhanced photorespiration due to stomatal closure, lack of intercellular CO2, enhanced oxygenation of RUBISCO and therefore stimulated release of H2O2 by glycolate oxidase in the peroxisome. CAT is needed to detoxify the released H2O2 and therefore inhibited CAT disturbs redox and ROS homeostasis under drought.

Wang and colleagues [180] compiled drought/dehydration-responsive proteins from both resurrection and common plants [180]. The comparison of tolerant with sensitive phenotypes highlights the role of the antioxidant system in drought tolerance. For instance, CAT, APX and SOD levels are up-regulated in the drought-tolerant CE704 genotype (maize), while CAT and APX levels decreased in the drought-sensitive genotype 2023 [182]. In wheat, TRX-h and glutathione S-transferase are selectively upregulated in the drought-tolerant genotype Khazar-1 [161].

It should be noted that dehydration tolerance depends on additional features of the plants apart from adjusting metabolism including the antioxidant system. Massive water loss usually causes mechanical disruption in hygrophytic and mesophytic plants, e.g., the rupture of the tonoplast/plasmamembrane/cell wall junctions. Such irreversible mechanical damage is prevented in resurrection plants such as *Craterostigma plantagineum* where the tissue shrinks proportionally to the water loss. Thus, special anatomical properties like leaf curling and structurally flexible vessels are important features of dehydration tolerance [183,184].

#### **6. Conclusion and Perspective**

Drought tolerance depends on conditional activation of the acclimation program during initial phases of water loss. This also applies for thallophytic and cormophytic resurrection plants which need a hardening period for full expression of the tolerance trait [183,185]. As pointed out in this review, different drought stress regimes and time points of analysis result in distinct states of the ROS and RNS network and the antioxidant defense system. In the initial phases of dehydration, the activation of the hardening program decisively involves the generation of ROS and RNS which assist in activating the redox regulatory network and appropriate gene expression and protein accumulation. It was out of focus of this review to describe the intimate link between ROS, RNS and hormone signaling like

salicylic acid and abscisic acid [186]. In the end ROS and RNS define a regulatory framework of the cell and contribute to link the stress impact to gene expression and whole plant performance [187].

At present our knowledge on specific subcellular ROS, RNS and redox patterns still falls short of the requirements for understanding the drought acclimation response in its entirety. Cell imaging with roGFP for glutathione redox state [188] and Hyper for H2O2 [189] will provide important insight on subcellular responses. In addition, in depth redox proteomics detecting the redox state of also low abundant proteins will provide a global view with subcellular resolution.

There is a need to assess the various PTMs in the proteome simultaneously. This is a challenge for current proteomics which for technical reasons often focuses on single or few PTMs only [190]. As functional readout of ROS and RNS, such approaches will realize the necessary temporal and spatial resolution since ROS and RNS partly antagonize each other. Nevertheless, the presence of both reactive species is necessary for full drought acclimation. Additionally, the reaction of NO with O2•− generates the highly reactive ONOO− which directly nitrates proteins. Cysteine oxidation and tyrosine nitrations are PTMs that change the activity of its target enzymes. Proteomics may tackle this challenge.

Along with the activation of the antioxidative system, other stress markers often increase during periods of progressive dehydration, e.g., H2O2 as indicator of redox imbalance, MDA as lipid oxidation product, glyoxylate linked to photorespiration, glutathione as antioxidant, glutamate and proline as precursor and compatible solute, and zeaxanthin with its role in photoprotection. The consensus of what defines drought tolerance is that many traits are needed to prevent biochemical or physiological impairment during water deficit. Several traits contribute to drought tolerance and include reduced water loss, build-up of osmotic potential, synthesis of compatible solutes, dissipation of excess energy, activation of antioxidant defense and repair systems, generation of sclerenchymatic tissue, strengthening the plasmamembrane-cell wall interaction and other mechanisms of growth adjustment such as differentiation of smaller leaves. The recovery from water depletion is affected by light intensity with often negative interference, i.e., slower recovery at high light.

Taken together, strategies to improve drought tolerance in crops need to target several metabolic pathways at the same time. Certainly, the activation of the antioxidative system following drought is one important goal. Attention should also be drawn to the pathways that are selected to increase drought tolerance. In the first instance, overexpressing of certain enzymes can lead to a beneficial increase in drought tolerance, but may delay germination and development for months and, thus, interfere with the growing season. Thus, biotechnological approaches should take into account the temporal and spatial signaling aspect in drought stress acclimation.

**Author Contributions:** Each author wrote a specific section of this review and commented on the whole manuscript. M.L.(Miriam Laxa) (Sections 1, 2, 4.2, 5; manuscript editing, coordination and formatting); M.L.(Michael Liebthal) (Section 3); W.T. and K.C. (Section 4); K.J.-D. (abstract; Section 6; manuscript editing).

**Funding:** Support of the own work by the Deutsche Forschungsgemeinschaft is gratefully acknowledged (Di 364/17-2; SPP 1710). The publication of this article was funded by the Open Access Fund of Bielefeld University.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **Abbreviations**



#### **References**


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