**Paraquat: An Oxidative Stress Inducer**

Ramiro Lascano1,2, Nacira Muñoz1,2, Germán Robert1,2, Marianela Rodriguez1, Mariana Melchiorre1, Victorio Trippi1,2 and Gastón Quero1,2 *1Instituto de Fitopatologia y Fisiologia Vegetal (IFFIVE-INTA), Córdoba, 2Cátedra de Fisiología Vegetal; Facultad de Ciencias Exactas, Físicas y Naturales; U.N., Córdoba, Argentina* 

#### **1. Introduction**

134 Herbicides – Properties, Synthesis and Control of Weeds

Walker, A. (1987) Evaluation of simulation model for prediction of herbicide movement and

Walker, A. (1983). The fate and significance of herbicide residue in soil. *Scientific horticulture*.

Weber, J. B. (1970). Mechanism of adsorption of s-triazines by clay colloids and factors

persistence in soil. *Weed Res*., Vol. 27, p. 143-152

affecting plant availability. *Residue Rev.*, v. 32, p. 93-130

Vol. 34, p. 35-47

Paraquat (1,1\_-dimethyl-4,4\_-bipyridinium dichloride), is a foliar-applied and non selective bipyridinium herbicides, and it is one of the most widely used herbicides in the world, controlling weeds in a huge variety of crops like corn, rice, soybean, wheat, potatoes; major fruits: apples, oranges, bananas; beverages: coffee, tea, cocoa; and processed crops: cotton, oil palm, sugarcane and rubber.

For a foliar absorbed herbicide to completely kill a plant, it must be capable of accessing the whole plant, as growing leaves and newly emerging roots. This often means that the herbicide not only needs to damage at the point of its absorption, but must also be translocated to parts of the plant not contacted by the herbicide during application.

Paraquat is a cation formed by two pyridine rings, each having a quaternary amine and thus charged 2+. Although the majority of herbicides are passively transported as noionic molecules, paraquat cation movement by diffusion across membrane lipid bilayer is unlikely. Transporter studies to explain paraquat compartment were made using several systems. ABC transporters, large membrane proteins which use ATP for the active transport of several compounds including paraquat have been described. Other groups of transporters are small antiporter proteins which exchange protons for some other molecules using the proton electrochemical potential gradient (Morymio et al., 1992, Yerushalmi, et al., 1995). In animal tissues it has been shown that paraquat transport occurs by carriers that also function as carriers of other molecules such as polyamines (Rannels et al., 1989, Jóri et al., 2007). Hart et al. (1992a 1992b) demonstrated that paraquat movement across plasma membrane root epidermal and cortical maize cells has a concentration-dependent kinetic and that the herbicide binds to cell wall, and its transport is facilitated by a carrier that normally functions in the movement of molecules that has a similar chemical structure or similar charge distribution such us diamines like putrescine and cadaverine. Using maize protoplast Hart et al. (1993) showed that paraquat uptake has similar concentration-kinetic to that observed in intact cells and the accumulation inside cells increase in a timedependent manner and is saturated after 10 min, although 50% of uptake occurs during the first 10 s. The saturable Km for paraquat uptake in maize cells and protoplasts was determined at 90 µM and 132 µM respectively, similarly the Km in rat lung was 70 µM

Paraquat: An Oxidative Stress Inducer 137

In green tissues under light, chloroplasts are the main intracellular source of ROS (Asada, 1999) and peroxisomes, through photorespiration, are other important ROS producers (del Río et al., 2006). While mitochondria, are the principal source of ROS in darkness and non green tissues. On the other hand, the NADPH oxidase complex, peroxidases and amino

1999). The peroxisomes produce H2O2 as byproduct of photorespiratory glycolate oxidase

xanthine oxidase and by electron transport chains in the peroxisomal membrane (del Río et

Various interconverting reactions occur among different ROS. Superoxide is spontaneously or enzymatically converted to H2O2 by disproportion mechanism and H2O2 and O2

Different ROS have different features. Hydrogen peroxide is a non radical, apolar molecule and, in consequence, it is a relatively stable compound with half-life around 1 ms. In plant tissues, its concentration could be in the micro to millimolar range. The half-lives of the other ROS are very short, ranging from nano to micro second, and then they are present at

Reactive oxygen species also have different reactivities. Hydrogen peroxide (Eo 1.77 V), not a highly reactive ROS *per se*, mainly oxidizes thiol groups, in presence of transition metal

ascorbate and NADPH, reduces metal ions and cytochrome C and reacts with protein Fe-S centers. Singlet oxygen is particularly reactive with conjugated double bonds of

types of macromolecular cellular components. The differential ROS reactivity means that they leave different footprints in the cell in the form of different oxidatively modified

Cellular membranes are the principal targets of ROS. Peroxidation of polyunsaturated fatty acids (PUFAs) is a common oxidative stress effect. Linoleic acid (18:2) and linolenic acid (18:3) are major fatty acid present in galactolips of thylakois and phospholipids of all membranes. PUFAs peroxidation generates mixtures of lipid hydroperoxides several aldehydes, e.g., 4-hydroxy-2-nonenal (HNE) and malondialdehyde (MDA), hydroxyl and keto fatty acids and oxidative modification in membrane protein. The consequences over the membrane function are the fluidity and selectivity decreases (Halliwell et al.,1999; Halliwell, 2006). Some of the PUFA peroxidation products act directly or after enzymatic modification

ROS induce mainly irreversible covalent modification on proteins. The reversible modifications on sulfur containing amino acid are very important in the redox or oxidative signaling. Cystein thiol groups are initially oxidized to disulfide and in further oxidation to sulfenic and sulfinic acid. The highest level of cysteine oxidation, cysteic acid seems to be irreversible and damaging (Ghezzi & Bonetto; 2003). Nitrosylation and glutathionylation are

as secondary messengers either, e.g. oxylipins (Muller et al., 2004).

OH through the Fenton reaction catalyzed by free transition metal ions

OH generation by Fenton reaction. Superoxide radical (Eo -0.33V) oxidizes

OH (Eo 2 V), the most oxidant ROS, reacts with all

•<sup>−</sup> at photosystem I (PSI) and 1O2 at

•<sup>−</sup> at complexes I and III (Asada,

•<sup>−</sup> is generated by

•<sup>−</sup> can

oxidases are major sources of apoplastic ROS (Sagi & Fluhr, 2006).

reaction, fatty acid β-oxidation and reaction of flavin oxidase, and O2

Primarily, the chloroplasts mainly produce O2

al., 2006).

interact to produce .

very low concentrations (Asada, 1999).

polyunsaturated fatty acids. Whereas .

components (Moller et al., 2007).

(Fridovich, 1986).

ions it catalyzes .

photosystem II (PSII), and the mitochondria produce O2

suggesting in both animal and vegetal tissues a carrier-mediated process (Rannels et al., 1985).

In order to investigate paraquat uptake, compartmentation and translocation, maize plantlets with their root immersed in paraquat solution for several loading periods were used (Hart et al., 1993). The lack of chloroplasts in roots provides a system to minimize the short-term phototoxic effect. The paraquat accumulation in the root vacuole was linear over a 24 h loading period. The vacuolar paraquat content, with respect to the total accumulated increased from 15% to 42% after 2 h and 24 h loading period, respectively. In contrast to the vacuole, total cytoplasmic paraquat content appeared to approach saturation whereas paraquat associated with the cell wall fraction remained relatively constant, suggesting that this phase is rapidly saturated. Even though paraquat is considered to be relatively immobile, linear paraquat (PQ) translocation occurred from roots to shoots and was estimated that approximately 50% of the paraquat effluxing from roots started translocation to shoots 5 h after the beginning of loading period (Hart et al., 1993b).

Paraquat acts as a redox cycler with a great negative reduction potential (E0 = – 0.446 V). This feature restricts its interaction with strong reductant compounds. When dication of paraquat (PQ 2+) accepts an electron from a reductant form the paraquat monocation radical (PQ +), which then rapidly reacts with oxygen (O2 Eo = 0.16 V) to initially produce superoxide radical (O2•−) (k 7.7 x 108 M-1 s -1) and subsequently the other reactive oxygen species (ROS) such as hydrogen peroxide (H2O2) and hydroxyl radical (OH).

In plants, paraquat is principally reduced within chloroplasts, where it acts as an alternative electron acceptor taking electron from Fe-S proteins of photosystem I; inhibiting the ferredoxin reduction, the NADPH generation, and also the regeneration of ascorbic acid. In consequence, paraquat is a potent oxidative stress inducer, because it greatly increases the ROS production and inhibits the regeneration of reducing equivalents and compounds necessary for the activity of the antioxidant system.

Paraquat also induces the increase of superoxide radical production in mitochondria, where complexes I and III are the major electron donors. For this reason paraquat has been widely used to induce mitochondrial oxidative stress in many experimental systems such as isolated mitochondria, cultured cells, and whole organisms including plants, *Saccharomyces cerevisiae, Caenorhabditis elegans, Drosophila melanogaster* and rodents (Cocheme & Murphy, 2008).

### **2. Generation and role of ROS**

Superoxide radical (O2 •−), singlet oxygen (1O2) hydrogen peroxide (H2O2) and hydroxyl radical (. OH) are highly reactive compounds that induce protein and pigment degradation, lipid peroxidation, nucleic acid damage, affecting key components of plant cell metabolism that can finally lead to cell death. These deleterious reactions triggered by ROS are known as oxidative stress phenomenon (Casano et al., 1994, 1997; Lascano et al., 1998, 1999).

Even though all ROS are highly reactive compounds their effects and plant responses depend on the ROS in question as well as on its concentration, site of production, interaction with other stress molecules and on the developmental stage and plant cell previous history.

suggesting in both animal and vegetal tissues a carrier-mediated process (Rannels et al.,

In order to investigate paraquat uptake, compartmentation and translocation, maize plantlets with their root immersed in paraquat solution for several loading periods were used (Hart et al., 1993). The lack of chloroplasts in roots provides a system to minimize the short-term phototoxic effect. The paraquat accumulation in the root vacuole was linear over a 24 h loading period. The vacuolar paraquat content, with respect to the total accumulated increased from 15% to 42% after 2 h and 24 h loading period, respectively. In contrast to the vacuole, total cytoplasmic paraquat content appeared to approach saturation whereas paraquat associated with the cell wall fraction remained relatively constant, suggesting that this phase is rapidly saturated. Even though paraquat is considered to be relatively immobile, linear paraquat (PQ) translocation occurred from roots to shoots and was estimated that approximately 50% of the paraquat effluxing from roots started translocation

Paraquat acts as a redox cycler with a great negative reduction potential (E0 = – 0.446 V). This feature restricts its interaction with strong reductant compounds. When dication of paraquat (PQ 2+) accepts an electron from a reductant form the paraquat monocation radical (PQ +), which then rapidly reacts with oxygen (O2 Eo = 0.16 V) to initially produce superoxide

In plants, paraquat is principally reduced within chloroplasts, where it acts as an alternative electron acceptor taking electron from Fe-S proteins of photosystem I; inhibiting the ferredoxin reduction, the NADPH generation, and also the regeneration of ascorbic acid. In consequence, paraquat is a potent oxidative stress inducer, because it greatly increases the ROS production and inhibits the regeneration of reducing equivalents and compounds

Paraquat also induces the increase of superoxide radical production in mitochondria, where complexes I and III are the major electron donors. For this reason paraquat has been widely used to induce mitochondrial oxidative stress in many experimental systems such as isolated mitochondria, cultured cells, and whole organisms including plants, *Saccharomyces cerevisiae, Caenorhabditis elegans, Drosophila melanogaster* and rodents (Cocheme & Murphy,

•−) (k 7.7 x 108 M-1 s -1) and subsequently the other reactive oxygen species (ROS)

•−), singlet oxygen (1O2) hydrogen peroxide (H2O2) and hydroxyl

OH) are highly reactive compounds that induce protein and pigment degradation,

lipid peroxidation, nucleic acid damage, affecting key components of plant cell metabolism that can finally lead to cell death. These deleterious reactions triggered by ROS are known as

Even though all ROS are highly reactive compounds their effects and plant responses depend on the ROS in question as well as on its concentration, site of production, interaction with other stress molecules and on the developmental stage and plant cell

oxidative stress phenomenon (Casano et al., 1994, 1997; Lascano et al., 1998, 1999).

to shoots 5 h after the beginning of loading period (Hart et al., 1993b).

such as hydrogen peroxide (H2O2) and hydroxyl radical (OH).

necessary for the activity of the antioxidant system.

**2. Generation and role of ROS** 

Superoxide radical (O2

previous history.

1985).

radical (O2

2008).

radical (.

In green tissues under light, chloroplasts are the main intracellular source of ROS (Asada, 1999) and peroxisomes, through photorespiration, are other important ROS producers (del Río et al., 2006). While mitochondria, are the principal source of ROS in darkness and non green tissues. On the other hand, the NADPH oxidase complex, peroxidases and amino oxidases are major sources of apoplastic ROS (Sagi & Fluhr, 2006).

Primarily, the chloroplasts mainly produce O2 •<sup>−</sup> at photosystem I (PSI) and 1O2 at photosystem II (PSII), and the mitochondria produce O2 •<sup>−</sup> at complexes I and III (Asada, 1999). The peroxisomes produce H2O2 as byproduct of photorespiratory glycolate oxidase reaction, fatty acid β-oxidation and reaction of flavin oxidase, and O2 •<sup>−</sup> is generated by xanthine oxidase and by electron transport chains in the peroxisomal membrane (del Río et al., 2006).

Various interconverting reactions occur among different ROS. Superoxide is spontaneously or enzymatically converted to H2O2 by disproportion mechanism and H2O2 and O2 •<sup>−</sup> can interact to produce . OH through the Fenton reaction catalyzed by free transition metal ions (Fridovich, 1986).

Different ROS have different features. Hydrogen peroxide is a non radical, apolar molecule and, in consequence, it is a relatively stable compound with half-life around 1 ms. In plant tissues, its concentration could be in the micro to millimolar range. The half-lives of the other ROS are very short, ranging from nano to micro second, and then they are present at very low concentrations (Asada, 1999).

Reactive oxygen species also have different reactivities. Hydrogen peroxide (Eo 1.77 V), not a highly reactive ROS *per se*, mainly oxidizes thiol groups, in presence of transition metal ions it catalyzes . OH generation by Fenton reaction. Superoxide radical (Eo -0.33V) oxidizes ascorbate and NADPH, reduces metal ions and cytochrome C and reacts with protein Fe-S centers. Singlet oxygen is particularly reactive with conjugated double bonds of polyunsaturated fatty acids. Whereas . OH (Eo 2 V), the most oxidant ROS, reacts with all types of macromolecular cellular components. The differential ROS reactivity means that they leave different footprints in the cell in the form of different oxidatively modified components (Moller et al., 2007).

Cellular membranes are the principal targets of ROS. Peroxidation of polyunsaturated fatty acids (PUFAs) is a common oxidative stress effect. Linoleic acid (18:2) and linolenic acid (18:3) are major fatty acid present in galactolips of thylakois and phospholipids of all membranes. PUFAs peroxidation generates mixtures of lipid hydroperoxides several aldehydes, e.g., 4-hydroxy-2-nonenal (HNE) and malondialdehyde (MDA), hydroxyl and keto fatty acids and oxidative modification in membrane protein. The consequences over the membrane function are the fluidity and selectivity decreases (Halliwell et al.,1999; Halliwell, 2006). Some of the PUFA peroxidation products act directly or after enzymatic modification as secondary messengers either, e.g. oxylipins (Muller et al., 2004).

ROS induce mainly irreversible covalent modification on proteins. The reversible modifications on sulfur containing amino acid are very important in the redox or oxidative signaling. Cystein thiol groups are initially oxidized to disulfide and in further oxidation to sulfenic and sulfinic acid. The highest level of cysteine oxidation, cysteic acid seems to be irreversible and damaging (Ghezzi & Bonetto; 2003). Nitrosylation and glutathionylation are

Paraquat: An Oxidative Stress Inducer 139

Superoxide dismutase (SOD) (E.C.: 1.15.1.1), ascorbate peroxidase (APX) (E.C.: 1.11.1.11), catalase (CAT) (EC 1.11.1.6), and glutathione reductase (GR) (E.C.: 1.6.4.2) are key antioxidant enzymes that modulate the concentration of two of the Haber/Weiss and

radical (Asada, 1999). Approximately, 80% of SOD, GR, and APX activity is located in the chloroplast (Asada, 1999). CAT activity is located in peroxisomes and mitochondria

multiple isoforms: copper/zinc (CuZn-SOD), iron (Fe-SOD) and manganese (Mn-SOD) (Bowler et al, 1992). In most plants, CuZn-SOD and Fe-SOD are present in the chloroplasts, CuZn-SOD in the cytosol and Mn-SOD in mitochondria (Casano et al., 1997; Scandalios, 1993). Degradation of H2O2, in the chloroplasts and in the cytosol is carried out by the ascorbate-glutathione cycle, which involves APX and GR activities (Lascano et al., 1999, 2003). APX has chloroplastic and cytosolic isoforms, and catalyses the conversion of H2O2 to

Reduced glutathione (GSH) and ascorbic acid are the most important soluble non-enzymatic antioxidants and in chloroplasts they are present at millimolar concentrations (Noctor & Foyer, 1998). Ascorbate acts as a ROS quencher and it is involved in the regenerations of tocopherol and violoxanthine depoxidase activity of xanthophylls cycle (Noctor & Foyer, 1998). Reduced glutathione is a tripeptide -glutamylcysteinyl glycine (-Glu-Cys-Gly) involved in: direct reaction with ROS, the regeneration of the ascorbate pool and as electron donor of glutaredoxins which are linked to type II peroxiredoxin activity. Likewise, GSH participates in the glutathionylation, a post-transcriptional modification of protein thiols groups that regulates the function of proteins like glyceraldehyde-3-phosphate dehydrogenease and thioredoxin activities (Michelet et al., 2005; Zaffagnini et al., 2007). The reduction of oxidized glutathione is NADPH-dependent and carried out by GR, a ubiquitous flavoenzyme with many isoforms, located in chloroplasts, cytosol, and

Other more recently identified components of enzymatic antioxidant system are peroxiredoxins and glutathione peroxidase, non-heme-containing peroxidase which activity

Plants as sessile organism are permanently exposed to changing environment that become stressful conditions affecting their growth, development and productivity. Tolerance to environmental stress is a major selection criterion in plant breeding. The cellular and molecular tolerance mechanisms of plants to different stresses have been intensively

Reactive oxygen species are produced as byproduct of normal aerobic metabolism and the life under aerobic conditions is strictly dependent on the presence of antioxidant system. Nowadays, it is widely accepted that the generation of ROS is enhanced under abiotic and biotic stress conditions. Depending on stress intensity and its associated-ROS levels the

Likewise, the positive response of the antioxidant system correlates, in part, with the tolerance to many different environmental stress conditions. ROS and antioxidant system

(Scandalios, 1994). SOD catalyses the disproportionation of O2

water using ascorbate as electron donor (Asada, 1999).

mitochondria (Lascano et al., 2001; Tanaka et al., 1994).

depend on cystein residues (Bryk et al., 2000; König et al., 2003).

**4. The use of paraquat in stress response studies** 

plant responses range from tolerance to death.

studied.

•- and H2O2, preventing the formation of the highly toxic .

OH

•- to H2O2, and is present in

Fenton reaction substrates, O2

other cystein thiol modification mediated by nitric oxide, reactive nitrogen species (RNS) and glutathione. RNS are generated by the interaction between nitric oxide and ROS. (Costa et al., 2003; Halliwell, 2006). Carbonylation, a common oxidative protein modification affecting particularly Arg, His, Lys, Pro, Thr, and Trp; and conjugation with peroxidation PUFA products, mainly with HNE, are other oxidative protein modifications (Shacter, 2000; Winger et al., 2005).

The generation of 8-Hydroxyguanine is the most common DNA modification induced by ROS. The nucleotide bases are attacked by. OH and 1O2 while H2O2 and O2 •− do not react at all (Wiseman & Halliwell, 1996). Chloroplastic and mitochondrial DNAs are into the two major source of ROS where potentially high rates of modification might occur (Thorslund et al., 2002). Another indirect oxidative modification to DNA is the conjugation of MDA with guanine (Jeong, 2005). The DNA oxidative modification could induce changes in cytosines methylation patterns, and then in the regulation of gene expressions. ROS-induced DNA modification seems to be a not completely random process (Halliwell, 2006).

Carbohydrates can be oxidatively modified by . OH, being the formic acid the main breakdown product of sugar oxidation (Isbell et al., 1973).

In spite of its toxic effects, increasing evidence indicates that ROS are signaling molecules that participate in many processes, such as cell cycle, cell elongation, cell death, plant growth and development, senescence, hormone signaling, responses to biotic and abiotic stress and in symbiotic interaction with microorganisms (Bustos et al., 2008; Mittler et al., 2004; Muñoz et al. 2011, submitted; Rodriguez et al., 2010). The H2O2 molecular properties make it a good second messenger that could cross membrane by diffusion or aquaporins. However, all ROS can act as signaling molecules directly or by oxidized product. NADPH oxidase complex, the main source of apoplastic ROS, has a key role in oxidative signaling (Sagi & Fhlur, 2004).

The dual role of ROS, as toxic or signaling molecules, depends on the ratio and subcellular location of its generation, thus the tight regulation of the steady-state level of ROS in different subcellular compartments has both signaling and oxidative damage protection purposes. The function of ROS as signaling molecules is intrinsically related to the interaction with non-enzymatic antioxidants, such as ascorbate and glutathione, which are redox buffers and also signal molecules *per se* (Foyer & Noctor 2005 a, 2005b).

The relationship among ROS, antioxidants, reducing equivalents, sugars, the redox state of chloroplastic and mitochondria electron transport chains are major determinants of the cellular redox state, which has a critical function in the environmental perception and modulation of defense, acclimation and tolerance responses (Foyer & Noctor, 2005 a; 2005b; Lascano et al., 2003; Melchiorre et al., 2009; Robert et al., 2009).
