Protoplasma DOI 10.1007/s00709-014-0636-x


Metal/metalloid stress tolerance in plants: role of ascorbate, its redox couple, and associated enzymes Naser A. Anjum & Sarvajeet S. Gill & Ritu Gill & Mirza Hasanuzzaman & Armando C. Duarte & Eduarda Pereira & Iqbal Ahmad & Renu Tuteja & Narendra Tuteja

Received: 14 January 2014 / Accepted: 11 March 2014 # Springer-Verlag Wien 2014

Abstract The enhanced generation of reactive oxygen species (ROS) under metal/metalloid stress is most common in plants, and the elevated ROS must be successfully metabolized in order to maintain plant growth, development, and productivity. Ascorbate (AsA) is a highly abundant metabolite and a watersoluble antioxidant, which besides positively influencing various aspects in plants acts also as an enigmatic component of plant defense armory. As a significant component of the ascorbate-glutathione (AsA-GSH) pathway, it performs multiple vital functions in plants including growth and development by either directly or indirectly metabolizing ROS and its products. Enzymes such as monodehydroascorbate reductase (MDHAR, EC and dehydroascorbate reductase (DHAR, EC maintain the reduced form of AsA pool Handling Editor: David Robinson N. A. Anjum (*) : A. C. Duarte : E. Pereira : I. Ahmad Centre for Environmental and Marine Studies (CESAM) and Department of Chemistry, University of Aveiro, 3810-193 Aveiro, Portugal e-mail: [email protected] I. Ahmad (*) Centre for Environmental and Marine Studies (CESAM) and Department of Biology, University of Aveiro, 3810-193 Aveiro, Portugal e-mail: [email protected] S. S. Gill : R. Gill Stress Physiology and Molecular Biology Lab, Centre for Biotechnology, MD University, Rohtak 001, India M. Hasanuzzaman Department of Agronomy, Faculty of Agriculture, Sher-e-Bangla Agricultural University, Dhaka 1207, Bangladesh R. Tuteja : N. Tuteja (*) International Centre for Genetic Engineering and Biotechnology (ICGEB), Aruna Asaf Ali Marg, New Delhi 067, India e-mail: [email protected]

besides metabolically controlling the ratio of AsA with its oxidized form (dehydroascorbate, DHA). Ascorbate peroxidase (APX, EC utilizes the reduced AsA pool as the specific electron donor during ROS metabolism. Thus, AsA, its redox couple (AsA/DHA), and related enzymes (MDHAR, DHAR, and APX) cumulatively form an AsA redox system to efficiently protect plants particularly against potential anomalies caused by ROS and its products. Here we present a critical assessment of the recent research reports available on metal/metalloid-accrued modulation of reduced AsA pool, AsA/DHA redox couple and AsA-related major enzymes, and the cumulative significance of these antioxidant system components in plant metal/metalloid stress tolerance. Keywords Ascorbate . AsA/DHA redox couple . Metal stress . Antoxidant defense system . Plant metal tolerance

Introduction Metals/metalloids, reactive oxygen species, and plants Though metals/metalloids [hereafter called “metal(s)”] are natural constituents of the earth’s crust, a drastic alteration in their geochemical cycles and biochemical balance has been arisen as a result of indiscriminate human activity-mediated rapid metal concentration enhancements in varied environmental compartments (Prasad 2004; Maksymiec 2007). Nevertheless, the human/animal food chain can be contaminated with these metals via the ingestion of plants or their products laden with metals that can be accumulated and bring adverse anomalies in the human/animal body over a period of time (Singh et al. 2011). However, once in the plant system, high levels of metal ions of plant significance (such as Co, Cu, Fe, Mn, Mo, Ni, Zn) and trace levels of toxic metals (Pb, Cd, Hg, As, Cr, Ag, Al, Cs, Sr, U) are known to impact negatively

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on plant growth, metabolism, development, and overall productivity, mainly as a result of accelerated reactive oxygen species (ROS) formation, albeit through different reactions (Candan and Tarhan 2003; Maksymiec 2007; Schröder et al. 2009; Gill and Tuteja 2010; Cuypers et al. 2013; Sytar et al. 2013). In fact, the relatively uncreative molecular oxygen is essential for the existence of aerobic organisms including plants. However, as a consequence of univalent reduction of molecular oxygen in various metabolic pathways localized in different cellular compartments (such as chloroplasts, mitochondria, and peroxisomes), the production of ROS (such as superoxide anion radicals, •O2−; hydrogen peroxide, H2O2; hydroxyl radicals, •OH) becomes a usual phenomenon under normal physiological conditions (Apel and Hirt 2004; Mittler et al. 2004). ROS are signaling molecules, which are involved in several physiological processes such as cell cycle, stress perception, gene regulation, programmed cell death, hypersensitive response, and senescence; and they are also able to trigger and/or orchestrate plants’ responses to various biotic and abiotic stress factors including metals (Mullineaux and Karpinski 2002; Scandalios 2002). Thus, in order to avoid potential anomalies caused by ROS and its products as well as to keep the growth, metabolism, development, and overall productivity at optimum, the intracellular level of ROS in plant cells must be tightly regulated and/or efficiently metabolized (Mittler et al. 2004; Gill and Tuteja 2010; Anjum et al. 2010; 2012a, b). Comprising both enzymatic (such as superoxide dismutase, SOD, EC; catalase, CAT, EC; glutathione reductase, GR, EC; peroxidase, POD, EC; ascorbate peroxidase, APX, EC; guaiacol peroxidases, GPX, EC and non-enzymatic (such as ascorbic acid, AsA; glutathione, GSH; tocopherols; phenolics) antioxidants, plants are able to counteract ROS and its reaction product-mediated potential impacts (Mittler et al. 2004; Gill and Tuteja 2010; Anjum et al. 2012b; Sytar et al. 2013). As a major component of the ascorbate-glutathione (AsAGSH) pathway, ascorbate (AsA) plays a significant role in maintaining equilibrium between the production and elimination of ROS, which in turn helps to avoid metabolic disorders and/or oxidative burst in cells caused by ROS and its products (Smirnoff 2000; Apel and Hirt 2004; Anjum et al. 2010, 2011; 2012a, b; Gill and Tuteja 2010). In the AsA-GSH pathway, AsA either directly interacts with ROS or it is consumed by the enzyme APX (EC reducing H2O2 to H2O and leading to concomitant generation of monodehydroascorbate (MDHA, a radical with a short lifetime) which can be subsequently disproportionated into dehydroascorbate (DHA) and/or back to AsA in chloroplasts and/or other cell compartments through the AsAregenerating system comprising dehydroascorbate reductase (DHAR, EC and monodehydroascorbate reductase (MDHAR, EC (Miyake and Asada 1994; Shigeoka et al. 2002; Anjum et al. 2010, 2012b).

During the last few decades, a tremendous progress has been made on the physiological and biochemical aspects of AsA, its redox couple (AsA/DHA), and related enzymes (DHAR, MDHAR, and APX) in context with their responses to a number of biotic and abiotic stresses in plants. However, the modulation of these traits in metal-stressed plants is invariable and inconsistent in the available literature (Gill and Tuteja 2010; Anjum et al. 2012b; Gest et al. 2012). Nevertheless, only a few studies and/or reviews have considered cross-talks on the plant responses to metal stress incorporating the aspects of AsA, AsA/DHA, and related major enzymes. Therefore, this work focuses mainly on the critical evaluation of the published data on the modulation of AsA, its redox couple (AsA/DHA), and AsA-regenerating (DHAR/MDHAR) and metabolism (APXs) enzymes to improve our understanding on the cumulative significance of these traits in the growth, metabolism, tolerance, and adaptation of plants to stress induced by metals. Additionally, a critical cross-talk on the previous aspects has been performed and the future research directions in the current context have been highlighted. Trace levels of heavy metal ions such as Co, Cu, Fe, Mn, Mo, Ni, and Zn are required for plant growth and development, but at supra-optimum supply, these ions readily impact the function of many enzymes and proteins, halt metabolism, and exhibit phytotoxicity (Schutzendubel et al. 2002; Krämer 2010). Therefore, besides considering the major toxic metals, recent reports available on the modulation of AsA, its redox couple, and related enzymes in plants under essential heavy metal ion exposure have also been discussed.

Ascorbate (AsA), dehydroascorbate (DHA), and monodehydroascorbate (MDHA) Ascorbate—occurrence, synthesis, and significance The organic molecule L-threo-hexenon-1,4-lactone, commonly known as L-ascorbic acid or vitamin C (AsA), is a multifunctional compound in both plants and animals. In plants, AsA stands second to tripeptide glutathione (GSH) in terms of its importance as a key antioxidant metabolite of the antioxidant defense system, redox buffer in plant cells, and also as a major player of key functions in plant growth, metabolism, development, and stress responses (Noctor and Foyer 1998; Smirnoff 2000; Pastori et al. 2003; Anjum et al. 2010, 2012b) (Fig. 1). AsA is present in the cytosol, chloroplasts, vacuoles, mitochondria, and cell wall, and it represents 10 % of the total soluble carbohydrate pool in favorable conditions (Noctor and Foyer 1998; Smirnoff and Wheeler 2000). While a very high level of AsA is evident in the cytosol, AsA can be present at much lower levels in the apoplast and thylakoid lumen (Foyer and Noctor 2011). Plant apoplast (aqueous solution that permeates the cell wall) has been shown to exhibit AsA as the major redox buffer (Pignocchi et al. 2006), and apoplatic AsA

Fig. 1 Major biological roles of ascorbate in plants

The Cofactors for Enzyme Activity

Cell Division

Plant Antioxidation Capacity

Shoot Apical Meristem Formation Cell Wall Metabolism and Cell Expansion ASCORBATE

Root Development Heavy Metal Evacuation and Detoxification


Role in Stress Defense

Regulation of Florescence

has been reported to participate in a variety of physiological phenomena, including cell division, cell elongation, and cell defense (reviewed by Horemans et al. 2000). In whole leaves and chloroplasts, AsA pool represents about 90 % of the reduced form under normal conditions (Foyer 1993; Smirnoff 2000). Particularly in chloroplasts, AsA has been reported to occur in the range of 20–300 mM, where it plays a central role in the process of photosynthesis (Foyer 1993; Smirnoff 2000). Camu-camu (Myrciaria dubia) and acerola (Malpighia glabra) have been reported to contain about 50 and 30 mg g−1 fresh weight of vitamin C, respectively (Justi et al. 2000; Badejo et al. 2009). Nevertheless, plant age transition from young to mature leaves has been evidenced to control the rate of AsA synthesis, recycling, and accumulation (Li et al. 2010). To date, an extensive research in plant science has been focused on deciphering the pathway of AsA biosynthesis and its significance in plant metabolism. In order to avoid the deviation from the theme of the present review as well as considering the space constraints, the informations pertaining to AsA biosynthesis in plants are succintly summarized herein. Many pathways and mechanisms have been studied and/or proposed for AsA biosynthesis, but no consensus has been reached. In plants, three pathways of AsA biosynthesis have been proposed: (a) through L-galactose (L-gal) (Wheeler et al. 1998); (b) from myo-inositol (Lorence et al. 2004); and (c) through galacturonic acid (Agius et al. 2003). Wheeler et al. (1998) proposed the first pathway which gained acceptance and is known as the Smirnoff-Wheeler (SW) pathway. Currently, the predominant AsA biosynthesis pathway in green plants via GDP-mannose and L-galactose is widely accepted (Smirnoff 2011). Herein, the sequential conversion of GDP-Lgalactose follows L-galactose-1-P, L-galactose, L-galactono1,4-lactone, and subsequently to L-ascorbate (Smirnoff 2011). The last step of AsA biosynthesis in SW pathway (conversion of L-galactono-γ-lactone to AsA) is catalyzed by L-galactono1,4-lactone dehydrogenase (GLDH) which is localized in the

Roles in Plant Growth and Development

Plant metal/metalloid stress tolerance and ascorbate significance

mitochondria (Siendones et al. 1999; Bartoli et al. 2000; Carlos et al. 2000). Then AsA is translocated into the cytosol, chloroplasts, vacuole, and apoplast, where it acts as a major redox buffer and plays vital roles in many cell wall processes (Smirnoff 1996; Horemans et al. 2000; Smirnoff et al. 2001). The regulation of L-ascorbate synthesis in plants is very complex, which occurs at the level of GDP-L-galactose phosphorylase and at possible additional steps (Linster and Clarke 2008). Thus far, major genes of the SW pathway have been characterized, including those encoding L-galactono-1,4lactonedehydrogenase (GalLDH) (EC (Imai et al. 1998), GDP- D -mannosepyrophosphorylase (GMP) (EC (Conklin et al. 1999), L-galactose dehydrogenase (GDH) (Gatzek et al. 2002), GDP-D-mannose-3, 5-epimerase (EC (Wolucka and VanMontagu 2003), L-galactose1-phosphatephosphatase (Laing et al. 2004), and GDP-Lgalactose phosphorylase (GGP) (Dowdle et al. 2007; Laing et al. 2007; Linster and Clarke 2008). A simplified schematic diagram of AsA biosynthesis and the interrelationships among AsA, DHA, MDHA, DHR, MDHAR, and APX in plants under metal/metalloid stress has been presented in Fig. 2. AsA has been recognized as a major solute undergoing turnover in all metabolically active plant tissues (Loewus 1999; Smirnoff et al. 2001). AsA has also been reported to play a role in cell wall biosynthesis, redox signaling and plant response modulation under pathogen (Conklin and Barth 2004), determination of flowering time (Barth et al. 2006; Kotchoni et al. 2009), regeneration of the reduced forms of GSH and NADP+ (e.g., in the highly oxidizing milieu of the photosynthesizing chloroplast) (Noctor and Foyer 1998; Mano et al. 2004; Foyer and Noctor 2009; Foyer and Shigeoka 2011), and in the protection of plasma membrane against oxidative damage (Wang et al. 2010) and ozone (Burkey et al. 2003; Frei et al. 2012). AsA is crucially involved in the regeneration of αtocopherol and zeaxanthin and the pH-mediated modulation of photosystem II (PS II) activity (Smirnoff 1996). AsA acts as a

N.A. Anjum et al.

D-Glucose Hexokinase


D-Glucose 6-phosphate

Phosphoglucose isomerase

GDP-D-mannose 3 ’,5 ’epimerase


L-Galactose 1-phosphate

D-Fructose 6-phosphate

GDP-D-mannose pyrophosphorylase

Phosphomannose isomerase

D-Mannose 6-phosphate

D-Mannose 1-phosphate

L-Galactose L-galactose-1-P phosphatase

L-galactose dehydrogenase

L-galactono-1,4lactone dehydrogenase


Metal/metalloid stress O2-.

Fig. 2 Schematic presentation of major pathway of ascorbate (AsA) in plants and the significance of the interrelationships among AsA, dehydroascorbate (DHA), dehydroascorbate reductase (DHAR), monodehydroascorbate (MDHA), monodehydroascorbate reductase

(MDHAR), and ascorbate peroxidase (APX) for the metabolism of reactive oxygen species (including H2O2) generated as a result of metal/ metalloid stress

Plant metal/metalloid stress tolerance and ascorbate significance

co-factor in the formation of hydroxyproline residues and as an antioxidant in plant apoplast, protecting it for example against tropospheric ozone (Takahama and Oniki 1992; Sanmartin et al. 2003). It also acts as a precursor for several other plant metabolites (Smirnoff 1996; Noctor and Foyer 1998; Debolt et al. 2007; Parsons and Fry 2012). AsA is required for transition from G1 to S phase in the cell cycle (Liso et al. 1988), cell proliferation of meristematic root cells (Herschbach et al. 2010), control of cell cycle progression (Potters et al. 2002), and for root growth and development (Liso et al. 2004). The modulation of the plasmalemma energetic state derived from the AsA-induced hyperpolarization and the activity of an intrinsic transplasmalemma AsA-regenerating enzyme has been proposed to control plant cell growth (reviewed by Córdoba and González-Reyes 1994). In addition, owing to AsA engagements in the synthesis of several growth regulators, such as ethylene, abscisic acids, and gibberellins where it functions as a co-factor of dioxygenases (enzymes playing a decisive role in the synthesis of these hormones), AsA significantly affects plant development (Dong et al. 1992; Liu et al. 1999; Arrigoni and De Tullio 2000; Lopéz-Carbonell et al. 2006) (Table 1). Ascorbate, dehydroascorbate, and monodehydroascorbate pools AsA has been considered of immense importance in the plant system where it performs key functions in growth, metabolism, development, and stress responses (Noctor and Foyer 1998; Smirnoff 2000; Pastori et al. 2003; Anjum et al. 2010, 2012b). AsA has also been considered much more than just an antioxidant (Arrigoni and De Tullio 2002). The maintenance of elevated redox state of AsA occurs at the expense of GSH in the Halliwell-Asada cycle. To this end, in isolated cells of Arabidopsis thaliana, the total AsA pool as well as its redox state did not vary during the early stages of Cd stress while the pool of GSH became more oxidized (Horemans et al. 2007). AsA pool may vary in different organs of the same plants under metal (such as Cu) exposure (Thounaojam et al. 2012). Metals have been extensively shown to modulate AsA pool in different plants, where the Cd-AsA pool interaction has been widely reported (Wu et al. 2004; Aravind and Prasad 2005;

Zhao et al. 2005; Romero-Puertas et al. 2007; Chao et al. 2010; Hasanuzzaman et al. 2012b; Shen et al. 2012). Decreased (Ceratophyllum demersum, Aravind and Prasad 2005; Brassica napus, Hasanuzzaman et al. 2012b; Pisum sativum, Romero-Puertas et al. 2007; Hordeum vulgare, Wu et al. 2004; Oryza sativa, Chao et al. 2010), increased (Brassica juncea, Mohamed et al. 2012; Sedum alfredii, Jin et al. 2008), and unchanged (B. juncea, Markovska et al. 2009) AsA pool has been evidenced in Cd-exposed plants. Considering dehydroascorbate (DHA) and monodehydroascorbate (MDHA) pools, AsA is oxidized in two sequential steps, first producing unstable radical MDHA, which subsequently disproportionates to form AsA and DHA and/or is reduced to AsA by NADP(H)-dependent enzyme MDHAR (Smirnoff 1996, 2000; Fotopoulos et al. 2010). DHA, highly unstable at pH>7 (Smirnoff 1996), is formed in cell metabolism as a consequence of AsA utilization, via spontaneous disproportionation of AsA-free radical (Arrigoni 1994; Paciolla et al. 2001). DHA can be decomposed to tartrate and oxalate (Noctor and Foyer 1998); however, to avoid this situation, DHA can be rapidly reduced to AsA by the enzyme DHAR using reducing equivalents from GSH (de Pinto and de Grara 2004; Sharma et al. 2012). Differential zonal values for intra- (symplastic) and extra- (apoplastic) cellular DHA content was reported by Córdoba-Pedregosa et al. (2003) in the roots of hydroponically grown Allium cepa. At every zone, DHA concentration was lower than the reduced form (AsA), but the redox status of the molecule (AsA/AsA+DHA ratio) remained similar along the A. cepa root axis. Novel insights into AsA catabolism have been recently provided by Parsons et al. (2011), where the authors invested the alternative pathways of DHA degradation in vitro and in plant cell cultures. DHA was proposed as a branch point in AsA catabolism which is either oxidized to oxalate and its esters or hydrolyzed to 2,3dioxo-L-gulonate and downstream carboxypentonates. The significance of ROS for the control of the oxidation/ hydrolysis ratio was also revealed. In these processes, oxalyl esters may be enzymatically hydrolyzed in vivo while the carboxypentonates may remain stable (Parsons et al. 2011). The studies on DHA transport/reduction in plants have been widely considered. In this context, an increased intracellular AsA content was reported in cultured cells as a result of DHA

Table 1 Summary of dehydroascorbate reductase-, monodehydroascorbate-, and ascorbate peroxidase-catalyzed reactions and sites involved therein Enzyme

Enzyme code

Major reactions catalyzed

Site of reaction



Chl, Cyt, Mit Chl, Cyt, Mit Chl, Cyt, Apo, Mit, Per

Adapted from Hasanuzzaman et al. (2012a) Chl chloroplast, Cyt cytosol, Mit mitochondria, Apo apoplast, Per peroxisome

N.A. Anjum et al.

transportation to the cells (Horemans et al. 1997, 1998; de Pinto et al. 1999; Potters et al. 2000). In intact Lupinus albus and A. cepa roots, DHA administration caused no DHA accumulation in the roots where DHA transportation inside the cells and the role of enzymatic and/or non-enzymatic mechanisms in the rapid reduction DHA into AsA were envisaged (Paciolla et al. 2001). Additionally, DHA-mediated inhibition of cell cycle progression was suggested as a result of DHA-accrued modulation of the redox state of SH-containing proteins (Pasciolla et al. 2001). GSH-independent DHA reduction mechanism has been reported following DHA influence on the plant cell cycle (Potters et al. 2004). DHA was shown to decrease the mitotic index in Nicotiana tabacum cultivar Bright Yellow 2 (BY-2) (de Pinto et al. 1999), whereas a slowed down cell cycle progression was evidenced when DHA was added in the G1 phase (Potters et al. 2004). In Cdexposed Arabidopsis plant cell cultures, impairment in DHA uptake was reported as the early response (Horemans et al. 2007). MDHA is the primary oxidation product of AsA and can act as an electron acceptor from PS II in vivo (Miyake and Asada 1992) and as both donor and acceptor of electron in transmembrane electron transport (Asard et al. 1995). Ascorbate/dehydroascorbate redox (AsA/DHA) status The cellular redox state regulation is a crucial factor for plant development and responsiveness to environmental stimuli, where ascorbate/dehydroascorbate (AsA/DHA) redox couple significantly contributes to the general redox homeostasis in the plant cell; hence, AsA/DHA redox couple is considered as the heart of the cell’s redox metabolism (de Pinto et al. 1999; Pastori and Foyer 2002; Foyer and Noctor 2003; de Pinto and de Gara 2004; Potters et al. 2010). In addition to the significance of the AsA redox state in the apoplast, the AsA/DHA ratio has been reported equally significant for the control of guard cell signaling and stomatal movement (Chen et al. 2003). Therefore, the changes in the AsA/DHA ratio are considered as a redox status indicator in plants (Yoshida et al. 2006; Foyer and Noctor 2011). At low concentrations, DHA has been shown to inhibit the activity of several enzymes in vitro, including malate dehydrogenase, fructose 1,6bisphosphatase (Morell et al. 1997), and hexokinase (Fiorani et al. 2000). Moreover, DHA administration in vivo was reported to inhibit root growth (Cordoba-Pedregosa et al. 1996), whereas enhanced growth was reported due to an increase in AsA content (Cordoba-Pedregosa et al. 1996; Arrigoni et al. 1997). Compared to redox status of the GSH pool, the AsA/ DHA ratio has been reported to be less affected by enhanced ROS availability. Because much of the DHA detected in tissues is localized in the apoplast rather than the cytosol, very high cytoplasmic AsA/DHA ratios simultaneously with low GSH/GSSG ratios are maintained presumably by efficient GSH-independent pathways of AsA regeneration and/or the

difference in the redox potential between the GSH/GSSG and AsA/DHA couples (reviewed by Foyer and Noctor 2011). Though the control of the lifetime of the ROS signal has been reported to be related also with AsA via its ROS scavenging role but in contrast to GSH, there is no evidence available that may confirm the involvement of the ratio of AsA to DHA in the transmission of ROS signals (review by Foyer and Noctor 2011). On the perspective of metal stress impacts on AsA/ DHA ratio, plant genotypes differing in their metal sensitivity exhibit differential AsA redox status. As-tolerant O. sativa cv. Triguna maintained a high AsA/DHA ratio as compared to AsA-sensitive O. sativa cv. IET-4786 under AsA (0–50 μM) (Tritpathi et al. 2012). Compared to wild-type Cd-tolerant O. sativa, mutant cadH-5 exhibited a relatively high AsA/ DHA ratio under Cd exposure (Shen et al. 2012). However, difference in metal and plant types may exhibit differential pools of AsA and DHA or the ratio of AsA/DHA. Al3+ stress caused decreases in the pools of reduced ascorbate (AsA) and total ascorbate (DHA+AsA) but increased DHA pool and AsA/DHA ratio in O. sativa seedlings (Sharma and Dubey 2007), whereas under Cd (10 μM) stress, 1.87-fold higher DHA/AsA ratio was depicted (vs. control) in C. demersum. Cu stress-accrued disturbed redox state of the cellular environment due to increased DHA/AsA ratio was considered as a major factor causing an accelerated senescence and poor growth in the Cu excess plants (Tewari et al. 2006). AsA recycling was proposed to be a potential mechanism for controlling the cellular redox state and the phenotypic performance of plants exposed to Ni (Saeidi-Sar et al. 2007).

Dehydroascorbate reductase (DHAR) and monodehydroascorbate reductase (MDHAR) As mentioned also in previous sections, a number of metabolic reactions in plants may result into the conversion/oxidation of the reduced form of AsA to MDHA, a semi-oxidized form also known as AsA-free radical, and DHA, the fully oxidized form. Nevertheless, higher plants utilize DHA reductase (DHAR) and MDHA reductase (MDHAR) for the reduction of oxidized or semi-oxidized forms, respectively, in order to ensure optimal concentrations of AsA, DHA, and/or MDHA in different tissues (Horemans et al. 2000; Paciolla et al. 2001; Smirnoff et al. 2001; Córdoba-Pedregosa et al. 2003). Therefore, DHAR and MDHAR are critical for the protection of cellular components against oxidative stress injury (Asada and Takahashi 1987). Salient features and metal-mediated modulation of both DHAR and MDHAR are discussed hereunder. Dehydroascorbate reductase (DHAR) DHAR is a monomeric thiol enzyme which is widely present in various plant tissues (Kato and Esaka 1999). Since 1976,

Plant metal/metalloid stress tolerance and ascorbate significance

DHAR activity originally was not believed to be present in chloroplasts and it was assumed that DHAR was reduced by GSH in a nonenzymatic reaction (Foyer and Halliwell 1976). However, in several subsequent studies until the year 2000, enzymes displaying DHAR activity were characterized (Foyer and Halliwell 1977; Hossain et al. 1984; Shimaoka et al. 2000). Additionally, the functions and occurrence of some of the previously found proteins with DHAR activities and also the significance of DHAR for AsA recycling were under debate (Foyer and Mullineaux 1998; Polle 2001). Now, it is well known that the expression of DHAR, responsible for regenerating AsA from an oxidized state, regulates the cellular AsA redox state, which in turn affects cell responsiveness and tolerance to environmental ROS (Chen and Gallie 2006). Thus, DHAR is a physiologically important reducing enzyme in the AsA-GSH recycling reaction for higher plants (Chen et al. 2003). Changes in DHAR expression have been reported to result into substantial alterations in both cytosolic and apoplastic AsA redox state, where DHAR overexpression increases leaf AsA redox state but its suppression results into an opposite effect (Chen et al. 2003; Chen and Gallie 2006). Additionally, the participation of DHARs in the regulation of transmembrane ion flux has been evidenced to provide a mechanism that couples ion transport to AsA redox state across the membrane (Foyer and Noctor 2011). Locato et al. (2009) and Hossain et al. (2010) are of opinion that an increase in DHAR activity could be a sort of feedback regulation mechanism occurring to improve AsA regeneration from DHA when AsA depletion occurs at its production sites due to enhanced ROS. Nevertheless, Chen et al. (2003) reported that overexpression of Triticum aestivum DHAR gene in N. tabacum and Zea mays resulted in 32- and 100-fold increase in DHAR enzyme activity in transgenic N. tabacum and Z. mays, respectively. Moreover, DHAR overexpression led to 2–4-fold increase in AsA content in Z. mays leaves and grains. The role of elevated DHAR activity has been reported in a number of plants in response to various ROS-inducing stresses (Lee et al. 2007; Hasanuzzaman et al. 2011a, b; 2012a, b; Hasanuzzaman and Fujita 2011, 2013). The available literature displays a differential modulation of DHAR activity in plants depending on the plant type and dose and exposure duration of metals (Table 2). However, in every case, an increased activity of DHAR resulted in enhanced regeneration of AsA and provided oxidative stress protection. A significantly increased DHAR activity has been reported in a number of plants including Populus × canescens roots (Schützendübel et al. 2002), Triticum durum root (Paradiso et al. 2008), B. juncea (Iqbal et al. 2010), and Vigna radiata (Anjum et al. 2011), whereas a significantly decreased DHAR activity was reported in Cd-exposed C. demersum (Aravind and Prasad 2005), N. tabacum (Islam et al. 2009), and B. napus (Hasanuzzaman et al. 2012b). Cd-mediated reduction in DHAR activity may

closely corroborate with the altered ratio of AsA to DHA, diminished activity of MDHAR, and increased nonenzymatic disproportionation of MDHA in Phaseolus vulgaris seedlings (Cuypers et al. 2000). Cd dose dependency of DHAR modulation (increase or decrease) has also been evident in a number of studies. For example, in Glycine max root, DHAR activity was significantly increased (41 % over control) under 50 μM CdCl2 treatments, but the activity was decreased by about 52 % with both higher concentrations of CdCl2 (100 and 200 μM) treatments (Balestrasse et al. 2001). In B. juncea, Cd was reported to decrease DHAR activity only at its high (30, 50, and 100 μM) concentrations, whereas 10 μM Cd caused a significant decrease in DHAR activity (Markovska et al. 2009). Plants exhibiting a differential Cd sensitivity may differ in DHAR activity. In this context, Cd-tolerant V. radiata genotype Pusa 9531 showed a relatively higher DHAR activity (46 % increase over control) when compared to Cd-susceptible cv. PS 16 (29.6 % increase over control) (Anjum et al. 2011). Enhanced DHAR and MDHAR activity was thought to help this plant to maintain AsA and DHA pools which in turn contributed to plant responses to Cd stress (Paradiso et al. 2008; Anjum et al. 2011). An inefficient turnover rate of AsA oxidation and reduction via AsA-GSH cycle (resulting into a decrease in AsA) has been evidenced in Cd-exposed B. napus seedlings (Hasanuzzaman et al. 2012b). No change in DHAR activity has also been reported in a few instances including the study on Cd-treated [0.5 mM Cd(NO3)2, 5 days] G. max seedlings (Aksoy and Dİnle 2012), where no change in DHAR activity was accompanied by a higher amount of lipid peroxidation. As3+-treated O. sativa exhibited exposure period and plant organ-dependent modulation in DHAR activity, where DHAR activity declined in the seedlings during 5–10 days, but during the later growth period of 15–20 days, As3+-treated seedlings displayed an increased DHAR activity compared to controls (Mishra et al. 2011). Moreover, approx. 42–79 and 50–15 % increases were revealed in DHAR activity, respectively, in the roots and shoots. Considering Ni stress-mediated modulation of DHAR activity, an increased DHAR activity was depicted in Ni-exposed O. sativa shoots and roots (Maheshwari and Dubey 2009) and B. juncea (Kanwar et al. 2012), suggesting that Ni activates the AsA-regenerating system to maintain the elevated level of AsA. A limited regeneration of AsA in chloroplast was revealed as a result of significantly decreased DHAR activity in Ni (100 mg Ni kg−1 soil) in Z. mays (Wang et al. 2009). Exposure period dependency in DHAR activity modulation was observed in Al [50 μM Al2(SO4)3]-exposed Cucurbita pepo roots, where increased DHAR was thought to maintain the AsA system in the reduced state which was confirmed by the unaffected redox state of AsA under Al treatment (Dipierro et al. 2005). The overexpression of DHAR was evidenced to maintain a high

N.A. Anjum et al. Table 2 Modulation of dehydroascorbate reductase, monodehydroascorbate, and ascorbate peroxidase activity in selected metal/metalloid-exposed plants Plant species

Metal/metalloid concentration(s)



10, 30, 50, and 100 μM of Cd, 5 days 25 and 50 μM Cd

46 % increase at 10 μM Cd, while 69 % decrease at 30, 50, and 100 μM of Cd The activity increased in tolerant cv. (Pusa Jai Kisan), while remained unchanged in sensitive cv. (SS2) By 24 and 43 % decrease upon exposure to 0.5 and 1.0 mM CdCl2 38 % decrease

Markovska et al. (2009)

Dehydroascorbate reductase Brassica juncea B. juncea

0.5 and 1.0 mM CdCl2, 48 h Ceratophyllum demersum 10 μM CdCl2, 7 days B. napus

Cucurbita pepo

50 μM Al2(SO4)3, 24 h

No changes, but contributed in AsA regeneration

Glycine max

0.5 mM Cd(NO3)2, 5 days

No change

G. max

CdCl2, 50, 100, and 200 μM

Hordeum vulgare Oryza sativa Nicotiana tabacum Triticum aestivum Vigna radiata

41 % increase at 50 μM, while 53 and 51 % decrease at 100 and 200 μM CdCl2 15, 30, or 60 μM CdCl2, 1–6 h Marked reduction in first segment, while increase in the second segment of roots 50 μM As3+, 20 days 42–79 % increased DHAR activity in roots and about 50–115 % increased activity in shoots 100 μM Cd Sharp decrease 0.25 and 0.5 mM Na2 HAsO4⋅7H2O, 48 h 100 mg Cd kg−1 soil

Iqbal et al. (2010) Hasanuzzaman et al. (2012b) Aravind and Prasad (2005) Dipierro et al. (2005) Aksoy and Dİnle (2012) Balestrasse et al. (2001) Zelinová et al. (2013) Mishra et al. (2011) Islam et al. (2009)

33 and 30 % decrease at 0.25 and 0.5 mM As

Hasanuzzaman and Fujita (2013)

46 and 30 % increase in Cd-tolerant cv. Pusa 9531 and Cd-susceptible cv. PS 16

Anjum et al. (2011)

Markovska et al. (2009)

Monodehydroascorbate reductase

B. napus

10, 30, 50, and 100 μM of Cd, 5 days 0.5 and 1.0 mM CdCl2, 48 h

C. demersum

10 μM CdCl2, 7 days

35, 310, 220, and 215 % increase at 10, 30, 50, and 100 μM of Cd, respectively By 16 and 32 % upon exposure to 0.5 and 1.0 mM CdCl2 34 % reduction

G. max

0.5 mM Cd(NO3)2, 5 days

No change

H. vulgare

15, 30, or 60 μM CdCl2, 1–6 h Marked reduction in first segment, while increase in the second segment of roots 50 μM As3+, 20 days 22–34 % increase in roots and about 9–61 % increase in shoots 100 μM Cd Sharp decrease

B. juncea

O. sativa N. tabacum T. aestivum

Hasanuzzaman et al. (2012b) Aravind and Prasad (2005) Aksoy and Dİnle (2012) Zelinová et al. (2013) Mishra et al. (2011) Islam et al. (2009)

No change

Hasanuzzaman and Fujita (2013)

Typha latifolia

0.25 and 0.5 mM Na2 HAsO4⋅7H2O, 48 h 10 μM CdSO4

30 % increase

Lyubenova and Schröder (2011)

T. latifolia

10 μM CdSO4

30 % increase

Lyubenova and Schröder (2011)

T. latifolia

100 μM PbCl2

250 % increase

Lyubenova and Schröder (2011)

V. radiata

100 mg Cd kg−1 soil

24 and 19 % increase in Cd-tolerant cv. Pusa 9531 and Cd-susceptible cv. PS 16

Anjum et al. (2011)

B. juncea

5 μM As

102–173 % increase

Khan et al. (2009)

B. juncea

25 μM As

59–148 % increase

Khan et al. (2009)

B. napus

39 and 43 % increase with 0.5 and 1.0 mM CdCl2

Hasanuzzaman et al. (2012b)

H. vulgare

0.5 and 1.0 mM CdCl2, 48 h 5 μM Cd, 25 days

Chen et al. (2010)

O. sativa

50 μM As3+, 20 days

T. latifolia

250 μM Na2HAsO4

89 % increase in tolerant genotype (Weisuobuzhi), while a slight decrease in sensitive (Dong 17) genotype 33–62 % increase in roots and 75–200 % increased activity in shoots 650 % increase

T. aestivum

24 and 34 % increase with 0.25 and 0.5 mM As

Hasanuzzaman and Fujita (2013)

T. aestivum

0.25 and 0.5 mM Na2 HAsO4⋅7H2O, 45 h 500 μM Fe

28 % decrease

Li et al. (2012)

V. radiata

100 mg Cd kg−1 soil

325 and 187 % increase in Cd-tolerant cv. Pusa 9531 and Cd-susceptible cv. PS 16

Anjum et al. (2011)

Ascorbate peroxidase

Mishra et al. (2011) Lyubenova and Schröder (2011)

Plant metal/metalloid stress tolerance and ascorbate significance

AsA level and confer Al tolerance in N. tabacum (Yin et al. 2010). Metal-mediated modulation in DHAR activity may vary with plant age and genotypes. In this context, adult Helianthus annuus exhibiting lower DHAR activity showed a lower Cd and Zn sensitivity than young seedlings showing higher DHAR activity (Nehnevajova et al. 2012). Monodehydroascorbate reductase (MDHAR) MDHAR (EC is a flavin adenine dinucleotide monomeric enzyme of the AsA-GSH cycle and is present as chloroplastic and cytosolic isozymes (Rizhsky et al. 2002; Yoon et al. 2004). MDHAR has also been reported in the mitochondrion (Deleonardis et al. 1995), peroxisome, and glyoxysome (Jiménez et al. 1997; Leterrier et al. 2005). Hossain and Asada (1985), Dalton et al. (1992), and Borraccino et al. (1986) purified cytosolic MDHAR from Cucumis sativus fruits, G. max root nodules, and Solanum tuberosum tubers, respectively. S. tuberosum mitochondrial MDHAR has been purified by De Leonardis et al. (1995), whereas Sano et al. (2005) reported a chloroplastic MDHAR from Spinacia oleracea. MDHAR activity varies in different stages of cell growth and plant tissues, leading to different AsA/MDHA ratios. In meristematic cells, MDHAR activity is very high, and consequently, a large amount of MDHA is reduced to AsA; in expanding cells, however, the MDHAR activity is relatively lower, and therefore, the accumulated MDHA is converted to a high level of DHA (Arrigoni 1994). There are also a plethora of reports available on cloning of MDHAR complementary DNAs (cDNAs) from a number of economically important plant species including P. sativum (Murthy and Zilinskas 1994; Leterrier et al. 2005), Brassica campestris (Yoon et al. 2004), Physcomitrella patens (Drew et al. 2007), and N. tabacum (Eltayeb et al. 2007) under varied environmental conditions. Based on the conservation of cytosolic MDHAR in the land plant lineage and the transcriptional upregulation under water deficiency, Lunde et al. (2006) suggested the essential role of evolution of cytosolic MDHAR in stress protection for land plants when they inhabited the dry terrestrial environment. MDHAR is the only known enzyme to use an organic radical (such as malondialdehyde) as a substrate and is also capable of reducing phenoxyl radicals which are generated by horseradish peroxidase with H 2O2 (Sakihama et al. 2000). Moreover, it is the only enzyme known to have a carbon-based radical as its substrate and is also known for its high specificity for MDHA as the electron acceptor and its preference for NADH rather than NADPH as the electron donor during AsA regeneration in plants (Hossain et al. 1984; Hossain and Asada 1985; Dalton et al. 1992; Murthy and Zilinskas 1994; De Leonardis et al. 1995; Asada 1999; Drew et al. 2007). Due to its involvement in AsA regeneration, MDHAR plays an important role in

maintaining the antioxidant properties of AsA. The catalytic reaction mediated by MDHAR is as follows: MDHAR

2 MDHA þ NADPH → 2 Ascorbate þ NADPþ : A great deal of research on different plants under environmental stresses has revealed the MDHAR regulatory role for oxidative stress tolerance and acclimation (Mittova et al. 2003; Hasanuzzaman et al. 2010, 2011a, b; 2012a, b; Hasanuzzaman and Fujita 2011, 2013). The activity of MDHAR in plants exposed to metals showed differential responses depending on the plant genotypes as well as metal dose and types and exposure duration (Table 2). MDHAR response was found dependent on metal type/dose, plant types/their organ, and exposure duration in a number of studies (Aravind and Prasad 2005; Jin et al. 2008; Paradiso et al. 2008; Anjum et al. 2011; Mishra et al. 2011; Nehnevajova et al. 2012; Hasanuzzaman and Fujita 2013; Lyubenova and Schröder 2011). For example, in T. aestivum roots, MDHAR activity remained unchanged after 3 days of Cd exposure, while an increased MDHAR activity was observed with a concomitant increase in AsA level after 7 days of Cd exposure (Paradiso et al. 2008). A significant increase in MDHAR activity was observed in Pinus sylvestris seedlings subjected to 5 and 50 μM Cd concentrations only after 96 h of treatment (Schützendübel et al. 2001). In C. demersum, Cd (10 μM CdCl2, 7 days) decreased MDHAR activity, whereas an enhanced tolerance of C. demersum to Cd exposure was revealed in terms of the restoration of MDHAR activity when Zn was supplemented to Cd-exposed plants (Aravind and Prasad 2005). Cd at 10 μM displayed no major change in MDHAR-mediated regeneration of AsA in S. alfredii ecotypes (hyperaccumulator and nonhyperaccumulator) (Jin et al. 2008). MDHAR response was different in two V. radiata genotypes exhibiting their differential Cd tolerance, where Cd-tolerant cv. Pusa 9531 showed a relatively higher MDAHR activity when compared to Cd-susceptible cv. PS 16 under 25–100 mg Cd kg−1 soil. A significantly elevated MDHAR activity has been evidenced extensively to protect plants against oxidative stress caused by different metals (Fecht-Christoffers et al. 2003; Sharma and Dubey 2007; Jin et al. 2008; Maheshwari and Dubey 2009; Anjum et al. 2011). MDHAR activity (and hence the extent of AsA regeneration) may also differ in different zones of plant roots under short and prolonged metal exposure. To this end, in H. vulgare under short-term (1–6 h) Cd (30 or 60 μM CdCl2) exposure, a transient decrease in MDHAR activity was observed at the zone immediately behind the root apex containing meristematic and elongation zone. However, in the second root segment containing the beginning of the differentiation zone, the activity of MDHAR increased after Cd treatment (Zelinová et al. 2013). In T. durum roots, Cd was reported to bring no change in MDHAR activity during short-term

N.A. Anjum et al.

exposure, whereas prolonged treatment (7 days) caused a significant elevation in MDHAR activity (Paradiso et al. 2008). In O. sativa seedlings, As3+ exposure for a period of 5–20 days led to a declined MDHAR activity, whereas a concomitant increase was observed during 10–20 days of As3+ treatment (25 and 50 μM) (Mishra et al. 2011). The rapid increase in MDHAR activity with 50 μM As3+ can be a mechanism for activating the AsA-generating system in order to counteract As3+-induced ROS (Mishra et al. 2011). Low and higher doses of the same metal impact AsA regeneration (in terms of MDHAR activity) differentially. In Typha latifolia, 100 μM of Pb caused a significant increase in MDHAR activity, whereas the exhibition of MDHAR activity up to the control values was depicted with 250 μM of Pb (Lyubenova and Schröder 2011). Nevertheless, metal speciation may also play a significant role in modulating the regeneration of AsA by differentially modulating MDHAR activity. Cr(VI) exposure resulted into a higher increase in MDHAR activity in V. radiata roots when compared to Cr(III) impacts. In addition, 24 h of exposure of V. radiata to Cr(III) (50 μM) led to a significant increase in MDHAR activity, whereas the same Cr concentration of Cr(IV) caused a significant increase only after 2 h of exposure (Karuppanapandian and Manoharan 2008).

Ascorbate peroxidases (APXs) A peroxidase using AsA as an electron donor was first proposed in 1976 (Foyer and Halliwell 1976). Subsequently, heme-containing homodimeric proteins—APXs (EC—have been reported in insects (Mathews et al. 1997), Trypanosoma cruzi (Boveris et al. 1980), and choroid and iris epithelium of bovine eye tissues (Boveris et al. 1980; Wada et al. 1998) and in higher plants, algae, and some cyanobacteria (Miyake et al. 1991; Takeda et al. 1998; Sano et al. 2001; Shigeoka et al. 2002; review by Dabrowska et al. 2007). Compared to catalase (another H2O2-metabolizing enzyme), APX has an affinity (with a Km for H2O2 in the micromolar range) for H2O2 at least a hundred times higher. Hence, despite the low concentrations of APX, due to its high affinity for H2O2, it is able to finely regulate this ROS (de Gara et al. 2010). APX has been identified in many higher plants, where the multigenic nature of this enzyme has been revealed. A variety of APX isozymes has been observed in the cytosol, chloroplast stroma, and thylakoids, where at the expense of AsA, these isozymes scavenge H2O2 and protect plant cells against potential destructive effects of H2O2 (Shigeoka et al. 2002). The reaction catalyzed by APX is as follows: APX

C6 H8 O6 ðAscorbateÞ þ H2 O2 ðHydrogen peroxideÞ → C6 H6 O6 ðDehydroascorbateÞ þ H2 OðWaterÞ:

At least five APX isoforms such as cytosolic (cAPX), mitochondrial (mitAPX), peroxisomal/glyoxysomal, stromal (sAPX), and thylakoid membrane‐bound (tAPX) in chloroplasts have been identified in plants (Asada 1992, 1999; Ishikawa et al. 1998; Noctor and Foyer 1998; De Leonardis et al. 2000; Shigeoka et al. 2002; Caverzan et al. 2012). In particular, similar to other peroxidases such as the yeast cyt-c peroxidase and guaiacol peroxidase in plants, the chloroplastic and cAPXs are hemoproteins containing protoporphyrin IX as a prosthetic group (Asada 1992, 1993). The two major isoforms of APX (cAPX and mAPX) have relatively higher halfinactivation times (≥1 h), while for mitAPX and chlAPX, it is less than 30 s (Caverzan et al. 2012). However, cAPX is stable under such condition (Amako et al. 1994). cDNA encoding a putative APX in the chloroplast thylakoid lumen has also been identified by Kieselbach et al. (2000) in Arabidopsis. Mittler and Zilinskas (1991) were the first to isolate and purify homogenous cAPX from P. sativum. cAPX has been reported as a key regulator of plant cell death (de Pinto et al. 2006). Nine genes were suggested as Apx genes in Arabidopsis where the cytosol is thought to harbor three, whereas peroxisome, thylakoid, and stroma are thought to harbor two Apx genes each (Chew et al. 2003; Mittler et al. 2004). In O. sativa, eight members of the Apx gene family encode two each of cytosolic and peroxisomal and three chloroplastic isoforms and one that is targeted to the mitochondria (Teixeira et al. 2006) (Fig. 3). Later, Rosa et al. (2010) reported the generation of transgenic O. sativa silenced for either both or each one of the cApx1 and Apx2 genes in order to investigate the importance of cApx isoforms on plant development and plant stress responses. Though the expression of APX genes occurs during normal plant development, abiotic stresses including toxic metals have been credibly reported to differentially modulate APX activity and gene expression in plants (Singh et al. 2008; Hasanuzzaman et al. 2011a, b; 2012b, c; Caverzan et al. 2012). Nevertheless, variability in APX activity and APX isoenzyme gene expression under the same stress conditions has been reported (Hossain et al. 2010; Hossain and Fujita 2012). Some of the APX isoenzyme genes are reported to be constitutively expressed for the immediate and efficient detoxification of H2O2 under normal and oxidative stress conditions (Ishikawa and Shigeoka 2008; Hossain et al. 2010; Hossain and Fujita 2012) (Table 2). Among the toxic metals, Cd is widely studied in plants and the modulation of APX activity under Cd stress is reported in various studies. The significance of Cd concentration applied as well as the plant age and genotypes has been widely reported to modulate APX activity. Increased APX activity under Cd exposure has been reported in a number of plants including B. juncea (Mobin and Khan 2007), Z. mays (Krantev et al. 2008), T. aestivum (Khan et al. 2007), Vigna mungo (Singh et al. 2008), B. campestris (Anjum et al. 2008), V. radiata (Anjum et al. 2011), and B. napus (Hasanuzzaman

Plant metal/metalloid stress tolerance and ascorbate significance

Arabidopsis thaliana


Oryza sativa

At1g07890/837304 At3g09640/820121 At4g32320/2127766

Cytosolic isoenzymes

Os03g0285700/4332474 Os07g0694700/4344397

At4g35000/829652 At4g35970/829751

Peroxisomal isoenzymes

Os04g0223300/4335202 Os08g0549100/4346347

At1g77490/844085 At4g09010/2122333

Thylakoid-bound isoenzymes

Os02g0553200/4329643 Os12g0178100/4351663 Os12g0178200/4351664

At4g08390/826396 At1g33660/2807587

Stromal/mitochondrial isoenzymes


et al. 2012b). However, in other plants such as H. vulgare roots, APX activity was reduced at a high concentration of Cd (Hegedus et al. 2001). The lower APX activity was also noted in C. sativus chloroplasts with increasing Cd concentration (Zhang et al. 2003). Balestrasse et al. (2001) reported that low Cd levels led to an increased APX activity in G. max roots and nodules, but the activity decreased with high Cd concentration. Brassica genotypes may differ in their responses to Cd stress in terms of APX activity. In this context, a significant decrease in APX activity was observed in B. juncea and B. napus subjected to Cd stress, but the reduction was more in B. campestris (Nouairi et al. 2009). Plants may exhibit a differential modulation of APX when exposed to a single metal or more than two metals at a time (Aravind and Prasad 2005; Khan et al. 2007). C. demersum and T. aestivum showed highly increased APX activity under Cd+Zn exposure when compared to Cd- or Zn-alone-treated plants, indicating their differential impact on the antioxidant system and ROS scavenging activities by Zn against Cd (Aravind and Prasad 2005; Khan et al. 2007). Similarly, APX activity was found higher in Sesbania drummondii seedlings treated with a combination of metals (Pb, Cu, Ni, and Zn) as compared to those treated with a single metal. For instance, APX activity showed a maximum increase in Pb+Cu+Ni+Zn treatment which was 112 % higher than the control (Israr et al. 2011). The modulation of APX activity also greatly depends on different organs of the metal-exposed plant. In Ni-exposed T. aestivum, the root and shoot displayed respectively decreased and increased APX activity (Gajewska and Sklodowska 2008). A time- and genotype-dependent response pattern for APX enzyme was observed in H. vulgare exposed to Cd stress (5 μM Cd) (Chen et al. 2010). The significant genotypic difference was observed only during 10 and 15 days of Cd exposure. The

Gene name and ID

Gene name and ID

Fig. 3 Ascorbate peroxidase genes in Arabidopsis thaliana and Oryza sativa (modified after Mittler et al. 2004; De Gara et al. 2010)

activity of APX in the relatively Cd-tolerant genotype Weisuobuzhi showed greater increase or less decrease than the sensitive genotype Dong 17. A completely inhibited APX activity was observed in T. latifolia under Pb exposure. However, in the same plant, higher concentrations of As and Cd inhibited APX activity to a lesser extent (Lyubenova and Schröder 2011). APX activity may differ in plants differing in metal sensitivity. A higher activity of APX was depicted in Cd-tolerant V. radiata cv. Pusa 9531 compared to Cdsusceptible cv. PS 16 with 100 mg Cd kg−1 (Anjum et al. 2011). This upregulation of APX activity was thought to reduce Cd-induced oxidative stress in Cd-tolerant cv. Pusa 9531 in terms of lesser increments in H 2 O 2 therein. Similarly, the occurrence of a higher APX activity in B. napus cv. Okai compared to the other cultivars (Mohican and Reg.Cob) under 0.75 mM Cd exposure was correlated with the greater Cd tolerance in cv. Okai (Touiserkani and Haddad 2012). Compared to young H. annuus, an increased APX activity in mature H. annuus exposed to Cd and Zn was an indication of the elevated use of AsA after a longer exposure (Nehnevajova et al. 2012). A relationship between Al toxicity and AsA metabolism has been reported in C. pepo roots (Dipierro et al. 2005) and O. sativa seedlings (Sharma and Dubey 2007). Al stress can also modulate the expression patterns of APX isoenzymes differentially in plant shoots and roots (Sharma and Dubey 2007). In this context, Sharma and Dubey (2007) observed two major activity bands of APX (APX 1 and APX 2) in enzyme preparations from shoots, whereas the authors detected only one band in chlAPX preparation from control as well as Al-stressed seedlings. In another study on O. sativa, the transcript levels of all OsAPX genes were notably enhanced upon 8 h of Al (20 mg Al kg−1) exposure (Rosa et al. 2010). Moreover, transgenic O. sativa

N.A. Anjum et al.

plants double-silenced for APX1 and APX2 (APX1/2 s plants) showed normal growth and development as well as enhanced tolerance to a higher dose of Al (Rosa et al. 2010). An increase in APX activity has been widely suggested to detoxify H2O2 and control its upregulation under metalinduced oxidative stress (Qureshi et al. 2005; Israr et al. 2006; Diwan et al. 2008). In B. juncea, a decreased APX activity at a high dose of As treatment (25 μM) was thought to be a result of insufficient availability of AsA since APX is labile at low AsA level and becomes very sensitive to inactivation by thiols and protein inhibitors (Khan et al. 2009). The maintenance of a higher chlAPX activity in O. sativa cv. Panta-12 under both controls as well as As3+ treatments (vs. O. sativa cv. Malviya-36) was proposed to serve as an important factor in mitigating As-induced oxidative damage (Mishra et al. 2011). Cu exposure for a long duration may cause a continuous increase in APX activity (Thounaojam et al. 2012). Moreover, a low dose of Cu may not be able to induce or inhibit APX activity during a passage of exposure time. In this context, Posmyk et al. (2009) reported that little or no change in APX activity was observed in terms of duration of exposure when the seedlings were treated with a low concentration of Cu (0.5 mM). In contrast, a linear increase in APX activity was observed under Cu (2.5 mM) stress in a time-dependent manner, which was much higher than the activity observed under 0.5 mM of Cu in B. oleracea seedlings (Posmyk et al. 2009). An increase in APX activity up to a certain limit in different plants or their organs has been proposed as the main factor responsible for avoiding oxidative stress caused by Ni (Gajewska and Sklodowska 2008; Kanwar et al. 2012) and Pb (Lamhamdi et al. 2011). Different levels of an essential metal such as Fe have also been reported to differentially impact APX activity. In T. aestivum, lower doses (100 and 300 μM) of Fe were evidenced to cause a significant elevation in leaf APX activity, whereas 500 μM Fe treatment caused a significantly decreased APX activity (Li et al. 2012). In other plants, Fe overload in bean plants was reported to rapidly induce cAPX expression (mRNA and protein) as a result of an enhanced production of reactive oxygen intermediates (Pekker et al. 2002).

Cross-talks, conclusions, and perspectives Due to the central position of AsA in cellular metabolism and its close relationship with AsA/DHA, DHAR, MDHAR, and APX, the AsA-dependent antioxidant defense system is the main player in metal-induced cellular responses. In the AsAGSH pathway, AsA is an important metabolite that helps plants to counteract the potential impacts of unmetabolized ROS and its reaction products. In fact, the effectiveness and fine-tuning between AsA-metabolizing (APX) and AsAregenerating (MDHAR and DHAR) enzymes and the

maintenance of AsA and DHA pools and AsA/DHA ratio may contribute to controlling metal-caused oxidative stress in plants (Anjum et al. 2010, 2012b). The AsA-metabolizing enzyme APX is highly sensitive to environmental changes and its activity is directly dependent on the AsA availability, where the APX isoenzymes become labile in the absence of AsA (Anjum et al. 2012b). In the AsA-GSH pathway, APX performs AsA-assisted catalysis of the reduction of H2O2 to H2O with simultaneous generation of MDHA, which if not rapidly reduced is converted back to AsA by the action of NADPH or NADH-dependent MDHAR or disproportionates nonenzymatically to AsA and DHA. DHA undergoes irreversible hydrolysis to 2,3-diketogulonic acid or is recycled to AsA by DHAR, which uses reduced GSH as the reductant. Therefore, in the absence of DHAR, DHA undergoes irreversible hydrolysis to 2,3-diketogulonic acid and DHAR allows the plant to recycle DHA, thereby recapturing AsA before it is lost (Asada 1992; Hegedus et al. 2001; Chen et al. 2003; Anjum et al. 2010, 2012b; Gill and Tuteja 2010; Martínez and Araya 2010). Although DHA is reduced to AsA by DHAR, traces of DHA are always present in plant samples and the ratio of AsA/DHA is relatively lower compared to the ratio of GSH/GSSG, especially under field conditions (Noctor et al. 1998). The oxidative and reductive status of AsA can represent the oxidative and reductive conditions in the cellular environment, where AsA concentration can change accordingly (Davey et al. 2000). Moreover, the AsA/DHA level has been used to gauge the redox state in plants (Yoshida et al. 2006). The presence of a high concentration of AsA has been considered necessary for redox cellular homeostasis under metal stress (Sobrino-Plata et al. 2009). Nevertheless, a high AsA/DHA in cells has been reported to ensure the proper functioning of AsA in the AsA-GSH pathway and in other physiological processes (Fotopoulos et al. 2010). Under Cd stress, a significantly decreased level of AsA but significantly increased level of DHA was considered as a result of suppressed glutathione-dependent DHAR activity and/or due to a decrease in AsA synthesis (Hatata and Abdel-Aal 2008). The importance of MDHAR and DHAR in regulating the AsA levels has been shown in transgenic plants by expressing enzymes involved in the recycling of oxidized AsA, including DHAR (Chen et al. 2003) and MDHAR (Eltayeb et al. 2007). The significance of overexpressed DHAR, but not of MDHAR, was evident for Al tolerance, where the maintenance of a high AsA level was also confirmed as essential toward acquiring Al tolerance (Yin et al. 2010). Increased MDHAR and DHAR activities helped Wedelia chinensis seedlings to combat As-induced phytotoxicity, as both enzymes are responsible for the AsA reduction in the AsA-GSH cycle (Talukdar and Talukdar 2013). The overexpression of MDHAR may affect AsA accumulation and increase the redox status of AsA toward reduction, because MDHAR functions upstream of DHAR in the AsA recycling pathway

Plant metal/metalloid stress tolerance and ascorbate significance

as reviewed by Ishikawa et al. (2006). Thus, the adoption of strategies for “enhanced AsA recycling” has been considered significant for increasing oxidative stress resistance ability in plants (Chen et al. 2003; Chew et al. 2003; Kwon et al. 2003). One of the most characteristic properties of APX which distinguishes it from guaiacol peroxidase, Cyt c peroxisome, and glutathione peroxidase is its instability in the absence of AsA (Pang and Wang 2010). APX in the glyoxysomal membrane can work in cooperation with MDHAR to oxidize NADH, regenerate AsA, detoxify H2O2, and protect the integrity of glyoxysomal proteins and membranes (Karyotou and Donaldson 2005). In addition, the significance of the membrane-bound APX/MDHAR in the detoxification of H2O2 produced in plant peroxisomes has been reported as a result of AsA-dependent reduction (Yamaguchi et al. 1995; Mullen and Trelease 1996; Karyotou and Donaldson 2005; Eastmond 2007). Since AsA is used as a substrate for APX activity, the decreased pool of AsA in cells has been thought as an obvious response (Paradiso et al. 2008). However, this is not the case always. For example, a decrease in AsA content was parallel with decreasing level of APX activity in Cd (5 and 50 μM)-exposed P. sylvestris seedlings (Schützendübel et al. 2001); As-mediated impaired photosynthesis in Luffa acutangula was related to decreased APX and subsequent AsA content (Singh et al. 2013), and Ni-mediated activation of AsA-regenerating system (in terms of elevated MDHAR and DHAR activity) in order to maintain the elevated level of AsA has been evidenced in O. sativa shoots and roots (Maheshwari and Dubey 2009). A difference in metal speciation may also affect the AsA-dependent plant antioxidant defense system under metal stress, where a differential response to AsA and H2O2 signaling by Cr(III) and Cr(VI) and AsA in combination with APX can be more effective in mitigating oxidative stress as against the other nonenzymatic antioxidant such as GSH (Karuppanapandian and Manoharan 2008). The present review attempted to develop an orchestrated understanding of AsA, its redox couple (AsA/DHA) and associated enzymes such as MDHAR, DHAR, and APX; and the mechanisms involved in plant tolerance to metals mediated by these traits. Numerous biological roles of this enigmatic, ubiquitous, and abundant metabolite has been highlighted in several physiological processes in plants, including growth, differentiation and metabolism, apoplastic and cytoplasmic signaling (Pourcel et al. 2007; Sharma et al. 2012), and plant metal tolerance (Romero-Puertas et al. 2007; Markovska et al. 2009; Chao et al. 2010; Anjum et al. 2012b; Hasanuzzaman et al. 2012b; Shen et al. 2012; Thounaojam et al. 2012). However, the literature critically appraised herein paints a clear picture of the physiological and biochemical aspects of ASA and its related enzymes such as APX, DHAR, and MDHAR in metal-stressed plants. Nevertheless, there seems a lack of reports on the exhaustive molecular genetics

of metal-accrued influence on the modulation of AsA, its redox couple (AsA/DHA), and related enzymes (APX, DHAR, MDHAR) in plants. Thus, the exhaustive studies should be performed in order to unveil the molecular genetics of metal-mediated regulation of biosynthesis, catabolism, recycling, and transport of AsA in particular, and DHA/AsA redox status and their tuning with DHAR, MDHAR, and APX in general. Such studies may help understand the regulatory mechanisms and approaches for manipulating plant AsA levels under a myriad of environmental stresses. Moreover, since apoplastic AsA is associated with a number of physiological phenomena such as cell division, cell elongation, and cell defense and its homeostasis in the apoplast is easily perturbed (Horemans et al. 2000), studies on the changes in apoplastic AsA to DHA ratio and AsA rapid transport across the plasmalemma under metal stress will be rewarding. Acknowledgments NAA (SFRH/BPD/84671/2012), ACD, EP, and IA are grateful to the Portuguese Foundation for Science and Technology (FCT) and the Aveiro University Research Institute/Centre for Environmental and Marine Studies (CESAM) for partial financial support. SSG, RG, RT and NT acknowledge the funds from CSIR, DST, and UGC, Government of India, New Delhi. The authors apologize if some references related to the main theme of the current article could not be cited due to space constraint. Conflict of interest The authors declare that they have no conflict of interest.

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metalloid stress tolerance in plants: role of ascorbate, its redox couple, and associated enzymes.

The enhanced generation of reactive oxygen species (ROS) under metal/metalloid stress is most common in plants, and the elevated ROS must be successfu...
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