Keywords

1 Introduction

In plant cells, the reactive oxygen species (ROS) scavenging is dependent on ascorbate (AsA) and glutathione (GSH), the two main non-enzymatic hydrophilic antioxidants and redox buffers (Noctor and Foyer 1998). Both antioxidant molecules act as signaling molecules in many cellular processes and are involved in basic metabolic reactions as well as in the protection of plant cells under environmental stress situations. Ascorbate and glutathione take part in the important ascorbate–glutathione (AsA–GSH) cycle , which plays an important role in the scavenging of H2O2 and in the recycling to the reduced forms of ascorbate and glutathione (AsA and GSH). These functions are catalyzed by APX (ascorbate peroxidase), DHAR (dehydroascorbate reductase), MDHAR (monodehydroascorbate reductase), and GR (glutathione reductase). DHAR is the enzyme that links both antioxidants in the AsA–GSH cycle (Fig. 14.1). APX activity depends not only on the AsA availability but also on GSH through the DHAR activity, a GSH-dependent enzyme that can regenerate AsA from DHA (dehydroascorbate). It is also true that the reaction catalyzed by MDHAR for AsA recycling is more economical for the plant cell, but under certain conditions, DHAR activation can be a good strategy to maintain ascorbate redox state.

Fig. 14.1
figure 1

Ascorbate–glutathione (AsA–GSH) cycle in plants. This cycle consists of a series of redox-coupled reactions whose main function is the scavenging of H2O2 and the recycling to the reduced forms of ascorbate and glutathione

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Glutathione is the major low-molecular-weight thiol in plants, and its role in plant defense and tolerance against abiotic and biotic stresses has been widely described. In 1998, Wingate et al. reported the importance of GSH for local resistance responses. Treatment of suspension-cultured cells of bean (Phaseolus vulgaris L.) with GSH resulted in a massive and selective induction of the transcription of defense genes encoding enzymes related to phytoalexin and lignin biosynthesis, as well as stimulation of genes encoding cell wall hydroxyproline-rich glycoproteins. GSH is considered the most important thiol antioxidant in plant–pathogen interactions (Kuźniak 2010), and a clear relation between GSH increases and pathogen resistance has been reported in different studies (Gullner et al. 1999; Kużniak and Sklodowska 2004; Zechmann et al. 2007a; Clemente-Moreno et al. 2010, 2013).

The redox state of glutathione plays an important role regulating the expression of defense genes. One of the most typical examples corresponds to the activation of NPR1 proteins as well as NPR1 gene expression. The reduction of NPR1 requires an increase in GSH contents, the NPR1 protein conformation being sensitive to cellular redox changes (Mou et al. 2003). Treatments with L-2-oxo-4-thiazolidine-carboxylic acid (OTC) increased GSH content and glutathione redox state, inducing the expression of the NPR1 gene in both healthy and Plum pox virus (PPV)-infected peach plantlets (Clemente-Moreno et al. 2012). In that regard, GSH seems to be the main antioxidant involved in the activation of plant defense genes (Wingate et al. 1988; Ghanta et al. 2011).

2 Plant–Virus Interaction

Different authors observed the importance of the glutathione content as well as the GSH-related enzymes in the physiological and biochemical responses of plants against virus infection (Hernández et al. 2016). The GSH contents , and therefore the redox state of glutathione , have been often associated with a resistance response to plant viruses (Table 14.1). In addition, the availability of the GSH precursors, cysteine, glycine, and glutamate, which can limit GSH synthesis, is also important in the plant–virus interaction (Zechmann et al. 2007a). In this sense, treatments aimed at increasing GSH levels, or the redox state of glutathione, also induced some kind of resistance in different plant–virus interactions. In 1999, Gullner et al. reported that the exposure of tobacco (Nicotiana tabacum) leaf discs to the cysteine precursor L-2-oxo-4-thiazolidine-carboxylic acid (OTC) led to a massive accumulation of GSH, as well as reduced tobacco mosaic virus (TMV) coat protein contents and decreased number of necrotic lesions and virus contents in TMV-inoculated tobacco leaf discs (Gullner et al. 1999). Similar results were also recorded by Zechmann et al. (2007a) in pumpkin (Cucurbita pepo) seedlings. These authors treated pumpkin seedlings with 1 mM OTC for 48 h previous to the infection with Zucchini yellow mosaic virus ( ZYMV ) and evaluated the symptoms of ZYMV disease. They observed a general delay, reduction, or complete suppression of symptom development in OTC-treated plants, depending on the time of infection and the severity of symptoms (Zechmann et al. 2007a).

Table 14.1 Response of glutathione and GSH-related enzymes in some plant–virus interactions
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Glutathione content is influenced by sulfate nutrition, and high sulfate supply correlated with increased levels of Cys (cysteine) and GSH (Kopriva and Rennenberg 2004; Király et al. 2012). In a recent work, Király et al. (2012) reported that TMV-resistant tobacco plants grown with sufficient sulfate developed less necrotic lesions response when compared with plants grown with sulfate deficiency. This enhanced virus resistance correlated with elevated levels of Cys and glutathione as well as the induction of glutathione S-transferase (GST) (Király et al. 2012).

In compatible interactions PPV–Prunus and PPV–pea , an imbalance in the antioxidant machinery at subcellular level is produced (Hernández et al. 2004, 2006, Diaz-Vivancos et al. 2008), and in all cases an inhibitory effect on some GSH-dependent enzymes was observed. For example, a decrease in soluble and chloroplastic DHAR, GR, or glutathione peroxidase (GPX) was noticed (Hernández et al. 2004, 2006; Diaz-Vivancos et al. 2008).

The treatment of pea and peach plants with 1 mM OTC, previous to the infection with PPV , resulted in a partial resistance response, in terms of PPV symptoms as well as in the percentage of leaves showing PPV symptoms (Clemente-Moreno et al. 2010, 2013). In pea plants, this response was correlated with a higher redox state of glutathione as well as with an increase in APX, POX, and GSH-related enzymes at the subcellular level (Clemente-Moreno et al. 2010). Interestingly, asymptomatic leaves of infected plants displayed a higher redox state of glutathione, a fact that could also play a role in the reduction of symptoms. In peach plants, OTC treatment, in addition to an increased plant growth, provides protection to the photosynthetic machinery and/or the chloroplast metabolism in PPV-infected plants (Clemente-Moreno et al. 2013). However, when OTC was applied to PPV-infected peach plantlets, the OTC treatments did not reduce the virus contents, although GSH levels were increased (Clemente-Moreno et al. 2012). In OTC-treated plantlets, an induction of NPR1 gene expression took place, mainly in PPV-infected plants, suggesting that GSH could play an important role in the NPR1 induction under a viral infection (Clemente-Moreno et al. 2012). Accordingly, a similar conclusion was reported by Ghanta et al. (2011) in tobacco plants.

The infection of apricot seeds by Prunus necrotic ringspot virus (PNRSV ) induced an oxidative stress that was parallel with a general decrease of all AsA–GSH cycle enzymes , including GSH-dependent enzymes such as DHAR and GR. This response was correlated with a more limited ability to eliminate H2O2 but also in the recycling of AsA and GSH in infected seeds (Amari et al. 2007).

Hakmaoui et al. (2012) studied the response of Nicotiana benthamiana plants against two different strains of Pepper mild mottle virus, the Italian ( PMMoV-I) and the Spanish (PMMoV-S) strains. The PMMoV-S was the most virulent, inducing dramatic symptoms, whereas plants infected with PMMoV-I were able to recover from their symptoms. This response was linked, among other factors, with the maintenance of GSH levels during the infection phase (up to 21 days). However, the plants infected with PMMoV-S strain showed a dramatic decrease in both total and reduced glutathione (Hakmaoui et al. 2012).

Cucumber mosaic virus ( CMV ) infection disrupted electron transport in chloroplast and mitochondria from cucumber and tomato plants. However, the net photosynthesis rate as well as the rate of mitochondrial complex I electron transport in mitochondria was less affected in tomato than in cucumber leaves (Song et al. 2009). This fact could be related with a better response of the antioxidant defenses in CMV-infected tomato plants than in cucumber , including an induction of DHAR in chloroplasts, mitochondria, and soluble fractions and GR in soluble fractions from tomato leaves. Nevertheless, in cucumber leaves, DHAR and GR only increased in mitochondria, whereas in chloroplasts a decrease in GR was recorded (Song et al. 2009).

3 Plant–Bacteria Interaction

A role for GSH and GSH-utilizing enzymes in the resistance against bacteria has been also suggested in different plant–bacteria interactions (Table 14.2). GSH mediates plant–bacteria interaction in both pathogenesis and symbiosis establishment.

Table 14.2 Response of GSH and GSH-related enzymes in some plant–bacteria interactions
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The Arabidopsis mutant pad2–1 showed an increased susceptibility to the bacterial pathogen Pseudomonas syringae (Glazebrook et al. 1996) as well as enhanced susceptibility to additional pathogens (Parisy et al. 2007). PAD2 encodes ɣ-glutamylcysteine synthetase (GSH1), the enzyme that catalyzes the first step of de novo GSH biosynthesis, suggesting that in Arabidopsis the maintenance of adequate levels of GSH is an important factor during P. syringae and other pathogen infections (Parisy et al. 2007). In addition, rax1–1, an Arabidopsis GSH1 mutant which accumulates less than 50% of wild-type GSH content, was shown to be more susceptible to avirulent strains of P. syringae (Ball et al. 2004). However, the cad2–1 mutant, with approximately about 30% of wild-type amounts of GSH, showed an unaltered disease resistance phenotype to virulent and avirulent strains of P. syringae (May et al. 1996).

The response of two different tomato cultivars against P.syringae pv. tomato is related with the glutathione levels and its redox state as well as the behavior of GSH-dependent enzymes (Kużniak and Sklodowska 2004). In that regard, the tomato A100 cultivar, susceptible to P. syringae, responded to the infection with a decrease in GSH and an accumulation of oxidized glutathione (GSSG) leading to a decrease in the redox state of glutathione. Under the same conditions, increases in GPX and GR were produced that was insufficient to keep the glutathione pool reduced (Kużniak and Sklodowska 2004). In contrast, the resistant tomato cultivar (Ontario), in addition to showing higher constitutive GSH levels than the susceptible cultivar, also maintained the glutathione pool homeostasis in response to P. syringae . Moreover, in response to the infection, the Ontario cultivar progressively increased GST activity (Kużniak and Sklodowska 2004). This enzyme not only plays an important role in the detoxification of organic hydroperoxides but also displays DHAR activity (Dixon et al. 2002). In that sense, the increase in GST was parallel with a decrease in the concentration of DHA, maintaining the AsA levels and thus increasing the redox state of ascorbate (Kużniak and Sklodowska 2004).

The treatment of apple plants with benzothiadiazole (BTH) , a SA analogue, limited the infection by Erwinia amylovora (Sklodowska et al. 2011). At short term (2 days post-inoculation, dpi), this response was correlated with an increase in GSH and GSSG, leading to a decrease in the GSH/GSSH ratio. After 7 dpi, BTH-treated plants showed a decline in GSH but a low increase in GSSG, about 20%, in relation to control plants. However, non-treated plants displayed a threefold increase in GSSG. In both cases, a decrease in GSH/GSSG ratio was produced, especially in non-treated plants, where the reduction in GSH/GSSG ratio was near four times. At long term (14 dpi), no significant changes in the redox state of glutathione occurred in any case. However, in this case, the BTH-induced resistance against the bacterial infection was not correlated with increases in GSH-dependent enzymes, such as GPX or GST (Sklodowska et al. 2011).

Additional evidence of the GSH role in plant–bacteria interaction comes from studies using transgenic plants. Tobacco plants overexpressing cytosolic Cu, Zn-superoxide dismutase (cytsod), and/or ascorbate peroxidase (cytapx) genes displayed a disease tolerance phenotype, with various levels of resistance, against bacterial wildfire caused by P. syringae pv. tabaci (Faize et al. 2012). Transgenic lines harboring cytapx and both transgenes showed the best response in terms of resistance. Inoculated transgenic lines displayed increased levels of GR activity when compared with wild-type inoculated plants (Faize et al. 2012), suggesting that the maintenance of adequate glutathione redox state could be an important factor during P. syringae infection. More recently, it has been reported that transgenic high-glutathione Nicotiana tabacum lines showed also an improved defense response against P. syringae, this response being modulated by the GSH redox potential (Matern et al. 2015).

The causal agent of bacterial wilt disease of plants is the bacteria Ralstonia solanacearum. This pathogen uses virulence effector proteins leading to the suppression of disease resistance responses to succeed in infection. Mukaihara et al. (2016) have described that the R. solanacearum effector protein RipAY is able to degrade GSH. This protein displays ɣ-glutamylcyclotransferase activity and due to its high GSH degradation activity could be considered as an effective mechanism to overcome pathogen plant defenses. Moreover, because GSH is also important for bacterial environmental stress tolerance and growth, RipAY displays a very interesting safety mechanism to avoid unwanted activation, and it is specifically activated by host eukaryotic thioredoxins (Mukaihara et al. 2016).

On the other hand, rhizobial bacteria interact with legume root to establish a symbiotic relationship leading to the formation of a new specialized organ, the nodule, which is capable of fixing atmospheric nitrogen. In root nodules, high level of GSH or homoglutathione (hGSH, GSH homolog present instead of or in addition to GSH in certain legumes) and the presence of an active AsA–GSH cycle have been reported (Becana et al. 2000; Dalton et al. 1986; Frendo et al. 1999). Thus, it has been suggested that GSH and hGSH protect nitrogen-fixing nodules against toxic oxygen species resulting from the active nodule metabolism and from varying physiological conditions, as well as from environmental challenges. In this sense, Dalton et al. (1991) reported an increase in ascorbate peroxidase and ascorbate-recycling enzymes (especially DHAR) and GR activities, as well as an increase in ascorbate and glutathione content in soybean nodules exposed to elevated ambient pO2, linking N2 fixation and antioxidative metabolism.

Moreover, during the nodulation process, an active root cell division is triggered in order to establish nodule primordia. Through the characterization of different A. thaliana GSH1 mutants, it has been showed that GSH plays a key role during root development (Diaz-Vivancos et al. 2015). The root meristemless1 (rml1) mutant, having only about 5% of the wild-type GSH levels (Schnaubelt et al. 2015), is not able to maintain cell division following germination. The cell cycle in rml1 is arrested in G1 phase of the cell cycle, being GSH the required factor to reactivate the cell division in the root apical cells (Vernoux et al. 2000). In addition, buthionine sulfoximine (BSO) , a GSH synthesis inhibitor, caused the arrest of root but not shoot development in wild-type seedlings (Schnaubelt et al. 2015). In Medicago truncatula, both BSO treatment and antisense glutathione synthetase genes in roots resulted in a decrease of (1) the average number of nodules in inoculated roots and (2) the expression of genes involved in the nodulation process, suggesting an important role for GSH in the symbiotic plant–bacteria interaction during the nodulation process (Frendo et al. 2005).

Taking together, all the reported evidences suggest that the maintenance of adequate levels of GSH is important for both the establishment of pathogen bacteria disease resistance and symbiotic plant–bacteria interactions.

4 Plant–Fungi Interaction

The effects of fungal infection on the glutathione metabolism in different cell compartments have been scarcely studied. Most of the information available has been obtained using crude extracts. In addition, the majority of information corresponds to interactions with a low range of fungi, including Botrytis cinerea, Fusarium oxysporum, or Trichoderma harzianum (Kuźniak and Skłodowska 1999, 2001, 2005; García-Limones et al. 2002; Bernal-Vicente et al. 2015) (Table 14.3).

Table 14.3 Response of glutathione and GSH-related enzymes in some plant–fungi interactions
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During a plant–fungi interaction , ROS can be generated by both the pathogen and/or the host plant. In the case of a necrotrophic fungus, ROS overproduction can be a strategy to kill the host tissue in the initial phase of infection (Tiedemann 1997). In such conditions, GSH seems to be the limiting factor for a proper functioning of the AsA–GSH cycle during the progression of the infection. In tomato plants, the infection by the necrotrophic fungus B. cinerea caused a progressive decrease in GSH contents, whereas GSSG was barely affected (Kuźniak and Skłodowska 1999). The fungal infection also affected GSH-dependent enzymes. In that regard, an increase in GR activity was produced in tomato leaves in order to try to maintain the redox state of the glutathione. However, a decrease in other GSH-dependent enzymes, such as DHAR , occurred (Kuźniak and Skłodowska 1999). The same authors studied the effect of B. cinerea in the antioxidative mechanisms in chloroplasts from tomato plants (Kuźniak and Skłodowska 2001). These authors reported that B. cinerea infection promoted senescence symptoms, the chloroplasts being one of the earliest cell compartments affected, as indicated by the loss of chlorophyll observed even after 1 dpi. This effect was correlated with a decrease in chloroplastic GSH and total glutathione pools as well as a decrease in GPX, an enzyme that participates in the reduction of lipid hydroperoxides by using GSH as reducing power (Asada 1999).

Years later, Kuzniak and Sklodowska (2005) studied the changes of the AsA–GSH cycle in different cell compartments (chloroplasts, mitochondria, and peroxisomes) in B.cinerea-infected tomato leaves . The oxidative stress caused by the fungal infection affected all cellular compartments, although the authors observed organelle-specific changes, such variations being masked when data were analyzed in whole-leaf extract. A general decrease in glutathione concentration occurred by the infection in different cell compartments from tomato leaves, mitochondria and peroxisomes being the most affected organelles. In chloroplasts and mitochondria, the total glutathione contents declined after 2–3 dpi, but in peroxisomes, this decrease started earlier, only 1 dpi (Kuzniak and Sklodowska 2005). The reduction in total glutathione was parallel with a significant increase in GSSG, especially in mitochondria and peroxisomes. As a result, in all cell compartments, the infection produced an important decrease in the GSH/GSSG ratios, appearing earlier in mitochondria and peroxisomes than in chloroplasts, showing lower GSH/GSSG ratio at the initial state of the infection phase (Kuzniak and Sklodowska 2005). GSH-dependent enzymes were also affected by B. cinerea infection in the different cell compartments studied. In this sense, the infection induced a decrease in DHAR activity in whole-leaf extracts, mainly from 2–4 dpi. The response of chloroplastic DHAR was somewhat different to that observed in mitochondria or peroxisomes. No important effect was produced in chloroplast, and even an important increase after 3 dpi occurred. However, mitochondrial and peroxisomal DHAR activities decreased after 3 dpi (Kuzniak and Sklodowska 2005). Regarding GR activity , an initial increase at 1 dpi was maintained during the infection period in whole-leaf extracts as well as in chloroplasts. In contrast, mitochondrial GR peaked at 3 dpi and then progressively decreased until the end of the infection period (5 dpi), whereas peroxisomal GR showed a decline only at 2 dpi. The authors suggested that the increases in GR can be an effective protection to avoid an excessive GSSG accumulation in order to maintain the redox state of glutathione (Kuzniak and Sklodowska 2005).

Fusarium oxysporum is another necrotrophic fungus that produces the fusarium wilt (a vascular wilt fungal disease) in many plant species, including tomato, pepper, melon, or legumes, among others. The information about the effect of F. oxysporum infection on the glutathione metabolism of higher plants is very scarce. In 2002, García-Limones et al. studied the possible role of ROS production in the development of the fusarium wilt disease in chickpea in compatible and incompatible interactions. However, these authors did not measure glutathione contents but only GR activity (among other antioxidant enzymes). The authors observed that the first symptoms appeared 15–17 dpi in the susceptible cultivar (cv. JG62). During this period, about 50% of plants were systemically infected. At the end of the disease development phase (20–22 dpi), more than 90% of susceptible plants showed vascular infection. In the case of the resistant cultivar (cv. WR315), no evidences of infection were observed (García-Limones et al. 2002). The authors found a constitutive GR activity much higher in stems than in roots in both chickpea cultivars. In infected plants, a transient increase in GR occurred only in roots from susceptible plants, and at the end of the disease development (20–22 dpi), a correlation among GR increase, H2O2-scavenging enzymes (APX, CAT), and H2O2-producing enzymes (SOD) took place. Although GR activity did not show significant changes in stems during the development of the fungal disease, in the resistant cultivar, GR activity levels were higher than the susceptible cultivar. In stems, APX, CAT, and SOD activities were induced only in susceptible plants during the disease development. All these responses led the authors to suggest that the lack of induction of antioxidant enzymes in the stem of resistant plants can denote a less efficient ROS scavenging defense and thus a higher ROS level accumulation that could be related with the resistance mechanism against F. oxysporum infection (García-Limones et al. 2002).

The addition of specific fungus such as T. harzianum to plant growth substrates can increase plant yield and also reduce plant diseases produced by other plant pathogens present in soils. However, the mechanisms of action of these biostimulant and biocontrol effects are not fully understood and knowledge about their influence in the antioxidative metabolism is very scarce. The inoculation with T. harzianum increased FW of melon plants grown in different organic substrates (Bernal-Vicente et al. 2015). This response was parallel with the increase of some GSH-dependent enzymes, such as DHAR and GST (Bernal-Vicente et al. 2015). More specifically, the combination of T. harzianum with either citrus or bentonite compost stimulated DHAR activity in melon leaves, whereas the combination of T. harzianum with either peat substrate or bentonite compost increased leaf GST activity (Bernal-Vicente et al. 2015). The increase in DHAR involves a higher AsA-recycling capacity, and according to Gong et al. (2005), ascorbate and GST seem to play key roles in plant growth and development.

Mycorrhizae may help plants to grow in semiarid ecosystems improving their response to the environmental changes that involve the progressive increase in atmospheric CO2 concentration in a climate change context (Terrer et al. 2016). Mycorrhizal inoculation increased the plant biomass in three Mediterranean shrubs, Olea europaea L. ssp. sylvestris, Retama sphaerocarpa (L.) Boissier, and Rhamnus lycioides L. This stimulant effect in plant growth was related with increased mineral content (N, P, K, Mg, Fe, Ca, etc.) as well as with increased antioxidant capacity, including GSH-dependent enzymes. The inoculation with the allochthonous arbuscular mycorrhizal (AM) fungus Glomus claroideum strongly increased DHAR in the three mentioned shrubs. In contrast, the inoculation with a mixture of native AM fungi produced a lower stimulation of DHAR activity (Alguacil et al. 2003). The presence of G. claroideum also increased GR activity in R. sphaerocarpa and R. lycioides, whereas the inoculation with the mixture of native AM fungi only raised GR activity in R. lycioides plants, producing even higher increases than G.claroideum. However, no effect in GR was observed in the shrub O. europaea (Alguacil et al. 2003).

Since the abovementioned experiment was carried out in semiarid conditions, the mycorrhizal-induced increases in antioxidant enzymes could be a strategy used by such shrubs to face the ROS overproduction under the environmental conditions assayed. Specifically, and as far as GSH-enzymes are concerned, the increase in DHAR can involve a better capability to recycle AsA, whereas GR, in addition to GSH recycling, may serve to provide NADP+ availability to accept electrons from the photosynthetic electron chain in order to minimize the reduction of O2 to O2 −. In conclusion, these authors suggested that the increase in antioxidant enzymes could be involved, at least partially, in the beneficial effect of mycorrhizal colonization on the performance of shrubs species grown under semiarid conditions (Alguacil et al. 2003).

Oxidative damage to biomolecules is one of the most important mechanisms triggering nodule senescence in stressed nodules (Becana et al. 2000). Mycorrhizal symbiosis can also protect plants against premature nodule senescence induced by drought situations, as observed in soybean plants (Porcel et al. 2003). This response seemed to be linked to a higher GR activity in roots and nodules in mycorrhizal plants. These authors proposed that the higher GR activity in roots and nodules of mycorrhizal plants has contributed to the protection of nodules from premature senescence (Porcel et al. 2003).

5 Changes in the Subcellular Compartmentalization of Glutathione Under Biotic Stress Conditions

As an antioxidant, glutathione is involved in detoxifying ROS through the ascorbate–glutathione cycle (Foyer and Noctor 2009, 2013). Changes in the subcellular contents of glutathione during biotic stimuli reflect the occurrence of compartment-specific defense mechanisms , which is associated with compartment-specific ROS accumulation and oxidative stress . Since virtually all plant pathogens cause ROS generation and oxidative stress, changes in subcellular glutathione contents are therefore valuable biotic stress indicators within plants during situations of pathogen attack. Whereas the role of glutathione and, by extension, of the antioxidative metabolism in different organelles to abiotic stress has been well reported, the data on the glutathione compartment-specific role during plant–pathogen interaction is poorly understood. The following lines summarize the findings on this subject, discussing the existing connection between subcellular accumulation of glutathione and ROS, and the documented functional differences of the subcellular compartments during biotic stress situations.

5.1 Apoplast

Glutathione concentrations in the apoplast constitute a minor portion of its total pool (Vanacker et al. 1998, 2000; Zechmann et al. 2008; Tolin et al. 2013). In leaf cells, the apoplast contains only 1–2% of the total cell glutathione (Foyer et al. 2001), although higher values has been reported in pea leaves (Hernández et al. 2001). Moreover, the glutathione homeostasis is easily alterable in the apoplast due to the absence of systems to regenerate the reduced glutathione form. Consequently the capacity of redox buffering in the apoplast is weaker than that found inside the cell (Horemans et al. 2000). These facts make the apoplast a sensor of changes in the environmental conditions (Tolin et al. 2013). In response to biotic stimuli, the low buffering capacity of the apoplast favors a rapid accumulation of ROS (Mittler 2002). Herein, ROS overproduction is one of the early events following pathogen attack, occurring mainly in the apoplast via plasma membrane NADPH oxidases (Torres et al. 2002; Suzuki et al. 2011) and cell wall peroxidases (Bindschedler et al. 2006). This has been reported in numerous plant–pathogen interactions as common event of plants’ hypersensitive response (HR) leading to programmed cell death (Lamb and Dixon 1997; Wojtaszek 1997; Jones and Dangl 2006).

5.2 Chloroplasts and Peroxisomes

Changes in glutathione contents in chloroplasts and peroxisomes are involved in plant defense against pathogens. P. syringae and B. cinerea caused enhanced accumulation of glutathione in Arabidopsis at early stages of infection, reaching 73% and 450% of control levels in chloroplast and peroxisomes, respectively. At later stages of infection, a pronounced decrease of glutathione in both cell compartments was accompanied by increased ROS accumulation (Großkinsky et al. 2012). This highlights the importance of glutathione during stress signaling. Similar results have been achieved in peroxisomes of tomato plants during B. cinerea infection, where the initial increase of glutathione was followed by a pronounced decrease and the disruption of the antioxidative system in peroxisomes (Kuźniak and Skłodowska 2005). Some authors have pointed out the existence of a connection between apoplastic and chloroplastic ROS signaling during the biotic response, in which the chloroplast may act as an amplifier of the signal from the apoplast (Joo et al. 2005; Vahisalu et al. 2010). As antioxidants protect the organelles by counteracting the level of ROS (Green et al. 2006), their contents in chloroplast can be decisive in the tuning of ROS signaling.

5.3 Mitochondria

Mitochondria possess a strong antioxidant system to protect them against the constant generation of ROS, in which GSH is of particular importance (Zechmann et al. 2008). In this sense, the drop of glutathione contents is associated with ROS accumulation and oxidative stress in this compartment, leading to the induction of programmed cell death (Vianello et al. 2007). Arabidopsis plants infected with B. cinerea displayed a strong drop of total glutathione content in mitochondria 48-h post-inoculation, which correlated with a strong increase of H2O2 in this compartment and the development of necrosis symptoms (Simon et al. 2013). In a similar way, the infection of N. tabacum with an incompatible strain of tobacco mosaic virus (TMV ) provoked a depletion of glutathione contents in mitochondria and the development of necrotic spots (Király et al. 2012). In tomato plants, B. cinerea produced a decrease of glutathione contents mainly in mitochondria, coupled with the accumulation of oxidized glutathione 48-h post-inoculation (Kuźniak and Skłodowska 2005).

5.4 Nucleus

Glutathione in nucleus plays essential roles in protection of DNA against oxidative damage, cell proliferation, DNA synthesis, and regulation of the nuclear matrix organization and proteins (Green et al. 2006; Díaz-Vivancos et al. 2010b; Go and Jones 2010). Glutathione also regulates the expression of genes involved in the activation of plant defense mechanisms (Han et al. 2013). The roles of glutathione in nuclei during pathogen attack are not fully understood. However it has been reported that there is a notable accumulation of glutathione in nuclei during early stages of viral, fungal, and bacterial infection. Such is the case of Arabidopsis plants infected with P. syringae (Großkinsky et al. 2012) and B. cinerea (Simon et al. 2013), leaves of Cucurbita pepo infected with ZYMV (Zechmann et al. 2005), and TMV-infected N. tabacum plants (Király et al. 2012). In Arabidopsis, increased GSH accumulation in the nuclei has been reported to be concomitant with decreased levels in the cytosol, followed by enhanced levels in the whole cell (Diaz-Vivancos et al. 2010a). Similarly, the increase of total glutathione in the nuclei of Arabidopsis plants infected with P. syringae (Großkinsky et al. 2012) and B. cinerea (Simon et al. 2013) was followed by a rapid accumulation of glutathione in the chloroplasts and cytosol. It was hypothesized that the initial accumulation of GSH in nuclei is perceived as a signal in order to increase its levels in the whole cell under stress conditions (Diaz-Vivancos et al. 2010a).

5.5 An Outlook of the Methods to Determine Subcellular Glutathione Concentrations

Determination of GSH and GSSG on the subcellular levels is technically challenging as the sample preparation itself can be perceived as a stress, thus altering the GSH levels. There are two major approaches to study the compartment distribution of glutathione in plants, presenting advantages and disadvantages inherent in both types of methods: (1) biochemical determination after subcellular fractionation of plant tissues and (2) microscopical visualization following glutathione labeling. Large amount of starting plant material and cross-contamination among fractions during organelle isolation are the major constraints of the biochemical determination. Moreover, the equivalence of the results obtained in vitro with the actual glutathione levels in vivo is, often, uncertain. Nevertheless, these methods allow the determination of glutathione in millimolar range, and glutathione redox state can be calculated through the measurement of both reduced and oxidized forms (Jiménez et al. 1997; Vanacker et al. 1998; Kuźniak and Skłodowska 2001; Ohkama-Ohtsu et al. 2007; Krueger et al. 2009). Regarding the microscopic approaches , they can be separated into light microscopical methods, in which glutathione is labeled with specific antibodies or dyes (Meyer and Fricker 2000; Müller et al. 2005; Zechmann and Müller 2010), fluorescence microscopy determination following labeling with redox-sensitive green fluorescent protein (GFP)(Meyer et al. 2007; Gutscher et al. 2008), and electron microscopy following immunogold labeling (Gao et al. 2012).Unfortunately, the antibody that is currently used for detecting glutathione cannot differentiate between the reduced and oxidized form (Zechmann et al. 2005).These methods allow determining the localization and concentration of glutathione in vivo in the different cell compartments in a more accurate way, which opens up new prospects in the study of glutathione dynamics in plant defense mechanisms. Main limitations of the microscopic techniques are intrinsic to the sample preparation and visualization, as mechanical separation of cells and tissues and exposure to light and dehydration under microscope can be perceived as a stress to the sample.

6 Conclusion and Future Perspectives

Biotic environmental stress situations lead to considerable yield drop causing important economic losses worldwide. One common consequence of exposure to stress conditions is the increased production of ROS. The most potentially deleterious effect of ROS is that at high concentrations they trigger genetically programmed cell suicide events. Far from being only damaging agents, ROS are also used by plants as second messengers in signal transduction cascades in a variety of process, their accumulation being crucial for plant development as well as defense (Foyer and Noctor 2013). In plants, ROS production and scavenging are intimately linked, and the balance between them will determine defense responses. Thus, the major low-molecular-weight antioxidants ascorbate and glutathione determine the specificity of this oxidative signaling. The tripeptide glutathione exerts a strong influence on plant responses against pathogens, not only as an antioxidant but also as a defense signaling compound. Due to its relatively high cellular concentration, GSH acts as ROS scavenger or sacrificial nucleophile. Although many other secondary metabolites can function similarly, GSH is distinguished from most of these compounds by three main characteristics: (1) the presence of specific enzymes that couple GSH to the oxidative metabolism, (2) the existence of relatively stable oxidizing form, and (3) the recycling of GSSG to GSH by high-capacity enzyme-based system.

Biological systems adapt to changing environments by reorganizing their cellular and physiological program with metabolites representing one important response level. Glutathione can thus be considered as multifunctional metabolite that is important in redox homeostasis and signaling as well as in developmental and defense reactions (Foyer and Noctor 2011). Changes in redox metabolism will inevitably modify much larger signaling network that integrates information from many pathways regulating plant growth and defense responses. The importance of GSH and GSH-related enzymes in reducing the incidence of plant pathogens and symptom development has been widely reported. In this sense, treatments inducing increases in GSH and/or the redox state of glutathione can be beneficial during plant–pathogen interactions. Nevertheless, how pathogens alter plant metabolism and biochemistry is not fully understood yet. New knowledge on this topic and the discovery of new products stimulating GSH redox homeostasis would lead to develop new strategies to achieve a durable tolerance against pathogens.