Redox Protein Thioredoxins: Function Under Salinity, Drought and Extreme Temperature Conditions

Abstract

Salinity, drought and extreme temperatures are environmental constraints that seriously alter plant growth and productivity. Oxidative and nitrosative stress are associated with these conditions and redox regulation is emerging as a key factor in the response of plants to such adverse environments. Thioredoxins (Trxs) are ubiquitous proteins present in the different cell compartments that control the structure and function of target proteins by reducing disulfide bridges in their redox active sites. The involvement of Trxs in the response of plants to abiotic stress is a subject of increasing interest due to the diverse target proteins that they regulate. In this chapter, we will first analyze the importance of salinity, drought and extreme temperatures as abiotic stress conditions in plant physiology. Furthermore, we provide information about the transcriptomic, genomic and enzymatic changes related to Trxs taking place under these adverse conditions, together with those observed in their protein targets. In this chapter we seek to unravel the specific roles of Trxs as redox sensors and their involvement in the ROS/RNS-mediated signal transduction.

1 Effect of Salt, Drought and Extreme Temperatures Stresses

The reduction in the availability and quality of cultivated lands and water resources, as well as extreme meteorological conditions, can induce different types of abiotic stresses, such as salinity, drought and extreme temperatures. These factors seriously affect plant development and limit agromonic performance by 65–85%, with a negative effect for fruit production (Shabala et al. 2006). Stress conditions can induce physiological, molecular and biochemical changes that alter several cellular processes in the plant, affecting membranes, osmotic equilibrium, activity of proteins and the establishment of an oxidative and/or nitrosative stress by the accumulation of reactive oxygen species and reactive nitrogen species (ROS/RNS). Salinity and drought are the most important conditions that strongly impair vegetal quality and total productivity in the world (Lobell et al. 2011). Hence, response mechanisms and adaptation strategies are currently one of the most important topics of study in plants (Munns and Tester 2008). Both stresses provoke an increase in the hydric and osmotic potential of the soil water, inducing a secondary oxidative stresses, while high salt concentration also induces ion toxicity (Zhu 2001). Although all salts may have negative effects on plant growth and reproduction, the term salt stress usually refers to the damage caused by NaCl. Salinity affects more than 800 million hectares of cultivated lands in the world and considering that one third of the population lives in lands irrigated with low quality water in zones of scarce pluviometry, salinity and drought could be the most frequent and severe stress conditions in the future (Munns and Tester 2008). Thus, the increase in the tolerance of crops to these adverse conditions may constitute a priority objective to assure food production in the coming decades.

Salinity in non-halophytic plants reduces growth, increases the relation of root to shoot systems, induces chlorosis by loss of chlorophyll, leads to wilting and, finally, may provoke plant death (Parida et al. 2004). Salt stress and water limitation negatively affect photosynthesis and respiration depending on the duration and severity imposed. The effects are related to stomatal and mesophyll limitations, affecting CO₂ diffusion and also photosynthetic biochemical limitations (Flexas et al. 2004; Chaves et al. 2009). Drought and salinity stress provoke a highly complex response, which involves the interplay of limitations at different sites and time scales in relation to plant development (Chaves et al. 2009). There are also several reported effects of salinity on plant respiration with specific rates being enhanced, diminished or unaltered (Khavari-Nejad and Chaparzadeh 1998; Epron et al. 1999; Koyro 2006), although measurements of in vivo electron partitioning between the alternative and cytochrome c pathways (AP, CP) have been carried out in pea leaves, showing a maintained alternative oxidase (AOX) activity under salinity, which may contribute to diminishing the further accumulation of ROS in mitochondria and reflects the presence of an active form of this protein (Martí et al. 2011). Plants have a high plasticity and can modulate their development and morphology adjusting physiological and biochemical mechanisms (Taji et al. 2004; Acosta-Motos et al. 2015). This includes the compartmentalization or exclusion of toxic ions and the synthesis of osmolytes, hormonal regulation (Gómez et al. 2004; Kosová et al. 2013; Lázaro et al. 2013) and/or antioxidant components to regulate the redox homeostasis, with several studies describing a correlation between tolerance to salinity and upregulation of specific antioxidant enzymes (Hernández et al. 1993, 2001; Mittova et al. 2003; Flowers and Colmer 2008). Respiration is also altered under drought situations and a strong increase in the division of electrons through the alternative pathway has been observed under severe water limitations in soybean (Ribas-Carbó et al. 2005). These effects were mainly due to a reduction and an increase in the activities of the CP and AP, respectively.

Almost all plants are exposed to daily changes of high and low diurnal and nocturnal temperatures, as well as seasonal changes that can become temperatures below zero degrees Celsius during the winter and extremely high during the summer. These thermal differences have been used throughout evolution to carry out essential processes such as the adjustment of circadian rhythms, dormancy and germination of seeds or flowering (Finch-Savage and Leubner-Metzger 2006; Kim et al. 2009; Thines and Harmon 2010). During adaptation to low temperatures, plants accumulate compatible solutes as proline, sugars and dehydration-related proteins (dehydrins) to prevent freezing damage and drought stress due to the lack of available liquid water (Korn et al. 2010). Under extreme temperatures, plants induce the expression of some heat stress proteins (HSP), also increased under drought and oxidative stress (Cho and Choi 2009; Banti et al. 2010), and in the adaptation and tolerance to heat, they also induce the accumulation of proline, enhance the antioxidant systems and increase the chaperone activity of some proteins (Frank et al. 2009). The response to extreme temperatures is closely related to changes in the structure and stability of proteins. Heat stress provokes the oligomerization of some proteins, and this modification induces the change from a disulfide reductase activity to molecular chaperone activity in some proteins (Lee et al. 2009; Park et al. 2009). As another example, low temperature enhances the DNA binding activity of transcription factor CBF2, which increases the expression of the most cold-inducible operons in plants, while this binding activity decreases when temperature rises (Ruelland et al. 2009).

The extreme temperature tolerance is characteristic of each species and dependent on the environmental conditions. In general, growth in a non-optimal temperature provokes the decrease in biomass and damage in tissues by alteration of physiological processes (Ruelland and Zachowski 2010) provoking important losses in the quality and productivity of sensitive fruits (Airaki et al. 2012). The effect of temperature on the enzymatic activities influences cell metabolism and changes may generate signaling pathways triggering cold/heat responses. The efficiency of enzymes during changing temperatures can be characterized by the Q₁₀-value—that is, the factor by which an enzyme’s activity is increased when the temperature rises by 10 °C. The fine interplay among enzymes and metabolites can be disturbed by these temperature changes. This value is used to measure the temperature-dependent photosynthetic efficiency. During low or high temperature, the most important changes are related to the reduction in RUBISCO activity, which is very temperature-sensitive (Kumar et al. 2009). In addition, under low temperatures, the limitation of the photosynthetic process is related to the lower return of inorganic phosphate (Pi) from sucrose synthesis for ATPase activity in the chloroplast (Yamori and von Caemmerer 2010). The imbalance in photosynthesis produces the accumulation of ROS in the chloroplast with almost all antioxidant systems being thermolabile (Panchuk et al. 2002).

The alteration of the membranes fluidity is important among the physiological changes occurring during adaptation to extreme temperatures. A decrease of temperature leads to a reorganization of unsaturated fatty acid composition causing an increase in rigidity with slower movements of cell membranes and a reduction in fluidity. By contrast, the increase of temperature causes a decrease in the rigidity of the membrane, increasing speed and fluidity. This reorganization is controlled in the plasmatic membrane by signaling cascades mediated by heat activation MAP kinases (HAMPK) and NAC type transcription factors (Fig. 1) (Seo et al. 2010; Sangwan and Dhindsa 2002).

Fig. 1

Signaling pathways in response to stresses. Heat and cold stresses affect the plasma calcium channels, and thus the MAPK cascade leading to gene expression. Secondary signals like ROS lead to stress tolerance

2 ROS and RNS Generation

The stress response is regulated by different signals including ROS and RNS that mediate post-translational modifications and redox balance among the different cellular compartments (Mittler et al. 2004; Lázaro et al. 2013; Foyer and Noctor 2016). The cell basal oxidative metabolism induces the production of ROS/RNS at low concentrations (Sies 2017); whilst, under salinity and drought stresses, a rise in their production and concentration takes place in the different organelles (Hernández et al. 1993, 1995; Gómez et al. 2004). This accumulation provokes metabolic disorders, cellular damage, and premature senescence or necrosis by oxidative damage in nucleic acids, proteins, lipids or pigments (Møller and Tester 2007; Habib et al. 2016; Jiménez et al. 1998; Ortiz-Espín et al. 2015). ROS include non-radical molecules like singlet oxygen (¹O₂) and hydrogen peroxide (H₂O₂), as well as free radicals, such as superoxide (${ {\text{O}}_{2}}^{ \cdot - }$) and hydroxyl radicals (^·OH). In addition, RNS include the free radicals nitric oxide (^·NO) and nitrogen dioxide (^·NO₂) and the molecules of peroxynitrite (ONOO⁻) and nitrite/nitrate $({{\text{NO}}_{2} }^{ - } /{{\text{NO}}_{3} }^{ - } )$. However, in recent years, ROS/RNS involvement has been reported to activate the stress response including antioxidant pathways (Suzuki et al. 2012; Noctor et al. 2014). In this way, these reactive species are considered to be cellular stress alarms and/or secondary messengers involved in the signal transduction under adverse conditions (Fig. 1). Among the multiple signaling cascades activated as a response to different abiotic stresses, like the osmotic one imposed by salinity and drought, together with Ca signaling and MAPK mitogen-activated protein kinase activation (Sanders et al. 2002; Wu et al. 2011), ROS production is a key event (Hasegawa et al. 2000). ROS signaling is mainly based on a balance between production and scavenging, which occurs in plants to maintain a balanced intercellular ROS concentration. However, relatively low levels of ROS are involved in stress tolerance, and, due to its high stability and permeability across membranes, H₂O₂ is still considered to be the best candidate as a signaling molecule. In fact, a maintained low level of endogenous H₂O₂ has been suggested to enhance tolerance to a number of abiotic stresses (Mittler 2002; Apel and Hirt 2004) and a constitutive increase of H₂O₂ in plants can improve multistress tolerance in plants (Van Breusegem et al. 2008; Niu and Liao 2016; Ortiz-Espín et al. 2017). However, the simultaneous induction of growth and stress tolerance by elevated ROS content is still under debate (del Río 2015). In fact, it has been reported that, as a result of an increased generation of ROS, plants reduce the transcript levels of certain carbohydrate metabolism and photosynthesis-related genes that suppress plant growth (Sakamoto 2004).

In the chloroplasts, the generation of ${{\text{O}}_2}^{.-}$ occurs at the level of photosystem I and II, ferredoxin NADP⁺-reductase and monodehydroascorbate reductase (Miyake et al. 1998). ${{\text{O}}_2}^{.-}$ can dismutate to H₂O₂ by superoxide dismutases (SODs) (Asada 2006) and this peroxide can generate.OH in the Fe–S centers through the Fenton reaction (Halliwell and Gutteridge 2015). In addition, ¹O₂ generation is produced in the chlorophyll excitation associated with the electron transport as a product of lipoxygenase (Triantaphylidès and Havaux 2009) (Fig. 2).

Fig. 2

Production of ROS in different subcellular compartments: mitochondria, chloroplast, apoplast, endoplasmic reticulum and peroxisomes. In mitochondria, the ${ {\text{O}}_{2}}^{ \cdot - }$ production is mainly at complex I and III during respiration. In the chloroplast and during photosynthesis, ¹O₂ and ${ {\text{O}}_{2}}^{ \cdot - }$are produced in the photoystem II (PS II) and photosystem I (PS I), repectively. In peroxisomes, ROS are acumulated during photorespiration and during β-oxidation of fatty acid by glycolate oxidase (GO) forming H₂O₂, directly by xanthine oxidase enzyme (XO) or monodehydroascorbate reductase (MDHAR) in the peroxisomal membrane. The NADPH oxidases in the plasma membrane and the cell-wall peroxidases are the main producing sources of ${ {\text{O}}_{2}}^{ \cdot - }$ and H₂O₂ in the apoplast. In the endoplasmic reticulum, the production of ROS is mainly in the cytochrome P450. The superoxide dismutase (SOD) enzymes eliminate the superoxide radical producing H₂O₂

Mitochondria are very active organelles that present an electron transport chain (ETC) responsible for respiration in which the electron gradient generates an electrochemical potential favoring ATP synthesis by ATP synthase. The mitochondrial metabolism contributes significantly to the total ROS production in the cell in the form of ${{\text{O}}_2}^{.-}$ (Noctor et al. 2007), with complex I and III as the main sites (Noctor et al. 2007), although under specific conditions they can be produced at the level of complex II in the reverse electron transport (Turrens 2003). Moreover, plant ETC contains complementary enzymes that do not contribute to the proton gradient and do not generate ATP. These pathways are NADP(H) dehydrogenases and alternative oxidase (AOX), which may function as security valves to limit the ROS generation, maintaining the ETC in an oxidized form (Møller and Sweetlove 2010; Millar et al. 2011). Interestingly, the alteration in the ROS level produced by mitochondria during abiotic stress can induce retrograde signaling between this organelle and the nucleus with an effect on plant acclimation (Woodson and Chory 2008) (Fig. 2).

Peroxisomes are organelles in which H₂O₂ is quite high as a result of an oxidative metabolism (del Río and López-Huertas 2016). The main sources of H₂O₂ are glycolate oxidase in photorespiration, acyl-CoA oxidasa in the β-oxidation of fatty acids, several flavin oxidases (such as urate and xanthine oxidases) and various SODs (Sandalio and Romero-Puertas 2015). The main sites of ${{\text{O}}_2}^{.-}$ production are xanthine oxidase in the matrix and an electron chain in the membrane composed of MDHAR, NADH and cytochrome b (del Río et al. 1992). In addition, ¹O₂ is produced in peroxisomes as well as in mitochondria and the nucleus (in a light independent manner) in non-photosynthetic tissues of Arabidopsis, and this generation is increased in the roots of plants subjected to biotic and abiotic stress in darkness. It has been suggested that this production may be a result of the reaction of ${{\text{O}}_2}^{.-}$ with H₂O₂ via a Haber-Weiss’ mechanism (Mor et al. 2014) (Fig. 2).

Related to RNS, NO is a lipophilic gaseous free radical and an important metabolite in plants involved in intra- and intercellular signaling, due to its capacity to diffuse among cellular membranes. NO can react with proteins, lipids and nucleic acids although its life time in vivo is quite short (around 10 s) (Pfeiffer et al. 1999). NO is involved in several processes such as seed germination, flowering, stomatal closure, maturation and senescence, growth regulation, hormonal signaling, stress response, Fe homeostasis and pathogen defense among others (Martí et al. 2011; Camejo et al. 2013; Liu et al. 2015; Shi et al. 2015; Wang et al. 2015b). NO can react with ${{\text{O}}_2}^{.-}$ to produce peroxynitrite that is able to mediate the nitration of Tyr (or, less frequently, Trp) residues in the proteins, thus altering their structure and activity (Dalle-Donne et al. 2005). Together with this post-translational modification (PTM), RNS also provoke S-nitrosylation and S-glutathionylation by nitrosoglutathione (GSNO) with influence on the protein function (De Pinto et al. 2013; Camejo et al. 2015). The subcellular localization of GSNO in pea mitochondria has been reported (Camejo et al. 2013; Corpas et al. 2013) and it is considered to be a vehicle of NO in the cell, allowing this molecule to expand its biological activity. Two major enzymatic pathways have been proposed as participating in the NO formation in plants: the reduction of nitrite to NO by a nitrate reductase (NR) and the oxidation of l-arginine to l-citrulline by a nitric oxide synthase (NOS)-like activity (Fröhlich and Durner 2011; Gupta et al. 2011). The latter activity was reported in plant peroxisomes, chloroplasts and in isolated root mitochondria (Corpas et al. 2001; Jasid et al. 2006; Gupta and Kaiser 2010) although the enzyme has not been characterized. Thus, RNS are involved in the peroxisomal metabolism, which suggests that these organelles may play a key function in the NO signal transduction. In mitochondria, there are several described sources of NO production. Under hypoxic conditions, a reduction of nitrite to NO by mitochondrial ETC has been reported to produce small amounts of ATP (Stoimenova et al. 2007) and also a mitochondrial nitrite-reducing activity (NR) has been detected in organelles isolated from roots at the site of cytochrome c oxidase (COX) and complex III and AOX, although a clear mechanism has only been established for COX under hypoxia (Igamberdiev et al. 2014; Yu et al. 2014). Targets of NO and its derivates in mitochondria are the ETC components inhibiting the CP, whereas the AP is only partially inhibited (Day et al. 1996; Martí et al. 2013). In addition, it has been shown that pea Mn-SOD was not inactivated by NO upon DETA NONOate treatment of mitochondria (Martí et al. 2013), and, together with the partial insensitivity of AOX to NO, represent important mechanisms to prevent deleterious effects on the respiratory activity during stress situations. Other examples of NO action are the inhibition by S-nitrosylation of the P protein of mitochondrial glycine decarboxylase (GDC) activity and Prx IIF, in which this PTM provoked a change in its peroxidase to chaperone activity (Palmieri et al. 2008; Camejo et al. 2015), and also the inhibition of peroxisomal catalase and glyoxylate oxidase involved in photorespiration, β-oxidation and ROS detoxification (Ortega-Galisteo et al. 2012).

Under different stress conditions including salinity, water deficit or extreme temperatures, ROS are generated at the cellular level, with possible negative consequences, by the induced oxidative stress (Hasegawa et al. 2000; Hernández et al. 1999, 2000). Early responses to salinity and drought include a reduction in stomatal conductance to avoid water loss (Chaves et al. 2009), in this way decreasing internal CO₂ content and carbon assimilation. In fact, during photosynthesis and under drought stress, there is a higher leakage of electrons to O₂ by the Mehler reaction in wheat and sunflower (Biehler and Fock 1996; Sgherri et al. 1996). In addition, the photorespiratory pathway is enhanced, especially when RuBP oxygenation is maximal, due to the limitation of CO₂ fixation, accounting for over 70% of the total H₂O₂ production under drought stress conditions (Noctor et al. 2002). The decline of the carboxylase reaction of Rubisco stimulates photorespiration, leading to H₂O₂ production in peroxisomes (Noctor et al. 2002) and desiccation and salinity have been shown to perturb the redox state, inhibit antioxidant mechanisms and increase ROS production in peroxisomes (del Río et al. 1996; Mittova et al. 2003). Elsewhere, mitochondria are described as key organelles in the ROS generation induced as a response to different stress conditions like salinity (Hernández et al. 1995; Gómez et al. 1999; Foyer and Noctor 2000) and drought (Loggini et al. 1999; Boo and Jung 1999). Thus, salinity increases respiratory rates, with the consequent electron leakage to O₂ in mitochondria (Miller et al. 2010). In addition, during drought, respiratory rate and mitochondrial ATP synthesis increased to compensate for the lower rate of chloroplast ATP synthesis, with the concomitant enhancement of mitochondrial ROS production (Atkin and Macherel 2009). Other important sources of ROS production are the plasma membrane-bound NADPH oxidase and the apoplastic diamine oxidase, which have been shown to be activated during salt stress and therefore to contribute to extracellular ROS propagation (Mittler et al. 2011). NaCl induced an oxidative stress in the apoplast of treated pea leaves related to the appearance of highly localized ${{\text{O}}_2}^{.-}$/H₂O₂-induced necrotic lesions in the minor veins (Hernández et al. 2000). During temperature stress, the rates and cellular sites of ROS generation play a central role in stress perception and protection. Heat stress causes impairments in mitochondrial functions and oxidative damage causing lipid peroxidation (Vacca et al. 2004). Cold stress has been shown to enhance the transcript, protein and activity of different ROS-scavenging enzymes (Suzuki and Mittler 2006) and low temperature stress induces H₂O₂ accumulation in Arabidopsis cells (O’Kane et al. 1996), while, in maize leaves, a differential distribution of H₂O₂ and antioxidants occurs between the bundle sheath and mesophyll cells (Pastori et al. 2000). Interestingly, ROS signature produced under conditions of stress combination is unique and provokes a specific set of physiological responses. As an example, cytosolic and not chloroplastic H₂O₂ in Arabidopsis APX1 mutants has been described to be important for acclimation to a combination of drought and heat stress (Koussevitzky et al. 2008).

Related to RNS, an enhancement in NO content has been observed in different species when grown under salinity, as in pea leaves, in which part of the NO localized in mitochondria is accompanied by an increase in mitochondrial GSNOR activity in response to short and long-term NaCl treatment, as well as presenting a higher number of nitrated proteins. In addition, the study of S-nitrosylation protein pattern indicated that PTMs seem to modulate respiratory and photorespiratory pathways, as well as some antioxidant enzymes, through differential S-nitrosylation/denitrosylation in control conditions and under salt stress (Camejo et al. 2013), pointing to these modifications being key events in the response. The production of NO in response to salt treatments has also been reported in olive trees with evidence of the establishment of nitrosative stress shown by an increase in total nitrosothiols (SNOs) and tyrosine-nitrated proteins (Valderrama et al. 2007). The exogenous application of NO donors to salt-treated maize plants increased the salt-stress tolerance by elevating the activities of the proton pump and the Na⁺/H⁺ antiport of the tonoplast. In Lupinus luteus, nitroprusside (SNP) treatment restored the inhibition of germination by salinity (Kopyra and Gwózdz 2003; Zhang et al. 2006). An induction of the antioxidant system has been shown in pretreatments of citrus plants with SNP when grown under salinity, parallel to a reduction in protein carbonylation and changes in the pattern of S-nitrosylated proteins, with all mechanisms providing major resistance to salinity stress (Tanou et al. 2009). Peroxisomes are also required for NO accumulation in the cytosol of root cells, which may be mediated by a putative calcium-dependent NOS activity, participating in the generation of peroxynitrite and in the increase of protein tyrosine nitration (Corpas et al. 2009). Furthermore, the exogenous application of NO donors reduced oxidative stress and increased the resistance to salinity in plants and germinating seeds (Kopyra and Gwózdz 2003). Under water limitations, the increase in NO production has been described in maize with a parallel induction of NOS and some antioxidant activities (Sang et al. 2008). Exogenous NO treatments also alleviated the water loss and the produced oxidative damage (Hao et al. 2008). NO generation has been observed in roots of cucumber seedlings under drought, accompanied by an increase in lipoxygenase activity, which was reduced by an exogenous NO application (Arasimowicz-Jelonek et al. 2009). Under low temperature stress, a decrease in NO has been described parallel to increases in GSH, peoxynitrite, GSNOR activity and nitrated proteins in pepper fruits (Airaki et al. 2012). In other plant species such as pea and Arabidopsis leaves, a rise of the NO content was observed, accompanied by an increase of SNOs, GSNOR activity and tyrosine-nitrated proteins in pea (Corpas et al. 2008; Cantrel et al. 2011). All these results support the existence of a link between low temperature stress and NO metabolism.

3 Control of ROS/RNS Under Stress

The redox homeostasis and oxidative stress are controlled by molecules and antioxidant systems, including enzymes such as superoxide dismutase isoforms (Fe-SOD, Cu/Zn-SOD and Mn-SOD) present in all cell compartments to scavenge ${{\text{O}}_2}^{.-}$ in a dismutase reaction that produces hydrogen peroxide (Sevilla et al. 1982; Hernández et al. 2001; del Río et al. 2003; Gómez et al. 2004). This H₂O₂ is scavenged by peroxisomal catalase in a high velocity and affinity reaction, by the ascorbate–glutathione (ASC–GSH) cycle and the thioredoxin/peroxiredoxin system. In the ASC–GSH cycle, the heme-containing ascorbate peroxidase (APX) scavenges H₂O₂ using reduced ASC that is oxidized to monodehydroascorbate (MDA) or dehydroascorbate (DHA). These oxidized forms are reduced by FAD-containing monodehydroascorbate reductase (MDHAR), and dehydroascorbate reductase (DHAR) using NADPH and GSH as reductants, respectively. The flavoprotein glutathione reductase (GR) is the enzyme in charge of the reduction of oxidized GSSG in a NADPH-dependent manner (Foyer and Halliwell 1976; Jiménez et al. 1997; Asada 1999).

The Trx/Prx system involves the redox proteins thioredoxin, peroxiredoxin and sulfiredoxin and is mainly in charge of the reduction of thiol residues in oxidized proteins present in the different cell compartments (Serrato and Cejudo 2003; Serrato et al. 2004; Lázaro et al. 2013; Calderón et al. 2017a, b). Disulfide generation is a reversible process in cells under optimal or slightly oxidant conditions protecting Cys residues from over-oxidation, which can lead to protein degradation, although it has been recently reported that the sulfinic groups in some proteins can be reverted by the action of sulfiredoxin (Srxs) (Iglesias-Baena et al. 2010). Moreover, functional changes derived from redox modifications can affect the transcriptional process, phosphorylation and other events involved in signaling, or can alter the metabolic fluxes by modification of the enzymatic activities (Reczek and Chandel 2015). The main systems based on the regulation of thiol-disulfide groups are related to proteins such as thioredoxins (Trxs) and glutaredoxins (Grxs), which, together with their roles in redox regulation, can be substrates of reductive enzymes such as peroxiredoxins (Prxs) or ribonucleotide reductases (RR) (Courteille et al. 2013). The redox state of the cell may depend then on the thiol and disulfide groups of the antioxidants such as GSH and the proteins Trx, Prx, Srx and Grx, among others. Moreover, it is considered that the redox state of the different cell compartments depends on the oxidized/reduced rates of GSH and Trx (Go and Jones 2010). In this chapter, we draw attention to the Trx system, mainly in its potential role in the response of plants to salinity, drought and extreme temperatures.

4 Thioredoxins in Higher Plants

Thioredoxins are small redox proteins (around 14 kDa) present in bacteria, Eukaria and in Archea (Holmgren 1985; Atkin and Macherel 2009) that are in charge of the reduction of specific disulfide bonds in many target proteins, in this way regulating their structure and function. Interestingly, new functions are being described for these proteins, such as a denitrosylase activity of a Trx h-type involved in plant immunity (Kneeshaw et al. 2014). Trxs in plants are especially numerous, and there are at least 10 types with more than 40 members present in different cellular compartments (Fig. 3), which are unified by the conserved small Trx domain. This consists of a four-stranded β sheet sandwiched by three α helices and an active-site sequence that contains two redox-active Cys residues in a conserved motif WCG/PPC. The number of isoforms seems to sustain plants with an additional antioxidant system, compared with yeast, which presents three isoforms, Escherichia coli with two Trxs, or mammals where only two types of Trx have been described: Trx1 and Trx2, in the cytosol and mitochondria, respectively (Schürmann and Jacquot 2000; Lillig and Holmgren 2007). Two distinct families of Trxs can be distinguished based on their amino acid sequences. Family I include proteins that contain one Trx domain, whereas Family II is composed of fusion proteins with one or more domains coupled to additional domains. In A. thaliana, there are 21 genes of typical thioredoxins in the Family I, which could be divided into eight classes (f, m, x, z, y, h, s and o), distributed in different cellular compartments (Fig. 3). The isoforms m, z, x and y are related to prokaryote organisms while the others are exclusively present in eukaryotic organisms (Gelhaye et al. 2004). Family II includes thioredoxin reductase C (NTRC), which contains an N-terminal thioredoxin reductase (NTR) and a C-terminal Trx domain, and which is considered to be an NADPH-dependent thioredoxin, and thioredoxin reductase protein (Pérez-Ruiz et al. 2006).

Fig. 3

Phylogenetic tree of in the thioredoxin protein family in different organisms. The phylogenetic tree was stimated using the full protein sequence with MEGA7. Prokaryote: Escherichia coli (Ec). Archea: Methanobrevibacter smithii. Red and green algae: Rhodocaete parvula (Rp), Chlamydomonas rehinartii (Cr). Angyosperms: Picea mariana (Pm). Gymnosperms: A. thaliana (At), Zea mays (Zm). Animals: Homo sapiens (Hs)

There are two different electron donor systems to reduce Trxs (Fig. 4): NADPH-dependent system, using the enzyme thioredoxin reductase A (NTRA), B (NTRB) and C (NTRC); and ferredoxin reductase (FDR)-dependent system, using the enzyme ferredoxin-thioredoxin reductase. All FDR-dependent Trxs are functional in the light phase while during the dark phase or low light intensity, these Trxs and their targets are oxidized and inactive (Scheibe 1981). However, NTRC is an especially important thioredoxin in chloroplast for the maintenance of the functions during the dark phase of the pentose phosphate pathway (Pérez-Ruiz et al. 2006). In A. thaliana the FDR-dependent isoforms present exclusively in chloroplast are m1, m2, m3, m4, f1, f2, x, y and z. Initially, the first Trxs identified in plants were the isoforms m and f in Spinacea oleracea, where they were described for their ability to activate the NADP-dependent malate dehydrogenase (NADP-MDH), preferentially by Trx m and fructose-1,6-bisphosphatase (FBPase) by Trx f (Buchanan 1980; Maeda et al. 1986). Other studies confirm that both Trxs in Pisum sativum can reduce in vitro the transmembrane channel Tic 110, in the inner envelope of chloroplasts, involved in the import of proteins synthesized in the cytosol (Balsera et al. 2009). It has been demonstrated that Trx m knockout mutant in Aspergillus nidulans presents a lethal phenotype, suggesting that this isoform is necessary and has a specific function. In potato, Trx m has been shown to be an inhibitor of glucose-6-P dehydrogenase of the oxidative pentose phosphate pathway (Wenderoth et al. 1997), and in Arabidopsis the four isoforms Trxm1, m2, m3 and m4 represent 70% of total chloroplastic thioredoxins (Okegawa and Motohashi 2015). Only the triple AtTrxm1/m2/m4 knockout mutant presented a reduced stability of the photosystem II (PSII) complex and elevated ROS content, suggesting redundancy among these isoforms. The three Trx m interacted in vitro with the particles D1, D2 and especially CP47, thus assisting the assembly of CP47 in PSII (Wang et al. 2013). In the triple mutant, CP47 accumulates as oxidized oligomers. On the other hand, AtTrxm4 and NtTrxm negatively regulate the enzymes NAD-MDH and PGR (protein gradient regulation complex) in vivo, controlling the photosynthetic alternative electron pathway via a mechanism distinct from direct upregulation of cyclic electron flow (Courteille et al. 2013). The AtTrxm3 is related to the meristem development and intracellular traffic of proteins, and the deletion of this gene provoked a lethal phenotype in seedlings (Benitez-Alfonso et al. 2009). In rice, the knockdown mutant of the m isoform provoked phenotypic alterations, chlorosis due to the loss of chlorophyll stability, increased the hydrogen peroxide content and presented the 2-Cys Prx in its oxidized form, suggesting that Trx m acts as antioxidant system (Chi et al. 2008). Actually, the Trx m isoforms have been connected with several functions: leaf and meristem development, chloroplast morphology, cyclic electron flow, traffic of proteins, antioxidant system, and tetrapyrrole synthesis and stability (Ikegami et al. 2007; Chi et al. 2008; Benitez-Alfonso et al. 2009; Luo et al. 2012; Courteille et al. 2013).

Fig. 4

Mechanism of reduction of the disulfide bond and denitrosylation of a protein substrate (target) by thioredoxin (Trx) and NADPH-dependent thioredoxin reductase (NTR) or FDR-dependent thioredoxin reductase systems. Blue arrows represent the electron transference

Regarding isoform f, in vitro studies have shown essential functions of isoform f1 and f2 on Calvin-Benson cycle target proteins (Michelet et al. 2013). In Arabidopsis, a Trxf1 knockout mutant decreased light-dependent reduction-mediated activation of starch metabolism and ADP-glucose pyrophosphorylase (AGPase) activity, but this deficiency did not provoke a different phenotype to wild type (Thormählen et al. 2013). The double mutant in Arabidopsis Trx f1/f2 decreased the growth under short day conditions but not in long day conditions, showing the same phenotype as wild type, and this supported the idea that Trx f is compensated for in chloroplasts by other Trx systems (Yoshida et al. 2015). Although mutants did not show an obvious phenotype, isoforms f are necessary for the reduction of some Calvin-Benson enzymes in the light phase such as FBPase, sedoheptulose-1,7-biphosphatase (SBPase), phosphoribulokinase (PRK), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and rubisco activase (Nikkanen et al. 2016) and this function cannot be compensated for by other FDR-dependent Trxs, other than NTRC (described in the text below). The isoform f has also been proposed to be the key regulator in ATP synthesis by the reduction of the subunit CF1y in the ATP synthase complex (Hisabori et al. 2013). On the other hand, the over-expression of Trx f in Nicotiana tabacum increased the biomass and enhanced the starch production (Sanz-Barrio et al. 2013).

Trx z is another chloroplast isoform, encoded by a genomic gene. It regulates the plastic-encoded polymerase (PEP), enhancing the expression of plastid genes, regulating two fructokinase-like proteins (FLNs) essential for PEP-dependent gene expression. Deletion of the Trx z provoked an albino phenotype with an inhibition of the chloroplast development (Arsova et al. 2010). On the other hand, in A. thaliana, Trx x and y and the chloroplastic drought induction stress protein (CDSP32) carry out an important protective function from oxidative stress: they can reduce 2-Cys Prx, which all supports the idea that the principal function of both isoforms is the control of the redox homeostasis in the chloroplast (Collin et al. 2003, 2004).

The only plastid-located NADPH-dependent Trx is NTRC. In fact, its location has been demonstrated in any kind of plastid—not only in chloroplasts. It is encoded by one gene and is present exclusively in oxygenic photosynthetic organisms, some cyanobacteria, algae and in all green higher plants. NTRC mRNA is present in shoot and root at similar levels but the protein is more abundant in shoots, especially in photosynthetic tissues from A. thaliana (Kirchsteiger et al. 2012), and it is probably regulated by alternative splicing (Nájera et al. 2017). As mentioned above, this Trx forms homodimers containing a TR and Trx domains: the reductase domain can reduce the disulfide bond of the Trx domain (Pérez-Ruiz and Cejudo 2009). NTRC is functional in light and dark phases (Serrato et al. 2004; Cejudo et al. 2014). The first studies in Oriza sativa showed its reduction activity on 2-Cys Prx (Pérez-Ruiz et al. 2006) with more affinity and efficiency than Trx z or CDSP32 (Pulido et al. 2010). In Arabidopsis, NTRC knockout mutant shows a hypersensitive genotype to biotic (Ishiga et al. 2012, 2016) and abiotic stress (Serrato et al. 2004; Chae et al. 2013) and in optimal conditions it presents a phenotype that is strongly dependent on the photoperiod, very different to the wild-type genotype, with an aberrant growth and loss of synthesis and stability of chlorophyll. All this is evidence of an important function for this Trx in the development of the chloroplast (Kirchsteiger et al. 2012). However, the over-expression of NTRC in Arabidopsis lacking a functional Trx domain (OE-SGPS line) rescued the phenotype in knockout mutants and this suggested that the reductase domain might reduce other chloroplast Trxs. However, when NTRC was over-expressed with the reductase domain inactivated (OE-SAIS line), the wild-type phenotype was not recovered, and only a small recovery in biomass was observed (Toivola et al. 2013). The over-expression of a completely functional NTRC protein conferred resistance to oxidative stress in Arabidopsis chloroplasts treated with methyl viologen compared with wild-type plants (Kim et al. 2017). Double and triple NTRC-Trxx and NTRC-Trxf1f2 knockout mutants have shown a lethal phenotype, with 50 and 95% of seedlings dead in soil the first day, respectively. This negative effect was palliated when the plants germinated and grew in a medium supplemented with sucrose such as carbon source, indicating the indirect regulation of FBPase by NTRC. This result suggests that the chloroplast´s cotyledon is not sufficient to supply the carbon necessary for the development of the new tissues (Ojeda et al. 2017). In other studies, Trx x was shown to be the sole target of NTRC (Yoshida and Hisabori 2016).

Trx o, h and s isoforms are NADPH-dependent (Suske et al. 1979; Florencio et al. 1988), using the enzymes NTRA, NTRB and NTRC (Reichheld et al. 2005). AtTrxo1 and o2 were predicted to be present in mitochondria in A. thaliana although only Trxo1 was localized in this organelle (Laloi et al. 2001), while, in P. sativum and tobacco BY-2 cells overexpressing PsTrxo1, this isoform presented a dual location in mitochondria and nuclei (Martí et al. 2009; Ortiz-Espín et al. 2015). In general, the information on this Trxo1 is scarce compared with other Trx types. In Arabidopsis, the lack of this Trxo1 does not result in an evident phenothype, but AtTrxo1 transcript levels have been found to be particularly high in dry seeds and cotyledons where they reached a maximum 36 h after imbibition with water, pointing to a role for this isoform in redox homeostasis during seed germination (Ortiz-Espín et al. 2017). This AtTrxo1 also regulates the mitochondrial TCA cycle repressing fumarase enzymatic activity and the citrate metabolism regulating ATP-citrate lyase acting as a direct regulator of carbon metabolism, in this way providing a mechanism for the coordination of cellular function (Daloso et al. 2015). It has also been demonstrated that citrate synthase is regulated by Trx in Arabidopsis, possibly constituting an important regulatory mechanism for the regulation of the TCA cycle in vivo (Schmidtmann et al. 2014). Trxo1 also regulates the electron transport process by reduction of a disulfide bond in the AOX dimer, which is crucial for AOX activation (Martí et al. 2009). Trxo1 is also involved in the antioxidant reducing activity of peroxiredoxin II F (PrxIIF) and sulfiredoxin in the mitochondria (Barranco-Medina et al. 2008; Iglesias-Baena et al. 2010). Throughout these interactions, Trxo1 is involved in the ROS detoxification in mitochondria (Lázaro et al. 2013; Sevilla et al. 2015). Other described targets of mitochondrial Trxo1 link this redox protein with cellular key processes like photorespiration and protein synthesis in pea plants (Martí et al. 2009). Related to its feasible role in the nucleus, it has recently been described that the over-expression of PsTrxo1 in TBY-2 cells caused cell viability to be maintained and alleviated oxidative stress induced by exogenous H₂O₂ treatments, producing an increase in catalase activity, a decrease in H₂O₂ and nitric oxide contents and the maintenance of the glutathione redox state (Ortiz-Espín et al. 2015). All these components together with Trxo1 are involved in the observed protection from the oxidative stress provoked by the treatment. This antioxidant role allows PsTrxo1 to control the redox status of specific target proteins in mitochondria and nuclei. Moreover, it has been recently described for the first time that Trxo1 interacts and regulates the redox state of PsPCNA (Proliferation Cellular Nuclear Antigen) in vivo and in vitro (Calderón et al. 2017b). Furthermore, PsTrxo1 over-expression in TBY-2 cells provoked an increase in the rate of cell proliferation with a higher percentage of the G2/M phase at the end of the culture growth coinciding with an upregulation of PCNA protein. This was accompanied by a higher mitotic index and a decrease in the total cellular glutathione content but maintained nuclear GSH accumulation. All these results have suggested that Trxo1 is involved in the cell cycle progression of TBY-2 cultures, possibly through its link with cellular PCNA and glutathione. The presence of thioredoxin in the nucleus is an interesting discovery. In animal cells, thioredoxins have been shown to interact with transcriptional factors whereas their function in the nucleus of plant cells has been much less studied (Sevilla et al. 2015). Trx h has been reported to accumulate in the nucleus of aleurone and scutellum cells of wheat seeds associated with oxidative stress during germination (Serrato et al. 2001; Serrato and Cejudo 2003). The first nuclear and functional Trx system with disulfide reduction capacity in the nucleus was described in Trypanosoma sp. and was named nucleoredoxin (Nrx). Nrxs are proteins composed of several domains where at least one is a Trx domain (Meyer et al. 2012). Mammalian cells have a unique Nrx but in angiosperm plants, there are multiple Nrx systems organized into three subfamilies. There are two NRX genes in Arabidopsis (NRX1 and NRX2), separated into subgroups I and II, respectively. Nrx1 has a typical Trx domain (WCG/PPC), while Nrx2 has an atypical Trx domain (WCRPC and WCPPF). Both proteins have a dual location in nucleus and cytosol but only Nrx1 has been shown to be reduced by NTRA (Marchal et al. 2014). Nrxs participate in the regulation of redox signaling and play an essential role in the maintenance of stem cells and establishment during early development (Funato and Miki 2010). In Arabidopsis, Nrx1 regulates the pollen tube growth during fecundation in the inner pistil (Qin et al. 2009); however, in poplar, Nrx1 was proposed as a regulator in the salicylic acid (SA) signaling pathway (Xue et al. 2013). Recently, Nrxs have been shown to play an interesting role as a protective mechanism of antioxidant systems controlling the status of ROS-scavenging enzymes such as catalase, and probably that of APX, MDHAR and DHAR, which have been identified among their possible target proteins. In this way, Nrxs could modulate H₂O₂ metabolism, protecting plant cells from oxidative stress imposed by environmental challenges (Kneeshaw et al. 2017).

Although it has been described that Trx h is located in cytosol and plasma membrane (Serrato et al. 2004), understanding its function and distribution is more intricate compared with other isoforms. In Arabidopsis, there are nine isoforms divided into three subgroups, depending on the sequence motif in the active centre. The first subgroup I includes h1, h3, h4 and h5, which are located in the cytosol, and reduced with high efficiency by NTRA (Ishiwatari et al. 1995). These isoforms are especially abundant in phloem sieve tubes, regulating the flow in vascular components and being involved in the plasmodesmata movements for the cell-to-cell communication (Gelhaye et al. 2004). The subgroup II is composed of the isoforms h2, h7 and h8 being h2 localized in mitochondria and reduced by NTRB (Gelhaye et al. 2004). Trxh8 is present only in callus tissue, whereas Trxh7 was identified in roots and reproductive plant organs (Reichheld et al. 2002). The subgroup III (Trxh9) is characterized by an N-terminal extension containing a conserved cysteine in the fourth position (Gelhaye et al. 2004) and is present in the plasma membrane where it regulates the malate valve interacting with NAD-MD (Hägglund et al. 2016). In Arabidopsis, the lack of Trxh9 results in a lethal phenotype (Meng et al. 2010). Nowadays, there are several functions described for Trx h. In cereals, they participate in reserve breakdown during seed germination (Wong et al. 2002), interacting with glutenins and gliadins in Triticum sp. and hordeins in Hordeum sp. (Yano et al. 2001). Other studies have demonstrated the implication in the mobilization of compounds and the protection from oxidative stress during the development and drying of seeds. In cereal seeds, this Trx is located in the inner nucleus of aleurone and scutellum seeds (Serrato and Cejudo 2003). In addition, in O. sativa, Trx h participates in the C4 metabolism through the interaction and regulation of PEPC-PK. As a new type of Trx in legume nodules, Trx s was described in the endoplasmic reticulum of Medicago truncatula nodules where it acts as an antioxidant system scavenging ROS and facilitating the nodulation process (Alkhalfioui et al. 2008).

In summary, Trxs are involved in several basic functions related to the regulation of specific protein targets and the implication in plant development and in the response to (a)biotic stresses. We will go on to discuss the role of Trxs in response to salinity, drought and extreme temperatures.

5 Functional Biochemistry of Trxs Mediated by ROS and RNS

The kinetic reaction of Trx in the disulfide exchange is faster than other molecules like GSH or DTT (Nikitovic and Holmgren 1996) and, as an example, the dissociation constant of insulin reduction by Trx in vitro is K₂ = 5 × 10⁴ M¹ s⁻¹ (Holmgren 1979). The reaction has two steps: first Trx reduces a disulfide bond, transferring the electron of the cysteine with less pK_a. Then, the other cysteine reduces this temporal bond, transferring other electron in a fast step (Holmgren 1995). Finally the target is reduced and the oxidized Trx is reduced by NTR or FTR enzymes in a closed system (Fig. 4).

Trx/NTR controls the S-nitrosylation process by transnitrosylation or denitrosylation of target proteins. Trx accepts or scavenges the NO radical and NTR restarts the thiol group or scavenges the NO (Fig. 4) (Kneeshaw et al. 2014). In animals, there are several studies, but the role of S-nitrosylation on Trxs in plants is quite limited. As an example, BjTrxh5 and AtTrxm5 have been shown to be unable to denitrosylate S-nitrosylated glyceraldehyde3-phosphate dehydrogenase in vitro (Lindermayr et al. 2005; Zaffagnini et al. 2013).

6 Role of Trx Under Salinity

Trxs are involved in plant tolerance to abiotic stress including salinity (Barranco-Medina et al. 2007; Pulido et al. 2009; Tripathi et al. 2009; Martí et al. 2011) and they have been reported to present a redox sensing and signal transduction function (Rouhier and Jacquot 2005) together with their participation in the repair of oxidized proteins during environmental stress (Dos Santos and Rey 2006). At the transcriptional level, transcriptomic analysis in poplar has revealed an inconsistent response to salt stress of the different Trx members (Ding et al. 2010). Regarding mitochondrial/nuclear Trxo1, an early induction of this gene expression concominant to an increase in the expression of its target Prx IIF has been reported in pea leaves 5 days after growth in the presence of 150 mM NaCl, pointing to an adaptive behavior (Martí et al. 2011). However, gene expression decreased while activity and protein levels increased at longer salt treatments, indicating a possible post-translational regulation of the protein and providing evidence for complex regulation in the response of plants to these stressful conditions. In fact, PrxII F was found to be S-nitrosylated under salinity conditions and this PTM provoked a switch from peroxidase to transnitrosylase activities, as demonstrated in in vitro experiments with recombinant PsPrxIIF. Interestingly, S-nitrosylation occurred in one or both Cys residues of the active site of the protein, depending on its oligomerization state. This process may function as a protective mechanism under oxidative and nitrosative stress, such as occurs under salinity. The S-nitrosylation under this condition and, as a consequence, its peroxidase activity may be reversed under more reducing conditions in which the Trx system operates (Camejo et al. 2015). A protective role for Trxo1 was also proposed, possibly via the redox modulation of its target protein AOX, which presented a higher capacity and activity in this condition. In addition, the analog of Trxo1 in mammalians Trx2 has been correlated with protection against oxidative-induced apoptosis (Chen et al. 2002; Tanaka et al. 2002). It has been reported that salt stress provoked an accumulation of PsTrx f and m1 transcripts (Fernández-Trijueque et al. 2012) but no correlation between mRNA and protein levels was observed, suggesting a significant post-transcriptional control such as that described for pea Trxo1 under salinity (Martí et al. 2011). On the other hand, salinity in imbibed Arabidopsis seeds usually produces a delay in germination although knockout AtTrxo1 mutant seeds had higher H₂O₂ content at the beginning of germination and a faster germination rate than those of the wild-type plants. This reflects a specific role for Trxo1 in the germination of seeds in the presence of NaCl, which may in turn be related to specific Trxo1 targets (Ortiz-Espín et al. 2015). In this adverse situation, Trxo1 could act as a possible sensor of saline stress and an inducer of H₂O₂ accumulation. Other Trxs, like the chloroplastic ones, respond to salinity during germination similarly to PsTrxm1, m2, m4 and f type and ZmTrxm1 m RNAs, which increased under this condition. Moreover, over-expression of PsTrxm1, PsTrxm2 and PsTrxf conferred resistance to salinity (Fernández-Trijueque et al. 2012), although it has been described that Trx m rather than Trx f-type is involved in the response to oxidative stress (Okegawa and Motohashi 2015). Also interesting is the hypersensibility of AtNTRC knockout mutants to saline stress (Serrato et al. 2004), suggesting that chloroplastic Trxs are involved directly or indirectly in tolerance to this stress condition. Another Trx involved is OsTrxh1, which responds with increased expression at the protein level under salinity (Zhang et al. 2011). This Trx is secreted into the apoplastic space and the knockout plants present a dwarf phenotype with fewer tillers, whereas its over-expression (Ovx) leads to a salt-sensitive phenotype in rice. Interestingly, both knockout and Ovx plants presented decreased ABA sensitivity during seed germination and seedling growth accompanied by an increased amount of H₂O₂ in the apoplast of knockout plants and a decreased amount in the Ovx plants compared with wild-type plants under saline conditions. All of this pointed to Trxh1 being involved in the regulation of the redox state of the extracellular space influencing plant development under salinity. Other Trx h types respond to salinity, increasing its protein content in the roots of salt-sensitive genotypes of tomato while decreasing it in more tolerant plants, suggesting a role for this Trx in responding to oxidative stress, which is more prolonged in sensitive cultivars (Manaa et al. 2011).

The involvement of Trxs in osmotic stress occurring under salinity and drought has also been demonstrated in Vitis vinifera through the presence of ABRE elements in the promoter regions of VvTrx h1, h2, h3, h4, and VvCxxS2 genes, which demonstrated that expression of drought- and salt-induced Trxs h is transcriptionally controlled by ABA, whose levels increased under stress (Haddad and Japelaghi 2014). Trx h protein has been reported to increase with salt stress during germination and early seedling growth (Cazalis et al. 2006) and during growth of grass pea (Chattopadhyay et al. 2011). Another family of Trx-like proteins, like CDSP32 a critical component of the defense system against oxidative damage, was found to accumulate in much higher levels in the halophytic plant Thellungiella halophila compared with the salt-sensitive A. thaliana (M’rah et al. 2007), pointing to the specificity of this redox protein.

One of the main effects of salt stress is the limitation of the vegetal production by the inhibition of photosynthesis. This inhibition may be due to stomata closure and limitation of intracellular CO₂ levels (Mäkelä et al. 1999; Yang and Lu 2005), but also to a decrease of PSII activity and electron transport (Mishra et al. 1991; Lu and Vonshak 2002; Rabhi et al. 2010). Trx m protein level has been described as being reduced by salt stress in Arabidopsis and in leaves of P. tenuiflora (Pang et al. 2010; Yu and Richardson 2011). AtTrxm1 and m2 positively regulate the synthesis of the PSII by reduction of the subunit CP47, hypothetically conferring resistance to salinity stress (Wang et al. 2013). In the photosynthesis process, the activity of the NDH (NADH-dehydrogenase) enzyme and the PGR increase in G. Cyrtoloba under salinity conditions (Yang et al. 2007). However, AtTrxm4 and NtTrxm negatively regulate the photosynthesis activity in vivo, interacting and inactivating the NADH-dehydrogenase enzyme and the PGR complex (Courteille et al. 2013). Although the CO₂ fixation is limited, the mRNA levels of FBPase and PRKase have been reported to decrease (Chaves et al. 2009). Nevertheless, an increase in protein level of HvSBPase and HvPRK has been noted in salt-tolerant barley plants whilst the opposite behaviour was shown to occur in plants sensitive to saline stress (Rasoulnia et al. 2011). As mentioned above, target proteins of the FTR/Trx system include some Calvin-Benson’s enzymes in light-dependent reactions, such as FBPase, PRK, GAPDH, Rubisco activase and SBPase (Raines 2003). AtTrxf1 positively regulates GAPDH; AtTrxf1 and AtTrxf2 regulate FBPase, SBPase and Rubisco activase; AtTrxm1 and AtTrxm2 control PRK and AtTrxm2; and AtTrxm4 adjusts SBPase activity. The CP12 (Calvin cycle Protein 12) is an inhibitor of PRK and GAPDH and is inactivated in vivo by the light-dependent Trx-mediated reduction of AtTrxf1, AtTrxm1 and AtTrxm2 (Michelet et al. 2013), which may have a role in the activity of their targets under salinity.

Other dysregulated redox processes under salinity conditions include the synthesis and balance of the reduction power of ATP/NADPH/NADH. In T. Aestivum and P. patens, the ATP synthesis in the chloroplast is strongly depressed under moderate and severe salinity stress by decreased activities of photosynthesis (P_N), Mg²⁺-ATPase and Ca²⁺-ATPase, which may contribute to the damage of PSII (e.g., reductions in Fv/Fm, FPSII and qP, and increase in NPQ) (Zheng et al. 2009; Wang et al. 2008). Trxf, SoTrxm and AtNTRC interact with the subunit A of the CF1-ATPase complex, positively regulating its expression in vivo (McKinney et al. 1978; Naranjo et al. 2016) and PsTrxf also activates and enhances the ATPase activity of pea (Luo et al. 2012). Interestingly, ATP synthase has shown to be activated in light conditions and inactivated in dark by redox-modulation through the thioredoxin system (Kohzuma et al. 2017). These authors have proposed that thiol modulation may act as a ‘redox switch’ so that the ATP synthase is fully inactivated in the dark to prevent ATP loss and fully active even in low light, therefore preventing the buildup of excessive proton motive force.

In the malate valve, the malic acid and oxaloacetic acid (OAA) represent a redox pair of compounds regulated by malate dehydrogenases (MDH). Exchange of both metabolites by membrane transport controls the balance of the reducing power, transferring the excess of equivalents from the photosynthesizing chloroplast to the Trx-regulated NADP-dependent MDH (Wolosiuk et al. 1977) to reduce OAA to malate, controlling the redox state of the chloroplast and playing an important role in the response to salt stress (Scheibe et al. 2005). An increase of chloroplast NADH-MDH mRNA level has been observed in Mesembryanthemum crystallinum under a brief salt stress (Cushman 1993). In a recent study, the activity of NAD-MDH increased in salt-resistant rice cultivars CSR-1 and CSR-3, specifically the chloroplastic NADP-MDH and mitochondrial NAD-MDH, while the activities were depressed in salt-sensitive cultivars when grown under salinity (Kumar et al. 2000). In A. thaliana, some Trxs are able to activate NADP-MDH in vitro, with the most efficient being Trx f1, followed by Trxs f2, m1, m2, m4, m3 and x (Collin et al. 2003). It has been reported that Trxf1 and f2 can fully activate NADP-MDH by cleaving two disulfide bonds, while Trxm2 and m4 partially activate the enzyme by the reduction of only one disulfide bond (Yoshida et al. 2015)—unlike in spinach plants where SoTrxf1 is more efficient than SoTrxf2 in the reduction of the enzyme (Yoshida and Hisabori 2016) (Fig. 5).

Fig. 5

Relationship among the different types of thioredoxins with the main processes altered during drought and salinity stresses: different Trxs described in chloroplast, mitochondria, cytosol, apoplast, nucleus and endoplasmic reticulum. Brown and blue colors indicate an up/down regulation, respectively, of genes or proteins; white color indicates absence of change under different stresses. The arrows and bars indicate the activation or inhibition, respectively of the processes: synthesis and stability of chlorophylls (CHL), synthesis of ATP (ATPsynthase), electron transport during photosynthesis (ET), Calvin-Benson cycle, malate dehydrogenase (MDH), starch synthesis, ROS production, alternative respiration pathway (AOX) and protein modulation

The metabolism of chlorophyll has been extensively studied under salt stress. In rice seedlings grown 72 h in 200 mM NaCl, the total chlorophyll accumulation was reduced by about 66% (Turan and Tripathy 2015), which may be attributed to decreased activities of chlorophyll biosynthetic pathway enzymes. In relation to Trx, it has been demonstrated in pea and Arabidopsis that PsTrxm, PsTrxf, AtTrxy1 AtTrxm2—and, with less efficiency, AtTrxf1, x and z—interact with Mg-chelatase, positively regulating its activity and indirectly the chlorophyll accumulation (Luo et al. 2012). By contrast, Hordeum vulgare Trx f and m interact in vitro with PAO (Pheophorbide A oxygenase), enhancing the chlorophyll breakdown (Bartsch et al. 2008).

Other described Trx-like proteins involved in the response to salinity include the TTL (tetratricopeptide thioredoxin-like) family, specifically TTL1. In fact, mutation of this protein in Arabidopsis results in a reduced tolerance to NaCl and osmotic stress, with the plants presenting a reduced root elongation, impaired osmotic response during germination and disorganization of the root meristem (Rosado et al. 2006). However, these authors described different roles for other TTL proteins, such as TTL1, 3 and 4, involved in the sensitivity to osmotic stress in roots, TTL4 in the tolerance to NaCl (a non-redundant function) while TTL2 did not present an specific role in these situations.

7 Role of Trx Under Drought

The link between drought stress and the redox state of thiols in proteins and nonprotein metabolites has been studied in different species. In Triticum sp., this stress condition provoked an increase in GSH content and GR activity (Zagdanska and Wisniewski 1996) induced by ROS production in an ABA-depending signaling pathway (Jahan et al. 2008). Other studies have corroborated the relation between redox thiol state, ROS production, drought and oxidative stress (Hajheidari et al. 2007) being the thioredoxin the responsible for oxidative stress protection and homeostasis (Buchanan 1980). Curiously, under cold stress, most Trx genes showed a downregulation while drought stress provoked an upregulation (Zagorchev et al. 2013). Under drought stress conditions, the most studied Trx is the atypical CDSP32, initially identified and located in the stromal membrane of Solanum tuberosum. This protein interacts and reduces 2-Cys Prx, which accumulated under drought and salt stresses (Rey et al. 1998; Collin et al. 2004). This protein has also been characterized in several plants, such as O. sativa, C. sinensis, A. thaliana, N. tabacum, H. vulgare and P. tricocarpa (Dubey 1999; Broin et al. 2002; Dietz 2003; Wijngaard et al. 2005; Chibani et al. 2009). Recently, it has been shown that it interacts with Prx Q, 2-Cys Prx, ATP synthase and methionine sulfoxide reductase (MSRB1 and MSRB2) in the chloroplast of potato plants under drought stress, indicating that the principal function may be to confer oxidative protection (Rey et al. 2007; Gama et al. 2008). Using knockout CDSP32 mutants, Broin and Rey (2003) corroborated that S. Tuberosum plants were hypersensitive to drought and oxidative stress, showing an increase in hydrogen peroxide and over-oxidation of 2-Cys Prx. In a drought-tolerant cultivar of Medicago sativa, the atypical Trx CDSP32 was upregulated compared with a sensitive cultivar and this inducible expression could be due to the hypomethylation of CDSP32 promoters (Sharma et al. 2017).

In Xerophyta viscosa, a resurrection plant, and in the moss Physcomitrella patens, both considered to be drought-tolerant models, a new peroxidase 1-Cys Prx was identified in the nucleus, suggesting an oxidative protection mechanism of DNA during dehydration events (Mowla et al. 2002; Wang et al. 2009). 1-Cys-Prx has been described in other plants like M. truncatula, where it increased during early development in somatic embryogenesis (Imin et al. 2005) and in A. thaliana during maturation drying of seeds and in dormant seeds (Aalen 1999; Stacy et al. 1999; Dietz 2003). Overexpression of O. sativa 1-Cys Prx in N. tabacum plants conferred oxidative and drought stress tolerance (Lee et al. 2000). Later on, it was demonstrated that Trx reduces the sulfenic state in this atypical Prx (Dos Santos and Rey 2006), concretely in wheat seeds, where Trxh1 and NADPH-NTR system reduced 1-Cys Prx in vitro—all of them co-localized in the nuclei of aleurone and scutellum cells of seeds (Pulido et al. 2009). In Xerophyta viscosa during desiccation events, an increase in 1-Cys Prx was accompanied by an increase in three Trxh orthologous in A. thaliana to Trxh1, h2 and h3. These results support the idea that this NTR/Trxh1/1-Cys Prx system plays an important role during drought stress and natural dehydration events. In other studies, Trx-dependent Prx protein was found to be oxidized under drought conditions, suggesting the important role of thioredoxin under this stress situation, due to the importance of its reductase activity (Gama et al. 2008; Rey et al. 2007).

Thioredoxin h type is another widely studied isoform in wheat under drought conditions, where it usually increases the expression of gene or protein isoforms in several species, (Fig. 5) such as Triticum aestivum (Hajheidari et al. 2007; Zhang et al. 2014), Oryza sativa (Gorantla et al. 2007), Zea mays (Kocsy et al. 2004), Solanum tuberosum (Watkinson et al. 2008), Glycine max (Ji et al. 2016), M. trucatula (Alkhalfioui et al. 2007), Vicia faba (Abid et al. 2015), Medicago sativa (Kang and Udvardi 2012), Populus euphratica (Bogeat-Triboulot et al. 2007) and Xerophyta viscosa (Colville and Kranner 2010). Initially, in Z. mays treated with PEG and subjected to osmotic stress (the early component in drought stress), the increase in two Trx h induced via an ABA-dependent pathway was described (Kocsy et al. 2004). Using tolerant and sensitive cultivars of T. aestivum, two TaTrxh orthologous types for A. thaliana Trxh3 and Trxh5 have been identified as belonging to the subgroup I, presenting the atypical active centre CPPC (Hajheidari et al. 2007). In the tolerant cultivar, Trxh5 expression and reduced state increased under drought conditions, while it decreased in the sensitive cultivar. Nevertheless, the expression of Trxh3 decreased under drought stress in both cultivars. Similarly, Alkhalfioui et al. (2007) showed an increase in MtTrxh3 and identified some targets for these Trx h in M. trucatula germinating seeds under drought conditions, such as the LEA protein dehydrin related to resistance to desiccation, LEA protein PM25 related to DNA protection during oxidative damage, and sorbitol 6-P dehydrogenase involved in osmotolerance. It has been proposed that Trx h maintains and stabilizes the redox state of thiols and structure in some proteins during fast rehydration processes (Alkhalfioui et al. 2007; Shahpiri et al. 2007). More recently, in two cultivars of V. faba with different sensitivity to drought stress, a differential expression of Trx h has shown to be higher in the tolerant cultivar under optimal and drought stress conditions than in the sensitive cultivar. A differential expression of genes related to drought stress has also been noted, including ATP synthase, chlorophyll a/bbinding protein, HSP and LEA (Abid et al. 2015). In this sense, the differential expression of proteins under osmotic stress in T. aestivum plants treated with PEG demonstrated that proteins Trx h and Trx m types increased under these conditions, accompanied by a rise in the protein level described in other studies as Trx-targets: ATP synthase, fructose bifosfatase, RUBISCO activase and transketolase (Zhang et al. 2014). In other studies, Trx h and three Trx m types orthologous to A. thaliana AtTrxh2 and AtTrxm1, AtTrxm2 and AtTrxm3 increased in M. sativa under drought conditions in the drought-tolerant cultivar compared with the drought-sensitive cultivar, accompanied by an increase in CDSP32 and tylakoidal Trx-like protein related to the synthesis of Cytochrome b6f, which transfers energy from PSII to PSI during photosynthesis, thereby avoiding the formation of superoxide radical and preventing oxidative damage in the chloroplast (Kang and Udvardi 2012). In relation to Trx m type, Vieira Dos Santos et al. (2007) corroborated in vitro, using recombinant Arabidopsis thaliana proteins, that AtTrxm1, AtTrxy2, AtCDSP32 and AtGrx can act as electron donor systems, reducing the methionine sulfoxide reductase B1 and B2 enzymes (MSRB1 and MSRB2) in charge of the reduction of methionine MetSO R-diastereomer and conferring protection against oxidative stress during drought or salt stress. In Hordeum vulgare under drought stress, Trx m was also found to increase in a tolerant cultivar compared with a sensitive cultivar. This differential expression of proteins was similar to that described for some of its targets, such as ATP synthase subunit CF1, glyceraldehyde 3-P dehydrogenase (GAPDH), RUBISCO activase, D1, CP47 and FtsH. This increase conferred resistance to drought stress in the tolerant cultivar related with ATP synthesis, carbon metabolism, photosynthesis and synthesis and stability of chlorophylls (Wang et al. 2015a). Related to this Trx type, using two drought-tolerant cultivars of Phaseolus vulgaris grown under drought stress, an increase in Trxm4 was parallel to an increase in some of its targets, such as GAPDH, RUBISCO activase, ATP synthase subunit CF1, Chlorophyll a/b binding protein, malate-dehydrogenase and glutamine synthase (Zadražnik et al. 2013).

In Solanun tuberosum under drought stress, Trxf2 and Trxh1 expression was found to increase during the early phase of stress and decreased when the plants were watered, similar to 2-Cys Prx and AGPase (ADP-glucose pyrophosphorylase)—both redox-regulated targets of Trx. The increase in these proteins is related to the inhibition of starch accumulation during drought stress in the tuber, and this inhibition could be necessary to sustain sucrose catabolism under stress conditions (Watkinson et al. 2008).

NTR has also been shown to respond to drought stress. Using two tolerant and sensitive cultivars of T. aestivum seedlings, the differential expression of protein and activity was enhanced under drought conditions in tolerant cultivars (Rakhra et al. 2015). Other Trx system involved include the atypical Trx NTRC, which has been associated with responses to drought, salinity, heat and oxidative stress (Serrato et al. 2004). Pulido et al. (2010) corroborated in vivo the decreased 2-Cys Prx gene and protein expression in knockout NTRC mutants, and it was shown that ASC and GSH were more oxidized compared with wild-type plants. In this work, it was also shown that NTRC is the most efficient thioredoxin to reduce 2-Cys Prx in vitro. Recently, Kim et al. (2017) demonstrated that NTRC over-expression in A. thaliana confers resistance to drought, salt and oxidative stresses while the knockout mutant proved to be hypersensitive to these conditions. The overexpressing mutant accumulated less ROS and showed a higher survival rate and a lower water loss compared with the knockout mutant under drought conditions. A differential increased expression of 2-Cys Prx and proteins of drought resistance (DREB2A and RD29A) was also observed in the overexpressing mutant. Curiously, the over-expression of NTRA in A. thaliana showed the same phenotype as NTRC with the same increase in expression of the named proteins (Cha et al. 2014). In fact, cytosolic NTR potential significance is less known due to functional redundancy (Reichheld et al. 2005). NTRA overexpressing plants displayed enhanced tolerance against drought stress, which was thought to be achieved by enhanced ROS scavenging systems, such as activated Prx, Cu,Zn-SOD and APX1 gene expression, revealing a ROS-regulatory function of NTRA (Cha et al. 2014).

Other atypical Trx recently related to drought stress are the nucleoredoxins. Using tolerant and sensitive cultivars of Sorghum bicolor, an increase in Nrx was shown during the stress conditions and recovery phase in the tolerant cultivar only. This result suggests that this Nrx could confer protection against the oxidative damage suffered in the nucleus during this stress condition (Jedmowski et al. 2014). In Cajanus cajan plants, an increase in the expression of Nrx orthologous to A. thaliana AtNrx3 under drought conditions also occurred (Sinha et al. 2016). Similarly, using two soybean tolerant to drought stress and two cultivars sensitive to drought stress, the increase in one Nrx and other thioredoxin-related redox-regulating proteins was shown only in the tolerant cultivars (Yu et al. 2016) (Fig. 5).

8 Role of Trx Under Extreme Temperatures

The knowledge of the role of thioredoxin under extreme temperatures is very limited. Thioredoxin and 2-peroxiredoxin transcripts have been described to be induced in Arabidopsis plants during a combination of drought and heat stress (Rizhsky 2004). Thioredoxin h may have a special role following moderate heat exposure in P. euphratica, since this species is able to survive under extreme temperatures, drought and salt stress. It has been reported that this Trx protein presented a prolonged accumulation upon heat stress, which may be related to its role in the oxidative stress response (Ferreira et al. 2006). It has been described that the promotor regions of Vitis vinifera Trx h family genes contain several known stress-responsive elements, such as MYB binding site (MBS) involved in drought response or heat-stress-responsive elements (HSEs). The presence of these putative regulatory elements suggests that Trxs h may respond to different environmental signals, including dehydration, salinity, heat or cold, among others. In fact, Trxs h (h1–5) were generally induced upon heat, drought and salt treatments, exhibiting generally higher level of transcripts than wild-type plants, depending on the severity of the stress (Haddad and Japelaghi 2014). Moreover, the overexpression of Trxh3 in Arabidopsis confers heat tolerance, indicating a role for this isoform under this stress. Using heat-treated cytosolic extracts and recombinant proteins expressed in E. coli, Park et al. (2009) demonstrated that AtTrxh3 has a dual function, it can act as a disulfide reductase protein or as a chaperone, and the duality was closely related with the quaternary structure of the protein. Usually chaperone activity is predominant in higher complexes (HMC), whereas disulfide reductase activity is associated with lower molecular weight (LMW) forms. Interestingly the oligomerization is reversible and inducible by an increase in temperature and redox status. Similarly, heat shock also reversibly regulates the oligomerization status of Arabidopsis AtTDX, a heat-stable and plant-specific thioredoxin (Trx)-like protein as well as AtNTRC, both with multiple functions depending on this oligomerization: disulfide reductase and foldase chaperone functions predominate in the LMW form, whereas a holdase chaperone function predominates in the HMW complexes (Lee et al. 2009; Chae et al. 2013). Interestingly, the overexpression of these proteins in Arabidopsis conferred increased heat shock resistance to plants, primarily via the holdase chaperone activity.

Low temperature also influences Trx expression. In this sense, transcripts of rice OsTrx23 in shoot and root tissues were described to increase after 24 h of chilling stress, which indicated that this Trx is involved in the cold stress response in rice seedlings. Interestingly, this cold-induced Trx has an inhibitory function on two stress-activated MAPKs of rice, proposing a new mechanism of redox regulation of MAPKs in plants (Xie et al. 2009). Banana (Musa acuminate) fruits are very sensitive to chilling injury and redox regulation is involved in the response to this stress situation in harvested fruits. It has been described that levels of gene expression of three Trxs (typical plasma membrane MaTrx6, atypical double located cytoplasmic/chloroplastic MaTrx9 and atypical cytoplasmic MaTrx12) by RT-qPCR revealed an upregulation of MaTrx12 in chilled banana fruits. Moreover, heterologous expression of MaTrx12 in cytoplasmic Trx-deficient Saccharomyces cerevisiae strain increased the viability under H₂O_2, suggesting an important role for this Trx12 in regulating redox homeostasis during chilling tolerance of harvested banana fruits (Wu et al. 2016).

9 Concluding Remarks

The link between redox regulation and ROS metabolism is a key element to control and optimize in the function of plant cell organelles in the response to abiotic stress. The studies reported in this chapter indicate that the thioredoxins present in different cell compartments play important roles in the response to salinity, drought and extreme temperatures in diverse plant species. Thioredoxins participate as signaling molecules and contribute to the redox state of the cell though the redox regulation of target enzymes involved in essential reactions in each cell compartment, and they participate in the response of plants to environmental constraints. The numerous target proteins redox-regulated by thioredoxins implicate them in essential processes such as photosynthesis, photorespiration, respiration, carbon metabolism, protein synthesis, DNA protection, gene transcription or post-translational regulation, among others. Increasing the knowledge about the specificity of the interactions of Trxs with their targets will help us to unravel the role of these proteins as redox sensors in the signaling process occurring under normal and stress conditions as response to salinity, water restriction and extreme temperatures.