Proteomics of stress responses in wheat and barley—search for potential protein markers of stress tolerance

Wheat (Triticum aestivum; T. durum) and barley (Hordeum vulgare) agricultural production is severely limited by various abiotic and biotic stress factors. Proteins are directly involved in plant stress response so it is important to study proteome changes under various stress conditions. Generally, both abiotic and biotic stress factors induce profound alterations in protein network covering signaling, energy metabolism (glycolysis, Krebs cycle, ATP biosynthesis, photosynthesis), storage proteins, protein metabolism, several other biosynthetic pathways (e.g., S-adenosylmethionine metabolism, lignin metabolism), transport proteins, proteins involved in protein folding and chaperone activities, other protective proteins (LEA, PR proteins), ROS scavenging enzymes as well as proteins affecting regulation of plant growth and development. Proteins which have been reported to reveal significant differences in their relative abundance or posttranslational modifications between wheat, barley or related species genotypes under stress conditions are listed and their potential role in underlying the differential stress response is discussed. In conclusion, potential future roles of the results of proteomic studies in practical applications such as breeding for an enhanced stress tolerance and the possibilities to test and use protein markers in the breeding are suggested.

During the past decades, the boom of high-throughput proteomics techniques has enabled the researchers to study plant proteome responses to various factors including stresses in a complex way. Despite numerous studies reporting identifications of a few thousand of proteins in plant samples, a complete description of plant proteome in a given tissue, developmental phase and environmental conditions still remains a great challenge (Jorrin-Novo et al., 2009).
Both abiotic and biotic stresses induce profound changes in plant proteomes aimed at an adjustment of metabolism to altered environment and an enhancement of plant stress tolerance. Plant stress response is a dynamic process and several phases with a unique proteome composition could be distinguished (Levitt, 1980;Larcher, 2003). Reviews on plant proteome responses to abiotic stresses (Kosová et al., 2011;Hossain et al., 2012) and pathogens (Sergeant and Renaut, 2010;Gonzalez-Fernandez and Jorrin-Novo, 2012) provide important overviews; however, several novel studies were published recently ( Table 1).
Most proteomic papers aimed at an investigation of plant stress responses are comparative studies that are based on comparison of proteome composition in stressed plants vs. control ones, and also in differentially-tolerant genotypes exposed to stress. Moreover, studies on the roles of subcellular proteomes such as chloroplast (Kamal et al., 2012) and mitochondrial (Jacoby et al., 2010(Jacoby et al., , 2013 proteomes as well as posttranslational modifications (PTMs) such as phosphoproteomics Zhang et al., 2014) in wheat exposed to stress have been published recently. Considering the increasing amount of proteomic data, it is arising necessary to mine the data published in various proteomic studies in order to identify key proteins involved in plant responses to a wide array of stress factors (dehydrative stresses-drought, osmotic stress, salinity, frost, heat) as well as proteins induced only at specific stress conditions (e.g., phytochelatins and heavy metal stress). An attempt has already been published regarding proteomic studies under salinity (Zhang et al., 2012). Moreover, comparison of proteome responses in differentially-tolerant genotypes may help researchers to identify key proteins underlying the differences in stress tolerance. The aim of this minireview is to summarize the major results obtained by proteomic studies in temperate cereal crops wheat and barley studied under abiotic and biotic stresses. Proteins affected by differential stress factors and proteins revealing a differential response between differentially-tolerant wheat and barley genotypes are discussed in a greater detail. Possibilities of utilization of proteins revealing a differential stress response between tolerant and sensitive genotypes as protein markers in breeding programs aimed at improvement of stress tolerance are suggested.

COMMON FEATURES OF STRESS RESPONSE AT PROTEOME LEVEL
Plant stress response is a dynamic process aimed at an enhancement of plant stress tolerance and an establishment of a novel homeostasis between plant and environment (Figure 1). Several

FIGURE 1 | A simplified scheme of plant cell response to an external stress stimulus leading to an activation of signaling cascades, changes in gene expression, activation of protein biosynthesis and degradation, profound changes in energy metabolism leading to an enhanced ATP biosynthesis and ROS production in chloroplasts and mitochondria resulting in ROS
induced signaling (RS). Changes in protein biosynthesis lead to an enhanced production of both protein and non-protein (metabolite) stress protective compounds including ROS scavenging enzymes and metabolites, which participate in an active plant stress acclimation response including a feedback regulation of stress-induced signaling, gene, and protein expression mechanisms.
phases of plant stress response could be distinguished including an alarm phase, an acclimation phase, a resistance phase, an exhaustion phase when stress is too severe or lasts too long, and a recovery phase after a cessation of the given stress factor (Levitt, 1980;Larcher, 2003;Kosová et al., 2011). At proteome level, profound alterations in protein relative abundance were found between stressed and control plants as well as between differential genotypes ( Table 1). During an alarm phase, stress induces profound alterations in proteins involved in cell signaling although these proteins are detected scarcely on 2DE gels due to their low abundance. An increase in 14-3-3 proteins as well as translationally controlled tumor protein homologs was detected in copperand water-stressed wheat (Kang et al., 2012;Ghabooli et al., 2013;Li et al., 2013;Alvarez et al., 2014) and barley (Wendelboe-Nelson and Morris, 2012) and genotype-specific responses of β subunit of heterotrimeric G protein were found in salt-stressed wheat (Peng et al., 2009). Phosphorylation plays an important role in abiotic and biotic stress responses as shown on several kinases (calciumdependent protein kinases CDPK, mitogen-activated protein kinases MAPK, sucrose non-fermenting-related kinases SnRK2), phosphatases (PP2C) and other signaling proteins (calmodulin 2-2) regulation Zhang et al., 2014). Stress acclimation represents an adaptive process aimed at an enhancement of plant stress tolerance. An active stress acclimation requires relatively high energy costs as indicated by profound alterations in energy metabolism. Practically all stresses induce an increased relative abundance of enzymes of carbohydrate catabolism such as glycolysis (glyceraldehyde-3-phosphate dehydrogenase GAPDH, triosephosphate isomerase TPI, enolase ENO), Krebs cycle (mitochondrial NAD + -dependent malate dehydrogenase (MDH; Vítámvás et al., 2012), aconitase (Jacoby et al., 2010(Jacoby et al., , 2013Budak et al., 2013) and components of mitochondrial ATP-synthase, namely β subunit of CF1 complex (Bahrman et al., 2004;Patterson et al., 2007;Vítámvás et al., 2012;Budak et al., 2013;Kosová et al., 2013b;Rollins et al., 2013;Xu et al., 2013) indicating an enhanced demand for energy. Regarding photosynthesis, an increase or a decrease in several photosynthetic proteins (proteins involved in primary photosynthetic reactions, carbon fixation, and Calvin cycle) have been observed depending on the severity of stress (Caruso et al., 2008(Caruso et al., , 2009Ye et al., 2013). A downregulation of photosynthesis reactions under severe stress is reflected at proteome level by a decrease in D1 and D2 proteins in photosystem II reaction center (RC PSII), proteins of oxygen evolving complex (OEC), a decrease in chlorophyll a-b binding proteins in both photosystem (PS) I and II, a decrease in Fe-S complex in PSI, a downregulation of RubisCO and key Calvin cycle enzymes phosphoglycerate kinase and phosphoribulokinase in cold-and waterlogging-treated winter wheat (Li et al., 2014), salt-treated durum wheat (Caruso et al., 2008) and in drought-treated barley (Ghabooli et al., 2013) while an increase in OEC protein OEE2 was found in salt-treated barley (Rasoulnia et al., 2011;Fatehi et al., 2012). In addition, an www.frontiersin.org December 2014 | Volume 5 | Article 711 | 9 increase in proteins with stimulating and protective functions such as RubisCO activase A (Bahrman et al., 2004;Caruso et al., 2008Caruso et al., , 2009Fatehi et al., 2012;Budak et al., 2013), a thermostable RubisCO activase B (Rollins et al., 2013), carbonic anhydrase (Caruso et al., 2008) and RubisCO large and small subunit binding proteins CPN60-α and CPN60-β was observed under various stresses (Caruso et al., 2008;Sarhadi et al., 2010;Kang et al., 2012;Budak et al., 2013;Kosová et al., 2013b;Xu et al., 2013). An increased demand on energy under stress acclimation corresponds with a decreased abundance of enzymes (fructokinase-2, sucrose synthase-1) involved in biosynthesis of energy-rich compounds such as starch and a decrease in storage proteins (11S seed storage protein 2-like, legumin-like protein; Vítámvás et al., 2012;Kosová et al., 2013b).
Stress represents an enhanced risk of protein damage due to imbalances in cellular homeostasis. Therefore, increased abundances of several proteins with chaperone and other protective functions have been reported. Extreme temperatures, but also drought, pathogens, and other stresses cause an enhanced risk of protein misfolding and they are thus associated with an enhanced accumulation of chaperones from HSP superfamily, namely HSP70 (Rampitsch et al., 2006;Li et al., 2013;Rollins et al., 2013), HSP100 (Clp protease; Ashoub et al., 2013), and small HSPs (Skylas et al., 2002;Majoul et al., 2004;Hajheidari et al., 2007), but also others such as chopper chaperone Hlaváčková et al., 2013), serpins (Yang et al., 2010b;Fercha et al., 2013Fercha et al., , 2014, and protein disulfide isomerase (Hajheidari et al., 2007;Vítámvás et al., 2012;Li et al., 2013). However, a decrease in HSP90 was reported under cold . Disorders in cellular metabolism under stress lead to an enhanced risk of oxidative damage. At proteome level, an increased abundance of several reactive oxygen species (ROS) scavenging enzymes was found practically under each kind of stress (Hajheidari et al., 2007;Ford et al., 2011). Plants try to reduce a risk of ROS formation by several ways. The major one represents a downregulation of photosynthesis reactions which is associated with a decrease in D1 and D2 proteins in photosystem II reaction center (RC PSII), proteins of OEC, RubisCO small subunit and key Calvin cycle enzymes phosphoglycerate kinase, phosphoribulokinase and transketolase (Caruso et al., 2008;Ford et al., 2011;Ashoub et al., 2013). Other indirect ways how to reduce ROS lie in a reduced uptake of metal ions, especially iron, which can act as catalyzers of ROS formation. A reduced level of protein IDI2 and dioxygenases IDS2, IDS3 involved in iron uptake and phytosiderophore biosynthesis was found by Witzel et al. (2009) in salt-treated barley roots while an increased level of these enzymes was found by Patterson et al. (2007) in barley grown under elevated boron.
Several stresses including drought, heat, salinity, cold, but also mechanical wounding, induce an enhanced accumulation of proteins belonging to LEA superfamily. Late embryogenesisabundant (LEA) superfamily includes at least five subclasses, the most important being LEA-II (dehydrins) and LEA-III proteins whose transcript and protein levels, and also phosphorylation level, have been reported to correlate with wheat and barley tolerance to low temperatures (Crosatti et al., 1995;Vágújfalvi et al., 2000Vágújfalvi et al., , 2003Vítámvás et al., 2007;Kosová et al., 2008cKosová et al., , 2013cSarhadi et al., 2010), drought (Labhilili et al., 1995;Brini et al., 2007) and other stresses.
Stresses also affect other aspects of cellular metabolism. An increased abundance of methionine synthase catalyzing formation of methionine or S-adenosylmethionine synthase (SAMS) catalyzing formation of S-adenosylmethionine (SAM) has been reported (Bahrman et al., 2004;Patterson et al., 2007;Witzel et al., 2009;Vítámvás et al., 2012;Kosová et al., 2013b;Xu et al., 2013). SAM represents not only a universal methyl donor in regulation of DNA heterochromatin formation and gene expression, but it is also a precursor of several stress-related metabolites as glycine betaine, polyamines, hydroxymugineic acids (phytosiderophore precursors; Mori and Nishizawa, 1987) and ethylene. Alterations in glutamine synthetase (GS) have been reported under drought (Ford et al., 2011;Kang et al., 2012) andcold (Hlaváčková et al., 2013) indicating an important role of nitrogen assimilation and proline biosynthesis in stress acclimation.
Stress affects cellular transport and membrane properties. An enhanced need for ion transport and thus an associated increase in plasma membrane and tonoplast ion transporters such as V-ATPase has been reported not only under salinity (Peng et al., 2009), but also under other stresses such as drought (Ghabooli et al., 2013), heat (Majoul et al., 2004) and osmotic stress (Ye et al., 2013;Zhang et al., 2014). Differential phosphorylation of several transport proteins such as aquaporins, H + -ATPase or

Frontiers in Plant Science | Plant Proteomics
December 2014 | Volume 5 | Article 711 | 10 monosaccharide sensing protein 2, was also reported in response to stress (Zhang et al., 2014). The effect of several stresses on cell wall remodeling is indicated by alterations in several enzymes involved in lignin metabolism such as caffeoyl-coenzyme A O-methyltransferase CCOMT indicating an important role of cell wall in plant stress response (Sugimoto and Takeda, 2009;Ghabooli et al., 2013). Long-term and regularly occurring stress factors such as cold during winter also affect plant development. At proteome level, significant changes in the level of small glycine-rich RNA-binding proteins (sGRPs) and in lectins, glycoproteins involved in saccharide signaling, were found in wheat (Rinalducci et al., 2011b;Kosová et al., 2013b). Ricin B lectin 2 was reported to be induced by cold in crowns of both winter barley (Hlaváčková et al., 2013) and winter wheat (Kosová et al., 2013b). Lectin VER2 was reported to accumulate in winter wheat shoot apex until vernalization (Yong et al., 2003;Rinalducci et al., 2011b). Differences in sGRPs and VER2 levels between spring and winter wheat growth habits indicate a differential response to cold within wheat germplasm (Kosová et al., 2013b).

PROTEINS REVEALING A DIFFERENTIAL RESPONSE BETWEEN STRESS-TOLERANT AND STRESS-SENSITIVE GENOTYPES
A differential ability of various wheat and barley genotypes to cope with several stresses is reflected also at protein level. Stresstolerant genotypes do not suffer from a disruption of energy metabolism when exposed to moderate stress levels; moreover, when exposed to stress, they can increase an abundance of key enzymes of energy metabolism to increase ATP production as indicated by a differential response observed in several photosynthesis-related proteins (RubisCO subunits, RubisCO activase), ROS scavenging enzymes as well as respiration (Krebs cycle) enzymes. Quantitative differences in Krebs cycle enzymes such as mitochondrial NAD + -dependent MDH between two differentially frost-tolerant winter wheats , in aconitase (Budak et al., 2013), thioredoxin h and glutathione-Stransferase (GST; Hajheidari et al., 2007;Sarhadi et al., 2010), lipoxygenase 1 and 2 (Alvarez et al., 2014) between differentially drought-tolerant wheats; in Cu/Zn-SOD, Mn-SOD (Ford et al., 2011;Xu et al., 2013), glyoxysomal MDH (gMDH; Ashoub et al., 2013), GST (Rasoulnia et al., 2011), class III peroxidase, catalase and lipoxygenase (Wendelboe-Nelson and Morris, 2012) between differentially drought-and salt-tolerant barleys; in Mn-SOD, MDH and aconitase between salt-treated wheat and wheat × Lophopyrum elongatum amphiploid (Jacoby et al., 2013), and a downregulation of MDH and isocitrate dehydrogenase in coldsensitive spring wheat (Rinalducci et al., 2011a) indicate a crucial role of mitochondrial respiration and ROS metabolism in stress acclimation. Along with these data, a differential abundance in storage proteins such as legumin-like protein between two differentially frost-tolerant winter wheats was found by Vítámvás et al. (2012) indicating a higher demand on energy ensured by storage compound degradation in the less-tolerant genotype. Moreover, tolerant genotypes can also afford to accumulate higher amounts of stress-protective proteins such as PR proteins (Witzel et al., 2014) and ABA-responsive proteins (Alvarez et al., 2014). A significant correlation between wheat WCS120 and barley DHN5 dehydrin relative accumulation and acquired frost tolerance (FT) determined as lethal temperature for 50 % of the sample (LT50) was reported for winter genotypes grown under both cold and moderate cold temperatures (Vítámvás et al., 2007(Vítámvás et al., , 2010Kosová et al., 2008cKosová et al., , 2013c. WCS120 and DHN5 can be thus considered promising FT markers.
Stress-tolerant and stress-sensitive genotypes or related plant species also reveal significant differences in proteins involved in regulation of cell cycle and plant development. Factor eIF5A2 does not only regulate translation inititation, but it is also known to participate in the regulation of cell cycle switch between cell proliferation and death (Thompson et al., 2004). Under salinity, a decreased abundance of eIF5A2 with respect to control was found in both salt-sensitive common wheat and salt-tolerant T. aestivum × Thinopyrum ponticum hybrid (Wang et al., 2008); however, a decrease in T. aestivum × Th. ponticum was much lower than in T. aestivum indicating a higher cell proliferation rate in the salttolerant hybrid. A differential abundance in lectin VER2 between cold-treated spring and winter wheat cultivars corresponds to a differential developmental response with a winter wheat revealing a developmental arrest while a spring wheat revealing a progression to reproductive phase as indicated at proteome and phytohormone levels (Kosová et al., , 2013b.

CONCLUSIONS AND FUTURE PERSPECTIVES
Both abiotic and biotic stress factors induce an active plant stress response including a profound reorganization of plant proteome. Comparative proteomic studies are usually carried out on a limited range of plant material due to their expensiveness and much of sophisticated work. However, they can significantly contribute to identification of novel proteins revealing a differential response in abundance or PTMs between differentially-tolerant genotypes and representing potential protein markers of stress tolerance.
The potential markers should be tested on a broad range of genotypes using simple protein quantification methods as ELISA or immunoblots which can be utilized by breeders. As an example, proteomic studies on cold-treated winter wheats resulting in an identification and testing of dehydrin proteins as FT markers can be given (Vítámvás et al., 2007(Vítámvás et al., , 2010. Recent publication of draft barley (The International Barley Genome Sequencing Consortium, 2012) and wheat (The International Wheat Genome Sequencing Consortium, 2014) genome sequences will significantly contribute to protein identification, sequentional characterization and preparation of specific antibodies which will stimulate further research and applications in breeding for an improved stress tolerance.

AUTHOR CONTRIBUTIONS
Klára Kosová has outlined the idea and prepared the text. Pavel Vítámvás and Ilja T. Prášil contributed to preparation, drafting, critical reading, and publication of the manuscript.

ACKNOWLEDGMENTS
The work was supported by projects from Ministry of Education, Youth and Sports of the Czech Republic (MEYS CZ) LD14064 and LD14087 as a part of of COST actions FA1204 and FA1208, www.frontiersin.org December 2014 | Volume 5 | Article 711 | 11 respectively. The work was also supported by an institutional project MZE RO0414 from Ministry of Agriculture of the Czech Republic (MZe CZ).