Stress Tolerance in Plants: A Proteomics Approach

Abstract

Both abiotic and biotic stresses adversely affect the plant growth and productivity including crop plants. The development of stress-tolerant plants will be greatly advantageous for modern agriculture in areas that are prone to such stresses. In recent times, several advances have been made towards identifying potential stress-related genes which are capable of increasing the tolerance of plants to both abiotic and biotic stresses. This interaction between biotic and abiotic stresses is controlled by hormone signaling pathways that may induce or antagonize one another, in particular that of abscisic acid. Specificity in multiple stress responses is further controlled by a range of biochemical and molecular mechanisms that act together in a complex regulatory network. Transcription factors, kinase cascades, and reactive oxygen species are key components of this cross talk as are heat shock factors and small RNAs. This review emphasis on elucidating the proteins associated with abiotic and biotic stresses in plants.

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Introduction

Plants need suitable physiological condition as well as essential nutrients to grow in the natural environment. As a consequence of their sessile growth habit, plants have adapted dynamic responses to these stresses at the physiological, biochemical, and molecular levels, thus enabling them to survive under different environmental conditions. Most of the crop plants grow in environments that are suboptimal, which prevents the plants from attaining their full genetic potential for growth and reproduction (Bray et al. 2000; Rockstrom and Falkenmark 2000). Abiotic stress factors such as heat, cold, drought, salinity, and nutrient stress have a huge impact on world agriculture, and it has been suggested that they reduce average yields by >50% for most major crop plants (Wang et al. 2003). Further to this, plants must defend themselves from attack by a vast range of pests and pathogens, including fungi, bacteria, viruses, nematodes, and herbivorous insects (Hammond-Kosack and Jones 2000). Each stress elicits a complex cellular and molecular response system implemented by the plant in order to prevent damage and ensure survival, but often at the detriment of growth and yield (Herms and Mattson 1992). Industrial activity combined with a low conscience of the consequences of environmental pollution during a long period created a worldwide problem of soil, air, and water contamination with various pollutants. Heavy metals are among the most widespread soil contaminants. Abiotic stresses usually cause protein dysfunction. The yield and quality of cereals are severely affected by heat stress in many countries (Treglia et al. 1999). Heat stress affects the grain yield and quality. Drought, soil salinity, and heavy metal significantly affect plant growth, development, and productivity, thus posing a severe threat to agriculture throughout the world. Among abiotic stresses, osmotic stress is one of the most severe, caused by drought, high salinity, and cold stresses in nature. Water is the most wide-ranging difficulty among abiotic stresses for production of crop in the world environment. Strategy is to obtain plants with higher performance under water stress conditions by identifying and modifying the molecular mechanisms that take place when the water availability becomes limiting. Plants, as sessile organisms, rely on proteomic plasticity to remodel themselves during periods of developmental change and to respond to biotic and abiotic stresses. In the last decade, methodological improvements have allowed comparative proteomic investigations of plants under stress which have allowed us to analyze biochemical pathways and the complex response of plants to environmental stimuli (Qureshi et al. 2007). More comprehensive approaches that include quantitative and qualitative analyses of gene expression products are necessary at the transcriptome, proteome, and metabolome levels. Abiotic stresses resulted the cellular dehydration, such as freezing and salt and water stress, often lead to similar changes in plant gene expression and metabolism (Cook et al. 2004; Kreps et al. 2002). The phytohormone abscisic acid (ABA) is produced under abiotic stress such as drought and high salinity. ABA is a key mediator in controlling plant response to abiotic stress by regulating stomatal closure and by triggering the activation of many stress-related genes, thereby increasing the tolerance of plants to the stresses. Many abiotic stress-responsive genes have been identified in plants, including rice and Arabidopsis, by using molecular techniques (Fowler and Thomashow 2002; Rabbani et al. 2003; Yamaguchi-Shinozaki and Shinozaki 2006; Nakashima et al. 2009). Stress-induced genes not only function to protect cells from abiotic stress through the production of important enzymes and metabolic proteins (functional proteins) but they also regulate signal transduction and gene expression in the stress response (regulatory proteins). Functional proteins contain hydrophilic proteins including dehydrins and “late embryogenesis abundant” (LEA) proteins and also enzymes that are required for the synthesis of osmoprotectants such as proline and sugars. Regulatory proteins that are activated in response to abiotic stresses, including transcription factors (TFs) such as DREBs (dehydration-responsive element-binding proteins), AREBs (ABA-responsive element-binding proteins), and NAC proteins, have been identified in Arabidopsis and rice (Yamaguchi-Shinozaki and Shinozaki 2006; Nakashima et al. 2009, 2012). Proteomics also makes an essential bridge between the transcriptome and metabolome (Wang et al. 2004; Gray and Heath 2005), complementing genomics research. Only by grouping all this information together is it possible to achieve a comprehensive and exhaustive analysis of the mechanism of plant defense against abiotic and biotic stresses. Upon several stress responses, protein, protein–protein interaction, and posttranslation modification have been also identified (Salekdeh et al. 2002b). Plants have evolved to live in environments where they are often exposed to different stress factors in combination. Being sessile, they have developed specific mechanisms that allow them to detect precise environmental changes and respond to complex stress conditions, minimizing damage while conserving valuable resources for growth and reproduction. Plants activate a specific and unique stress response when subjected to a combination of multiple stresses (Rizhsky et al. 2002). Keeping in the view of the above fact, the present review highlighted the abiotic and biotic stresses in plants in proteomic approach.

Abiotic Stress in Plants

Abiotic stresses limit the productivity and growth potential of plants. Phytohormone like abscisic acid (ABA) is a key mediator in controlling plant response to abiotic stress by triggering the activation of many stress-related genes, thereby increasing the tolerance of plants to the stresses. Stress-induced genes not only function to protect cells from abiotic stress through the production of important enzymes and metabolic proteins (functional proteins) but they also regulate signal transduction and gene expression in the stress response (regulatory proteins). Functional proteins are required for the synthesis of osmoprotectants such as proline and sugars. Regulatory proteins that are activated in response to abiotic stresses, including transcription factors (TFs) such as DREBs (dehydration-responsive element-binding proteins), AREBs (ABA-responsive element-binding proteins), and NAC proteins, have been identified in Arabidopsis and rice (Yamaguchi-Shinozaki and Shinozaki 2006; Nakashima et al. 2009). Nakashima et al. (2012) reported that NAC proteins are plant-specific transcription factors and more than 100 NAC genes have been identified in Arabidopsis and rice to date. Tran et al. (2010) reported that NAC transcriptional factors which constitute one of the largest families of plant specific and help to enhance tolerance against various abiotic stresses. The cDNA encoding a NAC protein was first reported as the responsive to dehydration 26 (RD26) gene in Arabidopsis (Yamaguchi-Shinozaki et al. 1992). Many NAC proteins, including Arabidopsis CUC2, have important functions in plant development. Some NAC genes are upregulated during wounding and bacterial infection (Collinge and Boller 2001; Mysore et al. 2002; Hegedus et al. 2003), whereas others mediate viral resistance (Xie et al. 1999). NAC proteins were thought to be transcriptional activators as the Arabidopsis ATAF1/2 proteins can activate the CaMV 35S promoter in yeast cells. The Arabidopsis AtNAM (NARS2) protein was confirmed by Duval et al. (2002) to function as a transcriptional activator in a yeast system. Kikuchi et al. (2000) reported eight NAC genes in rice (OsNAC1 to OsNAC8) which encode proteins with a single NAC domain. Each OsNAC gene has a unique tissue-specific expression pattern, suggesting this family regulates the development of rice. Ooka et al. (2003) performed a comprehensive analysis of NAC family genes in rice and Arabidopsis. They identified 75 predicted NAC proteins in full-length cDNA datasets of rice and 105 predicted genes in the Arabidopsis genome. Recently, Nuruzzaman et al. (2010) conducted a genome-wide analysis of the NAC transcription factor family in rice and Arabidopsis by investigating 151 nonredundant NAC genes in rice and 117 in Arabidopsis. Morishita et al. (2009) also reported that ANAC078 in the NAC group is responsive to a combination of high light and heat stress. They also demonstrated that ANAC078 regulates flavonoid biosynthesis, leading to the accumulation of anthocyanins under high-light conditions. Hu et al. (2006) reported that the overexpression of the stress-responsive SNAC1 gene increased drought and salt tolerance in rice. SNAC1 enhanced drought resistance in transgenic rice plants at the reproductive stage during growth in fields under severe drought stress without affecting yields (Hu et al. 2006). Nogueira et al. (2005) reported that the SsNAC23 gene, which is homologous to the rice OsNAC5, is associated with cold, herbivory, and water stress in sugarcane. Tran et al. (2009) identified 31 unigenes containing complete open reading frames encoding GmNAC proteins in soybean. Analysis of the C-terminal regulatory domain using a yeast one-hybrid system indicated that among the 31 GmNAC proteins, 28 have transcriptional activation activity. Among them, nine GmNAC genes are induced by dehydration stress with differential induction levels in both shoots and roots. Sperotto et al. (2009) reported that OsNAC5 expression is upregulated by natural (aging) and induced senescence processes. They suggested that OsNAC5 is a novel senescence-associated ABA-dependent NAC transcription factor. The stress-responsive NAC proteins, including OsNAC5, might also function during the process of senescence as well as stress tolerance mediated through ABA. Yang et al. (2011) identified a NAC transcription factor VND INTERACTING2 (VNI2) that mediates signaling cross talk between salt stress response and the process of leaf aging. VNI2 regulates the cold-regulated (COR) and responsive to dehydration (RD) genes by binding directly to their promoters. Overexpression of COR or RD led to prolonged leaf longevity, as observed in the VNI2-overexpressing transgenic plants. Mostafa Kamal et al. (2010a, b) identified GTP-binding proteins, which are involved in signal transduction mechanism in plant systems, and these proteins regulate a flow of kinases that play a vital role in environmental stress signal transduction, and also high temperature seems to upregulate the synthesis of GTP-binding proteins resulting in increased kinase activity (Grover et al. 2001). Some heat-upregulated proteins showed the similarities to elongation factors (EF) and eucaryotic translation initiation factors (eIFs). Heat shock involves changes in the expression patterns of the eIFs, the EF 1-beta, EF 1-alpha, EF Tu, and eIF (4A, 4B, 4E, 4E-1, 4E-2, 5A-1, 5A-2, SUII), in wheat leaves (Gallie et al. 1998). Calcium is a universal molecule in both animals and plants, and the transient increase in Ca²⁺ level during heat stress is well documented in plants. Heat shock triggers cytosolic Ca²⁺ bursts, which is transducted by Ca2+ binding proteins(CBP) such as calmodin (CaM), calcineurin (CBL), and annexin and then upregulates the expression of heat shock proteins (HSPs) (Liu et al. 2003). Tubulin proteins are coupled to GTP-binding proteins, which play a role in heat resistance in plant (Segal and Feldman 1996). Mostafa Kamal et al. (2010a, b) also identified serine carboxypeptidase, glucose-1-phosphate, glucose-6-phosphate, and S-adenosyl-methionine synthetase proteins. Different abscisic acid-responsive proteins, LEA protein such as chaperonin, cys peroxiredoxin, ethylene response, and elongation factor TU are responsible for drought stress (Mostafa Kamal et al. 2010a, b). They also observed that cyclin-dependent kinase like zinc finger, transcription factor like MYB, lipid transfer proteins and WRKY are effective for drought stress. Cyclophilin, aquaporin, and chitinase play an important role in cold (upregulated) stress of wheat as reported by Houde et al. (2006). The effects of dehydration, cold temperature treatment, and osmotic and salt stress on the expression of an abscisic acid-responsive protein kinase mRNA (PKABA1) were determined in wheat seedlings (Holappa and Simmons 1995). Mitogen has three protein kinases, MAPK (mitogen-activated protein kinase) and a ribosomal kinase homologue, increased markedly and simultaneously when plants were treated with low temperature (Zhang et al. 2006). GLPs (glucagon-like peptide) function primarily as superoxide dismutase (SOD) to protect plants from the effects of oxidative stress (Khuri et al. 2001). ABA is a central regulator of many plant responses to environmental stresses, particularly osmotic stresses (Chinnusamy et al. 2008; Cramer 2010; Hubbard et al. 2010; Kim et al. 2010). The essential components of ABA signaling include receptors (PYR/PYL/RCAR), protein phosphatases (PP2C), and protein kinases (SnRK2/OST1) (Ma et al. 2009; Park et al. 2009). The PYR/PYL/RCAR proteins were identified as soluble ABA receptors by two independent groups as reported by Park et al. (2009). Leung and Giraudat (1998) identified 2C-type protein phosphatases (PP2C) including ABI1 and ABI2 from the ABA-insensitive Arabidopsis mutants abi1-1 and abi2-1, and they act as global negative regulators of ABA signaling. In yeast, the well-documented central regulators of protein synthesis and energy are SnRK1 (Snf1/MAPK) (mitogen-activated protein kinase), TOR1, and GCN2 (Zaborske et al. 2010; Staschke et al. 2010; Smeekens et al. 2010). These proteins are largely controlled by the phosphorylation of enzymes; all three are protein kinases acting as key hubs in the coordination of metabolism during stressful conditions (Hey et al. 2010). In plants, target of rapamycin (TOR) activity is inhibited by osmotic stress and ABA (Deprost et al. 2007), and GCN2 activity is stimulated by UV light, amino acid starvation, ethylene, and cold stress (Lageix et al. 2008).

Water Stress

Drought is one of the major limiting factors of plant production worldwide. Plant adaptation to drought is the result of many different physiological and molecular mechanisms. Several studies have shown that physiological adaptations to water stress were associated with drought-induced proteins (Bray 1997). Proteomics has proved to be a powerful tool for the identification of proteins and mechanisms involved in drought response and tolerance (Riccardi et al. 1998; Kawasaki et al. 2000; Salekdeh et al. 2002a, b; Hajheidari et al. 2005). Out of 78 responsive proteins, 16 were identified, including proteins involved in the water stress response, the basic metabolic pathway, and lignification. Studies on mild drought stress in rice leaves showed that drought-induced changes in about 42 proteins were reversed completely within 10 days of rewatering. Molecular analyses of wild species such as drought-tolerant grasses would provide a better insight into genes and mechanisms by which plants may adapt to prolonged drought. Tolerant plants are able to maintain tissue water content, or survive a reduction in tissue water content, or recover more completely after rewatering (Cabuslay et al. 1999). The level of proline increased up to 20-fold in response to severe drought conditions as reported earlier (Pedrol et al. 2000; Clifford et al. 1998). Proline is considered as a compatible solute (Samaras et al. 1995) and an osmoprotectant (Serrano and Gaxiola 1994; Okuma et al. 2000). Proline may confer a protective effect by protecting or inducing stress-protective proteins (Khedr et al. 2003). They reported that severe salt stress caused an inhibition of the antioxidative enzymes catalase and peroxidase in Pancratium maritimum L., but the activity of these enzymes was also maintained significantly higher in the presence of proline. Therefore, plant cells require two different mechanisms which will enable the detoxification of excess ROS and fine modulation of ROS for signaling purposes. SOD acts as a first line of defense converting superoxide to the less toxic hydrogen peroxide molecules. The abundance of the cytosolic Cu-Zn SOD of Elymus elongatum increased up to twofold in response to drought. Cytosolic Cu-Zn SOD was shown to be upregulated in rice (Salekdeh et al. 2002b) and sugar beet (Hajheidari et al. 2005). The upregulation of this enzyme in plants grown under progressive stress reveals its important role in response to drought. The detoxification of H₂O₂ is accomplished with ascorbate peroxidase, glutathione peroxidase, catalase, and 2-Cys peroxiredoxin. Ascorbate peroxidase reduces H₂O₂ to water, with the concomitant generation of monodehydroascorbate (MDHA). MDHA is a radical with a short lifetime that disproportionates to ascorbate and dehydroascorbate (DHA). DHA is reduced to ascorbate by the action of DHA reductase, using glutathione as the reducing substrate. The most upregulated protein (4.8-fold) was identified as dehydroascorbate reductase suggesting its strong role in the detoxification of H₂O₂. Hydrogen peroxide (H₂O₂) is a key regulatory molecule in the response to stresses (Mittler 2002), and its ability to selectively induce a subset of defense genes like glutathione S-transferases and glutathione peroxidases without directly inducing other defense genes. The upregulation of both glutathione S-transferases and glutathione peroxidase may represent such co-expression mechanisms. Wehmeyer et al. (1996) suggested that small heat shock proteins (sHSPs) are among the several factors required for desiccation tolerance. Hajheidari et al. (2005) reported the upregulation of two sHSPs under drought stress in sugar beet grown in the field. These proteins showed sequence homology with abscisic acid- and stress-inducible protein (ASR). The upregulation of this gene has been also reported in response to drought stress in maize (Riccardi et al. 1998) and salt stress in a salt-tolerant rice genotype (Salekdeh et al. 2002a). Iusem et al. (1993) have reported that characterization of cDNA encodes ASR protein whose expression was activated by leaf water deficit and fruit ripening. Silhavy et al. (1995) identified a nuclear-targeting sequence motif in this gene isolated from Solanum chacoense Bitter which is extremely resistant to viruses, insects, and drought.

Phytohormone Stress

A variety of plant hormones, including salicylic acid (SA), jasmonate (JA), ethylene, and abscisic acid, have been implicated in mediating responses to a wide range of biotic and abiotic stresses (Diaz et al. 2002; Thomma et al. 1998; Audenaert et al. 2002). The roles of these hormones are dependent upon the particular host–pathogen interaction (Knoester et al. 1998). On the basis of the interactions of hormones in which resistant responses to biotrophs require SA, whereas responses to necrotrophs require JA and ethylene (Feys and Parker 2000), in some instances, these hormones are involved in determining the level of host basal resistance (Delaney et al. 1994). In case of tomato, ethylene, JA, and SA all independently contribute to its resistance to Botrytis cinerea (Diaz et al. 2002). The host plant actively regulates the Xanthomonas campestris pv. vesicatoria-induced disease response via the sequential action of at least three hormones (JA, ethylene, and SA), which promote expansive cell death of its own tissue (O’Donnell et al. 2003). Further, the effect of phytohormones is also regulated by other factors. For example, the MAPK kinase, EDR1, negatively regulates SA-inducible defenses (Frye et al. 2001), whereas MAPK 4 appears to differentially regulate SA and JA signals (Petersen et al. 2000). These findings also suggest that MAPK modulates cross talk between different plant defense pathways (Hammond-Kosacky and Parkerz 2003).

Osmotic Stress

Among abiotic stresses, osmotic stress is one of the most severe, caused by drought, high salinity and cold stresses in nature. Plants respond to osmotic stress at the morphological, anatomical, cellular, and molecular levels. To cope with osmotic-related stresses, plants have developed various responses such as production of osmolites for osmotic adjustment, synthesis of Na⁺/H⁺ antiporters for ion sequestration, and many other mechanisms (Bohnert et al. 1995). AtHKT1, which is a Na⁺ transporter, mediates osmolality balance between xylem vessels and xylem parenchyma cells (Snarpi et al. 2005). The operation of these responses usually requires three steps: osmotic stress recognition, signal transduction, and production of components for the physiological response (Tamura et al. 2003). In plants, a hybrid-type histidine kinase (AtTHK1) functions as an osmosensor and transmits the stress signal to a downstream mitogen-activated protein kinase (MAPK) cascade (Urao et al. 1999). Lu and Neumann (1999) reported that when rice seedlings were exposed to osmotic stress modulated by polyethylene glycol 6000, growth in emerging first leaves of the intact plant was inhibited. Early inhibition of leaf growth was not related to changes in root size, osmotic potential gradients, or cell wall-yielding characteristics in the leaf expansion zone of stressed seedlings. Deak and Malamy (2005) demonstrated that osmotic stress represses the formation of autonomous lateral roots from lateral root primordia of Arabidopsis, while lateral root initiation was not greatly affected. Abscisic acid (ABA) and a newly identified gene, LRD2, are involved in osmotic repression of lateral root formation. Further examination revealed that both ABA and LRD2 control root system architecture even in the absence of osmotic stress. The molecular mechanisms that mediated responses to environmental cues could also be regulators of intrinsic developmental programs in the root. Zonia and Mnnik (2004) investigated that tobacco pollen tube cell volume changes in response to osmotic perturbation by activation of the phospholipid signaling pathway. Several intermediates in the phospholipid signaling pathway were detected during pollen tube growth. Hypo-osmotic stress induced a rapid increase in phosphatidic acid and a decrease in phosphatidylinositol phosphate. The fact that these signaling molecules are present during normal growth and the mechanism for osmotic response involved components of the biomechanical networks driving pollen tube cell elongation. In osmotically stressed wheat coleoptiles, reduced rates of phenylalanine ammonia-lyase and tyrosine ammonia-lyase activities suppress phenylalanine biosynthesis, resulting in a reduced level of wall-bound ferulic acid. This decrease in wall-bound ferulic acid may lead to reduced levels of diferulic acid, an important contributor to maintaining cell wall extensibility (Wakabayashi et al. 1997). The osmotic potential of soil alters the depth of root systems, the rate of root elongation, and the number of lateral roots (van der Weel et al. 2000); however, there are only a few reports about phenotypic changes in rice under osmotic stress (Lu and Neumann 1999).

Zang and Komatsu (2007) reported that 15 proteins are changed in the basal part of rice leaf sheaths by mannitol treatment. The basal section of leaf sheath may undergo more subtle changes in protein expression than the distal end, where significant morphological changes are evident. They reported that 327 proteins were detected by digital image analysis. Among these, 15 proteins significantly responded to osmotic stress by up- or downregulation: 12 proteins increased in amount and 3 proteins decreased. Ten of the identified proteins are recognized as important components for stress response such as two proteasome degradation system-related proteins, five endoplasmic reticulum (ER)-related proteins, two proteins related to detoxification, and uroporphyrinogen decarboxylase, which is a cell death-related protein (Mock and Grimm 1997). Two proteins matched annotated sequences for putative proteins of unknown function and the three remaining proteins responding to osmotic stress were not similar to other proteins in existing databases.

The glyoxalase system is ubiquitous in nature and consists of two enzymes: glyoxalase I and glyoxalase II, which act coordinately to convert 2-oxoaldehydes into 2-hydroxyacids using reduced glutathione as a cofactor. The glyoxalase pathway involving glyoxalase I and glyoxalase II enzymes is required for glutathione-based detoxification of methylglyoxal. Methylglyoxal is a primary physiological substrate for glyoxalase I. Besides detoxification of methylglyoxal, the glyoxalase system could also play a role in providing tolerance under stress by recycling glutathione that would be “trapped” spontaneously by methylglyoxal to form hemithioacetal, thereby maintaining glutathione homeostasis (Creighyton et al. 1988). Transgenic plants overexpressing glyoxalase I showed significant tolerance to methylglyoxal and high salt (Veena and Sopory 1999; Singla-Pareek et al. 2003).

Heavy Metal Stress

Heavy metals (HMs) are among the most widespread soil contaminants which damage the physiological and metabolic processes. Certain heavy metals such as Cd, Hg, and Pb affect cell systems due to the increasing exposure of living organisms to these metals in the environment (DalCorso et al. 2008). The physiological and molecular basis of plant interactions with the environment has attracted considerable interest in recent years. Being sessile organisms, plants are constantly exposed during their life cycle to adverse environmental conditions that negatively affect growth, development, or productivity. The presence of toxic compounds, such as heavy metals (HMs), is one important factor that can cause damage to plants by altering major plant physiological and metabolic processes (Hossain et al. 2010; Villiers et al. 2011). Different metals and metalloids which are toxic to plants such as copper (Cu), iron (Fe), manganese (Mn), zinc (Zn), nickel (Ni), cobalt (Co), cadmium (Cd), and arsenic (As). Importantly, few HMs and transition metals such as sodium (Na), potassium (K), calcium (Ca), magnesium (Mg), Fe, Cu, Zn, Co, or Ni are essential micronutrients that are critically involved in the functional activities of large numbers of proteins involved in sustaining growth and development. However, at excess concentrations, these metal ions can become detrimental to living organisms, including plants. Although HMs are natural constituents of soils and occur naturally in the environment, contamination of soils by toxic metals and metalloids is of major concern worldwide (Villiers et al. 2011). Depending on their oxidation states, HMs can be highly reactive, resulting in toxicity of plant cells in many ways. At the cellular and molecular level, HM toxicity results in alterations of different plant physiological processes, including inactivation and denaturation of enzymes, proteins, blocking of functional groups of metabolically important molecules, displacement/substitution of essential metal ions from biomolecules and functional cellular units, conformational modifications and disruption of membrane integrity (Sharma and Dubey 2007), which is finally attributed to altered plant metabolism, inhibition of photosynthesis, respiration, and alerted activities of several key enzymes (Sharma and Dietz 2009; Hossain et al. 2010, 2012). In addition, HMs are known to disturb redox homeostasis by stimulating the formation of free radicals and reactive oxygen species (ROS) such as singlet oxygen (1O²), superoxide radicals (O^2•−), hydrogen peroxide (H₂O₂), and hydroxyl radicals (•OH) (Dubey 2011; Anjum et al. CR2402012). Recently, methylglyoxal (MG), a cytotoxic compound, was also found to increase in response various stresses including HMs (Yadav et al. 2005a, b). An increase in MG level in plant cells further intensifies the production of ROS by interfering with different plant physiological and metabolic processes such as inactivation of the antioxidant defense system (Hoque et al. 2010; Hossain et al. 2011a, b) and interfering with vital plant physiological processes such as photosynthesis (Saito et al. 2011). This increase in ROS and MG exposes cells to oxidative stress leading to lipid peroxidation, biological macromolecule deterioration, membrane dismantling, ion leakage, and DNA strand cleavage, and finally death of plants (Navari-Izzo 1998; Romero-Puertas et al. 2002; Barconi et al. 2011).

Heavy metals (HMs) are intracellularly chelated through the synthesis of amino acids, organic acids, GSH, or HM-binding ligands such as metallothioneins (MTs), phytochelatins (PCs), compartmentation fcc within vacuoles, and upregulation of the antioxidant defense and glyoxalase systems to counter the deleterious effects caused by ROS and methylglyoxal (Cobbett 2000; Yadav 2010; Hossain et al. 2012). A large number of recent studies in plants involving sensitive, tolerant, mutant, transgenic, and hyperaccumulator-adopting strategies in the fields of physiology, genomics, proteomics, and metabolomics suggest that GSH by itself and its related metabolizing enzymes, proteins, and peptides play a pivotal role in HM tolerance by controlling different plant physiological processes, including ROS and MG detoxification, heavy metal uptake, translocation, chelation, and detoxification. Yang et al. (2007) reported that a total of 17 Al-responsive proteins were identified, with 12 of those being upregulated and 5 downregulated. Among the upregulated proteins are copper/zinc superoxide dismutase (Cu-Zn SOD), GST, and S-adenosylmethionine synthetase 2, which are the consistently known Al-induced enzymes previously detected at the transcriptional level in other plants. More importantly, a number of other identified proteins including cysteine synthase (CS), 1-aminocyclopropane-1-carboxylate oxidase, G protein b subunit-like protein, abscisic acid- and stress-induced protein, putative Avr9/Cf-9 rapidly elicited protein 141, and a 33 kDa secretory protein are novel Al-induced proteins. Most of these proteins are functionally associated with signaling transduction, antioxidation, and detoxification (Hossain et al. 2012).

Mode of Action of Heavy Metals in Plant Cells

The toxicity of HMs is manifested in many ways when plant cells accumulate them at high levels. HMs are divided into two groups: redox active (Fe, Cu, Cr, Co) and redox inactive (Cd, Zn, Ni, Al, etc.). The redox active HMs are directly involved in the redox reaction in cells and result in the formation of O₂ ^•− and subsequently in H₂O₂ and OH production via the Haber-Weiss and Fenton reactions (Dietz et al. 1999; Schutzendubel and Polle 2002). Exposure of plants to redox inactive HMs also results in oxidative stress through indirect mechanisms such as interaction with the antioxidant defense system, disruption of the electron transport chain, or induction of lipid peroxidation. The latter can be due to an HM-induced increase in lipoxygenase (LOX) activity. Another important mechanism of HM toxicity is the ability of HMs to bind strongly to oxygen, nitrogen, and sulfur atoms (Nieboer and Richardson 1980). This binding affinity is related to free enthalpy of the formation of the product of the HM and ligand with low solubility of these products. Because of these features, HMs can inactivate enzymes by binding to cysteine residues. Many enzymes need cofactors to work properly for both HM ions (such as Fe²⁺, Mg²⁺, Cu²⁺, Ca²⁺) and organic molecules (such as heme, biotin, FAD, NAD, or coenzyme A). The displacement of one HM ion by another leads to the inhibition or loss of enzyme activities. Divalent cations such as Co²⁺, Ni²⁺, and Zn²⁺ displace Mg²⁺ in ribulose-1,5- bisphosphate-carboxylase/oxygenase (RuBisCO) and result in a loss of activity (Wildner and Henkel 1979; Van Assche and Clijsters 1986). Displacement of Ca²⁺ by Cd²⁺ in calmodulin, an important protein in cellular signaling, led to the inhibition of calmodulin-dependent phosphodiesterase activity in radish (Rivetta et al. 1997). Additionally, HMs cause membrane damage through various mechanisms, including the oxidation of and cross-linking with protein thiols, inhibition of key membrane protein such as H⁺-ATPase, or causing changes in the composition and fluidity of membrane lipids (Meharg 1993). Accumulation of MG, a cytotoxic compound, was found to increase in response to HM stress in plants due to impairment of the glyoxalase system that finally elicits oxidative stress by reducing the GSH content (Hossain and Fujita 2010; Hossain et al. 2011a, b, 2012).

Metallothioneins (MTs) are low-molecular-weight (4–8 kDa), Cys-rich, HM-binding, gene-encoded polypeptides that can bind heavy metals via the thiol groups of their Cys residues (Hamer 1986). Although the precise physiological function of MTs has not yet been fully elucidated, proposed roles include participation in maintaining the homeostasis of essential transition HMs, sequestration of toxic HMs, and protection against intracellular oxidative damage (Gasic and Korban 2006). Plant MTs are extremely diverse and have been subdivided into three classes based on the arrangement of the Cys residue (Zhou et al. 2006). The Cys-Cys, Cys-X-Cys, and Cys-X-X-Cys motifs (in which X denotes any amino acid) are characteristic and invariant for MTs. The organization or distribution of cysteine residues confers different MT isoforms and their ability to bind and sequester different HM ions for detoxification and homeostasis. The biosynthesis of MTs is regulated at the transcriptional level and is induced by several factors, including hormones, cytotoxic agents, and HMs, such as Cd, Zn, Hg, Cu, Au, Ag, Co, Ni, and Bi (Kagi 1991). Gene expression studies were performed to quantify mRNA levels in different tissues, at different developmental stages, and under stress conditions such as HM exposure. MT genes appear to be differentially regulated in a tissue-specific manner and in relation to the developmental stage and also in response to a number of stimuli, including heavy metals (Castiglione et al. 2007). Ahn et al. (2012) showed that three Brassica rapa MT genes (BrMT1, BrMT2, and BrMT3) are differentially regulated under various HM stresses.

Cold Stress

Plants are frequently facing different environmental variations. Among these constraints, low temperature is one of the most crucial factors that impair the distribution, growth, and productivity of crops or wild plant species. The mechanisms underlying cold acclimation and tolerance have been intensively studied over the past years, and numerous studies on plant response to cold stress have been reported (Ruelland et al. 2009). Cold weather can result in pollen sterility in plants at the reproductive stage of development. Several studies of plant transcriptomes during cold stress have been performed in rice (Chen et al. 2002a, b; Fowler and Thomashow 2002; Kawamura and Uemura 2003; Amme et al. 2006; Goulas et al. 2006; Cui et al. 2005; Yan et al. 2006; Hashimoto and Komatsu 2007). Hashimoto and Komatsu (2007) reported that some proteins are involved in cold stress, and these are the chilling stress-responsive proteins. In general, the level of mRNA does not always correlate well with the level of protein mainly due to posttranscriptional regulation (Yan et al. 2006). As a global study of the proteins comprising the proteome, proteomics can be playing an increasingly important role in addressing plant response to environmental changes. The proteomes of various plants in response to different environmental factors including drought, salt, heat, and heavy metal have been investigated (Ouerghi et al. 2000; Hajduch et al. 2001; Salekdeh et al. 2002a; Majoul et al. 2003). The differentially expressed proteins have been identified and well-documented stress-responsive proteins and some novel cold-responsive proteins. Plants differ in their cold response and cold-tolerant species may develop efficient strategies to adapt to chilling environment. Bressan et al. (2001) reported that the large-scale transcriptional studies were hindered by the absence of genome sequence information.

ROS-Scavenging Mechanisms in Cold Stress

Cold stress may disturb cellular redox homeostasis and promotes the production of reactive oxygen species (ROS) and reactive aldehydes (as 4-hydroxy-nonenal and methylglyoxal). Plants develop ROS-scavenging mechanisms to cope with the oxidative stress. Because chloroplast is one of the major sources of ROS, four proteins involved in chloroplast redox homeostasis, including 2-cys peroxiredoxin, chloroplast (BAS1), peroxiredoxin-2E, ferritin-1, and alanine-2-oxoglutarate aminotransferase 1 (GGT1) (Gao et al. 2009). BAS1 is involved in the detoxification of alkyl hydroperoxides and its gene expression is regulated under the control of the cellular redox state (Baier and Dietz 1997). Peroxiredoxin-2E reduces hydrogen peroxide and alkyl hydroperoxides with reducing equivalents provided through the thioredoxin or glutaredoxin system and was a component of chloroplast thioredoxin system (Bréhélin et al. 2003). Ferritin-1, an iron binding protein, is to protect plants from oxidative damage induced by manifold stresses (Deák et al. 1999). GGT1, an enzyme involved in photorespiration and photorespiration, has been suggested to be important for maintaining electron flow to prevent photoinhibition under stress conditions (Wingler et al. 2000). Methionine residues of proteins are a major target for oxidation by ROS and peptide methionine sulfoxide reductase plays protective roles in the cellular response to oxidative stress (Kwon et al. 2007). Aldo-keto reductases can detoxify lipid peroxidation products and glycolysis-derived reactive aldehydes that contribute significantly to cellular damages caused by abiotic stresses and improve scavenging capacity of the plant.

Cold stress may impose a great influence on the protein synthesis apparatus in plant. A chloroplast EF-G protein appeared in gel after 2, 5, and 24 days of cold treatment, but there was no spot at the corresponding position in gel of the control group. Because chloroplast EF-G is a housekeeping gene, there must be an EF-G protein spot elsewhere in gel of control group (Gao et al. 2009). It is postulated that posttranslational modification or alternative splicing may occur on EF-G in T. halophila under cold stress to maintain the normal operation of protein synthesis. Although, EF-G was previously reported to play a role in chloroplast biogenesis and development in Arabidopsis. PAB1 was reported to be involved in ubiquitin-dependent protein catabolic process. This may indicate that a high protein turnover rate is needed for plants to eliminate the misfolded polypeptides under cold environment (Amme et al. 2006; Goulas et al. 2006) and also in rice plant (Cui et al. 2005; Yan et al. 2006; Hashimoto and Komatsu 2007). They suggested that the cold stress responses in Thellungiella are similar, in general, to other plant species. Because Thellungiella is a cold-tolerant relative of Arabidopsis, comparison of the cold-induced rosette leaf proteome changes in Thellungiella, and Arabidopsis was performed to advance the understanding of the higher cold tolerance of Thellungiella. The identification of cold-responsive proteins in Thellungiella provides not only new insights into cold stress responses but also a good starting point for further investigation of their functions. However, considering the inability of 2-DE-based proteomic study to resolve membrane proteins and low abundant proteins, integrated transcriptomic, proteomic, and metabolomic approaches should be adopted to gain further insight into plant response to cold stress.

Biotic Stress in Plants

Plants responding to biotic stresses produce several protective compounds and proteins such as pathogenesis-related (PR) proteins, directly disease-related proteins, and other proteins. Biotic is one of the serious stresses affecting plant growth and productivity. A clear perceptive of the molecular mechanisms involved in plants response to biotic stress is of fundamental importance to plant science (Mostafa Kamal et al. 2010a, b). The study of PR proteins is also important for crop production due to the fact that many plant-derived pathogen-related proteins have been identified as members of PR protein families 2, 3, 4, 5, 8, 10, and 14 (Hoffmann-Sommergruber 2002). Among these are pathogenesis-related proteins coded by the host plant that accumulate in response to pathogen infection or other signals related to plant defense responses. Several PR proteins have been characterized at the molecular level and have shown to have antifungal activity in vitro (Datta and Muthukrishnan 1999) and show enzymatic activity such as β-1,3-glucanase and chitinase (PR2 and PR3, respectively), both involved in the degradation of microbial cell wall structural polysaccharides (Legrand et al. 1987) and PR4 and PR9, characterized by ribonuclease and peroxidase activity, respectively (Caporale et al. 2004). Proteomic approaches have been successfully used in discovering the resistance mechanisms in maize against kernel rot caused by Fusarium or Aspergillus (Chen et al. 2004; Chen and Chen 2002; Sonia et al. 2004). Mostafa Kamal (2010a,b) reported that methyl jasmonate (MeJA) and its free acid jasmonic acid (JA) collectively referred to as jasmonates are important cellular regulators involved in diverse developmental process, such as seed germination, root growth, fertility, seed ripening, and senescence. In addition, jasmonate activate plant defense mechanisms in response to insect-driven wounding and various pathogens (Creelman and Mullet 1997). Carmona et al. (1993) reported that different purothionins are active against plant pathogens both in vitro and in vivo. Gamma-1-purothionin showed a higher structural analogy with scorpion toxins and against insect defensins which also present the cystine-stabilized alpha helical (CSH) motif (Bruix et al. 1993) and gamma-2-purothionin inhibits protein translation in cell-free systems resulting in the exhibited plant toxins for pathogen (Colilla et al. 1990). Mostafa Kamal et al. (2010a) indicated that the antifungal activity has been associated with two immunochemically distinct proteins; the proteins are homologous with thaumatin- and pathogenesis-related proteins of the PR5 family. These proteins have intensely sweet properties of thaumatin; multiple unrelated defense functions against insect and fungal pests can now be associated with the family of thaumatin-homologous proteins (Hejgaard et al. 1991). Antimicrobial proteins (MBP-1) inhibit spore germination or hyphal elongation of several plant pathogenic fungi, including two seed pathogens of maize (Fusarium moniliforme Sheld. and Fusarium graminearum (Gibberella zeae (Schw.) Petsch)) and several bacteria, including a bacterial pathogen of maize (Clavibacter michiganense ssp. nebraskense) (Duvick et al. 1992). Wheat calcium-dependent protein kinase (CDPK) genes were found to respond to various biotic and abiotic stimuli, including cold, hydrogen peroxide (H₂O₂), salt, drought, powdery mildew (Blumeria graminis tritici, BGT), as well as phytohormones abscisic acid (ABA) and gibberellic acid (GA) (Li et al. 2008). Chitinases are important components of plant defense in response to attack by pathogens as F. graminearum (Li et al. 2001). Dilbirligi and Gill (2003) reported that many RGA sequences in wheat are identified as disease resistance gene. Ascorbate peroxidase, peroxidase, and glutathione (GSH)-dependent dehydroascorbate reductase accumulate early in grain fill. SGT1, a component of R-gene-triggered disease resistance, and serpin, a serine protease inhibitor, are also present and may protect the developing grain against various pathogens (Wong et al. 2004). The pathogen resistance proteins such as serpin; chitinase, which hydrolyzes the structural carbohydrate of fungal cell walls; barwin/PR-4 protein, which is induced by fungal pathogens and binds chitin; and xylanase inhibitor protein, which inhibits a fungal enzyme that degrades plant cell walls (Hurkman and Tanaka 2007). Zhang et al. (2008) reported that a total of 375 functional ESTs have been identified by SSH from a cDNA library of resistant soybean varieties associated with Phytophthora root rot. They also observed that system acquired resistance (SAR) induced by Phytophthora in soybean primarily depended on salicylic acid-mediated signaling pathways, which was different from resistance mechanisms in Arabidopsis. Nevertheless, the functions of differentially expressed proteins induced by Phytophthora are almost the same in soybean and Arabidopsis, including material metabolism enzymes and various regulatory factors; this suggests that the molecular basis of system acquired resistance against Phytophthora is much the same in soybean and Arabidopsis (Sun et al. 2008). Plant disease resistance genes usually have conserved motifs such as the nucleotide-binding site (NBS). Based on the NBS domain, two putative resistance genes (KR1 and KR4) were isolated from soybean. Sixteen identified proteins were potentially involved in protein degradation, defense signal transfer, oxidative stress, cell wall reinforcement, and energy and metabolism regulation (Yang, et al. 2010). In addition, differential expression was found by SSH in cDNA libraries from soybean varieties resistant to soybean cyst nematode (Cheng et al. 2007; Lu and Fang 2003).

Interaction with Insect and Pest with Stress

Trichoderma spp. have been known as biocontrol agents for the control of plant diseases (Harman et al. 2004). In many cases, the beneficial fungi may induce systemic resistance that is mediated by alterations in plant gene expression (Alfano et al. 2007; Shoresh et al. 2010). They also reported that the plant in association with Trichoderma strains enhanced plant growth, but the effects, as with other plant growth-promoting microbes (Gamalero et al. 2009), are greater when plants are under suboptimal conditions or biotic, abiotic, or physiological stresses (Yildirim et al. 2006). Several recent reports indicate that the fungi enhances tolerance to abiotic stresses during plant growth, in part due to improved root growth, improvement in water-holding capacity of plants (Harman 2000; Bae et al. 2009), or enhancement in nutrient uptake; whereas, in the absence of stress, plant growth may or may not be enhanced. Although molecular studies indicate greater expression of gene families involved in plant protection against abiotic stresses or oxidative damage in Trichoderma spp.-treated plants, no experimental evidence has been presented correlating enhanced tolerance of plants colonized with biocontrol fungi to these changes in molecular level. These fungi are frequently applied as seed treatments, where they may improve plant stands and induce long-term improvements in plant quality (Harman 2000, 2006).

Effects of Abiotic Stress on Plants: A Systems Biology

Recent advances in biotechnology have dramatically changed our capabilities for gene discovery and functional genomics. For the first time, we can now obtain a holistic “snapshot” of a cell with transcript, protein, and metabolite profiling (Umezawa 2011). Such a “systems biology” approach allows for a deeper understanding of physiologically complex processes and cellular function (Kitano 2002). Understanding the function of genes is a major challenge of the post-genomic era. While many of the functions of individual parts are unknown, their function can sometimes be inferred through association with other known parts, providing a better understanding of the biological system as a whole. High-throughput -omics technologies are facilitating the identification of new genes and gene function. In addition, network reconstructions at the genome scale are key to quantifying and characterizing the genotype to phenotype relationships (Feist and Palsson 2008).

Fundamentally, plants require energy (light), water, carbon, and mineral nutrients for growth. Abiotic stress reduces growth and yield below the optimum levels. Plant responses to abiotic stresses are dynamic and complex system (Skirycz and Inze 2010; Cramer 2010); they are both elastic (reversible) and plastic (irreversible). The plant responses to stress are dependent on the tissue or organ affected by the stress. Transcriptional responses to stress are tissue or cell specific in roots and are quite different depending on the stress involved (Dinneny et al. 2008). In addition, the level and duration of stress (acute vs. chronic) can have a significant effect on the complexity of the response (Tattersall et al. 2007). Water deficit inhibits plant growth by reducing water uptake into the expanding cells and alters enzymatically the rheological properties of the cell wall, for example, by the activity of ROS (reactive oxygen species) on cell wall enzymes (Skirycz and Inze 2010). In addition, water deficit alters the cell wall nonenzymatically, for example, by the interaction of pectate and calcium (Boyer 2009). Furthermore, water conductance to the expanding cells is affected by aquaporin activity and xylem embolism (Parent et al. 2009; Boursiac et al. 2008). With long-term stress, photosynthesis declines due to stomatal limitations for CO₂ uptake and increased photoinhibition from difficulties in dissipating excess light energy (Pinheiro and Chaves 2011). One of the earliest metabolic responses to abiotic stresses and the inhibition of growth is the inhibition of protein synthesis (Good and Zaplachinski 1994; Vincent et al. 2007) and an increase in protein folding and processing (Liu and Howell 2010). Energy metabolism is affected as the stress becomes more severe (e.g., sugars, lipids, and photosynthesis) (Cramer et al. 2007; Kilian et al. 2007). Thus, there are gradual and complex changes in metabolism in response to stress.

Regulatory Mechanism

The plant molecular responses to abiotic stresses involve interactions and cross-link with many molecular pathways (Takahashi et al. 2004). Reaction oxygen species (ROS) signaling in response to abiotic stresses and its interactions with hormones has been thoroughly reviewed (Mittler et al. 2010). ROS and RNS (reactive nitrogen species) form a coordinated network that regulates many plant responses to the environment. There are a large number of studies on the oxidative effects of ROS on plant responses to abiotic stress, but only a few studies documenting the nitrosative effects of RNS (Molassiotis and Fotopoulos 2011). Hormones are also important regulators of plant responses to abiotic stress. The two most important hormones are abscisic acid (ABA) and ethylene. ABA is a central regulator of many plant responses to environmental stresses, particularly osmotic stresses (Cramer 2010; Kim et al. 2010; Chinnusamy et al. 2008). There are slower responses to ABA involving transcriptional responses that regulate growth, germination, and protective mechanisms. Recently, the essential components of ABA signaling have been identified, and their mode of action was elaborated (Umezawa et al. 2010). The current model of ABA signaling includes three core components, receptors (PYR/PYL/RCAR), protein phosphatases (PP2C), and protein kinases (SnRK2/OST1). The PYR/PYL/RCAR proteins were identified as soluble ABA receptors by two independent groups (Ma et al. 2009; Park et al. 2009). The 2C-type protein phosphatases (PP2C) including ABI1 and ABI2 were first identified from the ABA-insensitive Arabidopsis mutants abi1-1 and abi2-1, and they act as global negative regulators of ABA signaling (Leung and Giraudat 1998). Yamaguchi-Shinozaki and Shinozaki (2006) reported that the transcriptional regulation of dehydration and salinity stresses have revealed both ABA-dependent and ABA-independent pathways. Cellular dehydration under water-limited conditions induces an increase in endogenous ABA levels that trigger downstream target genes encoding signaling factors, transcription factors, metabolic enzymes, and others. In the post-genomic era, comprehensive analyses using three systematic approaches have increased our understanding of the complex molecular regulatory networks associated with stress adaptation and tolerance. The first one is “transcriptomics” for the analysis of coding and noncoding RNAs and their expression profiles. The second one is “metabolomics” that is a powerful tool to analyze a large number of metabolites. The third one is “proteomics” in which protein and protein modification profiles offer an unprecedented understanding of regulatory networks. Protein complexes involved in signaling have been analyzed by a proteomics approach (Kaufmann et al. 2011). Hirai et al. (2004, 2007) identified MYB transcription factors regulating glucosinolate biosynthesis in Arabidopsis in response to S and N deficiency using an integrated transcriptomics and metabolomics approach. Genes and metabolites in glucosinolate metabolism were found to be coordinately regulated (Hirai et al. 2004). Co-expression analysis was used to identify two MYB transcription factors that positively regulate glucosinolate metabolism (Hirai et al. 2007). Mao et al. (2009) performed a gene co-expression network analysis of 1,094 microarrays of Arabidopsis using a nontargeted approach. They identified 382 modules in this network. The top three modules with the most nodes were photosynthesis, response to oxidative stress, and protein synthesis. Many of the modules also involved responses to environmental stresses. They constructed a cold-induced gene network from a subset of microarrays. Weston et al. (2008) used weighted co-expression analysis to define six modules for Arabidopsis responses to abiotic stress. Two hubs in the common response module were an ankyrin-repeat protein and genes involved in Ca signaling. They created a compendium of genomic signatures and linked them to their co-expression analysis.

Proteomics Approach in Biotic Stress

High temperature and drought, which often occur together during the growing season and likely contribute to poor kernel development, have been reported to increase growth of the fungus and toxin production (Payne 1998). Jones et al. (1981) reported that irrigating corn fields to reduce drought stress also reduced fungal infection and aflatoxin contamination. Irrigation not only relieved drought stress but also reduced soil temperature. Lower soil temperature was found to reduce aflatoxin contamination in peanut (Hill et al. 1983). Increased aflatoxin contamination was observed in drought-treated peanuts with increased soil temperatures (Cole et al. 1985). Dorner et al. (1989) also reported that a higher soil temperature favors Aspergillus flavus growth and aflatoxin production. A study on the effect of drought on peanut resistance to A. flavus by Wotton and Strange (1987) found that fungal colonization was inversely related to water supply as was aflatoxin production. Payne et al. (1986) also concluded that water stress appears to be a major factor affecting aflatoxin contamination. Studies of aflatoxin and fumonisin contamination of corn grown under high or moderate heat stress (Abbas et al. 2002; Chen et al. 2004) demonstrate that heat stress also plays an important role in the susceptibility of corn to both aflatoxin and fumonisin contamination. Tubajika and Damann (2001) compared ear rot and aflatoxin production between nine drought-tolerant and two aflatoxin-resistant corn lines under field and laboratory conditions. They found that drought-tolerant lines all had significantly lower levels of ear rot and aflatoxin contamination compared to the aflatoxin-resistant controls, when grown under drought conditions (Tubajika and Damann 2001). A proteomics approach was recently employed to identify proteins whose level of expression associated with kernel resistance against A. flavus infection and aflatoxin production (Chen et al. 2002a, b). They found over a dozen proteins, either unique or fivefold upregulated in resistant lines, have been identified and sequenced (Chen et al. 2002a, b, 2004). These proteins can be grouped into three categories based on their peptide sequence homology, that is, storage proteins, such as globulin 1, globulin 2, and late embryogenesis abundant proteins (LEA3, LEA14); stress-related proteins, such as an aldose reductase (ALD), a peroxiredoxin antioxidant (PER1), a cold-regulated protein, a water stress-inducible protein, an anionic peroxidase, a glyoxalase I protein (GLX I), and several small heat shock proteins (HSP); and antifungal proteins, which include a trypsin inhibitor and a pathogenesis-related protein. The majority of protein identified was stress-related proteins and highly hydrophilic storage proteins. These data suggest that kernel resistance may require not only the presence of high levels of antifungal proteins but also that of high levels of stress-related proteins and highly hydrophilic storage proteins. Storage proteins have been reported to play an important role in stress tolerance. Chen et al. (2002a, b) reported that the members of the LEA genes family have been associated with plant responses to many different stresses including drought, salt, cold, heat, and wounding (Thomann et al. 1992). Transgenic expression of an LEA protein from barley demonstrated increased tolerance to water and salt stress in rice (Xu et al. 1996). Some stress-related proteins have been reported to not only confer stress tolerance but also enhance disease resistance. The expression of heat shock proteins, especially the small HSPs under stress, has been widely studied (Vierling 1991) and shown to possess molecular chaperone activity (Jacob et al. 1993). Aside from heat stress, HSPs are also induced by other stresses such as cold, drought, or salinity (Sabehat et al. 1998). The role of glyoxalase in stress tolerance is also highlighted in a recent study using transgenic tobacco plants overexpressing a Brassica juncea glyoxalase I (Veena et al. 1999). Park et al. (2001) and Shin et al. (2002) also found that transgenic expression of the tobacco stress-inducible gene 1 (Tsi1) induced expression of several pathogenesis-related genes under normal conditions, resulting in improved tolerance to salt and pathogens. The mechanisms plants use to adapt to abiotic and biotic stresses have been widely studied in a number of plants.

Production of ROS in Plants

Current research effort has focused on the isolation of stress-responsive genes and their regulation as a means to understand the molecular events underlying the adaptation process. An increasing body of evidence suggests that a subset of plant responses to biotic and abiotic stress is shared, such as the generation of reactive oxygen species (ROS), the activation of mitogen-activated protein kinases (MAPKs), and hormone modulations (Mittler 2002) (Fig. 15.1). Organelles such as chloroplasts, mitochondria, and microbodies are a major source of ROS production in plant cells. Together with an extensive battery of oxidases, the plant cell is well armed for bountiful yet flexible ROS production. In chloroplasts, the primary sources of ROS production are the Mehler reaction and the antenna pigments (Asada and Takahashi 1987). Production of ROS by these sources is enhanced in plants by conditions limiting CO₂ fixation, such as drought, salt, and temperature stress, as well as by the combination of these conditions with high-light stress. In C3 plants, limiting CO₂ conditions can also activate the photorespiratory pathway (Foyer 2002). As part of this pathway, H₂O₂ is generated in peroxisomes by the enzymatic activity of glycolate oxidase. Production of H₂O₂ in microbodies can also occur during lipid catabolism as a side product of fatty acid oxidation. In mitochondria, over-reduction of the electron transport chain is the main source of O²⁻ production under specific stress conditions (Møller 2001). ROS in plant cells include the detoxifying reactions catalyzed by cytochromes in both the cytoplasm and the endoplasmic reticulum. They are thought to play a key role in ROS signaling and contain a multimeric flavocytochrome that forms an electron transport chain capable of reducing O₂ to O²⁻. Chemical inhibitors of mammalian NADPH oxidase (such as diphenyleneiodonium) have been shown to block or impair ROS production during biotic or abiotic stresses in plants (Allan and Fluhr 1997). In addition to NADPH oxidases, pH-dependent cell wall peroxidases, germin-like oxalate oxidases, and amine oxidases have been proposed to generate ROS at the apoplast (Bolwell and Wojtaszek 1997; Walters 2003). Although much attention has been given to NADPH oxidases and their possible role in cell signaling, other ROS-producing mechanisms in the mitochondria, apoplast, and peroxisome are likely to play a role in ROS signaling in response to different stimuli or developmental signals.

Fig. 15.1

Modulation of reactive oxygen species (ROS) signaling by the reactive oxygen gene network of plants (Published by Mittler et al. 2004)

Modulation of ROS Signaling

ROS production is recognized as a common event in plant response to biotic and abiotic stresses (Mittler et al. 2010; Lamb and Dixon 1997). The hypersensitive response (localized plant cell death at the infection site) to halt pathogen invasion during an incompatible host–pathogen interaction has also been reported to involve the production of ROS. Mittler et al. (2004) reported that calcium signaling is predominantly controlled in plants by storage and release; ROS signaling is controlled by production and scavenging. Different developmental or environmental signals feed into the ROS signaling network and perturb ROS homeostasis in a compartment-specific or even cell-specific manner. Perturbed ROS levels are perceived by different proteins, enzymes, or receptors and modulate different developmental, metabolic, and defense pathways. ROS can be generated by various enzymatic activities, of which the best studied are NADPH oxidases, and removed by an array of ROS-scavenging enzymes. The intensity, duration, and localization of the different ROS signals are determined by interplay between the ROS-producing and ROS-scavenging pathways of the cell. This process requires a tight mode of regulation and might involve amplification and/or feedback inhibition loops. In addition to regulating the intensity and duration of the different ROS signals, the ROS-scavenging pathways are also responsible for maintaining a low steady-state baseline of ROS on which the different signals can be registered. The reactive oxygen gene network therefore modulates the steady-state level of ROS in the different cellular compartments for signaling purposes as well as for protection against oxidative damage. It is possible that the use of ROS as versatile signaling molecules originated from their proposed use to sense stress. Most forms of biotic or abiotic stress disrupt the metabolic balance of cells, resulting in enhanced production of ROS. Simple organisms, such as bacteria or yeast, sense the enhanced production of ROS using redox-sensitive transcription factors and other molecular sensors, activate different ROS defense pathways, and regulate their metabolic pathways to lower the production rate of ROS (Costa and Moradas-Ferreira 2001; Georgiou 2002). The signal transduction pathway was demonstrated by Mittler et al. (2004) (Fig. 15.2).

Fig. 15.2

Model of the reactive oxygen species (ROS) signal transduction pathway. ROS can be detected by at least three mechanisms (ROS receptors, redox-sensitive transcription factors, and phosphatases). HSF heat shock factor, PDK phosphoinositide-dependent kinase, TF transcription factor (Published by Mittler et al. 2004)

Stress and Transcription Factors

Studies of transcriptional activation of some stress-responsive genes have also led to the identification of cis-acting elements ABRE (ABA-responsive element) and DRE (dehydration-responsive element)/CRT (C-repeat) that function in ABA-dependent and ABA-independent gene expression in response to stress, respectively (Seki et al. 2003). Transcription factors belonging to the ethylene-responsive element-binding factor family that bind to DRE/CRT were also isolated (Liu et al. 1998). The genes encoding these transcription factors are induced early and transiently in response to cold, and these transcription factors, in turn, activate the expression of target genes. Liu et al. (1998) reported that the transcription factors (DREB2A and DREB2B) are also induced by dehydration and promote the expression of various genes involved in drought stress tolerance. The expression of a new DNA-binding protein DBF1 that specifically interact with the DRE2 cis-element of a corn rab17 gene promoter is induced by ABA, dehydration, and high salinity (Kizis and Pages 2002). Another example of a transcription factor is calcium-dependent protein kinases (CDPKs). CDPKs are implicated as important sensors of Ca²⁺ flux in plants in response to stress (Ludwig et al. 2004). CDPKs are encoded by multigene families, and expression levels of these genes are spatially and temporally controlled throughout development. In addition, subsets of CDPKs are involved in signal transduction during stress including cold, salt, and drought or pathogen infection. A new transcription factor, BOS1 (Botrytis susceptible 1), was found to be required for both biotic and abiotic stress responses in Arabidopsis as reported by Mengiste et al. (2003). This complex network of interactions allows plants to respond in a highly specific fashion to the exact combination of environmental stresses encountered.

Identification of the Differentially Expressed Proteins

Many cold stress-responsive proteins have been identified (Gao et al. 2009). These proteins includeputative 2,3-bisphosphoglycerate-independent phosphoglycerate mutase, glycine-rich RNA-binding protein 7 (GRP7), ferritin-1, EF-G, phosphoglycerate kinase, ATP synthase CF1 beta chain, UDP-glucose pyrophosphorylase, cysteine proteinase inhibitor, RuBisCO small chain, carbonic anhydrase, and SAL1 phosphatase. New proteins such as isopropylmalate synthase, cytochrome b6–f complex iron–sulfur subunit, and SNF2 domain-containing protein/helicase domain-containing protein/zinc finger (C3HC4 type RING finger) family protein have been identified in response to cold stress. These novel cold-responsive proteins may play important roles in cold tolerance of Thellungiella halophila. The stress related proteins are involved in RNA metabolism and energy pathway, protein synthesis, folding and degradation as well as cell wall and cytoskeleton, metabolisms of nitrogen and sulfur, and signal transduction (Emanuelsson et al. 2000). The cold stress-responsive proteins linked to RNA metabolism, defense response, protein synthesis, and folding and degradation were all upregulated markedly during cold treatment. It suggests that these cellular processes were enhanced by exposure to cold stress. The identified protein involved in photosynthesis and energy pathway displayed diverse change patterns. T. halophila is a valuable model system for investigating the mechanisms involved in plant cold stress tolerance. Proteomic analysis of cold stress response in T. halophila can help to identify key regulators of cold tolerance in plants. Regulation of chloroplast functions under cold stress. The expression regulation of chloroplast proteins is of central importance in cold adaptation (Foyer et al. 1997), and a substantial portion (46%, 23/50) of the identified proteins were predicted to be localized in chloroplast. Eighteen of these have been identified to reside in the chloroplast by previous proteomic studies according to the Plant Proteome Database (PPDB, http://ppdb.tc.cornell.edu/) (Sun et al. 2009). These chloroplast-located cold stress-responsive proteins are associated with various aspects of chloroplast, including Calvin cycle and electron transport (discussed below), chloroplast RNA processing (putative RNA-binding protein cp29), and chloroplast protein synthesis and folding (EF-G and heat-shock protein 70), as well as chloroplast redox homeostasis (BAS1, Peroxiredoxin-2E and Ferritin- 1). At the same time, a chloroplast-localized carbonic anhydrase, which facilitates CO₂ move across the chloroplast envelope, was found to decrease in abundance after 5 and 24 days of cold treatment. The changes in Calvin cycle enzymes observed in this study might be associated with decrease in photosynthetic CO₂ assimilation, possibly resulting in decreased growth rate in the long term. Enhanced RNA metabolism, including RNA processing, transporting from nucleus to cytoplasm, and mRNA secondary structure stability may be impaired under cold stress, especially for the cold stress-induced defense-related transcripts (Zhu et al. 2007). Several RNA-binding proteins and helicase-like proteins were all found to be upregulated by cold stress in the present study, including GRP7, glycine-rich RNA-binding protein GRP1A (GRP1A), putative RNA-binding protein cp29, and SNF2 domain-containing protein/helicase domain-containing protein/zinc finger (C3HC4-type RING finger) family proteins indicating enhanced RNA metabolism may play an important role in cold tolerance of T. halophila. Among these cold-responsive nucleic acid-binding proteins, GRPs are suggested to play an important role in posttranscriptional regulation of gene expression in plants under various stress conditions (Mousavi and Hotta 2005). GRP7 has been demonstrated to play a role in the export of mRNAs from the nucleus to the cytoplasm under cold stress conditions in Arabidopsis (Kim et al. 2008). RNA-binding protein cp29 is a subunit of the photosystem II and its phosphorylation was reported to relate with the cold tolerance in maize (Mauro et al. 1997). A RNA-binding protein cp29 was proved to be induced by cold stress in Arabidopsis (Amme et al. 2006). The increase in two chloroplast RNA-binding proteins may help to enhance or maintain the chloroplast RNA synthesis under cold conditions. In addition, GRPs and chloroplast RNA-binding proteins are suggested to be part of the plant innate immunity system and increase in their abundance may promote the expression of immunity-related mRNA (Fu et al. 2007).

Conclusion

In conclusion, the proteomic analysis is a very useful tool for providing complex information about differences in the plant proteome during abiotic and biotic stresses. It is almost necessary to clarify the differential function of the individual stress-responsive NAC genes for the control of abiotic stress tolerance and the other biological processes including biotic stress tolerance and growth regulation in order to fully utilize the potential of NAC transcription factors. Large-scale transcriptome analyses coupled with microarray, proteomic, and metabolomic analysis of plants perturbed at the levels of individual or multiple components of the ROS network will be essential for future studies. In the future, a combination of reverse genetics, genomics, and proteomic approaches in various developmental stages and stress conditions will provide us with critical information to elucidate the functional differences of the stress-responsive NAC factors and their relationship in transcriptional control. It is a challenge for plant scientists/environmentalists/biotechnologists in the twenty-first century to develop stable multiple stress tolerance traits in agronomically important crop plants, thus improving yields particularly in areas with adverse environmental conditions and contributing to food security.