**Abiotic Stress Adaptation: Protein Folding Stability and Dynamics**

Martina Ortbauer

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/53129

## **1. Introduction**

Abiotic stress is best defined as any factor exerted by the environment on the optimal func‐ tioning of an organism. Abiotic stresses like heat, cold, freezing, drought salinity, flooding or oxidizing agents usually cause protein dysfunction [1]. Protein folding stability is un‐ doubtedly one of the most challenging problems in organisms undergoing stress conditions. Efficient protein repair systems and general protein stability facilitate survival upon sudden changes in the environment. As sessile organisms plants need to adopt quickly to overcome various environmental stresses during their lifespan. Recently, most emphasis is being di‐ rected towards an understanding of how plants recognize external conditions and initiate protective reactions such as mechanisms through which protein function is protected and maintained. Proteins are biological macromolecules involved in virtually every biological process in a living system. The roles played by proteins are varied and complex. Proteins are used for storage and transport of small molecules or ions and control the passage of mole‐ cules through the cell membranes essential for metabolic function [2]. Hormones, which transmit information and allow the regulation of complex cellular processes, are important regulators in responses to abiotic stress [3]. Enzymes act as catalysts and increase, with a re‐ markable specificity, the speed of chemical reactions essential to the organism's survival.

Protein function is dependent on its unique three-dimensional structure that is adopted by the initial folding of the polypeptide chains after translation. Encoded by DNA and synthe‐ sized on ribosomes as chains of hundreds of amino acids, each protein must find its charac‐ teristic and correct fold, rather than the countless alternatives, in order to function properly [4]. Folding into its native and active structure may involve one or more partially folded in‐ termediate states (Figure 1). It is not surprising that stress induced alterations in the physio‐ logical conditions may change the folding process and give rise to protein misfolding and

© 2013 Ortbauer; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 Ortbauer; licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

aggregation [5]. Folded proteins are generally much less prone to aggregation and degrada‐ tion but partially unfolded or intrinsically disordered regions of proteins can confer func‐ tional advantages, as they allow efficient interaction with binding partners and provide a mechanism for the regulation of cellular processes. Protein dynamics, meaning structural or conformational change with time, are an essential part of regulation of biological activity.

**Figure 1.** Protein folding involving a partially folded intermediate state. The two transition states (TS1, TS2) are sepa‐ rated by a metastable intermediate (I\*), modified from [6, 7]. The driving force for protein folding is the search for lower free energy states, separated by free energy barriers. The free energy of a protein in solution is highly depend‐ ent on temperature, pressure and solvent conditions

Many cellular processes are coupled to protein folding and unfolding, a process that is high‐ ly sensitive to rapid changes in environmental conditions such as denaturant concentration, temperature or pH. In determining the conformational properties of proteins, it is therefore important to include solvent and co-solvent conditions.

Protein conformation and activity can differ markedly between diluted and crowded envi‐ ronments. The diverse and highly specific function of proteins is a consequence of their so‐ phisticated, individual surface pattern regarding shape, charge and hydrophobicity that is a consequence of the three-dimensional structure of polypeptide chains. The stability of pro‐ teins results from a number of counteracting enthalpic and entropic contributions. Native states represent the most stable conformation under equilibrium. This does not necessarily mean that protein function is restricted to well-defined folded states. Internal dynamics play an important role in protein function. *In vivo* folding, catalytic function, transport and degra‐ dation of proteins all involve transitions between different conformations. Locally unfolded or disordered regions of a protein allow efficient interaction with binding partners and thus the regulation of cellular mechanisms. Identifying and defining the rules for protein folding and unfolding is fundamental for our understanding how living systems cope with abiotic stresses. Advanced experimental methods continue to be developed to elucidate the sheer complexity of protein folding and unfolding and the mechanisms of preserving functional folds under stress conditions.

## **2. Protein folding and abiotic stress**

aggregation [5]. Folded proteins are generally much less prone to aggregation and degrada‐ tion but partially unfolded or intrinsically disordered regions of proteins can confer func‐ tional advantages, as they allow efficient interaction with binding partners and provide a mechanism for the regulation of cellular processes. Protein dynamics, meaning structural or conformational change with time, are an essential part of regulation of biological activity.

**Figure 1.** Protein folding involving a partially folded intermediate state. The two transition states (TS1, TS2) are sepa‐ rated by a metastable intermediate (I\*), modified from [6, 7]. The driving force for protein folding is the search for lower free energy states, separated by free energy barriers. The free energy of a protein in solution is highly depend‐

Many cellular processes are coupled to protein folding and unfolding, a process that is high‐ ly sensitive to rapid changes in environmental conditions such as denaturant concentration, temperature or pH. In determining the conformational properties of proteins, it is therefore

Protein conformation and activity can differ markedly between diluted and crowded envi‐ ronments. The diverse and highly specific function of proteins is a consequence of their so‐ phisticated, individual surface pattern regarding shape, charge and hydrophobicity that is a consequence of the three-dimensional structure of polypeptide chains. The stability of pro‐ teins results from a number of counteracting enthalpic and entropic contributions. Native states represent the most stable conformation under equilibrium. This does not necessarily mean that protein function is restricted to well-defined folded states. Internal dynamics play an important role in protein function. *In vivo* folding, catalytic function, transport and degra‐ dation of proteins all involve transitions between different conformations. Locally unfolded or disordered regions of a protein allow efficient interaction with binding partners and thus the regulation of cellular mechanisms. Identifying and defining the rules for protein folding and unfolding is fundamental for our understanding how living systems cope with abiotic stresses. Advanced experimental methods continue to be developed to elucidate the sheer complexity of protein folding and unfolding and the mechanisms of preserving functional

ent on temperature, pressure and solvent conditions

4 Abiotic Stress - Plant Responses and Applications in Agriculture

folds under stress conditions.

important to include solvent and co-solvent conditions.

A striking feature of protein folding is that the overall mechanism follows simple physical rules, but examination in finer detail reveals a much greater complexity [8]. The protein structure-function paradigm has been reassessed with the discovery of partially unfolded or intrinsically disordered proteins that are fully functional. These proteins are widely distrib‐ uted in eukaryotes and fulfill crucial biological functions like transcriptional regulation, sig‐ nal transduction [9], enzyme catalysis and protein ligand interactions. They contain nativelike secondary structure elements but lack the tertiary interactions of folded proteins. One has to keep in mind that protein function is protected by stabilization of well-defined struc‐ tural regions but is largely dependent on protein motion and dynamics. NMR dynamic ex‐ periments indicate that protein conformational exchange spans a variety of time scales ranging from picoseconds to milliseconds [10]. Complete description of protein function, that may involve motion, requires an understanding of the molecular dynamics [11]. Many proteins form partially folded intermediate states along their folding-pathway. To search for correlations between function, structure and dynamics, it is essential to include all states formed at equilibrium [12, 13] in order to characterize protein dynamics under unfavorable environmental conditions.

Protein conformations and interconversion between different states are largely modified by internal and external signals such as ligand binding, phosphorylation or cleavage, molecular recognition or environmental changes [14]. *In vivo*, protein folding occurs spontaneously, meaning that proteins permanently exchange between folded, partially folded, locally un‐ folded and unfolded states during the period from protein synthesis until their degradation. According to the energy landscape theory, the free energy barriers connecting these states are small [15], suggesting that minor perturbations *in vivo* can have significant effects on the populations of different states and hence protein function. Intermolecular forces that drive protein folding generally stabilize both folded and unfolded states, but an altered balance in these forces can result in aggregation or misfolding to non-functional proteins [16]. Protein unfolding, misfolding and aggregation are a common threat to the living cell, especially when undergoing abiotic stress. To cope with stress, plants have developed various mecha‐ nisms to facilitate protein folding and to suppress protein misfolding.

## **3. Stability versus flexibility - How to protect protein function?**

Stabilizing proteins in their functional conformation is one of the great challenges in plant stress metabolism. Stress induced alterations in the structural and energy landscape of pro‐ teins affect and may inhibit both protein-ligand and protein-protein interactions. Small mol‐ ecules typically bind proteins in small cavities, whereas proteins recognize large surface areas [17]. Thus, protein function is a balancing act between structural stability and the con‐ formational flexibility needed for protein function. Protein stability results from stabilizing and destabilizing interactions of the polypeptide chains that slightly favor the folded state as compared to partially folded or unfolded states under physiological conditions (Figure 2).

**Figure 2.** Free energy model for protein folding and unfolding. Stabilization of the native state can be achieved by destabilization of the denatured state (D') or a more stabilized native state (N'). The free energy barrier (TS') may also be affected

The difference in the free energy (ΔG) of different states as measured from the reversible transition from the native to the denatured state is small [18]. Externally forced conforma‐ tional changes in the protein structure lead to a substantial decrease in its stability. The de‐ naturation process causes conformational destabilization by exposing hydrophobic residues to the solvent, normally deeply buried in the interior of a folded protein. The burial of non polar surfaces and the hydrophobic force is considered as the main driving force for protein folding and stability [19] as proteins become thermally more stable upon decreasing hydra‐ tion levels [20].

Evidence from proteins produced by hyperthermophil microorganisms, which are very thermostable and resistant to chemical denaturation, indicates that this resistance comes from lower protein flexibility and higher protein rigidity [21]. Thermostable proteins tend to be very rigid at mesophilic temperatures (10-450 C), but allow for greater flexibility at high temperatures, which is essential for their function in their thermophilic environment. It is as‐ sumed that intrinsic stability due to increased protein rigidity is important for thermal stabi‐ lization, since thermal motion decreases rigidity and enhances flexibility.

#### **3.1. Assisted folding under stress conditions**

Molecular responses to abiotic stress are complex and highly dependent on the level and du‐ ration of stress and on the tissue and organ that is affected. Sensing of environmental changes and transduction of stress signals triggers activation of molecular response mecha‐ nisms [22]. A general response to environmental stress conditions is the onset of stress pro‐ teins that facilitate protein folding and protect proteins from misfolding and aggregation. The targets for these so-called chaperons (heat shock proteins HSP, late embryogenesis abundant LEA proteins) are partially unfolded or misfolded proteins with stretches of hy‐ drophobic residues that are normally buried in the interior of the protein fold now exposed to the surface. Since aggregates of misfolded proteins can be very stable and the energy bar‐ riers towards the folded state can be of higher energy, chaperons assist the folding process by helping to overcome the energy barriers and to refold proteins from aggregates [23].

Transcription of many genes is up regulated under stress conditions. Among these genes, several code for stress-induced proteins that act to improve water movement through mem‐ branes (water channel proteins), detoxification enzymes or enzymes required for osmolyte biosynthesis [24]. Studies on plants reported that one of the initial responses to water deficit is the induction of osmolyte (Figure 3) production. Changes in protein expression levels are required to regulate osmolyte transport and distribution throughout the plant. The accumu‐ lation of low-molecular weight osmolytes (compatible solutes) is well known to protect mac‐ romolecular structure from stress-induced damage. Increased intracellular osmolyte concentrations on the other hand may affect protein structure and dynamics. Solvent and (co-)solvent conditions and protein solvent accessibility is of particular importance during stress periods because it influences ionic strength, pH values and affinity to certain molecu‐ lar groups on the protein surface.

**Figure 3.** Examples of organic co-solvents (osmolytes): uncharged sugars, polyols and betaines

**Figure 2.** Free energy model for protein folding and unfolding. Stabilization of the native state can be achieved by destabilization of the denatured state (D') or a more stabilized native state (N'). The free energy barrier (TS') may also

The difference in the free energy (ΔG) of different states as measured from the reversible transition from the native to the denatured state is small [18]. Externally forced conforma‐ tional changes in the protein structure lead to a substantial decrease in its stability. The de‐ naturation process causes conformational destabilization by exposing hydrophobic residues to the solvent, normally deeply buried in the interior of a folded protein. The burial of non polar surfaces and the hydrophobic force is considered as the main driving force for protein folding and stability [19] as proteins become thermally more stable upon decreasing hydra‐

Evidence from proteins produced by hyperthermophil microorganisms, which are very thermostable and resistant to chemical denaturation, indicates that this resistance comes from lower protein flexibility and higher protein rigidity [21]. Thermostable proteins tend to

temperatures, which is essential for their function in their thermophilic environment. It is as‐ sumed that intrinsic stability due to increased protein rigidity is important for thermal stabi‐

Molecular responses to abiotic stress are complex and highly dependent on the level and du‐ ration of stress and on the tissue and organ that is affected. Sensing of environmental

lization, since thermal motion decreases rigidity and enhances flexibility.

C), but allow for greater flexibility at high

be affected

tion levels [20].

be very rigid at mesophilic temperatures (10-450

6 Abiotic Stress - Plant Responses and Applications in Agriculture

**3.1. Assisted folding under stress conditions**

Accumulated osmolytes within the cells change the interaction of proteins with the solvent [25] by increasing (kosmotropic) or decreasing (chaotropic) the order of water. Kosmotropic, or so-called compensatory co-solvents are well-hydrated molecules with little tendency to aggregate, have no net charge, and strongly hydrogen-bond with water. They are preferen‐ tially solubilized within the bulk of water and preferentially excluded from the protein sur‐ face, which leads to a decrease in the water diffusion around the protein [26, 27]. Although molecules do not seem to directly interact with the protein surface, they modify protein sta‐ bility by altering solvent properties. According to the "water structure hypothesis" chaotrop‐ ic co-solvents increase the fraction of more dense species in the hydration water thereby destabilizing protein structure [28]. Molecules that stabilize the surface low-density water and increase the surface tension will stabilize the protein structure (kosmotropes).

Co-solvent effects that alter the water structure are not the sole driving force for increasing or decreasing protein stability. It also has to be considered that the interaction of a co-solvent with the protein surface may not be favorable and thus would destabilize a protein. Due to the fact that unfolded or denatured states comprise a higher solvent and co-solvent accessible surface area, the equilibrium tends to shift to the more compact folded state known as the "excluded volume effect". Among all the interactions that may stabilize or destabilize proteins, a main driving force for protein folding is "hydrophobic interactions". Hydrophobic forces will also be affected in the presence of co-solvents, partly depending on the ability of a solute to be exclud‐ ed or incorporated in the hydration shell of a certain protein [29].

Increases in temperature, pressure or osmotic stress alter the properties of protein conforma‐ tion and the hydration state. The free energy change resulting from folding or unfolding de‐ pends on the combined effects of the exposure of the interior and non-polar groups and their interaction with water, including changes in water-water interactions.

#### **3.2. Dynamics in enzymatic activity**

The ability to maintain protein performance under abiotic stress depends on intrinsic stabili‐ ty, chaperon activity, protein turnover and extrinsic stabilization through co-solvents (com‐ patible solutes). Molecular motion as well as protein flexibility and dynamics is highly linked to enzymatic activity, which is clearly dependent on the particular environment of a protein [30].

Hydration status and temperature are the main factors that contribute to the catalytic mech‐ anism. Hydration is necessary for enzyme catalytic function since dry enzymes are less func‐ tional, and below a threshold hydration level enzymes are inactive. Protein hydration may also be necessary for diffusion of substrate and product [31]. Temperature is a fundamental environmental stress, as flexibility and functionality of enzymes are highly temperature de‐ pendent. Low temperatures result in decreased catalytically activity, which is metabolically not favorable. Increases in the thermal energy will increase enzyme molecules that have the required energy for conformational changes into catalytically active enzymes, showing an increased catalytic rate (*k*cat). High temperatures, on the other hand, can cause the structure to become so loose that substrates and co-factors can no longer bind [32]. Extreme tempera‐ tures cause complete denaturation. Osmolyte (glycerol, sorbitol, xylitol, glucose, fructose, saccharose, proline, glycine betaine, *myo*-Inositol, pinitol, quercitol) protection of enzymes against heat-induced loss of activity has been extensively studied *in vitro* [33-35]. The partic‐ ular properties of a protein and the nature of the added osmolyte strongly influence protein thermal stability and enzyme activity. The ability to protect enzymes from heat induced ac‐ tivity loss varies between different osmolytes but preserving enzymes under heat stress seems to be a general feature for these osmolytes. Loss of enzymatic activity under high temperature treatment does occur but is always slower and at higher temperatures when compared to proteins without protective additives. Enzymatic activity tests demonstrate the function of osmolytes in preventing heat induced activity loss. To get further insights into folding stability and dynamics of proteins under stress conditions, more detailed analysis and extended methods are needed.

#### **3.3. Global conformational stability of proteins under stress**

ic co-solvents increase the fraction of more dense species in the hydration water thereby destabilizing protein structure [28]. Molecules that stabilize the surface low-density water

Co-solvent effects that alter the water structure are not the sole driving force for increasing or decreasing protein stability. It also has to be considered that the interaction of a co-solvent with the protein surface may not be favorable and thus would destabilize a protein. Due to the fact that unfolded or denatured states comprise a higher solvent and co-solvent accessible surface area, the equilibrium tends to shift to the more compact folded state known as the "excluded volume effect". Among all the interactions that may stabilize or destabilize proteins, a main driving force for protein folding is "hydrophobic interactions". Hydrophobic forces will also be affected in the presence of co-solvents, partly depending on the ability of a solute to be exclud‐

Increases in temperature, pressure or osmotic stress alter the properties of protein conforma‐ tion and the hydration state. The free energy change resulting from folding or unfolding de‐ pends on the combined effects of the exposure of the interior and non-polar groups and

The ability to maintain protein performance under abiotic stress depends on intrinsic stabili‐ ty, chaperon activity, protein turnover and extrinsic stabilization through co-solvents (com‐ patible solutes). Molecular motion as well as protein flexibility and dynamics is highly linked to enzymatic activity, which is clearly dependent on the particular environment of a

Hydration status and temperature are the main factors that contribute to the catalytic mech‐ anism. Hydration is necessary for enzyme catalytic function since dry enzymes are less func‐ tional, and below a threshold hydration level enzymes are inactive. Protein hydration may also be necessary for diffusion of substrate and product [31]. Temperature is a fundamental environmental stress, as flexibility and functionality of enzymes are highly temperature de‐ pendent. Low temperatures result in decreased catalytically activity, which is metabolically not favorable. Increases in the thermal energy will increase enzyme molecules that have the required energy for conformational changes into catalytically active enzymes, showing an increased catalytic rate (*k*cat). High temperatures, on the other hand, can cause the structure to become so loose that substrates and co-factors can no longer bind [32]. Extreme tempera‐ tures cause complete denaturation. Osmolyte (glycerol, sorbitol, xylitol, glucose, fructose, saccharose, proline, glycine betaine, *myo*-Inositol, pinitol, quercitol) protection of enzymes against heat-induced loss of activity has been extensively studied *in vitro* [33-35]. The partic‐ ular properties of a protein and the nature of the added osmolyte strongly influence protein thermal stability and enzyme activity. The ability to protect enzymes from heat induced ac‐ tivity loss varies between different osmolytes but preserving enzymes under heat stress seems to be a general feature for these osmolytes. Loss of enzymatic activity under high temperature treatment does occur but is always slower and at higher temperatures when compared to proteins without protective additives. Enzymatic activity tests demonstrate the

and increase the surface tension will stabilize the protein structure (kosmotropes).

ed or incorporated in the hydration shell of a certain protein [29].

8 Abiotic Stress - Plant Responses and Applications in Agriculture

**3.2. Dynamics in enzymatic activity**

protein [30].

their interaction with water, including changes in water-water interactions.

Circular dichroism (CD) spectroscopy has been introduced as a quick and valuable techni‐ que for examining the structure and stability of proteins in solution. CD is used for deter‐ mining whether a protein is folded and for characterizing its secondary structure (alphahelices, beta-strands) and some aspects of the tertiary structure (aromatic amino acids, disulfide bonds). Conformational changes during the acquisition of the native structure are measured in the near-UV (250-350) and far-UV (190-250). This technique has been used widely to determine the folding stability of proteins dependent on temperature, pH and un‐ der denaturant conditions [36, 37]. CD is a convenient tool to characterize the interactions between co-solvents and proteins and to find co-solvent conditions that increase the melting temperature or fully refold proteins after thermal unfolding. If the melting is fully reversi‐ ble, the melting temperature is directly related to conformational stability, and the thermo‐ dynamics of protein folding can be extracted from the data [38].

CD studies have been employed to investigate how osmolytes such as glycerol, trehalose and *myo*-Inositol affect the global folding of native proteins and its thermal unfolding proc‐ ess. CD signals arising from protein chromophors reflect an average of the protein popula‐ tion. The resulting spectrum is a sum of individual spectra arising from secondary structure elements present in the protein sample (Figure 4).

**Figure 4.** Circular dichroism spectra of Malate Dehydrogenase (insert) in 20mM NaP-buffer at pH 7.0. The far-UV spec‐ trum recorded from 260 to 190nm at 20oC displays a typical α-helical protein with two negative maxima at 208 and 222nm. Addition of 0.4M glycerol (–), *myo*-Inositol (––) or trehalose (- -) did not change protein secondary structure and did not show self-absorbance in this spectral region [39]

Thermally induced protein unfolding was monitored in the far-UV region by gradually in‐ creasing the temperature in the protein sample. Thermal denaturation curves were moni‐ tored at a fixed wavelength of 222nm (Figure 5) and acquired data were fitted to a simple thermodynamic unfolding model. The melting temperature, Tm (midpoint transition temper‐ ature) can be extracted from thermal denaturation curves, reflecting the global stability of the folded versus the unfolded protein. 0.4M glycerol, *myo*-Inositol and trehalose increased the melting temperature of malate dehydrogenase by 3 to 5o C as compared to proteins alone [39].

**Figure 5.** Thermally induced unfolding of Malate Dehydrogenase (•) in the presence of 0.4M glycerol (□), trehalose (▵), glucose (▪) and *myo*-Inositol (▴). The horizontal line indicates the midpoint transition temperature (Tm). Osmolytes and proteins were mixed to protein solution and equilibrated at room temperature prior to heating. Temperature pro‐ files at 222nm were recorded for 1 °C increments in the temperature range 20–90 °C at a heating rate of 1 °C min−1 [39]. Thermal unfolding measurements were set up in quartz cuvettes, placed into a Peltier controlled sample holder unit connected to a temperature probe to provide an accurate temperature record. Thermal unfolding curves were analyzed using a sigmoidal curve function according to (Equation 1) [40]:

$$\theta\_T = \left[ \frac{(m\_D \times T - b\_D) - (m\_N \times T - b\_{N\rangle})}{1 + (T \ / T\_m)^{m\_T}} + m\_N \times T - b\_N \right] \tag{1}$$

where θ<sup>T</sup> is the ellipticity at temperature T, mT is the slope of the curve within the transition region, and the inflection point of the curve the melting temperature Tm. At each tempera‐ ture bN and bD can be extrapolated from the pre- and post-transition baselines, (mN × T − bN) and (mD × T − bD), respectively. The fraction of unfolded protein can be calculated by sub‐ tracting these baselines (Equation 2):

$$f\_{\upsilon} = \frac{\theta\_T - \theta\_N}{\theta\_\upsilon - \theta\_N} = \frac{\theta\_T - (m\_N \times T - b\_N)}{(m\_D \times T - b\_D) - (m\_N \times T - b\_N)} \tag{2}$$

The stabilization of protein global folds through naturally occurring osmolytes seems to be a general mechanism. Other studies also reported increases in the midpoint transition temper‐ ature (ΔTm) of 2 to 18o C upon the addition of 0.1-2M glycerol, trehalose and sucrose meas‐ ured on various proteins [41-43]. Additionally, all proteins studied in the presence of osmolytes showed a remarkably retention of secondary structure at Tm relative to proteins alone. Retention of secondary structure in osmolyte solution was monitored even at temper‐ atures where proteins were fully unfolded when heated without additives.

ature) can be extracted from thermal denaturation curves, reflecting the global stability of the folded versus the unfolded protein. 0.4M glycerol, *myo*-Inositol and trehalose increased

**Figure 5.** Thermally induced unfolding of Malate Dehydrogenase (•) in the presence of 0.4M glycerol (□), trehalose (▵), glucose (▪) and *myo*-Inositol (▴). The horizontal line indicates the midpoint transition temperature (Tm). Osmolytes and proteins were mixed to protein solution and equilibrated at room temperature prior to heating. Temperature pro‐ files at 222nm were recorded for 1 °C increments in the temperature range 20–90 °C at a heating rate of 1 °C min−1 [39]. Thermal unfolding measurements were set up in quartz cuvettes, placed into a Peltier controlled sample holder unit connected to a temperature probe to provide an accurate temperature record. Thermal unfolding curves were

*m Tb*

) ( )( 1(/ ) *<sup>T</sup> D DN N T m N N m m Tb m Tb*

é ù ´- - ´- = ê + ´- ú ê ú <sup>+</sup> ë û

*TN T N N*

 q

*vN D D N N m Tb <sup>f</sup> m Tb m Tb*

The stabilization of protein global folds through naturally occurring osmolytes seems to be a general mechanism. Other studies also reported increases in the midpoint transition temper‐

ured on various proteins [41-43]. Additionally, all proteins studied in the presence of osmolytes showed a remarkably retention of secondary structure at Tm relative to proteins

where θ<sup>T</sup> is the ellipticity at temperature T, mT is the slope of the curve within the transition region, and the inflection point of the curve the melting temperature Tm. At each tempera‐ ture bN and bD can be extrapolated from the pre- and post-transition baselines, (mN × T − bN) and (mD × T − bD), respectively. The fraction of unfolded protein can be calculated by sub‐

> ( ) ( )( )


C upon the addition of 0.1-2M glycerol, trehalose and sucrose meas‐

*T T*

C as compared to proteins alone

(1)

the melting temperature of malate dehydrogenase by 3 to 5o

10 Abiotic Stress - Plant Responses and Applications in Agriculture

analyzed using a sigmoidal curve function according to (Equation 1) [40]:

q

*v*

qq

q q

tracting these baselines (Equation 2):

ature (ΔTm) of 2 to 18o

[39].

Studies on RnaseA previously showed that increases in ΔH<sup>m</sup> by the addition of trehalose re‐ sulted in a lower ΔCp-vlaue (heat capacity change). [41]. The heat capacity change, ΔCp, is a very sensitive thermodynamic parameter that correlates with the amount of the protein sur‐ face that is exposed to the solvent [44]. A decrease in ΔCp upon the addition of osmolytes reflects a lower surface exposed area and/or decreased exposure of hydrophobic groups to the solvent. Decreases in ΔCp may also result in flattening of thermal unfolding curves, leading to conformational stability over a wider range of temperature. This has shown to be an effective strategy for many mesophilic proteins.

The thermal stability of a protein is determined by the response to thermal energy, concern‐ ing globally and locally unfolding and the ability to refold into its active conformation. Ther‐ mal unfolding was shown to be highly reversible for thermostable proteins of hyperthermophilic organisms. The far-UV CD spectrum of the native protein was identical to that after heat denaturing and re-cooling [45]. Many mesophilic proteins, however, aggre‐ gate or precipitate after thermal unfolding making the unfolding process irreversible. Find‐ ing co-solvent conditions that facilitate refolding is as important as increasing the melting temperature. Facilitated refolding was observed for ribonuclease that undergoes a reversible denaturation in the presence of trehalose [46].

Taken together, these results from CD measurements reveal that osmolytes stabilize protein global folds under heat by supporting retention of secondary structure elements and aid in refolding of thermally unfolded proteins.

## **4. New insights into molecular dynamics of protein folding and unfolding from Nuclear Magnetic Resonance (NMR) spectroscopy**

Internal dynamics of proteins play an important role in their biological function. Proteins do not only exist in well-defined natively folded or fully unfolded states, but also in partially folded intermediate states. The conformational exchange between a folded state and partial‐ ly folded states is highly sensitive to changes in the environment such as temperature, pH, solvent and co-solvent conditions. In the plant cell, proteins are predestinated to function in environments crowded by macromolecules, metabolites and other co-solvents that facilitate protein folding under non-stress and stress conditions [47]. By measuring protein dynamics, it is therefore important to include (co-)solvent conditions (Figure 6). High-osmolyte accu‐ mulation upon stress conditions induces changes in the protein environment. Variable pro‐ tein folds may be affected slightly different according to their hydrophobic or hydrophilic surface properties, compactness, flexibility, hydrogen bonding patterns, excluded volume effects and the affinity of binding sites for co-solvents or the hydration water.

**Figure 6.** HSQC of the uniformly 15N-labeled KID-binding (KIX) domain of CREB-binding protein (CBP), residues 586-672 (black). Overlaid is a spectrum of KIX in the presence of 0.4M *myo*-Inositol (red) showing the intactness of the three-dimensional protein folds upon the addition of an osmolyte. Spectra were acquired on a Varian Inova 800MHz spectrometer at 26.9oC

Nuclear Magnetic Resonance (NMR) spectroscopy has greatly contributed to understanding of protein folding by characterizing protein conformation at the level of individual amino acid residues. NMR techniques can be used to monitor temperature dependence of folding and unfolding in order to determine their thermodynamic properties, measure sensitivity to denaturants and address solvent and co-solvent accessibility. NMR experiments provide in‐ formation at multiple sites within the protein, unlike spectroscopic techniques such as circu‐ lar dichroism that provide nonspecific information about aromatic side chains and averaged properties of the polypeptide backbone. Heteronuclear NMR relaxation and relaxation dis‐ persion experiments have emerged as powerful tool to study internal dynamics under a wide range of experimental conditions.

#### **NMR relaxation experiments**

Information about protein dynamics, extracted form heteronuclear NMR relaxation studies, is based on measurements of the longitudinal (T1) and transverse (T2) relaxation rate and the heteronuclear NOE, all sensitive for the motion of the N-H bond vector in the protein backbone [11]. Fast atomic motions on a picosecond to nanosecond (ps-ns) time scale are gained from the slower relaxation processes (R1, R2 and NOE) of nuclear spins, measured along the backbone and in the side chains using isotopically labeling (15N).

Relaxation data (T1, T2, NOE) can be interpreted according to the "model free" formalism in terms of the internal motional correlation time and an order parameter (S2) [48]. In the NMR experiment, order parameters (S2 ) report on the refinement of the N-H bond vector. The val‐ ue of S2 varies from 0 (no motional restriction) to 1 (complete motional restriction) [49]. Backbone segments in highly flexible parts of the protein, not restricted in their motion, have low S2 values, whereas rigid regions show typical high S2 values. Main chain 15N relax‐ ation data can be analyzed to yield S2 order parameters on a per residue basis (Figure 7).

**Figure 7.** Comparison of N-H order parameters (S2) of cold shock protein A (CspA) (red). Addition of 0.4M *myo*-Inositol (black) showed an overall increase in protein compactness by rigidification of former flexible parts of the protein (S2=0 flexible, S2=1 rigid)

Order parameter (S2 ) for cold shock protein A (CspA) showed an overall increase in the presence of the model osmolyte *myo*-Inositol. Residues in very flexible parts of the protein that have low motional restrictions tend to become more rigid and motional restricted upon the addition of the *myo*-Inositol. The overall protein compactness increases in the presence of the osmolyte, most profoundly observed in protein regions with high locally structural fluctuations.

#### **(CPMG)-type NMR relaxation dispersion experiments**

**Figure 6.** HSQC of the uniformly 15N-labeled KID-binding (KIX) domain of CREB-binding protein (CBP), residues 586-672 (black). Overlaid is a spectrum of KIX in the presence of 0.4M *myo*-Inositol (red) showing the intactness of the three-dimensional protein folds upon the addition of an osmolyte. Spectra were acquired on a Varian Inova 800MHz

Nuclear Magnetic Resonance (NMR) spectroscopy has greatly contributed to understanding of protein folding by characterizing protein conformation at the level of individual amino acid residues. NMR techniques can be used to monitor temperature dependence of folding and unfolding in order to determine their thermodynamic properties, measure sensitivity to denaturants and address solvent and co-solvent accessibility. NMR experiments provide in‐ formation at multiple sites within the protein, unlike spectroscopic techniques such as circu‐ lar dichroism that provide nonspecific information about aromatic side chains and averaged properties of the polypeptide backbone. Heteronuclear NMR relaxation and relaxation dis‐ persion experiments have emerged as powerful tool to study internal dynamics under a

Information about protein dynamics, extracted form heteronuclear NMR relaxation studies, is based on measurements of the longitudinal (T1) and transverse (T2) relaxation rate and the heteronuclear NOE, all sensitive for the motion of the N-H bond vector in the protein backbone [11]. Fast atomic motions on a picosecond to nanosecond (ps-ns) time scale are gained from the slower relaxation processes (R1, R2 and NOE) of nuclear spins, measured

Relaxation data (T1, T2, NOE) can be interpreted according to the "model free" formalism in terms of the internal motional correlation time and an order parameter (S2) [48]. In the NMR

Backbone segments in highly flexible parts of the protein, not restricted in their motion, have low S2 values, whereas rigid regions show typical high S2 values. Main chain 15N relax‐

varies from 0 (no motional restriction) to 1 (complete motional restriction) [49].

) report on the refinement of the N-H bond vector. The val‐

order parameters on a per residue basis (Figure 7).

along the backbone and in the side chains using isotopically labeling (15N).

spectrometer at 26.9oC

wide range of experimental conditions.

12 Abiotic Stress - Plant Responses and Applications in Agriculture

**NMR relaxation experiments**

experiment, order parameters (S2

ation data can be analyzed to yield S2

ue of S2

NMR relaxation dispersion methods have been introduced enabling studies on protein fold‐ ing under native conditions without the need for disturbing the equilibrium. Studying pro‐ tein folding and unfolding requires a thoroughly view of all states including the native state, folding intermediates and the unfolded state [12] as it is increasingly recognized that even small proteins fold via intermediates. Because these intermediates are low populated and short-lived (in the order of ms), their structural characterization has been a difficult task. In NMR relaxation dispersion experiments conformational exchange between a native ground state and low populated partially folded states can be characterized even if states are not visible in NMR spectra [50].

CPMG (Carr-Purcell-Meiboom-Gill)-type NMR relaxation dispersion techniques have been employed to investigate the site-specific conformational exchange processes of proteins on a microsecond-to-millisecond time scale that is highly sensitive to solvent and co-solvent con‐ ditions. These experiments are particular useful for simple two state exchange processes, providing information about the kinetics of the exchange process, the relative populations and structural features of invisible states in terms of NMR chemical shifts [51, 52]. Residues that undergo conformational exchange on the μs-ms time scale contribute to the effective transverse 15N relaxation rates (R2.eff). By measuring the increased contribution, Rex, to the ef‐ fective transverse relaxation rate as a function of CPMG pulse spacing relaxation, typical non-flat dispersion profiles are obtained (Figure 8).

**Figure 8.** Typical 15N relaxation dispersion profiles for KIX displaying residues Glu593 and Val635, recorded at static magnetic field strengths of 11.7T (red) and 18.8T (blue) at 26.9oC and in the presence of 0.4M *myo*-Inositol and 0.4M pinitol. Error bars represent uncertainties in relaxation rates. CPMG radio frequency field strengths, vCPMG, ranged from 40 to 960 Hz, relaxation delays were 50ms. Spectra were collected as series of two-dimensional data sets. Duplicate data sets were recorded at selected vCPMG values for error analysis. Peak intensities observed from 1H-15N spectra were converted into effective relaxation rates (R2.eff) and uncertainties in relaxation rates were calculated from repeat ex‐ periments. R2.eff were calculated by numerical modeling of magnetization evolution during the CPMG sequences. Fit curves were obtained by combining the dispersion of all residues in a collective fit to a two-state process

<sup>15</sup>N single quantum relaxation dispersion experiments were performed to characterize alter‐ ations in the two-site conformational exchange of the KID-binding (KIX) domain of CREBbinding protein (CBP) in the presence of osmolytes under native conditions. Conformational exchange of KIX 15N backbone resonances has been shown to be in the intermediate to slow time regime. CPMG-type relaxation dispersion data showed that under non-denaturing con‐ ditions, KIX permanently exchanges between its folded (native) ground state (G) and a par‐ tially unfolded high-energy state (E) that is populated to 3±0.2% at 26.9o C and pH 5.5. Relaxation dispersion experiments were performed for KIX and in the presence of 0.4M os‐ molytes (pinitol, *myo*-Inositol, quebrachitol, quercitol), operating at static magnetic field strengths of 11.7 and 18.8T at 26.9o C. 15N relaxation dispersion profiles were fit for each site individually (G↔E) to yield site-specific values of G→E and E→G rate constants (kGE and kEG) and differences in resonance frequencies between G and E states │Δωfit│. Dispersion profiles of all sites were then fit to a global two-site model assuming uniform values for kGE (ku) and kEG (kf ), but specific values for │Δωfit│ (Table1). Dispersion profiles (R2.eff/VCPMG) are dependent on kf and ku rate constants or the population of the unfolded state pE and the exchange rate constant (kex = kf + ku) and on chemical shift differences between the folded and unfolded state │Δω│[53].


**Table 1.** Two-site conformational exchange parameters of KIX. The response of R2.eff to vCPMG can be fitted to extract exchange parameters. A two-site exchange model (G↔E) was fit to 15N relaxation dispersion data, yielding sitespecific values of G→E and E→G rate constants (ku and kf). kf and ku are the first order rate constants for folding and unfolding transitions, calculated from global fits of 15N backbone relaxation experiments.

**Figure 8.** Typical 15N relaxation dispersion profiles for KIX displaying residues Glu593 and Val635, recorded at static magnetic field strengths of 11.7T (red) and 18.8T (blue) at 26.9oC and in the presence of 0.4M *myo*-Inositol and 0.4M pinitol. Error bars represent uncertainties in relaxation rates. CPMG radio frequency field strengths, vCPMG, ranged from 40 to 960 Hz, relaxation delays were 50ms. Spectra were collected as series of two-dimensional data sets. Duplicate data sets were recorded at selected vCPMG values for error analysis. Peak intensities observed from 1H-15N spectra were converted into effective relaxation rates (R2.eff) and uncertainties in relaxation rates were calculated from repeat ex‐ periments. R2.eff were calculated by numerical modeling of magnetization evolution during the CPMG sequences. Fit

<sup>15</sup>N single quantum relaxation dispersion experiments were performed to characterize alter‐ ations in the two-site conformational exchange of the KID-binding (KIX) domain of CREBbinding protein (CBP) in the presence of osmolytes under native conditions. Conformational exchange of KIX 15N backbone resonances has been shown to be in the intermediate to slow time regime. CPMG-type relaxation dispersion data showed that under non-denaturing con‐ ditions, KIX permanently exchanges between its folded (native) ground state (G) and a par‐

Relaxation dispersion experiments were performed for KIX and in the presence of 0.4M os‐ molytes (pinitol, *myo*-Inositol, quebrachitol, quercitol), operating at static magnetic field

individually (G↔E) to yield site-specific values of G→E and E→G rate constants (kGE and kEG) and differences in resonance frequencies between G and E states │Δωfit│. Dispersion profiles of all sites were then fit to a global two-site model assuming uniform values for kGE

), but specific values for │Δωfit│ (Table1). Dispersion profiles (R2.eff/VCPMG)

and ku rate constants or the population of the unfolded state pE and the

C. 15N relaxation dispersion profiles were fit for each site

+ ku) and on chemical shift differences between the folded

C and pH 5.5.

curves were obtained by combining the dispersion of all residues in a collective fit to a two-state process

tially unfolded high-energy state (E) that is populated to 3±0.2% at 26.9o

strengths of 11.7 and 18.8T at 26.9o

14 Abiotic Stress - Plant Responses and Applications in Agriculture

exchange rate constant (kex = kf

and unfolded state │Δω│[53].

(ku) and kEG (kf

are dependent on kf

The two-site conformational exchange of KIX between its natively folded ground state and a partially unfolded high-energy state, that represents the equilibrium analog of a folding in‐ termediate [54], was shown to be highly sensitive to the addition of osmolytes. NMR data showed that the composition of these two states differed between the protein in buffer alone and the osmolyte containing sample. Addition of 0.4M pinitol led to a decrease of more than 50% in the population of the partially unfolded state (pE). Accordingly, the first order rate constant for folding (kf ) increased from 574.4s-1 to 935.9s-1 in the presence of pinitol, while the rate constant for unfolding (ku) decreased (from 16.7s-1 to 12.7s-1). These data provide evidence that even under native conditions osmolytes shift the folding equilibrium towards the folded state. NMR relaxation experiments revealed that osmolytes play an important role on the structure of the folding intermediate, which is the main determinant for protein folding and dynamics. Even though intermediate states are extremely short-lived (in the or‐ der of ms), osmolytes greatly influence these states. A decrease in the population of the par‐ tially folded state is associated with a destabilization of this state relative to the folded state in the osmolyte containing sample. The interaction of the osmolyte with the protein surface is not favorable and therefore osmolytes are preferentially excluded from the protein sur‐ face. Osmolytes indirectly act by changing the properties of water surrounding the protein and hence modify protein-solvent interactions by altering the specific arrangement of the hydrophobic and hydrophilic residues. Folded states are relatively favored over (partially) unfolded states due to their compact structure and smaller surface exposed (solvent accessi‐ ble) area, leading to a net stabilization of the folded state even under native conditions. Ac‐ cumulation of high amounts of osmolytes does not seem to be useful under non-stress conditions as they influence protein conformation and dynamics, but they confer great ad‐ vances to enhance protein stability under stress conditions by counteracting the forces driv‐ ing protein unfolding. Compact folded conformations are generally less prone to unfolding, misfolding and aggregation that lead to loss of protein function. Increased conformational stability through osmolytes on the other hand allows for greater protein flexibility under elevated temperatures, since thermal motion decreases rigidity and enhances flexibility. This mechanism greatly contributes to preserve protein function under stress conditions in plants.

## **5. Biotechnology approaches for improved abiotic stress tolerance in plants**

Abiotic stress is one of the major causes of crop loss worldwide and restricts certain areas from productive agriculture and even less severe stress makes plants more susceptible to diseases and pests. As sessile organisms plants are exposed to various stresses during their lifespan. With increased understanding of the mechanisms of protein stabilization, advances have been made in genetically engineering more tolerant crop plants.

#### **5.1. Genetically engineering overproduction of osmolytes**

Progress is being made in genetically modifying plants to accumulate high amounts of os‐ molytes with the aim to enhance stress tolerance in plants. Transgenic plants have success‐ fully been engineered to accumulate metabolites such as proline, mannitol, glycine betaine and trehalose, which resulted in increased tolerance to various stresses [55-57]. In addition to lowering the osmotic potential and assisting in osmotic adjustment, osmolytes act as hy‐ droxyl radical scavengers and protect macromolecular structure. The accumulation of such metabolites in response to various stresses is a widely distributed phenomenon in the plant kingdom. Some important crop plants, however, are non-accumulators. Genetically intro‐ ducing mannitol, sorbitol, trehalose or *myo*-Inositol production in tobacco, *Arabidopsis* and rice, all species that do not synthesize these compounds naturally, produced enhanced toler‐ ance to salt and drought stress [58-60].

Recently, it has been shown that overexpression of rice (*Oryza sativa*) choline monooxyge‐ nase (OsCMO), the first enzyme in glycine betaine biosynthesis, enhances glycine betaine synthesis in transgenic tobacco plants and resulted in elevated tolerance to salt stress [61]. Although rice has been considered as typical non-accumulator of glycine betaine, this study revealed that the rice containing ortholog of CMO was fully functional in tobacco species. Enhanced tolerance toward salinity, heavy metal, oxidative stress and cold stress was also reported for transgenic tobacco plants when overexpressing rice cystathionine β-synthase [62] or cold regulated protein CbCOR15b transferred form *Capsella bursa-pastoris* [63]. Nu‐ merous reports show that introducing and enhancing abiotic stress tolerance by the transfer of one or more stress responsive genes between species would be an effective strategy to en‐ hance performance of crop plants in less-productive agricultural areas.

Another strategy for osmolyte overproduction and enhaced plant growth relies on site-di‐ rected mutagenesis. Δ<sup>1</sup> -Pyrroline-5-carboxylase synthase (P5CS), which is feedback inhibited by proline, has been mutated by site-directed mutagenesis, resulting in enzymes that were no longer inhibited. Plants expressing the mutated enzyme had twice the proline levels of WT-plants and exhibited increased tolerance to salt stress [64].

#### **5.2. Protein engineering**

Protein engineering approaches are being developed for the selection of protein mutations that increase protein stability. New stabilization strategies are based on random mutagene‐ sis and high-throughput screening for thermostability-improving mutations, functional screening or comparison of homologous proteins. Some proteins have been successfully sta‐ bilized by the introduction of structural elements from thermophilic and hyperthermophilic homologues [65]. However, the mechanisms underlying thermostability are diverse. Much research has been focused on understanding the stabilization of the hydrophobic core and internal structural elements of proteins [66, 67]. Recent research has also revealed that pro‐ tein surfaces have a strong influence on stability and, therefore, have to be taken into consid‐ eration. Surface residues are generally more flexible and the protein surface structure is less motional restricted than the compact core. Mutations in the protein surface are therefore supposed to largely affect protein stability and can be introduced to enhance protein stabili‐ ty. Much attention is paid to protein surface salt bridges, as it is known that surface salt bridges become more favorable with increasing temperature and hyperthermophilic pro‐ teins tend to have more salt bridges than their mesophilic homologues. Emphasis is made to investigate the contribution of surface salt bridges to enhanced protein stability under stress conditions.

Information from the protein biochemistry field will direct us toward an understanding of the rules for protein folding stability and dynamics with the goal to improve protein stabili‐ ty and stress tolerance in plants.

## **6. Conclusion**

**5. Biotechnology approaches for improved abiotic stress tolerance in**

have been made in genetically engineering more tolerant crop plants.

hance performance of crop plants in less-productive agricultural areas.

WT-plants and exhibited increased tolerance to salt stress [64].

**5.1. Genetically engineering overproduction of osmolytes**

16 Abiotic Stress - Plant Responses and Applications in Agriculture

ance to salt and drought stress [58-60].

rected mutagenesis. Δ<sup>1</sup>

**5.2. Protein engineering**

Abiotic stress is one of the major causes of crop loss worldwide and restricts certain areas from productive agriculture and even less severe stress makes plants more susceptible to diseases and pests. As sessile organisms plants are exposed to various stresses during their lifespan. With increased understanding of the mechanisms of protein stabilization, advances

Progress is being made in genetically modifying plants to accumulate high amounts of os‐ molytes with the aim to enhance stress tolerance in plants. Transgenic plants have success‐ fully been engineered to accumulate metabolites such as proline, mannitol, glycine betaine and trehalose, which resulted in increased tolerance to various stresses [55-57]. In addition to lowering the osmotic potential and assisting in osmotic adjustment, osmolytes act as hy‐ droxyl radical scavengers and protect macromolecular structure. The accumulation of such metabolites in response to various stresses is a widely distributed phenomenon in the plant kingdom. Some important crop plants, however, are non-accumulators. Genetically intro‐ ducing mannitol, sorbitol, trehalose or *myo*-Inositol production in tobacco, *Arabidopsis* and rice, all species that do not synthesize these compounds naturally, produced enhanced toler‐

Recently, it has been shown that overexpression of rice (*Oryza sativa*) choline monooxyge‐ nase (OsCMO), the first enzyme in glycine betaine biosynthesis, enhances glycine betaine synthesis in transgenic tobacco plants and resulted in elevated tolerance to salt stress [61]. Although rice has been considered as typical non-accumulator of glycine betaine, this study revealed that the rice containing ortholog of CMO was fully functional in tobacco species. Enhanced tolerance toward salinity, heavy metal, oxidative stress and cold stress was also reported for transgenic tobacco plants when overexpressing rice cystathionine β-synthase [62] or cold regulated protein CbCOR15b transferred form *Capsella bursa-pastoris* [63]. Nu‐ merous reports show that introducing and enhancing abiotic stress tolerance by the transfer of one or more stress responsive genes between species would be an effective strategy to en‐

Another strategy for osmolyte overproduction and enhaced plant growth relies on site-di‐

by proline, has been mutated by site-directed mutagenesis, resulting in enzymes that were no longer inhibited. Plants expressing the mutated enzyme had twice the proline levels of

Protein engineering approaches are being developed for the selection of protein mutations that increase protein stability. New stabilization strategies are based on random mutagene‐


**plants**

Abiotic stresses like desiccation, flooding, high salinity or extreme temperatures are com‐ mon threats to plants and the optimal function of their metabolism. Protein conformation and stability is dramatically affected by sudden changes in the environment, giving rise to protein unfolding, misfolding and aggregation. Finding the rules for protein folding and un‐ folding that lead to conformational stability is a matter of ongoing research. Folded states represent the most stable forms under native conditions, but partially folded states that al‐ low for efficient interaction with binding partners are of fundamental importance in biologi‐ cal activity. Studying protein stability under stress conditions has to take protein dynamics, meaning conformational changes of proteins with time, into consideration. Advances have been made in methods to study the conformational exchange in proteins and their folding stability under varying experimental conditions. Nuclear magnetic resonance spectroscopy techniques have been introduced to study the interconversion between folded and partially folded intermediate states. These short-lived, partially folded, states are extremely impor‐ tant for biological activity and play a major role in the energy landscape of proteins. NMR relaxation dispersion experiments revealed that such low populated intermediate folding states are strongly affected by solvent and co-solvent conditions. One of the early onsets of the stress response in plants is the accumulation of osmolytes that serve for osmotic adjust‐ ment and protect proteins by maintaining water at the protein surface where it is most need‐ ed. NMR dynamic measurements revealed that addition of osmolytes (*myo*-Inositol, pinitol, quebrachitol and quercitol) lead to a decreased population of the partially folded state by shifting the folding equilibrium towards the folded ensembles. Although osmolytes do not

directly interact with the protein surface, they alter protein surface properties by changing the water structure and hydrophobic interactions, thereby stabilizing the folded states rela‐ tive to unfolded states. Even under native conditions, osmolytes were shown to favor the compact folded structure over partially folded structures, consequently leading to altera‐ tions in the dynamics of these two states. Thermodynamic considerations assume that osmo‐ lytes act by raising the chemical potential of the partially unfolded state relative to the folded state, thereby increasing the (positive) Gibbs energy difference (ΔG) between folded and unfolded assemblies, thus favoring the folded state with the respect to the unfolded state. By stabilizing compact folded states over unfolded structures even under non-stress conditions, osmolyte accumulation exhibits a great potential to counteract the forces that lead to stress induced protein unfolding. High osmolyte accumulation in plants may not be useful under non-stress conditions as they tend to decrease protein globally and locally flex‐ ibility and increase protein overall rigidity. Increased rigidity and overall compactness, however, confer great advances under stress conditions. Compact structures are less prone to unfolding, misfolding, aggregation and degradation. Lower structural flexibility under ambient temperatures allows for greater flexibility under elevated temperatures since ther‐ mal motion decreases rigidity and enhances flexibility, which is essential for protein func‐ tion under stress conditions. Osmolyte production seems to be very effective strategy to adopt plants quickly and with a remarkable plasticity to various changes in their environ‐ ment. High osmolyte accumulation serves to suppress protein unfolding and misfolding, en‐ hances protein folding stability and facilitate the protein refolding process after complete denaturation. These lessions that we learned from plants and new insights from the protein biochemistry field are taken together for genetically engineering of more tolerant crop plants with the ultimate goal to improve yields in less productive agricultural land.

## **Acknowledgements**

This work was done in collaboration between the Department of Chemical Physiology of Plants and the Department of Biomolecular Structural Chemistry at the University of Vien‐ na. The author would like to thank Marianne Popp, Robert Konrat, Martin Tollinger and Karin Kloiber for supporting this research and the latter three for providing their expertise in Nuclear Magnetic Resonance Spectroscopy. The author is also grateful to Jürgen König for supporting this work.

### **Author details**

Martina Ortbauer

Address all correspondence to: martina.ortbauer@univie.ac.at

University of Vienna, Vienna, Austria

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directly interact with the protein surface, they alter protein surface properties by changing the water structure and hydrophobic interactions, thereby stabilizing the folded states rela‐ tive to unfolded states. Even under native conditions, osmolytes were shown to favor the compact folded structure over partially folded structures, consequently leading to altera‐ tions in the dynamics of these two states. Thermodynamic considerations assume that osmo‐ lytes act by raising the chemical potential of the partially unfolded state relative to the folded state, thereby increasing the (positive) Gibbs energy difference (ΔG) between folded and unfolded assemblies, thus favoring the folded state with the respect to the unfolded state. By stabilizing compact folded states over unfolded structures even under non-stress conditions, osmolyte accumulation exhibits a great potential to counteract the forces that lead to stress induced protein unfolding. High osmolyte accumulation in plants may not be useful under non-stress conditions as they tend to decrease protein globally and locally flex‐ ibility and increase protein overall rigidity. Increased rigidity and overall compactness, however, confer great advances under stress conditions. Compact structures are less prone to unfolding, misfolding, aggregation and degradation. Lower structural flexibility under ambient temperatures allows for greater flexibility under elevated temperatures since ther‐ mal motion decreases rigidity and enhances flexibility, which is essential for protein func‐ tion under stress conditions. Osmolyte production seems to be very effective strategy to adopt plants quickly and with a remarkable plasticity to various changes in their environ‐ ment. High osmolyte accumulation serves to suppress protein unfolding and misfolding, en‐ hances protein folding stability and facilitate the protein refolding process after complete denaturation. These lessions that we learned from plants and new insights from the protein biochemistry field are taken together for genetically engineering of more tolerant crop

18 Abiotic Stress - Plant Responses and Applications in Agriculture

plants with the ultimate goal to improve yields in less productive agricultural land.

Address all correspondence to: martina.ortbauer@univie.ac.at

This work was done in collaboration between the Department of Chemical Physiology of Plants and the Department of Biomolecular Structural Chemistry at the University of Vien‐ na. The author would like to thank Marianne Popp, Robert Konrat, Martin Tollinger and Karin Kloiber for supporting this research and the latter three for providing their expertise in Nuclear Magnetic Resonance Spectroscopy. The author is also grateful to Jürgen König

**Acknowledgements**

for supporting this work.

University of Vienna, Vienna, Austria

**Author details**

Martina Ortbauer


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## **Abiotic Stress in Plants and Metabolic Responses**

Saúl Fraire-Velázquez and Victor Emmanuel Balderas-Hernández

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/54859

## **1. Introduction**

The vast metabolic diversity observed in plants is the direct result of continuous evolutionary processes. There are more than 200,000 known plant secondary metabolites, representing a vast reservoir of diverse functions. When the environment is adverse and plant growth is affected, metabolism is profoundly involved in signaling, physiological regulation, and defense responses. At the same time, in feedback, abiotic stresses affect the biosynthesis, concentration, transport, and storage of primary and secondary metabolites. Metabolic adjustments in response to abiotic stressors involve fine adjustments in amino acid, carbohy‐ drate, and amine metabolic pathways. Proper activation of early metabolic responses helps cells restore chemical and energetic imbalances imposed by the stress and is crucial to acclimation and survival. Time-series experiments have revealed that metabolic activities respond to stress more quickly than transcriptional activities do. In order to study and map all the simultaneous metabolic responses and, more importantly, to link these responses to a specific abiotic stress, integrative and comprehensive analyses are required. Metabolomics is the systematic approach through which qualitative and quantitative analysis of a large number of metabolites is increasing our knowledge of how complex metabolic networks interact and how they are dynamically modified under stress adaptation and tolerance processes. A vast amount of research has been done using metabolomic approaches to (*i*) characterize metabolic responses to abiotic stress, (*ii*) to discover novel genes and annotate gene function, and, (*iii*) more recently, to identify metabolic quantitative trait loci. The integration of the collected metabolic data concerning abiotic stress responses is helping in the identification of tolerance traits that may be transferable to cultivated crop species. In this review, the diverse metabolic responses identified in plants so far are discussed. We also include recent advances in the study of plant metabolomes and metabolic fluxes with a focus on abiotic stress-tolerance trait interactions.

properly cited.

© 2013 Fraire-Velázquez and Balderas-Hernández; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is © 2013 Fraire-Velázquez and Balderas-Hernández; licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

## **2. Abiotic stresses and the impact on agriculture**

Today, in a world of 7 billion people, agriculture is facing great challenges to ensure a sufficient food supply while maintaining high productivity and quality standards. In addition to an ever increasing demographic demand, alterations in weather patterns due to changes in climate are impacting crop productivity globally. Warming and shifts in rainfall patterns caused an historically high \$10.3 billion in crop insurance payments to cover agriculture losses in 2011 in the U.S. [1]. Unfavorable climate (resulting in abiotic stresses) not only causes changes in agro-ecological conditions, but indirectly affects growth and distribution of incomes, and thus increasing the demand for agricultural production [2]. Adverse climatic factors, such as water scarcity (drought), extreme temperatures (heat, freezing), photon irradiance, and contamina‐ tion of soils by high ion concentration (salt, metals), are the major growth stressors that significantly limit productivity and quality of crop species worldwide. As has been pointed out, current achievements in crop production have been associated with management practices that have degraded the land and water systems [3]. Soil and water salinity problems exist in crop lands in China, India, the United States, Argentina, Sudan, and many other countries in Western and Central Asia. Globally, an estimated 34 million irrigated hectares are salinized [4], and the global cost of irrigation-induced salinity is equivalent to an estimated US\$11 billion per year [5].

A promising strategy to cope with adverse scenario is to take advantage of the flexibility that biodiversity (genes, species, ecosystems) offers and increase the ability of crop plants to adapt to abiotic stresses. The Food and Agricultural Organization (FAO) of the United Nations promotes the use of adapted plants and the selection and propagation of crop varieties adapted or resistant to adverse conditions [6]. Global programs, such as the Global Partnership Initiative for Plant Breeding Capacity Building (GIPB), aim to select and distribute crops and cultivars with tolerance to abiotic stresses for sustainable use of plant genetic resources for food and agriculture [7].

## **3. Plant responses to abiotic stress**

Through the history of evolution, plants have developed a wide variety of highly sophisticated and efficient mechanisms to sense, respond, and adapt to a wide range of environmental changes. When in adverse or limiting growth conditions, plants respond by activating tolerance mechanisms at multiple levels of organization (molecular, tissue, anatomical, and morphological), by adjusting the membrane system and the cell wall architecture, by altering the cell cycle and rate of cell division, and by metabolic tuning [8]. At a molecular level, many genes are induced or repressed by abiotic stress, involving a precise regulation of extensive stress-gene networks [9-11]. Products of those genes may function in stress response and tolerance at the cellular level. Proteins involved in biosynthesis of osmoprotectant compounds, detoxification enzyme systems, proteases, transporters, and chaperones are among the multiple protein functions triggered as a first line of direct protection from stress. In addition, activation of regulatory proteins (*e.g.*, transcription factors, protein phosphatases, and kinases) and signaling molecules are essential in the concomitant regulation of signal transduction and stress-responsive gene expression [12, 13]. Early plant response mechanisms prevent or alleviate cellular damage caused by the stress and re-establish homeostatic conditions and allow continuation of growth [14]. Equilibrium recovery of the energetic, osmotic, and redox imbalances imposed by the stressor are the first targets of plant immediate responses.

**2. Abiotic stresses and the impact on agriculture**

26 Abiotic Stress - Plant Responses and Applications in Agriculture

per year [5].

food and agriculture [7].

**3. Plant responses to abiotic stress**

Today, in a world of 7 billion people, agriculture is facing great challenges to ensure a sufficient food supply while maintaining high productivity and quality standards. In addition to an ever increasing demographic demand, alterations in weather patterns due to changes in climate are impacting crop productivity globally. Warming and shifts in rainfall patterns caused an historically high \$10.3 billion in crop insurance payments to cover agriculture losses in 2011 in the U.S. [1]. Unfavorable climate (resulting in abiotic stresses) not only causes changes in agro-ecological conditions, but indirectly affects growth and distribution of incomes, and thus increasing the demand for agricultural production [2]. Adverse climatic factors, such as water scarcity (drought), extreme temperatures (heat, freezing), photon irradiance, and contamina‐ tion of soils by high ion concentration (salt, metals), are the major growth stressors that significantly limit productivity and quality of crop species worldwide. As has been pointed out, current achievements in crop production have been associated with management practices that have degraded the land and water systems [3]. Soil and water salinity problems exist in crop lands in China, India, the United States, Argentina, Sudan, and many other countries in Western and Central Asia. Globally, an estimated 34 million irrigated hectares are salinized [4], and the global cost of irrigation-induced salinity is equivalent to an estimated US\$11 billion

A promising strategy to cope with adverse scenario is to take advantage of the flexibility that biodiversity (genes, species, ecosystems) offers and increase the ability of crop plants to adapt to abiotic stresses. The Food and Agricultural Organization (FAO) of the United Nations promotes the use of adapted plants and the selection and propagation of crop varieties adapted or resistant to adverse conditions [6]. Global programs, such as the Global Partnership Initiative for Plant Breeding Capacity Building (GIPB), aim to select and distribute crops and cultivars with tolerance to abiotic stresses for sustainable use of plant genetic resources for

Through the history of evolution, plants have developed a wide variety of highly sophisticated and efficient mechanisms to sense, respond, and adapt to a wide range of environmental changes. When in adverse or limiting growth conditions, plants respond by activating tolerance mechanisms at multiple levels of organization (molecular, tissue, anatomical, and morphological), by adjusting the membrane system and the cell wall architecture, by altering the cell cycle and rate of cell division, and by metabolic tuning [8]. At a molecular level, many genes are induced or repressed by abiotic stress, involving a precise regulation of extensive stress-gene networks [9-11]. Products of those genes may function in stress response and tolerance at the cellular level. Proteins involved in biosynthesis of osmoprotectant compounds, detoxification enzyme systems, proteases, transporters, and chaperones are among the multiple protein functions triggered as a first line of direct protection from stress. In addition,

Observed tolerance responses towards abiotic stress in plants are generally composed of stressspecific response mechanisms and also more general adaptive responses that confer strategic advantages in adverse conditions. General response mechanisms related to central pathways are involved in energy maintenance and include calcium signal cascades [15, 16], reactive oxygen species scavenging/signaling elements [17, 18], and energy deprivation (energy sensor protein kinase, SnRK1) signaling [19]. Induction of these central pathways is observed during plant acclimation towards different types of stress. For example, protein kinase SnRK1is a central metabolic regulator of the expression of genes related to energy-depleting conditions, but this kinase also becomes active when plants face different types of abiotic stress such as drought, salt, flooding, or nutrient depravation [20-24]. SnRK1 kinases modify the expression of over 1000 stress-responsive genes allowing the re-establishment of homeostasis by repres‐ sing energy consuming processes, thus promoting stress tolerance[24, 25]. The optimization of cellular energy resources during stress is essential for plant acclimation; energetically expensive processes are partially arrested, such as reproductive activities, translation, and some biosynthetic pathways. For example, nitrogen and carbon assimilation are impaired in maize during salt stress and potassium-deficiency stress; the synthesis of free amino acids, chlorophyll, and protein are also affected [26-28]. Once energy-expensive processes are curtailed, energy resources can be redirected to activate protective mechanisms. This is exemplified by the decrease in *de novo* protein synthesis in *Brassica napus* seedlings*, Glycine max, Lotus japonicas*, and *Medicago truncatula* during heat stress accompanied by an increased translation of heat shock proteins [29, 30].

## **4. Metabolic adjustments during stressing conditions: Osmolyte accumulation**

A common defensive mechanism activated in plants exposed to stressing conditions is the production and accumulation of compatible solutes. The chemical nature of these small molecular weight organic osmoprotectants is diverse; these molecules include amino acids (asparagine, proline, serine), amines (polyamines and glycinebetaine), and *γ*-amino-N-butyric acid (GABA). Furthermore, carbohydrates, including fructose, sucrose, trehalose, raffinose, and polyols (myo-inositol, D-pinitol) [12, 31], as well as pools of anti-oxidants such as gluta‐ thione (GSH) and ascorbate [32, 33], accumulate in response to osmotic stress. Common characteristics of these diverse solutes are a high level of solubility in the cellular milieu and lack of inhibition of enzyme activities even at high concentrations. Accumulation of compatible solutes in response to stress is not only observed in plants, it is a defense mechanism triggered in animal cells, bacteria, and marine algae, indicative of an evolutionarily conserved trait [34, 35]. Scavenging of reactive oxygen species (ROS) to restore redox metabolism, preservation of cellular turgor by restitution of osmotic balance, and associated protection and stabilization of proteins and cellular structures are among the multiple protective functions of compatible osmoprotectants during environmental stress [36-38].

A large amount of research has been done on the beneficial effects of compatible solutes on plant tolerance to environmental stress. Correlation between amino acid accumulation (mainly proline) and stress tolerance was described in the mid-1960s in Bermuda grass during water stress [39]. Since then, extensive work has proven that proline serves as an osmoprotectant, a cryoprotectant, a signaling molecule, a protein structure stabilizer, and an ROS scavenger in response to stresses that cause dehydration; including salinity, freezing, heavy metals, and drought (low water potential) [40, 41]. Proline oxidation may also provide energy to sustain metabolically demanding programs of plant reproduction, once the stress has passed [42].

Proline metabolism and its regulation are processes well characterized in plants. Proline is synthesized from glutamate in the cytoplasm or chloroplasts: Δ-1-pyrroline-5-carboxylate synthetase (P5CS) reduces glutamate to glutamate semialdehyde (GSA). Then GSA sponta‐ neously cyclizes into pyrroline-5-carboxylate (P5C), which is further reduced by P5C reductase (P5CR) to proline. Conversely, proline is catabolized within the mitochondrial matrix by action of proline dehydrogenase (ProDH) and P5C dehydrogenase (P5CDH) to glutamate. In an alternative pathway, proline can be synthesized from ornithine in a pathway involving ornithine δ-aminotransferase (OAT). Core enzymes P5CS, P5C, P5CR, ProDH, and OAT are responsible for maintaining the balance between biosynthesis and catabolism of proline. Regulation comes at transcriptional level of genes encoding the key enzymes. Transcriptional up-regulation of genes for P5CS and P5C to increase proline synthesis from glutamate and down-regulation of genes for P5CR and ProDH to arrest proline catabolism is observed during dehydration/osmotic stress [43]. Also, post-translational regulation of core enzymes is closely associated with proline levels and environmental signals. For example, the *Arabidopsis* P5CS1 enzyme is subjected to feedback inhibition by proline, controlling the carbon influx into the biosynthetic pathway [44, 45]. Considering that proline accumulation is associated with stress tolerance, that core enzymes regulate proline biosynthesis, and that these core enzymes are likely rate-limiting steps for its accumulation, logic dictates that overexpression of biosynthetic proline enzymes might increase the levels of the compatible solute and thus improve the tolerance in plants against abiotic stress. Several studies have tested this by overexpressing genes for P5CS or P5C enzymes in different plant species, reporting the expected rise in proline levels and the associated resistance to dehydration, salinity, or freezing [46-53]. Furthermore, deletion of genes coding ProDH [54] or P5CDH [55, 56], expression of a feedback-insensitive P5CS [45], or the overexpression of OAT [57, 58] increase the cellular levels of proline and osmoprotection to some abiotic stresses.

Comparable extensive work has been done for other compatible solutes such as γ-aminobu‐ tyric acid [59], glycine betaine [60], trehalose [61], mannitol, and sorbitol [36]; these solutes are efficient protectors against some abiotic stressors. Metabolic pathways for biosynthesis and catabolism of compatible solutes, their regulation, participant enzymes, and compartmental‐ ization are well characterized in most important plant species. This knowledge has led to strategies for improvement of plant tolerance involving the accumulation of those protective osmolytes in plants by expression of core biosynthetic enzymes or their improved derivatives, expression of related transporters, and deletion of osmolyte-consuming enzymes. These numerous studies have provided evidence that enhanced accumulation of compatible solutes correlates with reinforcement of plant resistance to adverse growth conditions.

## **5. Plant metabolomics and applications**

35]. Scavenging of reactive oxygen species (ROS) to restore redox metabolism, preservation of cellular turgor by restitution of osmotic balance, and associated protection and stabilization of proteins and cellular structures are among the multiple protective functions of compatible

A large amount of research has been done on the beneficial effects of compatible solutes on plant tolerance to environmental stress. Correlation between amino acid accumulation (mainly proline) and stress tolerance was described in the mid-1960s in Bermuda grass during water stress [39]. Since then, extensive work has proven that proline serves as an osmoprotectant, a cryoprotectant, a signaling molecule, a protein structure stabilizer, and an ROS scavenger in response to stresses that cause dehydration; including salinity, freezing, heavy metals, and drought (low water potential) [40, 41]. Proline oxidation may also provide energy to sustain metabolically demanding programs of plant reproduction, once the stress has passed [42].

Proline metabolism and its regulation are processes well characterized in plants. Proline is synthesized from glutamate in the cytoplasm or chloroplasts: Δ-1-pyrroline-5-carboxylate synthetase (P5CS) reduces glutamate to glutamate semialdehyde (GSA). Then GSA sponta‐ neously cyclizes into pyrroline-5-carboxylate (P5C), which is further reduced by P5C reductase (P5CR) to proline. Conversely, proline is catabolized within the mitochondrial matrix by action of proline dehydrogenase (ProDH) and P5C dehydrogenase (P5CDH) to glutamate. In an alternative pathway, proline can be synthesized from ornithine in a pathway involving ornithine δ-aminotransferase (OAT). Core enzymes P5CS, P5C, P5CR, ProDH, and OAT are responsible for maintaining the balance between biosynthesis and catabolism of proline. Regulation comes at transcriptional level of genes encoding the key enzymes. Transcriptional up-regulation of genes for P5CS and P5C to increase proline synthesis from glutamate and down-regulation of genes for P5CR and ProDH to arrest proline catabolism is observed during dehydration/osmotic stress [43]. Also, post-translational regulation of core enzymes is closely associated with proline levels and environmental signals. For example, the *Arabidopsis* P5CS1 enzyme is subjected to feedback inhibition by proline, controlling the carbon influx into the biosynthetic pathway [44, 45]. Considering that proline accumulation is associated with stress tolerance, that core enzymes regulate proline biosynthesis, and that these core enzymes are likely rate-limiting steps for its accumulation, logic dictates that overexpression of biosynthetic proline enzymes might increase the levels of the compatible solute and thus improve the tolerance in plants against abiotic stress. Several studies have tested this by overexpressing genes for P5CS or P5C enzymes in different plant species, reporting the expected rise in proline levels and the associated resistance to dehydration, salinity, or freezing [46-53]. Furthermore, deletion of genes coding ProDH [54] or P5CDH [55, 56], expression of a feedback-insensitive P5CS [45], or the overexpression of OAT [57, 58] increase the cellular levels of proline and

Comparable extensive work has been done for other compatible solutes such as γ-aminobu‐ tyric acid [59], glycine betaine [60], trehalose [61], mannitol, and sorbitol [36]; these solutes are efficient protectors against some abiotic stressors. Metabolic pathways for biosynthesis and catabolism of compatible solutes, their regulation, participant enzymes, and compartmental‐ ization are well characterized in most important plant species. This knowledge has led to

osmoprotectants during environmental stress [36-38].

28 Abiotic Stress - Plant Responses and Applications in Agriculture

osmoprotection to some abiotic stresses.

The traditional approach of enhancing the accumulation of a specific compounds in response to a determined stimulus, as done with compatible solutes, have resulted in some degree of tolerance in plants, and also demonstrates that the ability to redirect nutrients to imperative processes and the induction of adequate metabolic adjustments are crucial for plant survival during conditions of stress. However, this is a sectioned view of how plants regulate their entire metabolism in response to stressing conditions. In order to achieve a more comprehen‐ sive understanding, we must consider that plant metabolism is an intricate network of interconnected reactions. Plants have a high degree of subcellular compartmentation, a vast repertory of metabolites, and developmental stage strongly influences metabolism. Therefore, metabolic responses are complex and dynamic and involve the modification of more than one metabolite. Also, accumulation of a specific compound is not an absolute requirement indicative of a tolerance trait; adjustment of the flux through a certain metabolic pathway might be enough to contribute to stress tolerance [62]. Recently, it has been reported that plants modulate stoichiometry and metabolism in a flexible manner in order to maintain optimal fitness in mechanisms of storage, defense, and reproduction under varying conditions of temperature and water availability [63]. Furthermore, time-series experiments in *Arabidopsis thaliana* plants subjected to temperature and/or light alterations revealed that time-resolved metabolic activities respond more quickly than transcriptional activities do [64].

Traditional molecular approaches for tracing metabolic phenotypes in plants responding to abiotic stress have identified and manipulated specific genes or groups of genes in plant models. These have primarily been genes involved in early responses or in down-stream assembly of the response reaction. With the application of new powerful tools of molecular biology and bioinformatics, large collections of genes have been subjected to complete analysis. To arrive at a complete and comprehensive knowledge of physiology in the plant response to abiotic stress, researchers are embracing ionomic profiling, transcriptomic, proteomic and metabolomic analysis. A deep dissection of the biochemical pathways in plants facing stressing conditions requires integrative and comprehensive analyses in order to identify all the simultaneous metabolic responses and, more importantly, to be able to link these responses to specific abiotic stress. In this sense, metabolomics could contribute significantly to the study of metabolic responses to stress in plants by identifying diverse metabolites, such as the byproducts of stress metabolism, stress signal transduction molecules, and molecules that are part of the acclimation response [65].

The metabolome is the entirety of small molecules present in an organism and can be regarded as the ultimate expression of its genotype in response to environmental changes. Metabolomics is gaining importance in plant research in both basic and applied contexts. Metabolomic studies have already shown how detailed information gained from chemical composition can help us to understand the various physiological and biochemical changes occurring in the plants and their influence on the phenotype. The analytical measurement of several hundreds to thousands of metabolites is becoming a standard laboratory technique with the advent of "hyphenated" analytical platforms of separation methods and various detection systems. Separation methods include gas chromatography (GC), liquid chromatography (LC), and capillary electrophoresis (CE). Different types of mass spectrometry (MS), nuclear magnetic resonance (NMR), and ultraviolet light spectroscopy (UV/VIS) devices are utilized for detection. Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS) is a specialized technique often used in direct infusion (DI) mode for metabolomics analyses, as its high mass accuracy allows a separation solely based on this parameter. Each methodology offers advantages and disadvantages, and the method of choice will depend on the type of sample and metabolites to be determined, and the combination of analytical platforms [66].

GC and MS were the first pair of techniques to be combined, delivering high robustness and reproducibility. GC-MS remains one of the most widely used methods for obtaining metabolomic data because of its ease of use, excellent separation power, and its reprodu‐ cibility. The main drawback of GC-MS is that only thermally stable volatile metabolites, or non-volatile compounds that can be chemically altered to make them volatile, can be detected [67, 68]. NMR spectroscopy is a fingerprinting technique that offers several ad‐ vantages over high-throughput metabolite analyses, such as relatively simple sample preparation and the non-destructive analysis of samples. NMR can detect different classes of metabolites in a sample, regardless of their size, charge, volatility, or stability with ex‐ cellent resolution and reproducibility [69]. Labeling of metabolites with isotopes and sub‐ sequent NMR analysis is also useful for metabolic flux analysis and fluxomics as it allows tracking the selective signal enhancement of isotopologues [70]. Recent advances with high-throughput approaches using ultra-high-field FT-ICR-MS alone or in combina‐ tion with other tools of 'first pass' metabolome analysis as electrospray ionization mass spectrometry (ESI-MS) are expected to make inventory of the entire metabolome in a sin‐ gle sample possible in the near future [71, 72].

In metabolomics, the implicit objective is to identify and quantify all possible metabolites in a cellular system under defined states of stress conditions (biotic or abiotic) over a particular time scale in order to characterize accurately the metabolic profile [73]. But metabolome studies have some analytical limitations. It is important to have in mind that from the total amount of metabolites in a sample, only an informative portion can be reliably identified and quantified. In addition, metabolic networks in multicellular eukaryotes, specifically in plants, are chal‐ lenging because of the large size of the metabolome, extensive secondary metabolism, and the considerable variation in tissue-specific metabolic activity [74]. Therefore, experimental design and sample preparation need to be done with great care because environmental and experi‐ mental variation confer noticeable impact on the resulting metabolic profiles. This has been demonstrated in legumes in which a high proportion of nutritional and metabolic changes depend on non-controllable environmental variables [75].

Metabolomic analyses have been applied to the functional identification of unknown genes through metabolic profiling of plants in which some genes are up- or down-regu‐ lated, the discovery of biomarkers associated with disease phenotypes, the safety assess‐ ment of genetically modified organisms (GMOs), the characterization of plant metabolites of nutritional importance and significance in human health, and the discovery of com‐ pounds involved in plant resistance to biotic and abiotic stresses [76]. Metabolic profiles can be used as signatures for assessing the genetic variation among different cultivars or species of the same genotype at different growth stages and environments. The metabo‐ lite profile represents phenotypic information; this means that qualitative and quantita‐ tive metabolic measurements can be related to the genotypes of the plants to differentiate closely related individuals [77, 78]. Once the identification of individual metabolites is available, connections among metabolites can be established, and then metabolic profiles can be used to infer mechanisms of defense. Metabolic profiles will guide tailoring of genotypes for acceptable performance under adverse growth conditions and will be of help in design and development of crop plant cultivars best suited to sustainable agricul‐ ture [79, 80]. Metabolomics tools have been used to evaluate the impact of the genotype and the environment on the quality of plant growth in the study of interpecific hybrids between *Jacobaea aquatica* and *J. vulgaris* (common weeds native to Northern Eurasia). An NMR-based metabolomics profiling approach was used to correlate the expression of high and low concentrations of particular compounds, including phenylpropanoids and sugars, with results of quantification of genetically controlled differences between major primary and secondary metabolites [81]. In melon (*Cucumis melo* L.), metabolomic and el‐ emental profiling of fruit quality were found to be affected by genotype and environment [82].

## **6. Plant metabolomics and drought stress**

is gaining importance in plant research in both basic and applied contexts. Metabolomic studies have already shown how detailed information gained from chemical composition can help us to understand the various physiological and biochemical changes occurring in the plants and their influence on the phenotype. The analytical measurement of several hundreds to thousands of metabolites is becoming a standard laboratory technique with the advent of "hyphenated" analytical platforms of separation methods and various detection systems. Separation methods include gas chromatography (GC), liquid chromatography (LC), and capillary electrophoresis (CE). Different types of mass spectrometry (MS), nuclear magnetic resonance (NMR), and ultraviolet light spectroscopy (UV/VIS) devices are utilized for detection. Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS) is a specialized technique often used in direct infusion (DI) mode for metabolomics analyses, as its high mass accuracy allows a separation solely based on this parameter. Each methodology offers advantages and disadvantages, and the method of choice will depend on the type of sample and metabolites to be determined, and the combination of analytical platforms [66].

GC and MS were the first pair of techniques to be combined, delivering high robustness and reproducibility. GC-MS remains one of the most widely used methods for obtaining metabolomic data because of its ease of use, excellent separation power, and its reprodu‐ cibility. The main drawback of GC-MS is that only thermally stable volatile metabolites, or non-volatile compounds that can be chemically altered to make them volatile, can be detected [67, 68]. NMR spectroscopy is a fingerprinting technique that offers several ad‐ vantages over high-throughput metabolite analyses, such as relatively simple sample preparation and the non-destructive analysis of samples. NMR can detect different classes of metabolites in a sample, regardless of their size, charge, volatility, or stability with ex‐ cellent resolution and reproducibility [69]. Labeling of metabolites with isotopes and sub‐ sequent NMR analysis is also useful for metabolic flux analysis and fluxomics as it allows tracking the selective signal enhancement of isotopologues [70]. Recent advances with high-throughput approaches using ultra-high-field FT-ICR-MS alone or in combina‐ tion with other tools of 'first pass' metabolome analysis as electrospray ionization mass spectrometry (ESI-MS) are expected to make inventory of the entire metabolome in a sin‐

In metabolomics, the implicit objective is to identify and quantify all possible metabolites in a cellular system under defined states of stress conditions (biotic or abiotic) over a particular time scale in order to characterize accurately the metabolic profile [73]. But metabolome studies have some analytical limitations. It is important to have in mind that from the total amount of metabolites in a sample, only an informative portion can be reliably identified and quantified. In addition, metabolic networks in multicellular eukaryotes, specifically in plants, are chal‐ lenging because of the large size of the metabolome, extensive secondary metabolism, and the considerable variation in tissue-specific metabolic activity [74]. Therefore, experimental design and sample preparation need to be done with great care because environmental and experi‐ mental variation confer noticeable impact on the resulting metabolic profiles. This has been demonstrated in legumes in which a high proportion of nutritional and metabolic changes

gle sample possible in the near future [71, 72].

30 Abiotic Stress - Plant Responses and Applications in Agriculture

depend on non-controllable environmental variables [75].

The variable and often insufficient rainfalls in extended areas of rain-fed agriculture, the unsustainable groundwater use for irrigated agriculture worldwide, and the fast-growing demands for urban water are putting extreme pressure on global food crop production. The demand for water to sustain the agriculture systems in many countries will continue to increase as a result of growing populations [83]. This progressively worsening water scarcity is imposing hydric stress on both rain-fed and irrigated crops. Water deficiency stress induces a wide range of physiological and biochemical alterations in plants; arrestment of cell growth and photosynthesis and enhanced respiration are among the early affects. Genome expression is extensively remodeled, activating and repressing a variety of genes with diverse functions [11, 84]. Sensing water deficit and activation of defense mechanisms comes through chemical signals in which abscisic acid (ABA) plays a central role. ABA accumulates in tissues of plants subjected to hydric stress and promotes transpiration reduction via stomatal closure. Through this mechanism, plants minimize water losses and diminish stress injury. ABA regulates expression of many stress-responsive genes, including the late embryogenesis abundant (LEA) proteins, leading to a reinforcement of drought stress tolerance in plants [85]. Many questions remain unresolved concerning hydric stress-plant metabolic response: How does drought stress perturb metabolism in crop plants? How does hydric stress affect the metabolism of wild plants? What modern strategies of "omics" could be exploited to support future programs of crop breeding to lead to a more sustainable agriculture?

As previously described, one of the main mechanisms by which plants cope with water deficits is osmotic adjustment. These adjustments maintain a positive cell turgor via the active accumulation of compatible solutes. Traditionally, the analysis of metabolic responses to drought stress was limited to analysis of one or two classes of compounds considered as "role players" in the development of tolerance. Application of metabolomic approaches is providing a less biased perspective of metabolic profiles of response and also is aiding in the discovery of novel metabolic phenotypes. Unbiased GC-MS metabolomic profiling in *Eucalyptus* showed that drought stress alters a larger number of leaf metabolites than the previously reported in targeted analysis. Accumulation of shikimic acid and two cyclohexanepentol stereoisomers in response to drought stress was described for the first time in *Eucalyptus*. Also, the magnitude of metabolic adjustments in response to water stress correlates with the sensitivity/tolerant phenotype observed; drought affected around 30-40% of measured metabolites in *Eucalyptus dumosa* (a drought-sensitive specie) compared to 10-15% in *Eucalyptus pauciflora* (a droughttolerant specie) [86]. Similarly, critical differences in the metabolic responses were observed when drought-tolerant (NA5009RG) and drought-sensitive (DM50048) soybean cultivars were analyzed by 1 H NMR-based metabolomics. Interestingly, no enhanced accumulation of the traditional osmoprotectants, such as proline, soluble sugars as sucrose or myo-inositol, organic acids or other amino acids (except for aspartate), were detected in the leaves of either genotype during water stress. In contrast, levels of 2-oxoglutaric acid, pinitol, and allantoin were affected differentially in the genotypes when drought was imposed, suggesting possible roles as osmoprotectants [87]. In contrast to soybean, levels of amino acids, including proline, trypto‐ phan, leucine, isoleucine, and valine, were increased under drought stress in three different cultivars of wheat (*Triticum aestivum*) analyzed for 103 metabolites in a targeted GC-MS approach [88]. Metabolic adjustments in response to adverse conditions are transient and depend on the severity of the stress. In a 17-day time course experiment in maize (*Zea mays*) subjected to drought stress, GC-MS metabolic analysis revealed changes in concentrations of 28 metabolites. Accumulation of soluble carbohydrates, proline and eight other amino acids, shikimate, serine, glycine, and aconitase, was accompanied by the decrement of leaf starch, malate, fumarate, 2-oxoglutarate, and seven amino acids during the drought treatment course. However, as the water potential became more negative, between the 8th and 10th days, the changes in some metabolites were more dramatic, demonstrating their dependence on stress severity [89].

Accumulation of compatible solutes is an evolutionary conserved trait in bacteria, plants, animal cells, and marine algae. A recent GC-MS metabolomic analysis confirmed that the moss *Physcomitrella patens* also triggers compatible solute accumulation in response to drought stress. After two weeks of physiological drought stress, 26 metabolites were differentially affected in gametophores, including altrose, maltitol, L-proline, maltose, isomaltose, and butyric acid, comparable to metabolic adjustments previously reported in stressed *Arabidop‐* *sis* leaves. More interesting is the recent report of a new compound, annotated as EITTMS\_N12C\_ATHR\_2988.6\_1135EC44, with no previously mass spectra matching record, accumulated specifically in response to drought stress in this moss [90].

## **7. Plant metabolomics and salinity stress**

remain unresolved concerning hydric stress-plant metabolic response: How does drought stress perturb metabolism in crop plants? How does hydric stress affect the metabolism of wild plants? What modern strategies of "omics" could be exploited to support future programs of

As previously described, one of the main mechanisms by which plants cope with water deficits is osmotic adjustment. These adjustments maintain a positive cell turgor via the active accumulation of compatible solutes. Traditionally, the analysis of metabolic responses to drought stress was limited to analysis of one or two classes of compounds considered as "role players" in the development of tolerance. Application of metabolomic approaches is providing a less biased perspective of metabolic profiles of response and also is aiding in the discovery of novel metabolic phenotypes. Unbiased GC-MS metabolomic profiling in *Eucalyptus* showed that drought stress alters a larger number of leaf metabolites than the previously reported in targeted analysis. Accumulation of shikimic acid and two cyclohexanepentol stereoisomers in response to drought stress was described for the first time in *Eucalyptus*. Also, the magnitude of metabolic adjustments in response to water stress correlates with the sensitivity/tolerant phenotype observed; drought affected around 30-40% of measured metabolites in *Eucalyptus dumosa* (a drought-sensitive specie) compared to 10-15% in *Eucalyptus pauciflora* (a droughttolerant specie) [86]. Similarly, critical differences in the metabolic responses were observed when drought-tolerant (NA5009RG) and drought-sensitive (DM50048) soybean cultivars were

H NMR-based metabolomics. Interestingly, no enhanced accumulation of the

traditional osmoprotectants, such as proline, soluble sugars as sucrose or myo-inositol, organic acids or other amino acids (except for aspartate), were detected in the leaves of either genotype during water stress. In contrast, levels of 2-oxoglutaric acid, pinitol, and allantoin were affected differentially in the genotypes when drought was imposed, suggesting possible roles as osmoprotectants [87]. In contrast to soybean, levels of amino acids, including proline, trypto‐ phan, leucine, isoleucine, and valine, were increased under drought stress in three different cultivars of wheat (*Triticum aestivum*) analyzed for 103 metabolites in a targeted GC-MS approach [88]. Metabolic adjustments in response to adverse conditions are transient and depend on the severity of the stress. In a 17-day time course experiment in maize (*Zea mays*) subjected to drought stress, GC-MS metabolic analysis revealed changes in concentrations of 28 metabolites. Accumulation of soluble carbohydrates, proline and eight other amino acids, shikimate, serine, glycine, and aconitase, was accompanied by the decrement of leaf starch, malate, fumarate, 2-oxoglutarate, and seven amino acids during the drought treatment course. However, as the water potential became more negative, between the 8th and 10th days, the changes in some metabolites were more dramatic, demonstrating their dependence on stress

Accumulation of compatible solutes is an evolutionary conserved trait in bacteria, plants, animal cells, and marine algae. A recent GC-MS metabolomic analysis confirmed that the moss *Physcomitrella patens* also triggers compatible solute accumulation in response to drought stress. After two weeks of physiological drought stress, 26 metabolites were differentially affected in gametophores, including altrose, maltitol, L-proline, maltose, isomaltose, and butyric acid, comparable to metabolic adjustments previously reported in stressed *Arabidop‐*

crop breeding to lead to a more sustainable agriculture?

32 Abiotic Stress - Plant Responses and Applications in Agriculture

analyzed by 1

severity [89].

A current problem for crop plants worldwide, which will become more critical in the fu‐ ture, is salt stress imposed by salinity in soils due to poor practices in irrigation and over-fertilization, among other causes. Salt stress induces abscisic acid synthesis; abscisic acid transported to guard cells closes stomata, resulting in decreased photosynthesis, pho‐ to-inhibition, and oxidative stress. This causes an immediate inhibition of cell expansion, visible as general plant growth inhibition, accelerated development, and senescence [91]. To cope with salt stress plants implement strategies that include lowering of rates of pho‐ tosynthesis, stomatal conductance, and transpiration [92]. Sodium ion, by its similar chemical nature to potassium ion, competes with and inhibits the potassium uptake by the root. Potassium deficiency results in growth inhibition because this ion is involved in the capacitance of a plethora of enzyme activities in addition to its participation in main‐ taining membrane potential and cell turgor [91].

The metabolic perturbation in plants exposed to salinity involves a broad spectrum of metabolic pathways and both primary and secondary metabolism. For example, in a proteomic study in foxtail millet (cv. Prasad), 29 proteins were significantly up- or down-regulated due to NaCl stress, with great impact on primary metabolism. These proteins were classified into nine functional categories: cell wall biogenesis (lignin biosynthesis), among these were caffeic acid 3-O-methyltransferase and caffeoyl CoA 3-O-methyltransferase; photosynthesis and energy metabolism, which included proteins like cytochrome P450 71D9, phytochrome 1, photosystem I reaction center subunit IV B, and ATP synthase F1 sector subunit beta, among others; nitrogen metabolism, proteins like glutamine synthetase root isozyme 4, ferredoxindependent glutamate synthase, chloroplast precursor (Fd-GOGAT), and urease; carbohydrate metabolism, proteins such as UDP-glucose 4-epimerase GEPI42 (galactowaldenase) and betaamylase; and lipid metabolism including isovaleryl-CoA dehydrogenase 2 and aldehyde dehydrogenase [93].

Studies using metabolomic tools in plant models and plant crops have shown that the physiology in salt stress courses through a complex metabolic response including different systematic mechanisms, time-course changes, and salt-dose dependence. The biochemical changes involve metabolic pathways that fulfill crucial functions in the plant adaptation to salt stressing conditions. Time-course metabolite profiling in cell cultures of *A. thaliana* exposed to salt stress demonstrates that glycerol and inositol are abundant 24 h after salt stress exposure, whereas lactate and sucrose accumulate 48 h later. The methylation cycle, the phenylpropanoid pathway, and glycine betaine biosynthesis exhibit induction as a short-term response to salinity stress, whereas glycolysis and sucrose metabolism and reduction in methylation are long-term responses. Long-term salt exposure also causes a reduction in the metabolites that were initially responsive [94]. In tobacco plants treated with various doses of salt, 1 day of treatment with 50 mM NaCl induced accumulation of sucrose, and to a lesser extent glucose and fructose, through gluconeogenesis. Further stress (500 mM NaCl for another day) led to elevation of proline and even higher elevation in sucrose levels compared to the lower dose; at the same time, glucose and fructose levels decreased as transamination-related metabolites (asparagine, glutamine, and GABA) did. These data suggest that sugar and proline biosyn‐ thesis pathways are metabolic mechanisms for control of salt stress over one- to two-day periods (short-term). Proline continues to be observed at high levels at later stages (3 to 7 days under highly stressing concentrations of 500 mM NaCl) and sucrose decreases (although it remains at high levels compared to control). There are also significant elevations in levels of asparagine, valine, isoleucine, tryptophan, myo-inositol, uracil, and allantoin, and reductions in glucose, fructose, glutamine, GABA, malate, fumarate, choline, uridine, hypoxantine, nicotine, N-methylnicotinamide, and formate [95]. Similarly, in maize plants stressed with salt solutions ranging in concentration from 50 to 150 mM NaCl, the metabolic profile of the shoot extracts changes most dramatically compared to controls in the plants exposed to the highest salt concentration [96].

Another complexity in the metabolic perturbations in salt-stressed plants consists of tissuespecific response differences. In maize plants exposed to 50-150 mM NaCl saline solution, levels of sucrose and alanine were increased and levels of glucose decreased in roots and shoots. Other osmoprotectants exhibited differentiated behavior: GABA, malic acid, and succinate levels increased in roots, while glutamate, asparagine and glycine betaine were at higher concentrations in shoots. There were decreased levels of acetoacetate in roots and of malic acid and *trans*-aconitic acid in shoots. A progressive metabolic response was more evident in shoots than in roots [96].

In comparative ionomics and metabolite profiling of related *Lotus* species (*Lotus corniculatus*, *L. tenuis*, and *L. creticus*) under salt stress, the extremophile *L*. *creticus* (adapted to highly saline coastal regions) exhibits better survival after long-term exposure to salinity and is more efficient at excluding Cl from shoot tissue than the two cultivated glycophytes *L. cornicula‐ tus* and *L. tenuis* (grassland forage species). Sodium ion levels are higher in the extremophile than the cultivars under both control conditions and salt stress. In *L. creticus*, a differential homeostasis of Cl- , Na+ , and K+ is accompanied by distinct nutritional changes compared to the glycophytes *L. corniculatus* and *L. tenuis*. Magnesium and iron levels increase in *L. creti‐ cus* after salt treatment, but levels of potassium, manganese, zinc, and calcium do not. In nonstressed control plants, 41 metabolites are found at lower levels in *L. creticus* than in the two glycophytes, and 10 metabolites are at higher levels in *L. creticus*. These data demonstrate that each of these species has a distinct basal metabolic profile and that these profiles do not show a concordance with salt stress or salt tolerance. In salt stress conditions, 48 metabolites show similar changes in all species, either increasing or decreasing, with increased levels the amino acids proline, serine, threonine, glycine, and phenylalanine; the sugars sucrose and fructose, myo-inositol and other unidentified metabolites; and with decreased levels of organic acids such as citric, succinic, fumaric, erythronic, glycolic, and aconitic acid, including ethanolamine and putrescine, among others. Of note is that more than half of the metabolites affected by salt treatment are common among the three species, and only one-third of responsive metabolites in *L. creticus* are not shared with the glycophytes. Interestingly, the changes in the pool sizes of these metabolites are only marginal [97]. A few changes in the metabolic profile are extremophile-specific, but most salt-elicited changes in metabolism are similar. Other studies in glycophytes under salt stress indicate that organic acids and intermediates of the citric acid cycle tend to decrease [98]. Also in genus *Lotus*, model species (*L. japonicus*, *L. filicaulis*, and *L. burttii*) and cultivated species (*L. corniculatus*, *L. glaber*, and *L. uliginosus*) exhibit consistent negative correlation in the Cl levels in the shoots and tolerance to salinity, but metabolic profiles diverge amongst genotypes; asparagine levels are higher in the more tolerant geno‐ types. These results support the conclusion that Clexclusion from the shoots represents a key physiological mechanism for salt tolerance in legumes; moreover, an increased level of the osmoprotectant asparagine is typical [99]. In *L. japonicus*, which has a robust metabolic response to salt stress, levels of proline and serine, polyolsononitol and pinitol, and myoinositol increase [75].

All these studies demonstrate that the metabolic plant response to salinity stress is variable depending on the genus and species and even the cultivar under consideration. Differential metabolic rearrangements are in intimate correlation with genetic backgrounds. Furthermore, the plant physiology in salt stress with time proceeds through a complex metabolic response including different systematic mechanisms and changes. Inside a salt-stressed plant as a biological unit, different tissues respond differentially and in some cases the responses are even contrasting. From comparative ionomics studies, it is evident also that under salinity stress, differential homeostasis of ions as Cl- , Na+ , and K+ is correlated with distinct nutritional changes in extremophile and glycophyte species, even inside the same genus. Noticeable differences exist between plant species in the way they react to surpass the osmotic pressure imposed by high soil salt content through mechanisms such as tolerance, efficiency in salt exclusion, changes in nutrient homeostasis, and osmotic adjustment. From the aforementioned studies, metabolic markers in the response to high salinity in plants include glycine betaine, sucrose, asparagine, GABA, malic acid, aspartic acid, and *trans*-aconitic acid. In legumes, increases in levels of the amino acids asparagine, proline, and serine are notable as are increases in polyolsononitol, pinitol, and myo-inositol [75].

### **8. Plant metabolomics and oxidative stress**

were initially responsive [94]. In tobacco plants treated with various doses of salt, 1 day of treatment with 50 mM NaCl induced accumulation of sucrose, and to a lesser extent glucose and fructose, through gluconeogenesis. Further stress (500 mM NaCl for another day) led to elevation of proline and even higher elevation in sucrose levels compared to the lower dose; at the same time, glucose and fructose levels decreased as transamination-related metabolites (asparagine, glutamine, and GABA) did. These data suggest that sugar and proline biosyn‐ thesis pathways are metabolic mechanisms for control of salt stress over one- to two-day periods (short-term). Proline continues to be observed at high levels at later stages (3 to 7 days under highly stressing concentrations of 500 mM NaCl) and sucrose decreases (although it remains at high levels compared to control). There are also significant elevations in levels of asparagine, valine, isoleucine, tryptophan, myo-inositol, uracil, and allantoin, and reductions in glucose, fructose, glutamine, GABA, malate, fumarate, choline, uridine, hypoxantine, nicotine, N-methylnicotinamide, and formate [95]. Similarly, in maize plants stressed with salt solutions ranging in concentration from 50 to 150 mM NaCl, the metabolic profile of the shoot extracts changes most dramatically compared to controls in the plants exposed to the highest

Another complexity in the metabolic perturbations in salt-stressed plants consists of tissuespecific response differences. In maize plants exposed to 50-150 mM NaCl saline solution, levels of sucrose and alanine were increased and levels of glucose decreased in roots and shoots. Other osmoprotectants exhibited differentiated behavior: GABA, malic acid, and succinate levels increased in roots, while glutamate, asparagine and glycine betaine were at higher concentrations in shoots. There were decreased levels of acetoacetate in roots and of malic acid and *trans*-aconitic acid in shoots. A progressive metabolic response was more

In comparative ionomics and metabolite profiling of related *Lotus* species (*Lotus corniculatus*, *L. tenuis*, and *L. creticus*) under salt stress, the extremophile *L*. *creticus* (adapted to highly saline coastal regions) exhibits better survival after long-term exposure to salinity and is more

*tus* and *L. tenuis* (grassland forage species). Sodium ion levels are higher in the extremophile than the cultivars under both control conditions and salt stress. In *L. creticus*, a differential

the glycophytes *L. corniculatus* and *L. tenuis*. Magnesium and iron levels increase in *L. creti‐ cus* after salt treatment, but levels of potassium, manganese, zinc, and calcium do not. In nonstressed control plants, 41 metabolites are found at lower levels in *L. creticus* than in the two glycophytes, and 10 metabolites are at higher levels in *L. creticus*. These data demonstrate that each of these species has a distinct basal metabolic profile and that these profiles do not show a concordance with salt stress or salt tolerance. In salt stress conditions, 48 metabolites show similar changes in all species, either increasing or decreasing, with increased levels the amino acids proline, serine, threonine, glycine, and phenylalanine; the sugars sucrose and fructose, myo-inositol and other unidentified metabolites; and with decreased levels of organic acids such as citric, succinic, fumaric, erythronic, glycolic, and aconitic acid, including ethanolamine and putrescine, among others. Of note is that more than half of the metabolites affected by salt

from shoot tissue than the two cultivated glycophytes *L. cornicula‐*

is accompanied by distinct nutritional changes compared to

salt concentration [96].

evident in shoots than in roots [96].

34 Abiotic Stress - Plant Responses and Applications in Agriculture

, Na+

, and K+

efficient at excluding Cl-

homeostasis of Cl-

An increase in intracellular levels of ROS is a common consequence of adverse growth conditions. An imbalance between ROS synthesis and scavenging is caused in a manner independent of the nature of the stress; it is induced by both biotic and abiotic types of stress. Toxic concentrations of ROS cause severe damage to protein structures, inhibit the activity of multiple enzymes of important metabolic pathways, and result in oxidation of macromolecules including lipids and DNA. All these adverse events compromise cellular integrity and may lead to cell death [100, 101]. Normal cellular metabolic activity also results in ROS generation under regular growth conditions. Thus, cells sense uncontrolled elevation of ROS and use them as a signaling mechanism to activate protective responses [102]. In this context plants have developed efficient mechanisms for removal of toxic concentrations of ROS. The antioxidant system is composed of protective enzymes (*e.g*., superoxide dismutase, catalase, peroxidase, reductase, and redoxin) and radical scavenger metabolites (mainly GSH and ascorbate). GSH is an essential component of the antioxidant system that donates an electron to unstable molecules such as ROS to make them less reactive and also can acts as a redox buffer in the recycling of ascorbic acid from its oxidized form to its reduced form by the enzyme dehy‐ droascorbate reductase [103]. Organized remodeling of metabolic networks is a crucial response that gives the cells the best chance of surviving the oxidative challenge.

In *A. thaliana*, oxidative treatment with methyl viologen causes the down-regulation of photosynthesis-related genes and concomitant cessation of starch and sucrose synthesis pathways, meanwhile catabolic pathways are activated. These metabolic adjustments avoid the waste of energy used in non-defensive processes and mobilize carbon reserves towards actions of emergency relief such as the accumulation of maltose, a protein structure-stabilizer molecule [104]. A GC-MS metabolomic study, together with an analysis of key metabolic fluxes of cell cultures and roots of *A. thaliana* treated with the oxidative stressor menadione, revealed the similarities and divergences in the metabolic adjustments triggered in both culture systems. Inhibition of the tricarboxylic acid cycle (TCA) by accumulation of pyruvate and citrate is accompanied by a decrement of malate, succinate, and fumarate pools. This early (0.5 h) response was observed in both systems. Inhibition of TCA cycle concomitantly causes a decrement in the pools of glutamate and aspartate due to the inhibition of the synthesis of TCA-linked precursors 2-oxoglutarate and oxaloacetate, respectively. Another mutual early metabolic redistribution is the redirection of the carbon flux from glycolysis to the oxidative pentose phosphate (OPP) pathway. This is also reflected by the decrement in the glycolytic pools of glucose-6 phosphate and fructose 6-P, and the increment in the OPP pathway intermediates ribulose 5-phosphate and ribose 5-phosphate. Increased carbon flux through the OPP pathway might supply reducing power (via nicotinamide adenine dinucleotide phos‐ phate, NADPH) for antioxidant activity, since oxidative stress decreases the levels of the reductants GSH, ascorbate, and NADPH. After 2 and 6 h of stress progression, metabolic adjustments in response to oxidative stress are different in roots than in cell suspension cultures. In roots, pools of TCA cycle intermediates and amino acids are recovered. In contrast, in cell cultures, the concentrations of these metabolites remains depressed throughout the time course, indicating higher basal levels of oxidative stress in cell cultures. At the end of the treatment time (6 h), 39 metabolites, including GABA, aromatic amino acids (tryptophan, phenylalanine, and tyrosine), proline, and other amino acids, were significantly altered in roots. These results showed the broad spectrum of metabolic modifications elicited in response to oxidative stress and the influence of the biological system analyzed [105].

Redirection of carbon flux from glycolysis through the OPP pathway and subsequent increase in the levels of NADPH was also reported in rice cell cultures treated with menadione. CE-MS analysis of these rice cultures showed the depletion of most sugar phosphates resulting from glycolysis (pyruvate, 3-phosphoglyceric acid, dihydroxyacetone phosphate, fructose-6 phosphate, glucose-1-phosphate (G1P), G6P, G3P, phosphoenolpyruvate) and TCA-organic acids (2-oxoglutarate, aconitate, citrate, fumarate, isocitrate, malate, succinate) and increases in the levels of OPP pathway intermediates (6-phosphogluconate, ribose 5-phosphate, ribulose 5-phosphate). Incremental increases in the biosynthesis of GSH and intermediates (*O*-acetyl-L-serine, cysteine, and γ-glutamyl-L-cysteine) are also observed in the menadione-treated rice cell cultures [106].

## **9. Perspectives**

developed efficient mechanisms for removal of toxic concentrations of ROS. The antioxidant system is composed of protective enzymes (*e.g*., superoxide dismutase, catalase, peroxidase, reductase, and redoxin) and radical scavenger metabolites (mainly GSH and ascorbate). GSH is an essential component of the antioxidant system that donates an electron to unstable molecules such as ROS to make them less reactive and also can acts as a redox buffer in the recycling of ascorbic acid from its oxidized form to its reduced form by the enzyme dehy‐ droascorbate reductase [103]. Organized remodeling of metabolic networks is a crucial

In *A. thaliana*, oxidative treatment with methyl viologen causes the down-regulation of photosynthesis-related genes and concomitant cessation of starch and sucrose synthesis pathways, meanwhile catabolic pathways are activated. These metabolic adjustments avoid the waste of energy used in non-defensive processes and mobilize carbon reserves towards actions of emergency relief such as the accumulation of maltose, a protein structure-stabilizer molecule [104]. A GC-MS metabolomic study, together with an analysis of key metabolic fluxes of cell cultures and roots of *A. thaliana* treated with the oxidative stressor menadione, revealed the similarities and divergences in the metabolic adjustments triggered in both culture systems. Inhibition of the tricarboxylic acid cycle (TCA) by accumulation of pyruvate and citrate is accompanied by a decrement of malate, succinate, and fumarate pools. This early (0.5 h) response was observed in both systems. Inhibition of TCA cycle concomitantly causes a decrement in the pools of glutamate and aspartate due to the inhibition of the synthesis of TCA-linked precursors 2-oxoglutarate and oxaloacetate, respectively. Another mutual early metabolic redistribution is the redirection of the carbon flux from glycolysis to the oxidative pentose phosphate (OPP) pathway. This is also reflected by the decrement in the glycolytic pools of glucose-6 phosphate and fructose 6-P, and the increment in the OPP pathway intermediates ribulose 5-phosphate and ribose 5-phosphate. Increased carbon flux through the OPP pathway might supply reducing power (via nicotinamide adenine dinucleotide phos‐ phate, NADPH) for antioxidant activity, since oxidative stress decreases the levels of the reductants GSH, ascorbate, and NADPH. After 2 and 6 h of stress progression, metabolic adjustments in response to oxidative stress are different in roots than in cell suspension cultures. In roots, pools of TCA cycle intermediates and amino acids are recovered. In contrast, in cell cultures, the concentrations of these metabolites remains depressed throughout the time course, indicating higher basal levels of oxidative stress in cell cultures. At the end of the treatment time (6 h), 39 metabolites, including GABA, aromatic amino acids (tryptophan, phenylalanine, and tyrosine), proline, and other amino acids, were significantly altered in roots. These results showed the broad spectrum of metabolic modifications elicited in response

response that gives the cells the best chance of surviving the oxidative challenge.

36 Abiotic Stress - Plant Responses and Applications in Agriculture

to oxidative stress and the influence of the biological system analyzed [105].

Redirection of carbon flux from glycolysis through the OPP pathway and subsequent increase in the levels of NADPH was also reported in rice cell cultures treated with menadione. CE-MS analysis of these rice cultures showed the depletion of most sugar phosphates resulting from glycolysis (pyruvate, 3-phosphoglyceric acid, dihydroxyacetone phosphate, fructose-6 phosphate, glucose-1-phosphate (G1P), G6P, G3P, phosphoenolpyruvate) and TCA-organic acids (2-oxoglutarate, aconitate, citrate, fumarate, isocitrate, malate, succinate) and increases

Metabolome analysis has become an invaluable tool in the study of plant metabolic changes that occur in response to abiotic stresses. Despite progress achieved, metabolomics is a developing methodology with room for improvement. From a technical perspective, further developments are required to improve sensitivity for identification of previously uncharac‐ terized molecules and for quantification of cellular metabolites and their fluxes at much higher resolution. This will allow the identification of novel metabolites and pathways and will allow linkage to responses to specific stresses, and, therefore, increase our level of knowledge of the elegant regulation and precise adjustments of plant metabolic networks in response to stress.

Another challenging task is the integration of metabolic data with data from experiments profiling the transcriptome, proteome, and genetic variations obtained from the same tissue, cell type, or plant species in response to a determined environmental condition. Integrated information can be used to map the loci underlying various metabolites and to link these loci to crop phenotypes, to understand the mechanisms underlying the inheritance of important traits, and to understand biochemical pathways and global relationships among metabolic systems. Elucidation of the regulatory networks involved in the activation/repression of key genes related to metabolic phenotypes in response to determined abiotic stress is becoming possible. Transcription factors (TFs) are central player in the signal transduction network, connecting the processes of stress signal sensing and expression of stress-responsive genes. Thus engineered TFs have emerged as powerful tools to manipulate complex metabolic pathways in plants and generate more robust metabolic phenotypes [107, 108].

Metabolic networks are highly dynamic, and changes with time are influenced by stress severity, plant developmental stage, and cellular compartmentalization. Since metabolic profiling only reveals the steady-state level of metabolites, detailed kinetics and flux analyses will support a better understanding of metabolic fluctuations in response to stress [109]. Genome-scale models (GSM) are *in silico* metabolic flux models derived from genome anno‐ tation that contain stoichiometry of all known metabolic reactions of an organism of interest. Construction of detailed GSMs applied to plant metabolism will provide information about distribution of metabolic fluxes at a specific genotype, a determined developmental stage, or a particular environmental condition. This detailed knowledge of the metabolic and physio‐ logical status of the cell can be used to design rational metabolic engineering strategies and to predict required genetic modifications to obtain a desired metabolic phenotype such as optimized biomass production, increased accumulation of a valuable metabolite, accumula‐ tion of a metabolite of response towards abiotic stress, or modification of metabolic flux through a specific pathway of significance [110]. Recently advances have been made in this field. For example, in rice, by using four complementary analytical platforms based on highcoverage metabolomics, molecular backgrounds of quality traits and metabolite profiles were correlated with overall population structure and genetic diversity, demonstrating that quality traits could be predicted from the metabolome composition, and that traits can be linked with metabolomics data. Results like these are opening the doors to modern plant breeding programs [111].

Once a metabotype (metabolic phenotype) is confirmed to strengthen the tolerance to a particular abiotic stressor, the next challenge will be the transfer of this metabolic trait to a nonadapted plant species of interest. Engineering of more tolerant plants will then require the efficient integration and expression of one to several transgenes in order to modify an existent metabolic pathway or reconstruct a new complete one. Development and optimization of protocols for robust transformation of nucleus, mitochondria, and chloroplasts must be made available for higher plants including economically important crops; this will open new opportunities for plant metabolic engineering [112]. Future research progress on these topics will lead to novel strategies for plant breeding and elevating the health and performance of crops under adverse growth conditions to keep up with the ever-increasing needs for food and feed worldwide.

## **10. Conclusions**

Metabolomics is the comprehensive and quantitative analysis of the entirety of small molecules present in an organism that can be regarded as the ultimate expression of its genotype in response to environmental changes, often characterized by several simultaneous abiotic and biotic stresses. Results obtained from a number of metabolomic studies in plants in response to different abiotic stresses have shown detailed relevant information about chemical compo‐ sition, including specific osmoprotectants, directly related to physiological and biochemical changes, and have shed light on how these changes reflect the plant phenotype. Metabolomic studies are impacting both basic and applied research. Metabolomic studies will generate knowledge regarding how plant metabolism is differentially adjusted in relation to a specific stress and whether metabolic adjustments are stress specific or common to different types of stress. These studies will also reveal how metabolic pathways coordinate their fluxes and enzymes activities in order to strength their cellular energy requirements under stressing conditions. In an applied context, metabolomic approaches are providing a broader, deeper, and an integral perspective of metabolic profiles in the acclimation plant response to stressing environments. This information will reveal metabotypes with potential to be transferred to sensitive, economically important crops and will allow design of strategies to improve the adaptation of plants towards adverse conditions. Ultimately, design strategies will consider plant metabolism as a whole set of interconnected biochemical networks and not as sections of reactions that lead to the accumulation of a final metabolite. The task is challenging as it must take into account that reactions to stress course through a complex metabolic response, including different systematic mechanisms, time-course changes, and stress-dose dependen‐ ces. Moreover, there are differences among plant tissues, and, as expected, marked differences between plants at the genus and species levels, exposing intimate correlation with genetic backgrounds. Nevertheless, the application of more advanced metabolomics tools will lead to new knowledge that will accelerate the design and the improvement of plant breeding projects, that surely will lead to the next generation of crops for specific applications in particular circumstances to cope with abiotic and biotic stress on agricultural crops worldwide.

## **Author details**

field. For example, in rice, by using four complementary analytical platforms based on highcoverage metabolomics, molecular backgrounds of quality traits and metabolite profiles were correlated with overall population structure and genetic diversity, demonstrating that quality traits could be predicted from the metabolome composition, and that traits can be linked with metabolomics data. Results like these are opening the doors to modern plant breeding

Once a metabotype (metabolic phenotype) is confirmed to strengthen the tolerance to a particular abiotic stressor, the next challenge will be the transfer of this metabolic trait to a nonadapted plant species of interest. Engineering of more tolerant plants will then require the efficient integration and expression of one to several transgenes in order to modify an existent metabolic pathway or reconstruct a new complete one. Development and optimization of protocols for robust transformation of nucleus, mitochondria, and chloroplasts must be made available for higher plants including economically important crops; this will open new opportunities for plant metabolic engineering [112]. Future research progress on these topics will lead to novel strategies for plant breeding and elevating the health and performance of crops under adverse growth conditions to keep up with the ever-increasing needs for food and

Metabolomics is the comprehensive and quantitative analysis of the entirety of small molecules present in an organism that can be regarded as the ultimate expression of its genotype in response to environmental changes, often characterized by several simultaneous abiotic and biotic stresses. Results obtained from a number of metabolomic studies in plants in response to different abiotic stresses have shown detailed relevant information about chemical compo‐ sition, including specific osmoprotectants, directly related to physiological and biochemical changes, and have shed light on how these changes reflect the plant phenotype. Metabolomic studies are impacting both basic and applied research. Metabolomic studies will generate knowledge regarding how plant metabolism is differentially adjusted in relation to a specific stress and whether metabolic adjustments are stress specific or common to different types of stress. These studies will also reveal how metabolic pathways coordinate their fluxes and enzymes activities in order to strength their cellular energy requirements under stressing conditions. In an applied context, metabolomic approaches are providing a broader, deeper, and an integral perspective of metabolic profiles in the acclimation plant response to stressing environments. This information will reveal metabotypes with potential to be transferred to sensitive, economically important crops and will allow design of strategies to improve the adaptation of plants towards adverse conditions. Ultimately, design strategies will consider plant metabolism as a whole set of interconnected biochemical networks and not as sections of reactions that lead to the accumulation of a final metabolite. The task is challenging as it must take into account that reactions to stress course through a complex metabolic response, including different systematic mechanisms, time-course changes, and stress-dose dependen‐ ces. Moreover, there are differences among plant tissues, and, as expected, marked differences

programs [111].

38 Abiotic Stress - Plant Responses and Applications in Agriculture

feed worldwide.

**10. Conclusions**

Saúl Fraire-Velázquez and Victor Emmanuel Balderas-Hernández

\*Address all correspondence to: sfraire@prodigy.net.mx

\*Address all correspondence to: balderas.victor@gmail.com

Laboratorio de Biología Integral de Plantas y Microorganismos, Unidad Académica de Cien‐ cias Biológicas, Universidad Autónoma de Zacatecas, Campus II UAZ, Colonia Agronómica, Zacatecas, Zacatecas, México

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