**3. Plant metabolite responses to individual abiotic stresses**

Metabolite responses to individual abiotic stresses such as drought, salinity or heat have been widely studied, and comprehensive reviews on this topic can be found in the literature [24, 31]. In this section, we describe recent applications of MS-based metabolomics approaches to study plant responses to individual abiotic stresses, namely drought, salt and heat stress, highlighting the identification of stress-responsive metabolites that ultimately contribute for the development of plants with enhanced abiotic stress-tolerance.

#### **3.1. Metabolite responses to drought stress**

Drought is a well-studied abiotic stress, and one major limiting factor in agriculture worldwide [35–37]. This stress condition leads to huge reductions in crop yields mainly derived from a series of morpho-physiological changes such as reduction in shoot growth [38], decreases in photosynthesis and transpiration rates as a direct consequence of abscisic acid (ABA)-mediated leaf stomata closure [36, 37] as well as changes in signalling pathways [36] and transcriptional and posttranscriptional regulation of several stress-related genes [39, 40]. In addition, plant metabolism is also readjusted under drought stress conditions through the accumulation of osmolytes or compatible solutes [41, 42]. These small molecules can accumulate at high concentrations in the cell without inhibiting cellular metabolism, and comprise, for example, soluble sugars and sugar alcohols such as glucose, sucrose and mannitol; the raffinose family oligosaccharides (RFOs) such as raffinose, stachyose and verbascose, amino acids and polyamines. Because of this osmolyte accumulation, a decrease in the osmotic potential of the cell is observed and the turgor pressure is maintained as the cell uptakes water, thereby help in stabilising membranes, enzymes and proteins, or maintaining cell turgor by osmotic adjustment. In addition, osmolyte accumulation also confers protection against oxidative damage by decreasing the levels of reactive oxygen species (ROS), which in turn, helps re-establish cellular redox balance. Consequently, osmotic adjustment is commonly recognised as an effective factor of drought tolerance in several plants to enable water uptake and the maintenance of plant metabolic activity, hence, growth and productivity as the water potential decreases [36, 37]. Drought stress has been widely reported to increase the production of ROS in different cellular compartments (i.e., oxidative stress) [43]. However, this oxidative stress has shown to lead to the formation of specific peptides that might counterbalance the accumulation of ROS upon abiotic stress conditions [44]. Nevertheless, ROS species are known to interact with proteins, lipids and DNA during abiotic stress episodes, and thus impair the normal function of cells [45–47].

to its poor sensitivity and poor dynamic range relative to mass spectrometry (MS) [29, 30], MS-based analytical tools are the most widely used in plant metabolomics. Among them, powerful chromatographic techniques such as liquid chromatography-mass spectrometry (LC-MS) and gas chromatography-mass spectrometry (GC-MS) have been extensively used to obtain comprehensive information of the plant metabolome in a wide range of plant species [24, 31]. Regardless the analytical platform of choice, great attention must be paid to the experimental design. In plant metabolomics, an adequate and well-studied experimental design should address different environmental and experimental variables such as (i) plant tissue harvest, (ii) metabolic quenching and (iii) metabolite extraction methods. In addition, randomisation procedures throughout all the experimental workflow should be taken into account to minimise potential sources of experimental errors [32, 33]. A detailed discussion of sample preparation workflows and MS-based analytical platforms typically used in plant metabolomics experi-

Metabolite responses to individual abiotic stresses such as drought, salinity or heat have been widely studied, and comprehensive reviews on this topic can be found in the literature [24, 31]. In this section, we describe recent applications of MS-based metabolomics approaches to study plant responses to individual abiotic stresses, namely drought, salt and heat stress, highlighting the identification of stress-responsive metabolites that ultimately contribute for

Drought is a well-studied abiotic stress, and one major limiting factor in agriculture worldwide [35–37]. This stress condition leads to huge reductions in crop yields mainly derived from a series of morpho-physiological changes such as reduction in shoot growth [38], decreases in photosynthesis and transpiration rates as a direct consequence of abscisic acid (ABA)-mediated leaf stomata closure [36, 37] as well as changes in signalling pathways [36] and transcriptional and posttranscriptional regulation of several stress-related genes [39, 40]. In addition, plant metabolism is also readjusted under drought stress conditions through the accumulation of osmolytes or compatible solutes [41, 42]. These small molecules can accumulate at high concentrations in the cell without inhibiting cellular metabolism, and comprise, for example, soluble sugars and sugar alcohols such as glucose, sucrose and mannitol; the raffinose family oligosaccharides (RFOs) such as raffinose, stachyose and verbascose, amino acids and polyamines. Because of this osmolyte accumulation, a decrease in the osmotic potential of the cell is observed and the turgor pressure is maintained as the cell uptakes water, thereby help in stabilising membranes, enzymes and proteins, or maintaining cell turgor by osmotic adjustment. In addition, osmolyte accumulation also confers protection against oxidative damage by decreasing the levels of reactive oxygen species (ROS), which in turn, helps re-establish cellular redox balance. Consequently, osmotic adjustment is commonly recognised as an effective factor of drought tolerance in several plants to enable water uptake and the maintenance of plant metabolic activity, hence, growth and productivity

**3. Plant metabolite responses to individual abiotic stresses**

the development of plants with enhanced abiotic stress-tolerance.

ments can be found elsewhere [24, 34].

114 Plant, Abiotic Stress and Responses to Climate Change

**3.1. Metabolite responses to drought stress**

Comprehensive omics studies have been reported to investigate plant responses to drought stress [42, 48–50]. An interesting study developed by Gechev and collaborators [51] addressed the molecular mechanisms of desiccation in *Haberlea rhodopensis* through transcriptomics and metabolomics approaches. The complementary use of GC-TOF-MS and LC-MS metabolite analyses revealed significant accumulation in the levels of the soluble sugars sucrose and maltose as wells as of the RFOs stachyose and verbascose in *H. rhodopensis* plants upon dehydration. Furthermore, and together with transcriptomics, these results were associated to *H. rhodopensis* ability to survive under dehydration conditions [51].

A similar comprehensive metabolomics approach was applied to study the resurrection plant *Selaginella lepidophylla* [52]. Metabolite profiles from ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) and GC-MS analysis revealed an accumulation of metabolites involved in the glycolytic pathway (glucose-6-phosphate, fructose-6-phosphate and pyruvate) as well as in the TCA cycle (2-oxoglutarate, succinate, fumarate and oxaloacetate) in hydrated *S. lepidophylla* plants. In parallel, the accumulation of the sugar alcohols sorbitol, myo-inositol and mannitol was related to the desiccation mechanisms developed by resurrection plants, which involve water uptake or loss during the rehydration/ dehydration cycle [52].The moderate long-term drought stress effects was investigated in 21 rice cultivars (*Oryza sativa* L. ssp. indica and japonica) through physiological, gene expression and GC-TOF-MS metabolite profiling analysis [53]. Overall, this comprehensive study revealed that in rice, drought conditions induce an accumulation of spermine, thereby leading to a coordinated adjustment of polyamine metabolism which is in agreement with an osmoprotectant role of this metabolite under drought stress [53].

Meyer and co-workers [54] analysed at transcriptional, physiological and metabolite levels the responses to soil drying of the perennial C4 grass and biofuel crop, *Panicum virgatum* L. (switchgrass). In this study, genes associated with C4 photosynthesis were down-regulated during drought, while C4 metabolic intermediates have shown to accumulate. GC-TOF-MS data revealed that the abundance of 13 primary metabolites was significantly affected by the drought treatment and that most of these compounds also accumulated amino acids (>32 fold), monosaccharides (>14-fold) and organic acids (>four-fold) [54]).

GC-TOF-MS metabolite profiling in the leaves and roots of two barley (*Hordeum vulgare* L.) genotypes, with contrasting drought tolerance, revealed approximately 100 drought stressresponsive metabolites with amino acids being the most affected metabolite class. Together with proteomics data, this study indicated that the proteins and metabolites that have shown to accumulate in the susceptible variety also revealed elevated constitutive accumulation levels in the drought-resistant line. Moreover, the accumulation of several carbohydrates was affected in tissues of both genotypes subjected to drought [55].

In sunflower (*Helianthus annuus* L.), molecular mechanisms to drought tolerance were recently addressed through the characterisation and integration of transcriptional and metabolic data. GC-TOF-MS analysis allowed detecting 54 primary metabolites, including different amino acids, organic acids, sugars and sugar alcohols. This analysis revealed that most of the amino acids showed lower levels under drought with exception to proline, tyramine, glycine, malonate and γ-aminobutyrate (GABA), which accumulated upon drought conditions. On the other hand, glycolysis and tricarboxylic acid cycle (TCA) metabolites as well as all the detected carbohydrates showed higher levels under drought conditions. Overall, these results indicated the putative role of these metabolites during stress response in sunflower [56].

have a comparable response to salinity [64]. A similar scenario was observed for *Arabidopsis thaliana* (salt-sensitive) and its distant relative *Thellungiella halophila* (salt-tolerant), both accu-

Plant Metabolomics in a Changing World: Metabolite Responses to Abiotic Stress Combinations

http://dx.doi.org/10.5772/intechopen.71769

117

Among crops, an interesting study on barley (*Hordeum vulgare* L.) cultivars that differed in salt-stress tolerance were analysed for their metabolite response to long-term salt stress [66]. While the most tolerant cultivar Sahara showed elevated levels of hexose phosphates and TCA cycle intermediates, the levels of these metabolites remained unaffected during salinity stress in the less-tolerant cultivar Clipper [66]. In another study, wild barley showed to be more salttolerant than cultivated barley by accumulating more carbohydrates (sucrose, trehalose and raffinose) and proline in its roots than its cultivated counterpart, therefore demonstrating an improved ability to regulate osmotic stress [67]. Rice represents one of the most-sensitive cereal crops; however, a GC-TOF-MS analysis revealed lower levels of TCA cycle intermediates and other organic acids in the roots of more-tolerant rice cultivars than in those more sensitive. On the other hand, accumulation of amino acids was detected in the salt-tolerant rice cultivars [68].

A modern metabolomics approach based on two complementary highly sensitive approaches, namely GC- and LC-coupled to a triple quadrupole mass spectrometer (GC-QqQ-MS and LC-QqQ-MS), was applied for the quantitative profiling of a wide range of metabolites from two chickpea (*Cicer arietinum* L.) cultivars with contrasting responses to salt stress. While the GC-QqQ-MS metabolite profiling approach allowed to quantitatively analyse 48 primary metabolites, ranging from sugars and sugar phosphates to organic acids, the LC-QqQ-MS approach allowed to quantitatively measure 28 biogenic amines and amino acids. Furthermore, this complementary approach indicated that the metabolic differences between the two contrasting cultivars relied on metabolites involved in carbon metabolism, TCA cycle as well as amino acid metabolism [69]. A better elucidation of the physiological and biochemical processes of a salt-resistant maize (*Zea mays* L.) hybrid was achieved with GC-TOF-MS metabolite profiling analysis. By comparing a salt-sensitive and a salt-resistant maize hybrid, Richter and co-workers [70] could observe the accumulation of neutral sugars (glucose, fructose and sucrose) in the leaves of the salt-sensitive hybrid and regard these metabolites accumulation as a salt-resistance adaptation. In addition, both hybrids showed a strong decrease in the lev-

Actinorhizal plants are a group of perennial dicotyledonous angiosperms. These plants are not only of economic importance (production of wood and derivatives), but are also highly resilient to extreme environments. *Casuarina glauca*, the model actinorhizal plant, is characterised by its ability to establish symbiosis with nitrogen-fixing *Frankia* bacteria and can thrive under extreme salinity conditions [71, 72]. However, until now, only few reports investigating the mechanisms underlying salt stress tolerance in actinorhizal plants are available, and most of these studies are not broad enough to grasp the complexity of the response. To better understand *C. glauca* ability to tolerate high levels of salinity, Jorge and collaborators [74] have pioneered a metabolomics study to investigate the impact of salt stress in *C. glauca* nodulated

200, 400 and 600 mM [NaCl]) [73]. GC-TOF-MS metabolite profiling data revealed major metabolite divergences in amino acid metabolism in both plant groups (NOD+ and KNO<sup>3</sup>

+) plants subjected to different salinity levels (0 control,

+).

els of TCA cycle intermediates [70].

(NOD+) and non-nodulated (KNO<sup>3</sup>

mulating proline and soluble sugars (fructose, glucose, sucrose and raffinose) [65].

Another interesting study investigated osmoadaptation to drought stress in leaves and roots of cowpea (*Vigna unguiculata* L. Walp.) through analysis of photosynthetic traits, water homoeostasis, inorganic ions and primary and secondary metabolites. In this study, physiological and metabolite changes were shown to develop in parallel while drought/recovery responses revealed a progressive acclimation of the cowpea plant to stress. GC-TOF-MS analysis and subsequent multifactorial analyses indicated allocation of high quantities of amino acids, sugars and proanthocyanidins into roots, which were linked to their role in growth and initial stress perception. From the 88 metabolites detected, proline, galactinol and a quercetin derivative, were those that most responded to drought. In addition, these metabolites accumulated differently in roots, but similarly in leaves, suggesting a more conservative strategy to cope with drought in the aerial parts of cowpea plants [57].

#### **3.2. Metabolite responses to salt stress**

Soil salinity significantly reduces crop yields, being considered a global problem that affects approximately 20% of irrigated land [58]. The effects of salt stress in plants occur in two different sequential stages. In a first stage, the plant perceives osmotic stress, which reduces the plant's ability to uptake water, decreases cell turgor and leads to the accumulation of ROS in the cells. Subsequently, a second stage is initiated by an over accumulation of Na<sup>+</sup> and Cl<sup>−</sup> ions that severely affect key plant physiological processes including photosynthesis, plasma membrane stability and cellular metabolism [59]. Consequently, plant growth and fertility are reduced, and premature senescence occurs [59, 60]. Plant susceptibility or tolerance to salt stress strongly depends on the mechanisms used by the plant to detoxify ROS species within the cells and exclude Na<sup>+</sup> ions from the roots or to compartmentalise these ions in the vacuoles [59, 61]. To cope with salt stress, plants adjust their metabolic status, and although this metabolic adjustment widely differs among salt-tolerant species, several common salt-stress metabolite responses are found within the plant kingdom [62, 63].

According to their salt tolerance, plants are usually divided in glycophytes (salt-sensitive) and halophytes (salt-tolerant). For glycophytic plants, there is an increasing evidence that amino acids, sugars, sugar alcohols and tricarboxylic acid (TCA)-cycle intermediates, form the core of metabolite adjustments to salinity stress [24, 31, 62]. On the other hand, for halophytic or extremophile plants, the pre-accumulation and differential response of osmoprotectant metabolites varies among plant species. Interestingly, a comparative study using both saltsensitive and salt-tolerant Lotus species has demonstrated that around 50% of all metabolites have a comparable response to salinity [64]. A similar scenario was observed for *Arabidopsis thaliana* (salt-sensitive) and its distant relative *Thellungiella halophila* (salt-tolerant), both accumulating proline and soluble sugars (fructose, glucose, sucrose and raffinose) [65].

In sunflower (*Helianthus annuus* L.), molecular mechanisms to drought tolerance were recently addressed through the characterisation and integration of transcriptional and metabolic data. GC-TOF-MS analysis allowed detecting 54 primary metabolites, including different amino acids, organic acids, sugars and sugar alcohols. This analysis revealed that most of the amino acids showed lower levels under drought with exception to proline, tyramine, glycine, malonate and γ-aminobutyrate (GABA), which accumulated upon drought conditions. On the other hand, glycolysis and tricarboxylic acid cycle (TCA) metabolites as well as all the detected carbohydrates showed higher levels under drought conditions. Overall, these results indi-

cated the putative role of these metabolites during stress response in sunflower [56].

with drought in the aerial parts of cowpea plants [57].

**3.2. Metabolite responses to salt stress**

116 Plant, Abiotic Stress and Responses to Climate Change

the cells and exclude Na<sup>+</sup>

Another interesting study investigated osmoadaptation to drought stress in leaves and roots of cowpea (*Vigna unguiculata* L. Walp.) through analysis of photosynthetic traits, water homoeostasis, inorganic ions and primary and secondary metabolites. In this study, physiological and metabolite changes were shown to develop in parallel while drought/recovery responses revealed a progressive acclimation of the cowpea plant to stress. GC-TOF-MS analysis and subsequent multifactorial analyses indicated allocation of high quantities of amino acids, sugars and proanthocyanidins into roots, which were linked to their role in growth and initial stress perception. From the 88 metabolites detected, proline, galactinol and a quercetin derivative, were those that most responded to drought. In addition, these metabolites accumulated differently in roots, but similarly in leaves, suggesting a more conservative strategy to cope

Soil salinity significantly reduces crop yields, being considered a global problem that affects approximately 20% of irrigated land [58]. The effects of salt stress in plants occur in two different sequential stages. In a first stage, the plant perceives osmotic stress, which reduces the plant's ability to uptake water, decreases cell turgor and leads to the accumulation of ROS

ions that severely affect key plant physiological processes including photosynthesis, plasma membrane stability and cellular metabolism [59]. Consequently, plant growth and fertility are reduced, and premature senescence occurs [59, 60]. Plant susceptibility or tolerance to salt stress strongly depends on the mechanisms used by the plant to detoxify ROS species within

oles [59, 61]. To cope with salt stress, plants adjust their metabolic status, and although this metabolic adjustment widely differs among salt-tolerant species, several common salt-stress

According to their salt tolerance, plants are usually divided in glycophytes (salt-sensitive) and halophytes (salt-tolerant). For glycophytic plants, there is an increasing evidence that amino acids, sugars, sugar alcohols and tricarboxylic acid (TCA)-cycle intermediates, form the core of metabolite adjustments to salinity stress [24, 31, 62]. On the other hand, for halophytic or extremophile plants, the pre-accumulation and differential response of osmoprotectant metabolites varies among plant species. Interestingly, a comparative study using both saltsensitive and salt-tolerant Lotus species has demonstrated that around 50% of all metabolites

ions from the roots or to compartmentalise these ions in the vacu-

and Cl<sup>−</sup>

in the cells. Subsequently, a second stage is initiated by an over accumulation of Na<sup>+</sup>

metabolite responses are found within the plant kingdom [62, 63].

Among crops, an interesting study on barley (*Hordeum vulgare* L.) cultivars that differed in salt-stress tolerance were analysed for their metabolite response to long-term salt stress [66]. While the most tolerant cultivar Sahara showed elevated levels of hexose phosphates and TCA cycle intermediates, the levels of these metabolites remained unaffected during salinity stress in the less-tolerant cultivar Clipper [66]. In another study, wild barley showed to be more salttolerant than cultivated barley by accumulating more carbohydrates (sucrose, trehalose and raffinose) and proline in its roots than its cultivated counterpart, therefore demonstrating an improved ability to regulate osmotic stress [67]. Rice represents one of the most-sensitive cereal crops; however, a GC-TOF-MS analysis revealed lower levels of TCA cycle intermediates and other organic acids in the roots of more-tolerant rice cultivars than in those more sensitive. On the other hand, accumulation of amino acids was detected in the salt-tolerant rice cultivars [68].

A modern metabolomics approach based on two complementary highly sensitive approaches, namely GC- and LC-coupled to a triple quadrupole mass spectrometer (GC-QqQ-MS and LC-QqQ-MS), was applied for the quantitative profiling of a wide range of metabolites from two chickpea (*Cicer arietinum* L.) cultivars with contrasting responses to salt stress. While the GC-QqQ-MS metabolite profiling approach allowed to quantitatively analyse 48 primary metabolites, ranging from sugars and sugar phosphates to organic acids, the LC-QqQ-MS approach allowed to quantitatively measure 28 biogenic amines and amino acids. Furthermore, this complementary approach indicated that the metabolic differences between the two contrasting cultivars relied on metabolites involved in carbon metabolism, TCA cycle as well as amino acid metabolism [69]. A better elucidation of the physiological and biochemical processes of a salt-resistant maize (*Zea mays* L.) hybrid was achieved with GC-TOF-MS metabolite profiling analysis. By comparing a salt-sensitive and a salt-resistant maize hybrid, Richter and co-workers [70] could observe the accumulation of neutral sugars (glucose, fructose and sucrose) in the leaves of the salt-sensitive hybrid and regard these metabolites accumulation as a salt-resistance adaptation. In addition, both hybrids showed a strong decrease in the levels of TCA cycle intermediates [70].

Actinorhizal plants are a group of perennial dicotyledonous angiosperms. These plants are not only of economic importance (production of wood and derivatives), but are also highly resilient to extreme environments. *Casuarina glauca*, the model actinorhizal plant, is characterised by its ability to establish symbiosis with nitrogen-fixing *Frankia* bacteria and can thrive under extreme salinity conditions [71, 72]. However, until now, only few reports investigating the mechanisms underlying salt stress tolerance in actinorhizal plants are available, and most of these studies are not broad enough to grasp the complexity of the response. To better understand *C. glauca* ability to tolerate high levels of salinity, Jorge and collaborators [74] have pioneered a metabolomics study to investigate the impact of salt stress in *C. glauca* nodulated (NOD+) and non-nodulated (KNO<sup>3</sup> +) plants subjected to different salinity levels (0 control, 200, 400 and 600 mM [NaCl]) [73]. GC-TOF-MS metabolite profiling data revealed major metabolite divergences in amino acid metabolism in both plant groups (NOD+ and KNO<sup>3</sup> +). Subsequent multivariate statistical analysis allowed concluding that modifications in the metabolite levels of neutral sugars, proline and ornithine revealed to be central in conferring tolerance to high levels of salinity in *C. glauca.* Furthermore, the same study also concluded that the main differences observed in the metabolite pool between NOD+ and KNO<sup>3</sup> + plants not only rely on the impact of the salt stress itself [73], but also on the disruption of the symbiotic activity of *C. glauca* NOD+ plants at early salt stress exposure (i.e., 200 mM [NaCl]) [74].

(*Agrostis Stolonifera* L.) [89]. Upon exogenous application of GABA, metabolite profiling data revealed an accumulation in the levels of six amino acids (glutamic acid, aspartic acid, alanine, threonine, serine and valine), five organic acids (aconitic acid, malic acid, succinic acid, oxalic acid and threonic acid), five sugars (sucrose, fructose, glucose, galactose and maltose) and two sugar alcohols (mannitol and myo-inositol). Together with physiological measurements, this study suggested that the GABA-induced heat tolerance in creeping bentgrass might result from three main factors (i) balance of photosynthesis and transpiration, (ii) improvement of the ascorbate-glutathione cycle and (ii) maintenance of osmotic adjustment. Furthermore, an increase in the levels of metabolites involved in the GABA shunt (glutamic acid, GABA and alanine) was suggested to act as an intermediate supplier to feed the TCA cycle during a long-term heat stress,

Plant Metabolomics in a Changing World: Metabolite Responses to Abiotic Stress Combinations

http://dx.doi.org/10.5772/intechopen.71769

119

Plant abiotic stress studies typically deal with the comparison of a few genotypes (tolerant versus sensitive species) grown under controlled conditions, followed by the analysis and identification of differential responses to the imposed stress. Yet, these conditions are unlikely to reproduce field conditions in which a range of abiotic stresses is likely to occur simultaneously. Abiotic stress combinations, such as those involving drought and salinity, salinity and heat as well as drought and extreme temperature or high light intensity are the most commonly reported stress combinations in field conditions [17, 90]. Pioneering abiotic stress combination studies, that involved drought and heat stress, were performed in tobacco (*Nicotiana tabacum* L.) and in the model plant *A. thaliana*. These studies revealed that the molecular responses to this stress combination are unique and should not be regarded as the sum of the responses from each individually applied stress [17, 91, 92]. Afterwards, significant studies have been performed to elucidate the plant molecular responses to several abiotic stress combinations that include

thereby maintaining metabolic homeostasis [89].

**4. Plant metabolite responses to abiotic stress combinations**

drought, salt, extreme temperatures, heavy metals, UV-B, high light, ozone, CO<sup>2</sup>

individually. On the other hand, combinations of drought and ozone, high CO<sup>2</sup>

dant mechanisms or the synthesis of osmolytes [90, 92, 94–98].

tion and biotic stresses (e.g., pathogen attack) [17, 93]. Likewise, these studies also reported that each stress combination requires specific plant molecular responses. Among them, specific physiological responses as well as specific regulatory transcripts, proteins and metabolites were found for each stress combination under study. Having said this, plant responses to combined stresses require an orchestration of specific metabolic and signalling responses such as antioxi-

In 2006, Mittler [16] developed an intuitive diagram denominated "*Stress Matrix*" in which the result of a positive and/or negative interaction between two different stress combinations on plant growth, yield and physiological traits can be easily described [16]. Since then, this matrix has been updated several times [17, 93, 99] (**Figure 2**). According to **Figure 2**, most abiotic stress combination studies include drought or salinity as one of the main stress conditions. Stress combinations between drought and heat, salinity and heat, ozone and salinity, ozone and heat, nutrient stress and drought, nutrient stress and salinity (to name a few) were reported to have a higher negative impact on plant development than when each different stress component is applied

, soil compac-

with ozone, salt

#### **3.3. Metabolite responses to heat stress**

Heat stress is often defined as the rise in temperature beyond a threshold level (usually 10–15°C) above ambient temperature, for an enough period of time, to cause irreversible damage to plant growth and development. The impact of heat stress depends not only on the temperature intensity but also on its duration and rate of increase [75, 76].

When a plant perceives exposure to heat stress, a series of cellular and molecular responses are known to be initiated, such as increased fluidity of lipid membranes, inactivation of key enzymes in some organelles (chloroplasts and mitochondria) and protein denaturation and aggregation. The ability of some plants to grow, develop and give profit under these circumstances is defined as heat tolerance. In plants, the heat stress response (HSR) pathway has been extensively studied [77–79]; however, a more comprehensive understanding of this pathway remains unclear [76].

Heat tolerance has been widely reported in the literature as being mediated by the synthesis of stress-related proteins, also known as heat shock proteins (HSPs) [77, 80]. This class of proteins has shown to confer heat tolerance by reducing the impact of high temperatures in photosynthesis, in carbon assimilate partitioning, in water and nutrient use efficiency as well as in keeping membrane stability [81–83]. General plant cellular and molecular responses to heat stress have been thoroughly reviewed elsewhere [75, 76, 79, 84, 85].

Metabolomics studies on plants subjected to heat stress have reported the accumulation of osmolytes, namely soluble sugars, glycine-betaine and proline [86]. In addition, high temperatures have been reported to disrupt sugar metabolism and proline transport during male reproductive development in tomato (*Solanum lycopersicum* L.) [87].

Du and co-workers [88] applied a GC-MS metabolite profiling approach to identify metabolites associated with differential heat tolerance between two grass species, namely C4 bermudagrass and C3 Kentucky bluegrass [88]. In both grass species, 36 heat stress-responsive metabolites were identified, ranging from organic and amino acids to sugars and sugar alcohols. However, most of these metabolites showed higher accumulation in bermudagrass when compared with Kentucky bluegrass. Among the differentially accumulated metabolites, this study reported seven sugars (sucrose, fructose, galactose, floridoside, melibiose, maltose and xylose), a sugar alcohol (inositol), six organic acids (malic acid, citric acid, threonic acid, galacturonic acid, isocitric acid and methyl malonic acid) and nine amino acids (asparagine, alanine, valine, threonine, GABA, isoleucine, glycine, lysine and methionine) [88].

Using a similar GC-MS metabolic profiling approach, Li and co-workers [89] investigated whether increased GABA levels could improve heat tolerance in cool-season creeping bentgrass (*Agrostis Stolonifera* L.) [89]. Upon exogenous application of GABA, metabolite profiling data revealed an accumulation in the levels of six amino acids (glutamic acid, aspartic acid, alanine, threonine, serine and valine), five organic acids (aconitic acid, malic acid, succinic acid, oxalic acid and threonic acid), five sugars (sucrose, fructose, glucose, galactose and maltose) and two sugar alcohols (mannitol and myo-inositol). Together with physiological measurements, this study suggested that the GABA-induced heat tolerance in creeping bentgrass might result from three main factors (i) balance of photosynthesis and transpiration, (ii) improvement of the ascorbate-glutathione cycle and (ii) maintenance of osmotic adjustment. Furthermore, an increase in the levels of metabolites involved in the GABA shunt (glutamic acid, GABA and alanine) was suggested to act as an intermediate supplier to feed the TCA cycle during a long-term heat stress, thereby maintaining metabolic homeostasis [89].
