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

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

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].

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

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

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

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

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

+ plants

that the main differences observed in the metabolite pool between NOD+ and KNO<sup>3</sup>

temperature intensity but also on its duration and rate of increase [75, 76].

heat stress have been thoroughly reviewed elsewhere [75, 76, 79, 84, 85].

reproductive development in tomato (*Solanum lycopersicum* L.) [87].

**3.3. Metabolite responses to heat stress**

118 Plant, Abiotic Stress and Responses to Climate Change

pathway remains unclear [76].

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 drought, salt, extreme temperatures, heavy metals, UV-B, high light, ozone, CO<sup>2</sup> , soil compaction 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 antioxidant mechanisms or the synthesis of osmolytes [90, 92, 94–98].

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

factors are the most representative in the field. In addition, they are the primary environmental stresses that determine the distribution and productivity of plants [91, 100]. Following the pioneering studies of the effects of combined drought and heat stress in tobacco and *A. thaliana* [91], many similar studies have been carried out in several other plant species and crops [16, 17, 90, 93, 101]. One interesting study is that of Obata and collaborators [102] who aimed at dissecting the metabolite responses induced by drought, heat and the combination of both stresses in 10 tropical maize hybrids. Through the integration of physiological and metabolomics data, this study identified promising metabolite marker candidates [102]. Under drought stress, GC-TOF-MS analysis of maize leaves revealed the accumulation of several amino acids (isoleucine, valine, threonine, 4-aminobutanoate, glycine and serine) as well as the accumulation of the sugar alcohol *myo*-inositol. On the other hand, when both drought and heat stress were combined, metabolite responses could be predicted from the sum of individual stresses

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Metabolite changes under this stress combination were also assessed in the fleshy herbaceous plant Purslane (*Portulaca oleracea* L.) [103]. In total, GC-TOF-MS analysis allowed detecting 37 primary metabolites. Of these, fructose, galactose and xylitol were only detected in control plants; alanine, sorbose, glucose and heptulose were only detected in drought stress-treated plants; glycine, threonine and asparagine were only detected in heat stress-treated plants, while isoleucine and phenylalanine were only found in combined stress-treated plants. On the other hand, propionic acid, gluconic acid, mannose and urea were detected in both individual and combined stress-treated plants. Overall, this study allowed to conclude that the main strategies adopted by purslane to survive drought, heat, and combined drought and heat stress, involves the accumu-

lation of osmoprotectant metabolites and an increase in the antioxidative system [103].

metabolism are essential in soybean to cope with drought and heat stress conditions [104].

With increasing earth surface temperatures, it is very likely that regions of high surface salinity, where evaporation dominates, will become more saline [2]. Therefore, it is of great interest to study plant's physiological and metabolite responses to harsh environments where drought and salt stress are occurring simultaneously. However, only a few studies under this context have been performed [105–108]. Among them, only one study addressed maize metabolite responses induced by a combination of drought and salt stress [107]. Indeed, under its natural habitat of irrigated and dry land agricultural lands, maize is exposed to the combined

maize leaves revealed that metabolite responses of drought and salt stress differed from those

H NMR-based metabolomics analysis of

**4.2. Metabolite responses to combined drought and salt stress**

stresses of water deficiency and soil salinity [107]. 1

The impact of combined drought and heat stress has also been evaluated in the crop plant soybean (*Glycine max* L.) through a comprehensive MS-based metabolomics approach comprising LC- and GC⁻MS analysis [104]. This approach allowed identifying 266 putative metabolites, including primary and secondary metabolites. Subsequent statistical analysis revealed that combined drought and heat stress induced a differentially accumulation of several metabolites in soybean leaves, such as sugars, amino acids and lipids. Moreover, individual stresses (i.e., drought or heat) affected key metabolites involved in different pathways such as glycolysis, TCA cycle, the pentose phosphate pathway and starch biosynthesis. That said, this study demonstrated that sugar and nitrogen

as only a few specific responses could be observed [102].

**Figure 2.** Intuitive "*Stress Matrix*" showing the result of a positive (light grey) and/or negative (dark grey) interaction between two different stress combinations on plant growth, yield and physiological traits. Striped-pattern square indicates a not well-studied species specific-interaction (might be positive and/or negative) (adapted from [16, 17, 97, 103]).

or high light were shown to have a favourable effect on plants as compared to when each different stress component is applied individually [17, 99]. Interestingly, the combination of salinity and heat stress has shown to provide both positive and negative interactions. These conflicting results suggest that the positive or negative effects of a stress combination could be dependent on the plant genotype, species and/or timing and intensity of the different stresses involved. Considering the increased number of heat waves and rising seawater levels expected for the next decades [2], the study of plant metabolite responses to salt and heat stress in a wide range of species is therefore predicted to become increasingly relevant in the current climate change context.

#### **4.1. Metabolite responses to combined drought and heat stress**

The effect of drought and heat stress on plant growth and development is currently the most wellstudied abiotic stress combination [16, 17, 90], mainly because these two environmental-stress factors are the most representative in the field. In addition, they are the primary environmental stresses that determine the distribution and productivity of plants [91, 100]. Following the pioneering studies of the effects of combined drought and heat stress in tobacco and *A. thaliana* [91], many similar studies have been carried out in several other plant species and crops [16, 17, 90, 93, 101]. One interesting study is that of Obata and collaborators [102] who aimed at dissecting the metabolite responses induced by drought, heat and the combination of both stresses in 10 tropical maize hybrids. Through the integration of physiological and metabolomics data, this study identified promising metabolite marker candidates [102]. Under drought stress, GC-TOF-MS analysis of maize leaves revealed the accumulation of several amino acids (isoleucine, valine, threonine, 4-aminobutanoate, glycine and serine) as well as the accumulation of the sugar alcohol *myo*-inositol. On the other hand, when both drought and heat stress were combined, metabolite responses could be predicted from the sum of individual stresses as only a few specific responses could be observed [102].

Metabolite changes under this stress combination were also assessed in the fleshy herbaceous plant Purslane (*Portulaca oleracea* L.) [103]. In total, GC-TOF-MS analysis allowed detecting 37 primary metabolites. Of these, fructose, galactose and xylitol were only detected in control plants; alanine, sorbose, glucose and heptulose were only detected in drought stress-treated plants; glycine, threonine and asparagine were only detected in heat stress-treated plants, while isoleucine and phenylalanine were only found in combined stress-treated plants. On the other hand, propionic acid, gluconic acid, mannose and urea were detected in both individual and combined stress-treated plants. Overall, this study allowed to conclude that the main strategies adopted by purslane to survive drought, heat, and combined drought and heat stress, involves the accumulation of osmoprotectant metabolites and an increase in the antioxidative system [103].

The impact of combined drought and heat stress has also been evaluated in the crop plant soybean (*Glycine max* L.) through a comprehensive MS-based metabolomics approach comprising LC- and GC⁻MS analysis [104]. This approach allowed identifying 266 putative metabolites, including primary and secondary metabolites. Subsequent statistical analysis revealed that combined drought and heat stress induced a differentially accumulation of several metabolites in soybean leaves, such as sugars, amino acids and lipids. Moreover, individual stresses (i.e., drought or heat) affected key metabolites involved in different pathways such as glycolysis, TCA cycle, the pentose phosphate pathway and starch biosynthesis. That said, this study demonstrated that sugar and nitrogen metabolism are essential in soybean to cope with drought and heat stress conditions [104].

#### **4.2. Metabolite responses to combined drought and salt stress**

or high light were shown to have a favourable effect on plants as compared to when each different stress component is applied individually [17, 99]. Interestingly, the combination of salinity and heat stress has shown to provide both positive and negative interactions. These conflicting results suggest that the positive or negative effects of a stress combination could be dependent on the plant genotype, species and/or timing and intensity of the different stresses involved. Considering the increased number of heat waves and rising seawater levels expected for the next decades [2], the study of plant metabolite responses to salt and heat stress in a wide range of species is therefore predicted to become increasingly relevant in the current climate change context.

**Figure 2.** Intuitive "*Stress Matrix*" showing the result of a positive (light grey) and/or negative (dark grey) interaction between two different stress combinations on plant growth, yield and physiological traits. Striped-pattern square indicates a not well-studied species specific-interaction (might be positive and/or negative) (adapted from [16, 17, 97, 103]).

The effect of drought and heat stress on plant growth and development is currently the most wellstudied abiotic stress combination [16, 17, 90], mainly because these two environmental-stress

**4.1. Metabolite responses to combined drought and heat stress**

120 Plant, Abiotic Stress and Responses to Climate Change

With increasing earth surface temperatures, it is very likely that regions of high surface salinity, where evaporation dominates, will become more saline [2]. Therefore, it is of great interest to study plant's physiological and metabolite responses to harsh environments where drought and salt stress are occurring simultaneously. However, only a few studies under this context have been performed [105–108]. Among them, only one study addressed maize metabolite responses induced by a combination of drought and salt stress [107]. Indeed, under its natural habitat of irrigated and dry land agricultural lands, maize is exposed to the combined stresses of water deficiency and soil salinity [107]. 1 H NMR-based metabolomics analysis of maize leaves revealed that metabolite responses of drought and salt stress differed from those caused by drought and salt stress applied individually. Additionally, subsequent multivariate statistical analysis allowed identifying those metabolites that specifically responded to the combined stress, namely two TCA cycle intermediates (citrate and fumarate) and four amino acids (the branched chain amino acids—valine, leucine and isoleucine, and the aromatic amino acid—phenylalanine) [107].

analysis of the impact of combined stresses in plants. Researchers must regard simultaneous multiple climate change factors, which sum will play a key negative influence on global agriculture, as a new state of stress in which the exposed plant might require differential responses

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C. António gratefully acknowledges support from Fundação para a Ciência e a Tecnologia (FCT) through the FCT Investigator Program (IF/00376/2012/CP0165/CT0003) and from the ITQB NOVA research unit Green-IT "Bioresources for sustainability" (UID/Multi/04551/2013). T.F. Jorge acknowledges FCT for the PhD grant (PD/BD/113475/2015) from the ITQB NOVA

Plant Metabolomics Laboratory, Instituto de Tecnologia Química e Biológica António Xavier,

[1] VijayaVenkataRamana S, Iniyanb S, Goic R. A review of climate change, mitigation and adaptation. Renewable & Sustainable Energy Reviews. 2012;**16**:878-897. DOI: 10.1016/j.

[2] Core Writing Team IPCC. Climate Change 2014: Synthesis Report. Contribution of Working Groups I, II and III to the Fifth Assessment Report of the Intergovernmental

[3] Gray SB, Brady SM. Plant developmental responses to climate change. Developmental

[4] Smith M. An ecological perspective on extreme climatic events: A synthetic definition and framework to guide future research. Journal of Ecology. 2011;**99**:656-663. DOI:

[5] Becklin KM, Anderson JT, Gerhart LM, Wadgymar SM, Wessinger CA, Ward JK. Examining plant physiological responses to climate change through an evolutionary

from those induced by a stress alone. Further research in this area is therefore critical.

International PhD program "Plants for Life" (PD/00035/2013).

Universidade Nova de Lisboa (ITQB NOVA), Oeiras, Portugal

Panel on Climate Change. Geneva: IPCC; 2014. 151 p

10.1111/j.1365-2745.2011.01798.x

Biology. 2016;**419**:64-77. DOI: 10.1016/j.ydbio.2016.07.023

lens. Plant Physiology. 2016;**172**:635-649. DOI: 10.1104/pp.16.00793

\*Address all correspondence to: antonio@itqb.unl.pt

**Acknowledgements**

**Author details**

**References**

rser.2011.09.009

Tiago F. Jorge and Carla António\*

#### **4.3. Metabolite responses to combined salt and heat stress**

Up to date, studies on the combined effects of salt and heat stress in plants have revealed both positive and negative interactions on plant growth, yield and physiological traits (**Figure 2**). In wheat, the combination of salt and heat stress enhanced the transpiration rate, which in turn, was already induced by heat stress itself. On the other hand, this stress combination also promoted a higher uptake of Na<sup>+</sup> ions by the plant [109, 110].

The effects of the combination of salt and heat stress were evaluated in tomato plants (*Solanum lycopersicum* cv. Optima) [111]. This stress combination was observed to induce a specific response by the plants through the accumulation in the levels of glycine betaine and trehalose, both well-known for their osmoprotectant roles. The accumulation of glycine betaine and trehalose was associated to the maintenance of a lower Na<sup>+</sup> :K<sup>+</sup> ratio, thereby leading to a better performance of the cell water status and photosynthesis when compared to the salt stress alone [111].

To the best of our knowledge, metabolomics studies aiming at dissecting metabolite responses induced by salt and heat stress are scarce, highlighting the need for further research in this area.
