**2. Metabolomics—a key** *omics* **tool to study plant responses to abiotic stress**

since the industrial revolution that human-kind activities (e.g., fossil fuel burning) have also contributed to the release of significant amounts of greenhouse gases (GHGs) namely CO<sup>2</sup>

401 μL L−1 since the industrial revolution, and consequently, the average global temperatures to rise by 0.85°C. Moreover, global warming has been reported to be highly correlated with ocean thermal expansion and loss of glacier mass, which ultimately reflected the observed global mean sea level rise of 0.19 ± 0.02 m over the period 1901–2010 [2]. By the end of the

at least 700 μL L−1 and global temperatures are expected to rise at least 4°C. Consequently, higher surface temperatures, longer and frequent heat waves and intense extreme precipitation events are very likely to occur in many regions around the globe. The consequences from climate change cannot be totally avoided, but without additional mitigation efforts beyond those already in place today, warming by the end of the twenty-first century will lead to very

Extreme climate change events expose plants to stressful environmental conditions that are outside of their physiological limits, and beyond the range by which they are already adapted [3]. Studies aiming at assessing the impact of climate change in plant ecosystems revealed that plant community responses occur at three sequential levels in which (i) climate change immediately impacts plant individuals at the morpho-physiological level, (ii) the community response is affected because of demographic changes in species abundances and (iii) the mortality or loss of species leads to their replacement by novel species within the community [4–6]. Although some studies have contributed to a better understanding of plant ecosystem responses to climate change, this research field is still emerging. A comprehensive discussion on this topic falls outside the scope of this chapter, and detailed information can be found elsewhere [4–13]. Responses by individual plant species to climate change have been indirectly studied through the assessment of the strategies and mechanisms by which they cope with adverse environmental conditions, that is, abiotic stresses. Abiotic stresses in plants comprise a multitude of environmental factors such as water (drought, flooding and submergence), temperature (high and low), light (high and low), radiation (UV-B and UV-A), salinity and nutrients, heavy metals, among others. These environmental (stress)factors negatively affect plant growth and development, and trigger a series of high-complex adaptive responses initiated by stress perception, signal transduction and the activation of many stress-related genes and metabolites [14, 15]. However, under natural environmental conditions, plants are routinely exposed to a combination of different abiotic stresses, and therefore, must develop different strategies to cope with a multitude of environmental factors. The latter gains more relevance under climate change scenarios, and therefore, there has been an increasing interest in understanding the molecular and biochemical mechanisms that underlie plant responses to abiotic stress combinations [16, 17]. Many studies, at both physiological and biochemical levels, have been performed to study plant responses to different stress combinations namely drought, salt, extreme temperatures and biotic stresses. Interestingly, these studies demonstrated that a plant response to a combined stress is unique, and should not be regarded as the sum of the responses from each

twenty-first century, unmatched climate changes are envisaged with CO<sup>2</sup>

ments have reported that the global atmospheric CO<sup>2</sup>

112 Plant, Abiotic Stress and Responses to Climate Change

high risk of severe and irreversible impacts globally [2].

O as well as fluorinated gases to the atmosphere [1]. Indeed, climate change assess-

CH<sup>4</sup> , N<sup>2</sup> ,

concentrations of

concentration has increased from 270 to

Over the past decade, plant metabolomics has undoubtedly become a powerful research tool to study the biochemical mechanisms underlying plant growth and development in the context of plant metabolite responses to abiotic stress, particularly drought, flooding, salinity and extreme temperatures (heat and cold). In fact, metabolomics itself, together with the other *omics* technologies (genomics, transcriptomics and proteomics) has accelerated our understanding of the complex molecular interactions in biological systems [18–24] (**Figure 1**). Moreover, unlike other *omics* tools, metabolomics has the advantage of not being dependent on the availability of organism-specific genome information for data analysis [25–27]. The main goal of plant metabolomics is to provide a non-biased characterisation of the total metabolite pool of a plant tissue in response to its environment. This metabolite pool includes a wide range of metabolites with diverse physical properties, from ionic inorganic compounds to biochemically derived hydrophilic carbohydrates, organic and amino acids, and a range of hydrophobic lipid-related compounds. Indeed, it is estimated that more than 200,000 different primary and secondary metabolites exist in the plant kingdom over a large dynamic range in concentrations that can vary from femtomolar to millimolar [28]. While, primary metabolites are fundamental for plant growth and development, being highly conserved in their molecular structures and abundances throughout the plant kingdom, secondary metabolites help plants communicating with the environment and widely differ across species. Due to such metabolite diversity, current plant metabolomics studies often combine multiple analytical tools in an effort to acquire more comprehensive metabolite coverage from a complex biological plant sample. One powerful analytical tool is nuclear magnetic resonance (NMR); however, due

**Figure 1.** *Omics* technologies have accelerated our understanding of the complex molecular interactions in biological systems.

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 experiments can be found elsewhere [24, 34].

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

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

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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

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

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-

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

to *H. rhodopensis* ability to survive under dehydration conditions [51].

osmoprotectant role of this metabolite under drought stress [53].

fold), monosaccharides (>14-fold) and organic acids (>four-fold) [54]).

affected in tissues of both genotypes subjected to drought [55].

the normal function of cells [45–47].
