Climate change constitutes a real threat to the global landscape. Current climate models predict an increased occurrence of coastal floods associated to sea level rise and long-term droughts associated to changes in the intra- and inter-year rainfall variability. Under natural environmental conditions, plants are routinely exposed to abiotic stresses, and must develop different strategies to cope with this multitude of climate change factors. Mass spectrometry (MS)-based plant metabolomics approaches are finding an increasing number of applications to investigate the molecular and biochemical mechanisms that underlie plant responses to changing environments. These studies provide a promising basis for facilitating our understanding of the plant’s flexibility to reconfigure central metabolic pathways (i.e., carbon, nitrogen and energy metabolism) as well as the degree by which plants tolerate and/or are susceptible to a climate change scenario. In this chapter, we will provide an update on the recent MS-based metabolomics strategies to study plant responses to drought, salt and heat stress as well as combinations thereof. We will describe how these stresses activate and coordinate several different signalling pathways, for example, through the synthesis of osmolytes.
- plant metabolomics
- drought stress
- salinity stress
- heat stress
- stress combination
- climate change
- mass spectrometry
Climate change can be defined as a statistically significant variation in the weather pattern or in its variability during a long-term period . The causes of climate change have been mainly associated to (i) internal environmental processes and (ii) anthropogenic activities that lead to changes in the chemical composition of the atmosphere . Natural climate variability itself is not enough to explain the unforeseen weather changes in the last decades. In fact, 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 CO2, CH4, N2O as well as fluorinated gases to the atmosphere . Indeed, climate change assessments have reported that the global atmospheric CO2 concentration has increased from 270 to 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 . By the end of the twenty-first century, unmatched climate changes are envisaged with CO2 concentrations of 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 high risk of severe and irreversible impacts globally .
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 . 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, 5, 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, 5, 6, 7, 8, 9, 10, 11, 12, 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 applied stress alone. Additionally, when different stresses are combined, they might require synergistic or antagonistic responses that are largely controlled by, sometimes, opposing signalling pathways [16, 17]. In this chapter, we will provide an update on recent studies of plant responses to drought, salt and heat stress as well as combinations thereof. We will describe how these abiotic stress combinations activate and coordinate several different signalling pathways, for example, through the synthesis of osmolytes, in order to ensure plant survival.
2. Metabolomics—a key
omicstool to study plant responses to abiotic stress
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
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, 36, 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 , 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  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) . 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 . 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, 46, 47].
Comprehensive omics studies have been reported to investigate plant responses to drought stress [42, 48, 49, 50]. An interesting study developed by Gechev and collaborators  addressed the molecular mechanisms of desiccation in
A similar comprehensive metabolomics approach was applied to study the resurrection plant
Meyer and co-workers  analysed at transcriptional, physiological and metabolite levels the responses to soil drying of the perennial C4 grass and biofuel crop,
GC-TOF-MS metabolite profiling in the leaves and roots of two barley (
In sunflower (
Another interesting study investigated osmoadaptation to drought stress in leaves and roots of cowpea (
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 . 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+ and Cl− ions that severely affect key plant physiological processes including photosynthesis, plasma membrane stability and cellular metabolism . 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+ 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 salt-sensitive and salt-tolerant Lotus species has demonstrated that around 50% of all metabolites have a comparable response to salinity . A similar scenario was observed for
Among crops, an interesting study on barley (
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 (
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.
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, 78, 79]; however, a more comprehensive understanding of this pathway remains unclear .
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, 82, 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 . In addition, high temperatures have been reported to disrupt sugar metabolism and proline transport during male reproductive development in tomato (
Du and co-workers  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 . 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) .
Using a similar GC-MS metabolic profiling approach, Li and co-workers  investigated whether increased GABA levels could improve heat tolerance in cool-season creeping bentgrass (
4. Plant metabolite responses to abiotic stress combinations
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 (
In 2006, Mittler  developed an intuitive diagram denominated “
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 well-studied 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
Metabolite changes under this stress combination were also assessed in the fleshy herbaceous plant Purslane (
The impact of combined drought and heat stress has also been evaluated in the crop plant soybean (
4.2. Metabolite responses to combined drought and salt stress
With increasing earth surface temperatures, it is very likely that regions of high surface salinity, where evaporation dominates, will become more saline . 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, 106, 107, 108]. Among them, only one study addressed maize metabolite responses induced by a combination of drought and salt stress . 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 . 1H 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) .
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+ ions by the plant [109, 110].
The effects of the combination of salt and heat stress were evaluated in tomato plants (
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.
5. Concluding remarks
Climate change disturbs a number of variables that determine how much plants can grow and develop. Extreme temperatures, elevated CO2 together with a decrease in water availability and changes to soil conditions will essentially make it more challenging for plants to thrive. Overall, climate change is expected to decline the growth and development of plants, particularly with reference to agricultural systems. Declining plant growth also dramatically changes the habitats that are necessary for many species to survive. Undoubtedly, under the current threat of climate change, it is urgent to address the molecular and biochemical mechanisms that underlie plant responses to several abiotic stresses and combinations thereof. However, a complete understanding of plant responses to climate change is best obtained if data is integrated at several levels, including morpho-physiological and developmental studies as well as molecular studies that comprise the so-called
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 International PhD program “Plants for Life” (PD/00035/2013).