Keywords

6.1 Legume Family: Agronomic Relevance

A major source of protein in the human diet is of animal origin. The production of beef and mutton is based on natural pastures or supplementation based on grains (feedlot). Sown pastures can be monospecific or may be ultrasimple, simple or complex of different species of the same botanical family or a family of different botanical blends. Within the latter group, are mixtures of grasses and legumes.

From the point of view of human and animal consumption, legumes belonging to the subfamily Papilonideae are relevant. This includes seeds and forage legumes such as peanut, beans, chickpea, broad beans, lentils, soybean, among others. Some species of the genus Medicago, Lotus and Adesmia can be used as forage or green manure, thus enhancing the contents of nitrogen in the soils.

Forage legumes have been widely spread in the world due to the great agronomic importance that they possess. The species of this plant family are an invaluable component of pastures, mainly due to their ability to fix atmospheric nitrogen through symbiotic association with several bacteria collectively called rhizobia. Second, legumes have a high nutritional value, especially proteins and minerals (Ca+2 and Mg+2), which makes them essential for the production of forage. Legume crops also play a critical role as main protein sources in vegetarian diets. Tolerance to environmental abiotic stress is one of the ways to improve the productivity of legumes and aid in harnessing their potential nutritional value. Identification of biochemical and physiological characters which contribute to improve the yield in legumes under limiting conditions is a main objective of plant breeders for agricultural and cattle-rearing regions. Thus, this chapter intends to provide an understanding of the mechanisms involved in the combined stress-tolerance responses in legumes.

6.2 Environmental Stresses Induce Varied Plant Responses

Plants are frequently subjected to stress—environmental condition that adversely affects the growth, development and productivity thereof. Biotic stress can be imposed by organisms such as viruses, bacteria and fungi, while abiotic stress can be due to an excess or deficit in some environmental factor. Among the environmental conditions that cause damage are excess water, water deficit, soil salinity, extreme temperatures, insufficient mineral nutrients in the soil and high- or low-light radiation (Bohnert and Sheveleva 1998; Bray et al. 2000).

Resistance or susceptibility to stress depends on the species, genotype and stage of development of the plant. Resistance mechanisms can be grouped into two categories—those that prevent exposure to stress and the other that results in tolerance. Certain morphological features such as sunken stomata and deep roots are examples of resistance mechanism that can prevent stress. However, other mechanisms of resistance are achieved by acclimation, i.e. the maintenance of internal homeostasis of the various organelles in response to changing environmental factors (Bray et al. 2000).

Plants acclimate to manage the different types of stress triggering a wide range of responses from the perception of stress at the cellular level, leading to the activation of a very large number of genes. Key components of the stress response are the stimulus itself, transducers, signal molecules, transcription regulators, responsive genes that trigger morphological, biochemical and physiological adaptation involved in this situation. In turn, the duration and severity with which stress is imposed determine how the plant will respond (Pastori and Foyer 2002; Bray et al. 2000).

Unlike resistance to biotic factors, resistance to water stress and other abiotic factors, despite being clearly genetic, is not a result of the action of a specific gene (Zhu et al. 1997). The ability of plants to withstand water stress is a multigenic trait and biochemical pathways responsible for products or processes that improve the overall strength can act additively, and also synergistically (Bohnert et al. 1995).

It is reported that several genes responsive to water stress not only perform their functions protecting cells by producing metabolically important proteins under water deficit but also in the regulation of genes involved in signal transduction in response to stress. Thus, these gene products are classified into two groups: The first group includes proteins that are involved in stress tolerance such as channel proteins involved in the movement of water across membranes, enzymes necessary for the biosynthesis of osmolytes, proteases and macromolecules that can protect membranes, among others. The second group includes factors involved in the regulation of signal transduction and gene expression, such as protein kinases, transcription factors and 14-3-3 proteins, among others (Bray 1997; Shinozaki and Yamaguchi-Shinozaki 1997).

Higher temperatures primarily affect photosynthesis, in particular CO2 assimilation because Rubisco activation is inhibited. Plants exposed to excessive temperatures have specific metabolic cellular response characterized by low protein synthesis, and induction of the synthesis of heat shock proteins (HSPs). In addition to altering the pattern of gene expression, the high temperature can damage cellular structures such as organelles and cytoskeleton (Bray et al. 2000; Tang et al. 2007).

Water stress and high temperatures interact strongly with each other and have opposite effects on photosynthesis. For example, in response to high temperature, plants open their stomata to cool their leaves by transpiration, but if there is also water deficit condition, plants would not be able to open the stomata and hence leaf temperature will increase (Rizhsky et al. 2002). While both types of stress have been extensively studied individually, few studies (Lu and Zhang 1999; Rizhsky et al. 2002; Rizhsky et al. 2004) focused on impacts of combined heat and water stress—a common situation prevailing under field conditions. It is possible that combination of these stress factors can alter the metabolism of the plant differently, compared to when a single stress is imposed (Xu and Zhou 2006).

6.2.1 Plants Response to Water Stress

Water deficit is one of the most widespread environmental factor stresses that occurs when the transpiration rate exceeds the absorption of water from the root system. Water deficit at the cellular level may result in an increase of solute concentration, changes in cell volume, disruption of water potential gradient, turgor loss, loss of membrane integrity and protein denaturation. The ability of the plant to respond to water deficit and survive depends on mechanisms that involve the integration of cellular responses throughout the plant (Bray et al. 2000).

Water deficit is a common plant environmental stress that dramatically limits growth and development. Water stress can trigger a significant decrease in crop productivity and quality, especially evident in grain and forage legumes. Lotus japonicus is a well-established model legume closely related to forage legumes such as Lotus corniculatus, Lotus tenuis and Lotus uliginosus (Choi et al. 2004; Díaz et al. 2005a). Alfalfa is a legume species with great plasticity that can succeed in semiarid, subhumid and humid regions and for that reason is called the “queen of forage legumes”. However, it requires well-aerated and deep soils and is morphologically and physiologically adapted to withstand prolonged water deficiencies. In marked contrast to their drought-tolerant nature, these plants are very sensitive to a lack of oxygen that is common in flooding soils.

Legumes are typically subjected to a variety of different environmental stresses such as water stress. At the cellular level, this stress induces overproduction of reactive oxygen species (ROS; Fig. 6.1), such as hydrogen peroxide (H2O2), superoxide radical (O2 ●−) and hydroxyl radical (●OH), which are responsible for oxidative damage associated with stress (Dat et al. 2000). Plants respond to stress using different enzymatic and non-enzymatic antioxidant systems. Oxidative stress responses may involve increased activity of superoxide dismutase (SOD), catalase (CAT) and ascorbate–glutathione cycle activities such as glutathione reductase (GR) or ascorbate peroxidase (APX), which can confer greater tolerance against a specific environmental stress (Sade et al. 2011). Increased levels of non-enzymatic soluble antioxidants including glutathione (GSH), ascorbic acid and tocopherols are also produced in response to water stress-induced oxidative stress (Feng et al. 2004). Plant antioxidant defence systems normally provide adequate protection against ROS damage under optimal growth conditions. The generation of higher levels of ROS may overcome the defence provided by these systems and result in oxidative stress (Mittler 2002; Noctor and Foyer 1998; Valderrama et al. 2006). Cellular damage caused by oxidative stress includes lipid peroxidation, which increases in various tissues during water stress and is also a common marker of oxidative stress (Sade et al. 2011).

Fig. 6.1
figure 1

ROS production in the chloroplast. Chl chlorophyll, Chl* excited chlorophyll. PSI photosystem I, PSII photosystem II. Cyt cytochrome, PQ plastoquinone, PC plastocyanin. Superoxide (O2 ●−) can be produced by electron transfer to oxygen. Hydrogen peroxide (H2O2) is produced from superoxide by spontaneous dismutation or SOD activity. Hydroxyl radicals (●OH) are produced from hydrogen peroxide by homolysis or Fenton reaction in the presence of Fe3+. Singlet oxygen is generated from oxygen by energy transfer from excited chlorophylls

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In response to water deficit, plant cells also accumulate low-molecular-mass compounds termed compatible solutes, mainly proline, glycine betaine, sugars and polyols, in the cytoplasm to control the ionic balance in the vacuoles (Parida and Das 2005). Among these solutes, proline has been associated with different functions, such as being a free radical scavenger, a cell redox balancer, a cytosolic pH buffer and a stabilizer for subcellular structures, especially during osmotic and salt stresses (Szabados and Savouré 2010).

During drought establishment, plants exhibit a decrease in stomatal conductance with the consequent decrease in CO2 assimilation. Stomatal closure has been considered as the main reason for the inhibition of photosynthesis under drought. However, it was demonstrated that limiting stomatal water losses is not so important to maintain photosynthetic activity. For example, it has been observed in leaves of various species, reductions in photosynthesis occur without apparent effects on stomatal conductance (Teskey et al. 1986; Hutmacher and Krieg 1983), suggesting that factors independent of stomatal behaviour impact photosynthesis in plants subjected to drought.

The use of split root system has helped in gaining knowledge about the impact of drought on the process of nodulation in legumes (Larrianzar et al. 2014). Nodule number is mainly regulated at the systemic level through a signal which is produced by nodule/root tissue, translocated to the shoot and transmitted back to the root system. This process involves shoot Leu-rich repeat receptor-like kinases. In contrast, local and systemic mechanisms regulate nitrogenase activity in nodules (Esfahani et al. 2014). Under drought and heavy metal stress, the regulation is mostly local, whereas the application of exogenous nitrogen seems to exert a regulation of nitrogen fixation both at the local and systemic levels (Marino et al. 2007).

6.2.2 Response of Plants to Heat Stress

High temperature at early sowing resulted in poor crop establishment due to failure of seed germination, emergence and reduced vigour (Khalaffalla 1985; Weaich et al. 1996). In such situations, avoidance mechanisms, such as transpiration, leaf rolling, hairiness or wax layers, may play a role in dissipating the heat load. However, in general, transpiration is the most important heat-dissipating system through latent heat loss (Kramer 1983).

Plants exposed to high temperatures, at least 5 °C above their optimal growing conditions, exhibit cellular and metabolic responses required for the plants to survive under this condition (Guy 1999). These effects include changes in the organization of organelles, cytoskeletal reorganization and membrane functions, accompanied by a decrease in the synthesis of some proteins and overexpression of HSPs, the production of phytohormones such as abscisic acid (ABA) and antioxidants and other protective molecules (Bita and Gerats 2013; Maestri et al. 2002; Bray et al. 2000). Under heat stress, about 5 % of plant transcripts (∼ 1500 genes) are up regulated, twofold or more (Rizhsky et al. 2004; Larkindale and Vierling 2008; Finka et al. 2011). A significant fraction of these transcripts encode heat-induced chaperones. For example, 88 out of 1780 in Arabidopsis thaliana, and 117 out of 1509 in wheat, are associated with HSP-based protection mechanism (Liu et al. 2008; Ginzberg et al. 2009; Bokszczanin and Fragkostefanakis 2013). There are many transcripts-encoding proteins involved in calcium signalling; protein phosphorylation; phytohormone signalling; sugar and lipid signalling and metabolism; RNA metabolism; translation, primary and secondary metabolisms; transcription regulation and responses to different biotic and abiotic stresses (Mittler et al. 2012; Huve et al. 2011). Changes in ambient temperature are sensed by plant sensors positioned in various cellular compartments. The increased fluidity of the membrane leads to activation of lipid-based signalling cascades and to an increased Ca2+ influx. Signalling by these routes leads to the production of osmolytes and antioxidants as a response to heat stress. This stress also brings about changes in respiration and photosynthesis and thus leads to a shortened life cycle and diminished plant productivity (Barnabás et al. 2008).

The early effects of heat stress comprise of structural alterations in chloroplast–protein complexes and reduced activity of enzymes (Ahmad et al. 2010). The photochemical modifications in the carbon flux of the chloroplast stroma and those of the thylakoid membrane system are considered the primary sites of heat injury (Wise et al. 2004), as photosynthesis and the enzymes of the Calvin–Benson cycle, including ribulose 1,5-bisphosphate carboxylase (Rubisco) and Rubisco activase are very sensitive to low increases of temperature, and it is suggested to be one of the primary determinants of heat-dependent reduction in photosynthesis (Maestri et al. 2002; Morales et al. 2003). Heat inactivation of Rubisco is reversible (Salvucci and Crafts-Brandner 2004; Kim and Portis 2005). However, moderate heat stress has been shown to alter the thylakoid permeability and electron transport (Schrader et al. 2007; Zhang and Sharkey 2009), and this inhibition of electron transport is associated with enhanced membrane permeability, disorganization of photosystem II (PSII) and antenna tertiary structure, and disruption of the water splitting and oxygen evolving system (Huve et al. 2011). Other specific responses of heat stress on photosynthetic membranes include the swelling of grana stacks and an aberrant stacking. Such structural changes are accompanied by ion leakage from leaf cells exposed to heat and changes in energy allocation to the photosystems (Wahid and Shabbir 2005; Allakhverdiev et al. 2008). The maintenance of cellular membrane function under heat stress is thus essential for sustained photosynthetic and respiratory performance (Chen et al. 2010). The detrimental effects of heat on chlorophyll and the photosynthetic apparatus are also associated with the production of ROS (Guo et al. 2007). By increasing chlorophyllase activity and decreasing the amount of photosynthetic pigments, heat stress ultimately reduces the plant photosynthetic and respiratory activity (Sharkey and Zhang 2010).

Homeostasis, in general, including biosynthesis and compartmentalization of metabolites, is disturbed in high-temperature-challenged plant tissues (Maestri et al. 2002). Among the primary metabolites, accumulating in response to heat stress are proline, glycine betaine or soluble sugars (Wahid 2007).

Heat stress results in the misfolding of newly synthesized proteins and the denaturation of existing proteins. Protein thermostability is provided in part by chaperones (Ellis 1990). In this sense, the exacerbation of combined heat and other stress could be due to the loss of function of some enzymes that are overexpressed in response to other stress.

6.3 Effect of Water Stress–Heat Stress Combination on Different Plant Processes

L. corniculatus and Trifolium pratense are legumes used in agriculture as a forage source. These species are both perennial herbaceous plants used in temperate grassland and can be nodulated by rhizobia. Nevertheless, lotus is better suited to soils with water restriction and has a superior tolerance to water stress (Peterson et al. 1992). In the field, mainly during summer, these plants are commonly exposed to environmental stresses such as water stress and high temperatures, which in fact are considered to be the most important environmental factors limiting plant growth and development (Berry and Bjorkman 1980; Yordanov et al. 1986; Sinsawat et al. 2004).

6.3.1 Proline Accumulation

The accumulation of proline is known to be a good indicator of water stress in L. corniculatus (Díaz et al. 2005b). However, the responses to combination of stresses are not a mere additive effect of the single stresses. For example, some plants that tend to accumulate proline in water stress conditions replace it with sucrose as the major osmoprotectant when subjected to a combination of water stress and heat stress (Rizhsky et al. 2004). In L. corniculatus water stress and heat individually produce proline accumulation, but concomitant imposition of both stresses produced a higher accumulation of proline. In contrast, Trifolium Pratense-accumulated proline in water stress conditions but not under heat stress and the imposition of the combined stress produced only a slight increase in proline concentration compared to unstressed plants (Signorelli et al. 2013b). Thereby, for L. corniculatus, proline accumulation is a parameter that can be used as a stress marker to assess water stress and heat stress conditions, as well as the combination of both. However, proline accumulation in legumes cannot always be considered a good indicator of stress condition when two or more stresses are present. It is also known that proline accumulation under heat stress decreases the thermotolerance of the plant, probably because of an enhancement in the production of ROS via the Pro/P5C cycle (Lv et al. 2011). In T. pratense, it was suggested that blocking proline accumulation might be a strategy to avoid self-toxicity during heat stress (Signorelli et al. 2013b). This hypothesis correlated with the lipid peroxidation estimated by thiobarbituric reactive substances (TBARS), as T. pratense did not show an increase in lipid peroxidation under heat conditions. Moreover, T. pratense has a lower lipid peroxidation content than L. corniculatus when water stress and heat stress are combined—a treatment in which L. corniculatus accumulates the highest levels of proline.

On the other hand, it has been demonstrated that proline can act as an osmolyte under severe dehydration (Verslues and Sharp 1999). The non-accumulation of proline and the greater leaf area of T. pratense are important disadvantages of this species compared to L. corniculatus when water loss must be prevented. In a comparative analysis of L. corniculatus and T. pratense subjected to water stress and heat, it was observed that T. pratense did not survive 5 days of combined stress, while lotus was still alive (Signorelli et al. 2013b). In concordance, higher dry-matter yield was observed in L. corniculatus compared to T. pratense under field conditions subjected to summer water stress (Peterson et al. 1992).

6.3.2 Oxidative Stress

Most stresses induce ROS and alter the antioxidant–enzymatic response (Mahalingam and Fedoroff 2003). However, little is known about how two or more stresses affect the ROS production and the antioxidant response. Alterations induced by water and heat stress on antioxidant response and oxidative damage in the model legume L. japonicus (Sainz et al. 2010), in the forage legumes L. corniculatus and T. pretense has been reported (Signorelli et al. 2013b).

SOD is the main enzymatic system responsible for cell detoxification and is well documented in several plant species to increase in response to water deficit and heat stress (Alscher et al. 2002). In L. corniculatus, the activity of Mn-SOD and Fe-SOD increased as a consequence of water stress and combined stress (Fig. 6.2), but it did not change under heat stress (Fig. 6.2). In the related model specie L. japonicus, Cu/Zn-SOD immunodetection and the isoenzyme-specific activity assays confirmed that high-temperature treatment provoked a reduction in the Cu/Zn-SOD protein content and activity. This is consistent with a failure to convert O2 ●− to H2O2 in the combined heat–drought condition. Additionally, in spite of the decreased Cu/ZnSOD in the high-temperature treatment, the accumulation of O2 ●− remains low, and this is likely because high temperature does not induce accumulation of this ROS (Sainz et al. 2010).

Fig. 6.2
figure 2

SOD activity under drought and combined heat and drought stress. a SOD isoforms profile. C control; D drought; H heat at 42 °C; D + H drought + heat at 42 °C. 40 and 200 mg of protein were loaded in L. corniculatus and T. pratense, respectively. The gel is the most representative of three replicates of native gels. b Total in vitro SOD activity. C control; D drought; H heat at 42 °C; D + H drought + heat at 42 °C. One unit of SOD was defined as the amount of enzyme that inhibits the rate of cytochrome c reduction by 50 %. Bars indicate the relative standard deviation. (Figure modified from Signorelli et al. 2013b)

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In T. pratense, however, no changes were observed in the activities of any SOD isoforms. The results of the quantitative enzyme activity assay demonstrated that total SOD activity is 2.6-fold greater in L. corniculatus than in T. pratense, and it is affected by the stress treatments. Heat did not modify the SOD activity in L. corniculatus, but the combination with water stress led to same level activity observed under water stress (Fig. 6.2). T. pratense showed a slight increase in the SOD activity by heat stress and combined stress (Fig. 6.2). In this case, for both legumes the response of SOD activity in the combined stress was the addition of responses in the individuals’ stresses. It could be concluded that if one of the stresses that produce the induction of SOD activity is present, the induction of SOD activity will be warranted in the combined stress. In L. japonicus, heat stress led to a decrease on Cu/Zn-SOD contents, which also was observed under a combination of heat and water deficit (Sainz et al. 2010).

In L. corniculatus, CAT activity only increases during the combination of water stress and heat. However, in T. pratense, CAT enzyme activity increased with reference to control in response to water deficit, heat stress and combined stress, although no differences were observed among these stresses. In T. pratense, it was observed that any stress was able to induce CAT activity and the combination of both stresses did not lead to an additive effect on the enzyme activity. For L. corniculatus, it seems that any individual stress is not sufficient to induce CAT activity; however, the combination of stresses led to the induction of CAT, suggesting that more than one signal is required to induce this enzyme. In L. japonicus, the combination of heat and water deficit led to an increase in CAT activity, that was much higher than the activity observed when the stressors were imposed individually (Sainz et al. 2010).

Interestingly, the APX activity in L. corniculatus was inhibited by water stress condition, while in T. pratense, this activity was inhibited only in the combined stress treatment. This enzyme is inactivated by nitration (Begara-Morales et al. 2014), which is reported to occur under several abiotic stresses (Corpas et al. 2013). For example, for L. japonicus, a closely related species, it was observed that water deficit induces a nitro-oxidative stress that was also reducing APX activity (Signorelli et al. 2013c). We speculate that the different stressful situations are also inducing nitro-oxidative stress in these plants, and this could explain the decay in enzyme activity.

Both L. corniculatus and T. pratense leaves showed O2 ●− accumulation only in the water deficit–heat stress combination, as was previously observed in the model legume L. japonicus (Sainz et al. 2010). The higher SOD activity in water stress conditions with respect to controls, would allow this species to deal with the O2 ●− induced mainly by water stress. However, the increase of Mn-SOD and Fe-SOD isoform activity by water stress was lost under high-temperature conditions, resulting in an increase of O2 ●− in the combined treatment. In L. japonicus, similar results were obtained with Cu/Zn-SOD, showing that deleterious effects of heat stress on SOD activity might be a general response for this legume genus (Sainz et al. 2010). The differences detected between both species are mainly explained by changes in the Cu/Zn- SOD isoforms. In T. pratense, H2O2 accumulation showed the same pattern; however, in L. corniculatus, the highest accumulation of ROS was observed under water deficit. These results clearly demonstrate that combination of stress situations cannot be always considered the additive responses of individual stresses.

L. corniculatus showed an increase in TBARS content as a consequence of water deficit, heat stress and a combination of these. But T. pratense did not produce any increase in TBARS content under heat stress. As proline antioxidant protection function under stress conditions is now in discussion (Signorelli et al. 2013a), the absence of proline accumulation in T. pratense may be an advantage under heat stress by avoiding the Pro/P5C cycle which, as previosuly mentioned, could result in higher ROS production via the Pro/P5CS cycle (Lv et al. 2011). However, proline accumulation might be critical under combined stress because the osmolyte function seems to be important when water stress is established.

6.3.3 Photosynthesis

Water stress and heat combination affects the rate of photosynthesis due to an increase in photoinhibition, a process that can be enhanced when more types of abiotic stress coexist (Takahashi and Murata 2008). Under stress conditions, the possibility of overexcitation of PSII increases. This can cause a decline in the photosynthetic rate as the process of photoinhibition increases due to the necessity to dissipate, through nonradiative processes, the excess of absorbed energy (Takahashi and Murata 2008; Baker 2008). Because the capacity of photoprotection is limited, certain conditions can lead to damage and loss of active PSII reaction centres. Under severely high temperatures, in combination with water stress, the photosynthetic apparatus is the primary site of damage. On the contrary, photosystem I is more resistant to heat than PSII (Sayed et al. 1989; Hu et al. 2004; Havaux 1993). Once photoinhibition is established, the PSII reaction centre is simultaneously repaired via removal, synthesis and replacement of degraded D1 protein (Ohad et al. 1984; Kyle and Ohad 1986), a protein of reaction centre of PSII (Fig. 6.1). The observed photoinhibitory damage is the net result of a balance between photodamage and the repair process (Samuelsson et al. 1985; Lidholm et al. 1987; Shyam and Sane 1989). Several studies have reported a good correlation between changes in chlorophyll fluorescence parameters in response to environmental stresses, such as heat, chilling, freezing and salinity (Bonnecarrére et al. 2011; Smillie and Hetherington 1983; Yamada et al. 1996; Hakam et al. 2000). Others authors have linked the decrease in the maximum quantum yield of PSII (F V/F M) to the physical dissociation of the PSII reaction centres that lead to photoinhibition, and this assay was used to identify tolerant wheat cultivars (Abdullah et al. 2011).

In L. corniculatus, no changes of the maximum quantum efficiency, evaluated as F V/F M, were observed in any treatment until the 5th day, when the combined treatment showed a significant decrease in the F V/F M parameter. In contrast, in T. pratense, this fluorescence parameter slightly decreased from the 1st day in the heat and combined treatment, but no changes were observed under water stress conditions.

L. corniculatus showed a slight decrease in the amount of D1 protein after water stress treatment. However, there was no decrease in the protein content when the control and heat conditions were compared. The D1-complex profile of T. pratense was also analysed, and the western blot showed a very different result when compared with the L. corniculatus profile. The total D1 protein content in T. pratense did not change in any treatment, but a difference was found in the ratio between the free protein and the complex form. In the treatments where heat was involved, an increase in free D1 protein together with a decrease in the D1-complex form was evident, but it should be considered that this result might be a consequence of the high hydrophobicity of these complexes, which makes their isolation difficult. Regarding D2, in L. corniculatus, the results were similar to those observed with D1; namely, when water stress was present in the treatments, a reduction in the amount of D2 protein was observed. Surprisingly, in the combined treatment, the D2 protein was not detected. In contrast, in T. pratense, no significant changes were observed in D2 protein levels.

The chlorophyll fluorescence parameter that was evaluated showed that L. corniculatus had a significant decrease in the maximum quantum efficiency at the end of the combined treatment. The low F V to F M ratio indicated photoinhibition, a process that can act as determinant of plant performance during a stress condition (Abdullah et al. 2011). In T. pratense, only a small decrease in the F V/F M values was observed from the 1st day and in the treatments involving heat stress.

Analysis of PSII proteins in the two legumes with contrasting water stress responses shows an effect that is stress- and species-specific. The D1 and D2 subunit content is decreased in L. corniculatus in both treatments involving water stress, showing certain adaptability in response to water stress. Interestingly, the decrease in the D2 levels was pronounced in the combined treatment, and this is well correlated with the decrease in the maximum quantum efficiency, suggesting the presence of a disassembling process. The D2 subunit is of particular interest because it represents the initial point for the assembly of the PSII as a whole (de Vitry et al. 1989; Komenda et al. 2004; Minai et al. 2006). The expression of the gene that encodes the D2 subunit of the PSII reaction centre is regulated posttranscriptionally by an RNA-binding protein (Schwarz et al. 2007). Modifications induced by the stress in this post-transcriptional regulation could be a possible explanation for the absence of D2 in L. corniculatus subjected to the combined stress treatment.

In T. pratense, the total content of D1 and D2 did not change, but we observed an increase in the free form of D1 in treatments involving heat. One possible explanation is that the turnover of D1 is taking place in the heat treatments, and this is evident based on the increase of free D1 together with a reduction of the D1–D2 complex, as well as a decrease in the F V/F M values.

6.4 Waterlogging and Salinity: A Combined Stress in Legumes

Salt stress is certainly one of the most serious environmental factors limiting the productivity of crop plants (Ashraf and O’Leary1999). Salinity reduces the ability of plants to take up water, causing rapid reductions in growth rate, along with an array of metabolic changes identical to those caused by water stress (Munns 2002).

High salt concentration in the external solution of plant cells produces several deleterious consequences. First, salt stress causes an ionic imbalance (Niu et al. 1995). The homeostasis of not only Na+ and Cl− but also K+ and Ca+2 ions is disturbed (Rodriguez-Navarro 2000; Hasegawa et al. 2000; Serrano et al. 1999). As a result, plant survival and growth will depend on adaptations that re-establish ionic homeostasis, thereby reducing the duration of cellular exposure to ionic imbalance. Second, high concentrations of salt impose a hyperosmotic shock by decreasing water and causing loss of cell turgor. This negative effect in the plant cell is thought to be similar to the effects caused by drought. Third, reduction of chloroplast stromal volume and generation of ROS, in salt-induced water stress, are also thought to play important roles in inhibiting photosynthesis (Price and Hendry 1991). On the molecular level, these responses are manifested as changes in the pattern of gene expression (Maggio et al. 2002).

The process of salinization results from the interaction between climate, geomorphology, hydrology, land use and surface water properties and dynamics of the salts. Regions with salinity are frequently associated with geographical localization with inundation events; thus it is not infrequent that salt and flood stress occurs simultaneously.

Salinity and waterlogging interact adversely to reduce production of crops and pastures, as very few species used in agriculture can tolerate the combination of both stresses (Barrett-Lennard 2003). Moreover, annual pasture legumes are particularly sensitive to combined salinity and waterlogging (Bennett et al. 2009).

One of the most important consequences of energy limitation under anoxia is altered redox state of the cell. Under low oxygen pressure conditions, the intermediate electron carriers in electron transport chain become reduced, affecting redox-active metabolic reactions. Therefore, for maintaining redox homeostasis cells need to regulate NADH to NAD ratio under flooding (Chirkova et al. 1992). Saturated electron transport components, the highly reduced intracellular environment and low-energy supply are the factors favourable for ROS generation. The consequences of ROS formation depend on the intensity of the stress as well as on the physicochemical conditions in the cell (i.e. antioxidant status, redox state and pH). As was mentioned for other stresses, ROS accumulation may cause damage to different cell structures and biomolecules. H2O2 production during O2 deprivation was observed in the plant cells (Blokhina et al. 2001), and its degradation was found to play an important role in waterlogging tolerance in non-legume plants (Lin et al. 2004).

A trait that is essential for root survival during water logging or flooding is the development of aerenchyma (Armstrong 1979). Aerenchymas are cortical airspaces that provide a low-resistance internal pathway for the movement of O2 from the shoots to the roots, where it is consumed in respiration and may also reoxidize the rhizosphere (Armstrong 1970; Armstrong 1971, 1979). In legumes, aerenchyma may also be important for supplying O2 and N2 to root nodules (Walker et al. 1983; James et al. 1992; Zook et al. 1986; Pugh et al. 1995). Tolerance of Melilotus siculus to waterlogging is associated with the production of a highly porous phellem, a type of secondary aerenchyma, on taproots and upper lateral roots (Verboven et al. 2011).

Studies with plant species sensitive or tolerant to flooding–salt stress combination have shown that the rate of transport of Na+ and Cl− to the shoot is critical to define the response. The ions transport rate increases significantly under combined stress in comparison with salinity alone (Barrett-Lennard 2003). For more tolerant species, there is only small or even no increase in shoot Na+ and Cl− in response to combined salinity and waterlogging (Colmer and Flowers 2008), presumably due to better root aeration. Moreover, in perennial legumes such as Trifolium repens L. (Rogers and West 1993) and Liolaemus tenuis (Teakle et al. 2007), high root porosity was associated with better shoot ion regulation under combined salinity and waterlogging. Comparisons of annual pasture legumes in growth, ion regulation and root porosity demonstrate that M. siculus has exceptional tolerance to combinations of salinity and waterlogging (Teakle et al. 2012). Enhanced root aeration would avoid energy deficits that could impair ion transport processes in roots, which determines delivery of Na+ and Cl− to shoots via the xylem (Barrett-Lennard 2003; Teakle et al. 2007; Colmer and Flowers 2008). Thus, traits of importance for tolerance to combined salinity and waterlogging are likely to include high root porosity, leading to decreased shoot Na+ and Cl− concentrations.

6.5 Metabolic Changes in Responses to Stress Combination

It is well known that the effect of a combination of different stresses on plants can be quite different from those generated when plants are subjected to individual types of stress (Rizhsky et al. 2002). Table 6.1 represents a summary of how the combination of different stresses affects some parameters in legumes.

Table 6.1 Effects of stress combination on main parameters studied in legumes
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With reference to antioxidant responses, different patterns are observed when more than one stress is imposed. However, it seems that in most cases the addition of other stress did not alter the response. It implies that the signal molecules that induce the expression of antioxidant enzymes probably are the same in different stresses and so the imposition of both stresses is redundant. In other cases, the effect of simultaneous stresses produces deleterious effects. For example, for APX and CAT, one stress produces the induction of the activity (or at least a normal level of activity), but the imposition of two stresses could produce a more nitrosative condition in the cell leading to the nitration of the enzyme, which is known to decrease the activity of these enzymes.

Among the oxidative stress markers, synergistic effect was the most commonly observed response. Most stresses are accompanied by an increment of ROS production, and the source of ROS is different for different stresses (Mahalingam and Fedoroff 2003; Wrzaczek et al. 2013). Thus, when more than one stress is present, it induces ROS from different organelles, and hence the total ROS tends to be higher in combined stress scenarios. Less commonly, a negative correlation or an unchanged response is observed. In one case of negative correlation observed for H2O2, it was suggested that the reduction in SOD activity in combined stress as opposed to in single stress was responsible for the lower H2O2 in the former. In the other case, induction of CAT activity only in the combination of stress was suggested to be the cause of lower H2O2 levels.

Photosynthetic activity does not show a defined pattern, maybe due to lack of information. Even with the limited data, it can be seen that in all the cases examined, D1 was unchanged by the imposition of combined stresses. D2 protein had a synergistic effect in combined stress. It is important to point out that in drought and heat stress were considered in these studies, and some of these responses were observed in T. pratense and in two related species such as L. japonicus and L. corniculatus. Other species should be evaluated to see the conservation in the response of D2, which is suggested to disassemble to induce inhibition of photosystem activity, and protect cells from oxidative damage caused by its own activity.

6.6 Forage Legumes Field Productivity and Combined Environmental Stress

Legumes have a high level of productive diversification and flexible utilization. The same species can be usefully exploited for different purposes such as soil protection from erosion; green manure crop; mulching; cover crop in vineyards, orchards and firebreak lines; high quality honey production; landscape enhancement and medicinal use. Consequently, forage legumes were adapted to a wide range of soil types, climatic conditions and management systems (Sánchez-Díaz 2001).

Legumes, as many other crops, have been bred to maximize productivity (forage or grain). But this productivity is always affected by adverse environmental factors. Perennial forage legumes are a good model to analyse the responses of adaptability of plants under field conditions. This is because during the whole plant growth and development cycle, plants are subjected to various types of abiotic stresses, both singly and in combinations.

Low temperatures and periods of water saturation in soils are common during the winters in many regions and in the other side periods of low water regime combined with high temperatures are common during summers. To these we must add other combinations of stresses such as periods of high radiation or toxic ions (Na+ or heavy metals) produced by changes in the physicochemical conditions of the soils.

Further, abiotic stress can affect the legume plants at different developmental stages. So legumes growing under field conditions must have adaptation process triggered by stress in seedling, vegetative or reproductive stages. For example, for seedling emergence, the optimal conditions in the field are established at the end of winter (Fig. 6.3).

Fig. 6.3
figure 3

Seedlings emergence of L. tenuis during a typical of temperate zones from south hemisphere (Ayala and Carámbula 2009)

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Legumes are adapted to different environmental conditions by setting the developmental stages, such as reseedling capacity that is an important characteristic for the perpetuation of L. corniculatus. Yield of L. corniculatus during 3 years with seed set and without seed set, reveal the importance in reseedling (Fig. 6.4, Ayala and Carámbula 2009).

Fig. 6.4
figure 4

Seasonal dry matter production of L. corniculatus under two-seed set management. With seed set (solid line) and without seed set (dashed line). (Ayala and Carámbula 2009)

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This suggests that the tolerance in reproductive stages should be accompanied by physiological responses to deal with water restriction and high temperatures. Another key physiological mechanism in the survival of legumes is their ability to mobilize carbohydrates to storage tissues that can be located on the crown, root or rhizome (Castillo et al. 2012). However, it is critical that the photosynthetic activity remains active during the stress period to achieve significant accumulation of sugars allowing regrowth of the shoot after stress.

6.7 Breeding Approaches for Improving Tolerance to Combined Abiotic Stresses

Selection for one abiotic stress tolerance in the field is very challenging due to interactions among the different stresses. Thus, the only strategy to identify the traits to be applied in field for breeding tolerant genotypes is by performing experiments under controlled environment conditions. Regardless of the screening method, a key objective for plant breeders is to develop an effective set of stress combination markers that can be used to improve legume crop species. Controlled environmental conditions allow the dissection of each one of different stress effect and the identification of principal targets affecting plant tolerance. Breeding for stress tolerance requires efficient screening procedures, identification of key traits in diverse donor or tolerant lines and understanding their inheritance and molecular genetics. Statistical package applied to plant breeding will facilitate the identification of markers in a multi-trait multi-environment way (Malosetti et al. 2004).

Several quantitative trait locus (QTL) studies relating to various abiotic stress tolerances have already been reported showing it is possible to improve and accelerate the breeding process in plant species without sequenced genomes (Chandra et al. 2004). In order to transfer these traits, classical breeding requires the establishment of rapid and cost-effective screening procedures and implementing these using breeding approaches such as association mapping or genomic selection procedures.

For the complete sequencing of the different important legumes, genome opens the possibility of fine mapping of the QTLs. In this perspective, gene identification for combined stress tolerance in legumes using genetic map information and genome data is an achievable goal (Heffner et al. 2009; Hirayama and Shinozaki 2010).

Phenotypic and physiological characterization along with RNA sequencing analysis of plants subjected to drought, heat, salt, flooding stress or their combination would confirm that the simultaneous imposition of different types of stress presents unique but varied aspects that includes alteration of respiration rate, decreased photosynthesis, stomatal closure, high leaf temperature and redox homeostasis. Thus, deep phenotyping methodologies, genome-based selection and massive RNA sequencing technologies emerge as a promising avenue for the development of multiple abiotic stress-tolerant crops.