The Physiology of Abiotic Stresses

Chapter 3 The Physiology of Abiotic Stresses Paulo C. Cavatte, Samuel C. V. Martins, Leandro E. Morais, Paulo E. M. Silva and Fábio M. DaMatta Abstract Plants are often exposed to several adverse environmental conditions that potentially generate stress and thus negatively affect their growth and productivity. Understanding the physiological responses of crops to stress conditions is essential to minimizing the deleterious impacts of stress and maximizing productivity. Therefore, there is urgent need for more scientific research to increase our understanding of the physiological behavior of crops in response not only to a specific type of stress but also to multiple interacting stressors, such as water, and thermal stresses. The proper assessment of this information may result in important tools for monitoring the most promising genetic material in plant breeding programs. In this chapter, the plant strategies associated with satisfactory growth and yield under abiotic stress conditions are discussed, with emphasis in tropical environments. In addition, the state of the art on the physiology of the major abiotic stresses (drought, salinity, heat, nitrogen and phosphorus deficiencies and aluminum toxicity) and possible strategies to develop cultivars with P. C. Cavatte
S. C. V. Martins
L. E. Morais
P. E. M. Silva
F. M. DaMatta (&) Departamento de Biologia Vegetal, Universidade Federal de Viçosa, Avenida Peter Henry Rolfs s/n, Viçosa-MG, 36570-000, Brazil e-mail: [email protected] P. C. Cavatte e-mail: [email protected] S. C. V. Martins e-mail: [email protected] L. E. Morais e-mail: [email protected] PauloE. M. Silva e-mail: [email protected] R. Fritsche-Neto and A. Borém (eds.), Plant Breeding for Abiotic Stress Tolerance, DOI: 10.1007/978-3-642-30553-5_3, Ó Springer-Verlag Berlin Heidelberg 2012 21 22 P. C. Cavatte et al. satisfactory productivity in stressful environments using a physiological approach are summarized. Keywords Aluminum Breeding Salinity Stress Yield


Drought
Heat
Nitrogen
Phosphorous

3.1 Introduction Because of the present scenario of global climate changes and considering that major advances in agriculture were designed for environments favorable to the ‘‘Green Revolution’’, crop performance under adverse conditions in marginal environments, which has often been overlooked, is currently the subject of constant debate. Plants are often exposed to several harsh environmental conditions that potentially generate stresses. Stresses trigger a wide range of plant responses, from altered gene expression and cellular metabolism to changes in growth rates and crop yields. The duration, severity, and rate at which a stress is imposed all influence how a plant responds. A response may be triggered directly by a stress, such as water deficit, or may result from a stress-induced injury, such as loss of membrane integrity (Bray et al. 2000). The indubitable importance of abiotic stresses to agriculture can be easily perceived taking into consideration that altogether abiotic factors provide the major limitations to crop yield worldwide, and can reduce average productivity of annual crops by 51–82 %, depending on the crop (Bray et al. 2000). Understanding the physiological responses of crops to stress conditions is, thus, essential to minimizing the deleterious impacts of stress and maximizing productivity. Therefore, there is urgent need for more scientific research to increase our understanding of the physiological behavior of crops in response not only to a specific type of stress but also to multiple interacting stressors, such as water and thermal stresses. The proper assessment of this information may result in important tools for monitoring the most promising genetic material in plant breeding programs. In this chapter, the plant strategies associated with satisfactory growth and yield under abiotic stress conditions are discussed, with emphasis in tropical environments. In addition, the state of the art on the physiology of the major abiotic stresses [(drought, salinity, heat, nitrogen (N) and phosphorus (P) deficiencies, and aluminum toxicity)] and possible strategies to develop cultivars with satisfactory productivity in stressful environments using a physiological approach are summarized herein. 3.2 Water Deficit In recent years, losses related to drought have been the main challenge for grain production, and it is the environmental factor that most limits crop productivity worldwide, especially in semi-arid regions. Drought events have become stronger 3 The Physiology of Abiotic Stresses 23 in recent decades and are most likely associated with climate changes due to global warming. According to climate projections, this situation tends to be exacerbated, and thus, the availability of water for irrigation is expected to be reduced (IPCC 2007). The demand for food, however, is growing significantly, such that more water will be necessary for agricultural activities in the future. In this context, the use of water (consumption) and efficiency of its use (water use efficiency, WUE; i.e., amount of water consumed to produce a given amount of biomass) are key parameters in areas affected by water deficit. Indeed, one of the major goals of modern agriculture is to achieve increased crop productivity using less water (‘‘more crop per drop’’). For purposes of this chapter, drought must be defined. In a broad sense, drought is a multidimensional phenomenon, including water deficits in not only the soil but also the atmosphere, which is primarily determined by relative humidity and air temperature. In this chapter, drought will be considered as a meteorological phenomenon, described as a sufficiently long period of imbalance between precipitation and evapotranspiration, that is capable of promoting the depletion of soil water and causing water stress in plants, thereby reducing their growth and/or yield. 3.2.1 Plants Strategies Against Water Deficit The productivity of plants with a water deficit depends on climate and soil conditions, which affect the quantity of water available and how the water is used by the plant. The responses to limited water availability are diverse with respect to the plant species involved and/or the severity and duration of the water deficit. Basically, there are three strategies by which plants can grow and develop properly in environments with water restrictions: (i) drought escape, which can be observed in short cycle crops and which allows the plants to complete their productive cycle before the water deficit becomes severe; (ii) drought avoidance, which, for example, reduces transpiration or increases the absorption of water; and (iii) drought tolerance, which involves some protoplasmic tolerance. Each of these strategies is associated with costs and benefits, which vary according to the species, the environment, the technological resources of the farmer, and the goals of the plant breeding program (Tardieu 2005). The adaptation of plants to water deficit is a very complex process, involving morphological, physiological, biochemical, and molecular alterations (Passioura 1997). As reasoned by DaMatta (2003), in the short-term soil drought, yield reduction could be related to reduced stomatal conductance (physiological variable linked to the degree of stomata opening) and concomitantly lower photosynthetic rates, but in the long term, a smaller leaf area (due to decreased leaf size and production, and higher rates of leaf senescence) and an altered assimilate partitioning between plant structures and organs could be more directly responsible for decreased crop yield. Additional reductions in stomatal conductance are to be expected as internal water deficits develop and, as a consequence, stomatal 24 P. C. Cavatte et al. limitations to photosynthesis should be exacerbated with the progression of drought, thus further contributing to limit crop yield. Under moderate water deficit conditions, decreases in leaf area are not necessarily accompanied by decreases in photosynthetic rates on a leaf area basis, but if drought progresses further, strong decreases in photosynthetic rates per unit leaf area are often observed (DaMatta 2003). Another problem associated with reduced stomatal conductance (and thus reduced transpiration rate) is increased leaf temperature, which has possible consequences on higher maintenance respiration and photorespiration rates, which further contributes to decrease crop productivity. In addition to factors leading to a lower transpiration rate, the adaptation to water deficit involves a greater capacity for water absorption by the plant. This process can be accomplished with genotypes that have more robust root systems (deep and branched), which can better exploit deeper soil water reserves (Pinheiro et al. 2005). Indeed, a hallmark of plants subjected to water stress is an increase in the biomass allocated to the root system. 3.2.2 Physiological Strategies for Productivity Increases Under Water Deficit Conditions Under water stress conditions, the yield (Y) of a crop can be described by the equation (Passioura 1977): Y = E 9 WUE 9 HI, where E = transpired water, WUE = water use efficiency, and HI = harvest index (i.e., relationship between the biomass of the organ of commercial interest and the total plant biomass). Large increases in productivity through breeding programs occurred by increasing the harvest index. For most annual species grown, however, HI is close to its maximum value, and additional increases in crop yields must necessarily be accomplished through increased biomass accumulation. Therefore, the challenge is to increase the capacity of the plant to produce a larger amount of dry matter per area unit. In the case of breeding programs for drought conditions, this increased biomass production must be combined with lower water consumption or increased WUE (Tambussi et al. 2007). WUE can be estimated easily and quickly using a small amount of dry biomass, which release 12CO2 and 13CO2 while undergoing combustion in a mass spectrometer. The resulting data can be used to estimate the carbon isotopic discrimination (D13C), and the WUE can then be integrated overtime (Farquhar et al. 1989). The high negative correlation between D13C and WUE has been experimentally demonstrated in a large number of species (Condon et al. 2002; Monneveux et al. 2007). Thus, D13C has been a widely used parameter in breeding programs to select genotypes with higher productivity under drought conditions (Ehleringer et al. 1993). Reducing the stomatal conductance, increasing the photosynthetic capacity or even combining both factors may lead to a higher WUE. A reduction in stomatal conductance is not favorable in breeding programs aimed at increasing 3 The Physiology of Abiotic Stresses 25 productivity because it entails less CO2 influx and lower photosynthetic rates and, therefore, less biomass accumulation. Thus, the great challenge is to increase the photosynthetic capacity under conditions of low stomatal conductance (Tambussi et al. 2007). Several strategies have been proposed to increase the photosynthetic capacity of cultivars. One of these strategies involves CO2 concentrator mechanisms, such as the one found in species that utilize C4 metabolism. Another strategy would be to increase the CO2 specificity of the enzyme ribulose1,5-bisphosphate carboxylase/oxygenase (Rubisco), thus reducing losses related to photorespiration in C3 plants (Parry et al. 2005). The increase in the mesophyll conductance is directly associated with increases in photosynthetic rates without the need for increased stomatal conductance. Finally, another strategy involves increasing the specific leaf mass because such an increase represents a greater amount of photosynthetic apparatus per leaf unit area. At a cellular level, osmotic adjustment has been considered as the most striking acclimative response of plants subjected to water deficit (Blum 2005), which is characterized by a net solute increase in cells. These solutes, named compatible osmolytes, are organic compounds (e.g., proline, sugar alcohols, sorbitol, mannitol) that do not interfere with cell metabolism. The osmotic adjustment has two main functions: (i) maintaining cell turgor in the presence of negative water potential, which allows the process of cell elongation to be maintained in addition to allowing relatively larger stomatal aperture and (ii) providing a greater capacity for water uptake by the roots, which would also contribute in the maintenance of higher stomatal conductance. Concomitantly, osmotic adjustment leads to the maximization of the photosynthetic process, resulting in relatively greater biomass accumulation. Thus, genotypes with greater potential for osmotic adjustment tend to be more tolerant and, therefore, more productive under drought conditions (Ludlow et al. 1990; Morgan et al. 1992; Moinuddin and Khanna-Chopra 2004). Additional factors, such as excessive temperature and high irradiance, are common under drought conditions. These factors explain why drought is considered to be a multidimensional stressor (DaMatta 2003). With a reduction in photosynthesis, the use of incident solar radiation decreases, leading to the accumulation of reducing power (NADPH) and a reduction in the pool of the final acceptor (NADP+) of the electron transport chain. This excess reducing power may lead to an over-reduction of the electron transport chain. In this process, electrons may escape and, in turn, react with molecular oxygen to form the so-called reactive oxygen species (ROS; e.g., superoxide anions, hydrogen peroxide, and hydroxyl radicals), which can damage plant metabolism through processes such as lipid peroxidation and protein and nucleic acid oxidation. These oxidative damages can be observed in leaves as chlorotic areas, which in many cases progress to necrosis, leading to leaf abscission. Genotypes with higher antioxidant activity, both enzymatic (increased activity of enzymes, such as superoxide dismutase, catalase, ascorbate peroxidase, and glutathione reductase) and non-enzymatic (e.g., glutathione and ascorbate), tend to be more tolerant to drought, showing less oxidative damage compared with genotypes with less robust antioxidant systems. This high antioxidant capacity confers increased cellular protection, especially for 26 P. C. Cavatte et al. the photosynthetic machinery, and maintenance of leaf area, which consequently results in increased production, even under severe water deficits (Lima et al. 2002; Posch and Bennett 2009). 3.2.3 Difficulties and Advances in the Development of Drought-Tolerant Cultivars The progress performed in the last 100 years, in relation to obtaining droughttolerant cultivars, has been very weak in most breeding programs, regardless of the species considered, in contrast to the improvements made with respect to other biotic and abiotic stresses (Tardieu 2005). In addition, from a physiological point of view, many experiments have been conducted in greenhouses or growth chambers with plants in small pots, a condition that causes the plants to be exposed to drastic and swiftly imposed drought. Thus, the responses obtained do not entirely reflect the field situation, and therefore, the comparative results should be treated with caution. Moreover, in many cases, the genotypic comparative analyses have been performed with varying hydration degrees and, hence, are not strictly comparable. Consequently, much of the drought tolerance information generated by physiologists has limited utility for the crop breeder because such information cannot be converted to adequate indices to predict the behavior of genotypes with varying levels of tolerance to water stress. The dissociation between basic information and the requirements of applied (or applicable) information is an additional bottleneck for advancements leading to the acquisition of more drought-tolerant materials. In brief, drought tolerance is the result of several traits that are expressed differently and concomitantly, depending on a variety of factors, including the severity and rate of water deficit imposition, the age and nutritional status of the plant, the soil type and depth, the fruit load status and the atmospheric evaporative demand. Therefore, the adoption of only one strategy for adaptation to drought is certainly inadequate for any type of environment (Sambatti and Caylor 2007). Obviously, numerous genes affect the aforementioned conditions, and therefore, the expected progress in increased drought tolerance through genetic engineering is disappointing. Moreover, only a small proportion of genes involved in plant responses to water deficit are known. Simulating the interaction between genes in complex systems is virtually impossible: for example, a study of 15 genes with three alleles each would generate 14 million genotypes, far too many to be analyzed in detail, particularly without the risk of misinterpretation (Tardieu 2005). In any event, there is great need to conduct multidisciplinary research involving molecular, physiological, and classic genetic improvement to enhance crop performance in drought-prone regions. Recent advances in the transcriptome, proteome, and metabolome have allowed an integrated view of the expression of thousands of genes and their products involved in drought tolerance (Tuberosa and Salvi 2006). In the long term, these improvements will provide a better 3 The Physiology of Abiotic Stresses 27 understanding of the polygenic nature of drought tolerance. The adequate selection of indices and parameters for the identification of genetic material with increased drought tolerance has resulted in satisfactory increases in the yield of some crop species. Improved WUE, more robust root systems, osmotic adjustments, and increased activity of the antioxidant system are some of the features that should be focused on in breeding programs aiming to increase production under water deficit conditions. Recently, Muuns et al. (2010) pointed out that changes in spectral reflectance and fluorescence related to altered light harvesting and biochemistry caused by oxidative stress may be easily monitored (Munns et al. 2010); a combination of spectral reflectance, fluorescence, and thermal sensing can, thus, provide a powerful tool for large population screening to identify genotypes with improved tolerance to drought stress (Chaerle et al. 2009). 3.3 Salinity More than 800 million hectares worldwide are estimated to be affected by high soil salinity (FAO 2005). In addition, intensive agriculture and inadequate management practices are causing substantial salinization of agricultural areas. A large proportion of the cultivated regions have become saline due to increased irrigation. When irrigation water contains a high concentration of solutes and there is no possibility of unloading the accumulated salts into a drainage system, these salts can quickly reach levels that are harmful to crops. Thus, salinity is a major threat to the sustainable irrigation necessary to meet the increasing human population demands for food. The importance of breeding programs aiming to increase tolerance to drought and salt stress is therefore inherent. 3.3.1 Salinity Tolerance Plants are commonly divided into two distinct groups according to the degree of salinity tolerance. Halophyte plants are native to saline soils and complete their life cycle in saline environments, and thus, they are more tolerant to high salt concentrations in the soil. Conversely, glycophytes, or non-halophyte plants, are unable to tolerate the same concentration of salt as halophytes. There is substantial genetic variation regarding salt tolerance both in halophyte and glycophyte plants. The accumulation of salts in the soil causes a significant reduction in water potential, affecting the water balance of plants and requiring the development of more negative water potentials to maintain the water potential gradient, which allows the movement of soil water into the plant (Munns and Tester 2008). This osmotic effect of dissolved solutes is similar to soil water deficit, and the initial plant responses to excessive levels of salt are the same as described above for water deficit. 28 P. C. Cavatte et al. In addition to the osmotic effect, salinity leads to an ionic imbalance caused by a high sodium (Na+) concentration. Metabolic disturbances generated by Na+ accumulation in cells are partly due to competition with potassium (K+) for the active sites of enzymes (Blumwald et al. 2000) and ribosomes (Tester and Davenport 2003). K+ is the activator of over 50 plant metabolic enzymes and cannot be replaced by Na+ in this function. Thus, high concentrations of Na+ or a high Na+/ K+ ratio will affect several critical metabolic processes. High concentrations of specific ions, especially Na+ and Cl-, can cause a nutritional imbalance in plants, which frequently involve the deficiency of ions, such as K+, Ca2+, Mg2+, NO3 , and PO4- (Läunchli and Epstein 1990). The plant response to salinity develops according to the limiting factor to growth, and this effect involves an osmotic or ionic imbalance caused by Na+ excess. Typically, the first phase of salt stress is represented by the osmotic effect that occurs rapidly from the moment that the salt concentration around the roots reaches the tolerance threshold of the species (approximately 40 mM NaCl for most species). In cereals, the first phase of salt stress is marked by a reduction in the number of tillers, whereas in dicotyledons, the main effect is reductions in leaf size and branch number (Munns and Tester 2008). The second stress phase occurs more slowly and involves the per se toxicity of Na+ ions inside the cell, which is reflected by the accumulation of salt in older leaves, which usually die. The mechanisms of salinity tolerance are connected to several processes: the recovery of ionic equilibrium and cellular homeostasis, the osmotic adjustment to maintain water absorption by the plant, and the prevention or mitigation of oxidative stress that occurs in the same way as in water deficit. Osmotic adjustment may involve the accumulation of inorganic ions (Na+ and Cl-), as typically observed in halophytes, or via the synthesis of compatible osmolytes, a response usually observed in glycophytes (Ashraf 2004). Another type of adaptation to salt stress involves the ability to remove excess salt by specialized salt glands; ions are transported to these glands, where the salt is crystallized and rendered no longer harmful to the plants (Thomson et al. 1988). For the recovery of cell homeostasis as a function of increased Na+ levels, the regulation of several carriers involved in the maintenance of appropriate Na+ levels in the cytosol and the involvement of the salt overly sensitive (SOS) pathway play important roles. The SOS signaling pathway was discovered in Arabidopsis and comprises three key components: SOS3, a sensor of Ca2+; SOS2, a serine/threonine protein kinase; and SOS1, a Na+/H+ antiporter (exchanger) present in the plasma membrane (Türkan and Demiral 2009). The presence of Na+ in the apoplast is perceived by an as yet unknown sensor that alters the levels of cytoplasmic Ca2+, activating SOS3 and forming the SOS2/SOS3complex. This complex is responsible for activating SOS1 and the Na+/H+ antiporter NHX1 in the tonoplast membrane. These transporters enable Na+ sequestration in the vacuole and its subsequent extrusion from the cell, reducing the amount of Na+ in the cytosol. The SOS2/SOS3 complex is also involved in the regulation of the gene expression of the aforementioned transporters and of the activity of vacuolar H+-ATPase and H+-PPase, which are responsible for providing H+ ions for NHX1 3 The Physiology of Abiotic Stresses 29 activity (Chinnusamy et al. 2005a). Another primary mechanism of salt tolerance, conserved in both monocotyledons and dicotyledons, is performed by HKT transporters, which are responsible for maintaining a high K+/Na+ ratio in the leaves. Indeed, a high K+/Na+ ratio can be a good marker for salt tolerance (Hauser and Horie 2010). The transporter AtHKT1;1 is responsible for Na+ removal via the xylem vessels in parallel to the stimulatory effect of K+ loading, thus controlling the concentration of these two elements to maintain them in a favorable ratio. The mechanisms mentioned above require an efficient supply of energy in the form of ATP, reducing power and carbon skeletons, either to maintain ATPases or to synthesize compatible osmolytes. This high energy demand has highlighted the role of mitochondrial respiration as an integrator of important mechanisms involved in salt tolerance (Jacoby et al. 2011). Genotypes tolerant to salt stress have been shown to have a more robust mitochondrial antioxidant system, which would be responsible for maintaining the integrity of the energy efficiency required for the acclimation to high salinity conditions. In addition, the ROSs formed in the mitochondria have a signaling role, which may be responsible for orchestrating antioxidant responses in other plant parts. The mitochondrion itself also plays a role in the osmotic adjustment because proline catabolism occurs within mitochondria, with proline abundance affecting the regulation of enzymes involved in mitochondrial catabolism (Jacoby et al. 2011). The extent of the mechanisms used by salt-tolerant species may differ even intraspecifically. In maize, for example, vacuolar Na+ compartmentalization occurs in some but not all strains, and conversely, a higher K+/Na+ ratio and lower Na+ content in the tissues are apparently essential characteristics for the tolerance of maize to salinity. In some species, such as Brassica napus, however, the inverse situation occurs because the most tolerant genotypes have higher Na+ concentrations in the aboveground parts but in parallel with a higher accumulation of proline and K+ (Moller and Tester 2007). To elucidate the mechanisms associated with salt tolerance, several transgenic approaches have already been performed, although there are few studies in which comparisons are made properly or under field conditions typically experienced by crops (Flowers 2004; Ashraf and Akram 2009). Another complicating factor is the evaluation of the importance of each mechanism involved in salt tolerance. Discussing several examples of salinity tolerance, Ashraf (2004) concluded that the identification of the physiological and biochemical indicators for each species individually is more reliable than the use of generic selection markers due to the variability of the responses at the inter and intraspecific levels. Likewise, caution should be applied when extrapolating results found in model plants, such as Arabidopsis, for use in other species because the tolerance strategies are different among groups. Several Arabidopsis ecotypes are known to be more salt tolerant via the accumulation of Na+, whereas most cereals show the opposite response, with an increased tolerance via decreased Na+ levels in the tissues (Moller and Tester 2007). The polygenic nature of salt tolerance has resulted in difficulties for the development of improved varieties through conventional genetic improvement, 30 P. C. Cavatte et al. although classical plant breeding has had greater success in the production of new varieties more tolerant to salinity than transgenic breeding strategies (Ashraf and Akram 2009). Therefore, the most effective strategy for increasing salinity tolerance remains the use of the genetic variability within crops. The domestication of halophytes also plays an important role in the use of saline areas, and the use of some agronomic practices, such as increasing the levels of Ca2+ in the soil, have been reported to soften the salt effects in some crops (Cimato et al. 2010). 3.4 Heat Stress The threshold temperature (upper and/or lower), which varies both inter and intraspecifically, refers to the daily average temperature that causes a reduction in growth. The temperature increase above the upper limit for a period of time sufficient to cause irreversible damage to plant growth and development is defined as heat stress, which is a complex function of the intensity, duration, and rate of temperature increase (Peet and Willits 1998). Plants are subjected to heat stress under the following conditions: (i) the air temperature is high, and the plants receive energy by the transfer of sensible heat; (ii) solar radiation incident on the ground raises the temperature above the air temperature; and (iii) solar radiation-induced heating and the inability to dissipate heat can rapidly warm the leaves (up to 15 °C above the air temperature or more), particularly in leaves that have a low transpiration rate and are frequently subjected to high temperatures (Singsaas et al. 1999). Heat stress can affect crop yield at any time from sowing to grain maturity, but it is the time around flowering, when the number of grains per land area is established, and during the grain-filling stage, when the average grain weight is determined, that high temperatures have the most impact on the final harvestable crop, as found in cereals (Morison and Lawlor 1999; Barnabás et al. 2008). Thus, understanding how environmental factors signal phenological processes such as flowering will be extremely relevant for future food production, since a large part of food comes as grains/seeds. In this context, any change in flowering time could affect not only seed production but also food composition (DaMatta et al. 2010). 3.4.1 Plant Response and Morpho-Physiological Alterations to High Temperatures Thermal stress increases the kinetic energy and movement of the molecules composing biological membranes, leading to the relaxation of chemical bonds, and providing fluidity to the lipid bilayer. The denaturation of proteins and/or the increase of unsaturated fatty acids also contribute to this process. These changes 3 The Physiology of Abiotic Stresses 31 increase membrane permeability, which is demonstrated by an increased loss of electrolytes. This behavior indicates a decreased thermostability of the cell membrane, which is used as an indirect measurement of heat stress tolerance in several species, such as cotton (Ashraf et al. 1994), sorghum (Marcum 1998), beans (Ismail and Hall 1999), and barley (Wahid and Shabbir 2005). The membrane properties can be adjusted to increase the temperature range tolerated by plants, such as through changes in fatty acid composition. These properties may be linked with the tolerance of photosynthesis to high temperature stress. In Arabidopsis plants grown at high temperatures, the total lipid content in the membranes decreased by half, and the ratio of unsaturated/saturated fatty acids decreased by one-third (Somerville and Browse 1991). Thus, the relationship between the membrane and thermal stability at high temperatures can vary between genotypes and can be used as an important selection criterion for heat stress tolerance. In some species, however, heat tolerance does not correlate with the degree of lipid saturation, suggesting that other factors in addition to membrane thermal stability limit growth at high temperatures. Proteins are easily denatured by high temperature. As a result, organisms have a set of heat shock proteins (HSPs) that are synthesized in response to high temperature and designed to prevent or reverse the effects of heat on protein denaturation. The expression of HSPs and heat shock factors (proteins that stimulate the expression of HSPs and other heat tolerance genes) increases thermal tolerance in many plants (Sun et al. 2002). Thus, various studies on thermal tolerance have evaluated the expression pattern of HSPs. High temperatures can cause considerable pre- and post-harvest damages, including leaf, twig and stem burns, leaf senescence and abscission, root growth inhibition and fruit discoloration, and damage, which eventually reduce production. For example, the growth of maize coleoptiles is reduced at 40 °C and ceases at 45 °C (Weaich et al. 1996). In sugarcane grown at high temperatures, the internodes are shorter, and there is greater tillering, early senescence, and a reduction in total biomass (Ebrahim et al. 1998). In rice, high soil temperatures (above 37 °C) between the final tillering stage and panicle initiation reduce the production, filling, and quality of grains (Arai-Sanoh et al. 2010). The development of pollen and anthers is also particularly sensitive to heat (Sato et al. 2006). In tomatoes, microsporogenesis is very sensitive to high temperatures, which can compromise fruiting at the anthesis phase (Sato et al. 2000). In beans, there were substantial reductions in the number of pods and in seed production and a 50 % inhibition of pollen tube viability at temperatures above 37/27 °C day/night (Prasad et al. 2002). During reproduction, a short period of heat stress can cause significant increases in the number and opening of flower buds and in the rate of floral abortion (Young et al. 2004). Furthermore, grain quality in maize is compromised by starch, protein, and oil content reductions (Wilhelm et al. 1999). In wheat, high temperatures during grain filling can change the quality and other physicochemical properties of the flour (Perrotta et al. 1998), including the protein content (Wardlaw et al. 2002). A reduction in the number of grains per spike also occurs at maturity in wheat (Ferris et al. 1998). 32 P. C. Cavatte et al. The reduction in grain weight in response to heat stress during the initial stage of filling can be partially attributed to the small number of cells in the endosperm, while the subsequent stress reduces starch synthesis due to the reduced availability of assimilates for the grain or direct effects on the synthesis processes in the grains (Yang et al. 2004). Photosynthesis is limited at temperatures above 35 °C due to the decreased activity of Rubisco. In addition, stomatal closure due to high evaporative air demand, which normally occurs at high temperatures, contributes to the reduction of photosynthesis. Most plants have a considerable capacity to adjust photosynthetic performance to their growth temperatures. Part of that capacity can be assigned to the properties of Rubisco, which is very thermostable. Notably, the rate of carboxylation may increase at 40 °C or above but at a lower speed than the oxygenation rate; consequently, higher photorespiration rates are frequent at high temperatures, which ultimately reduce carbon gain. Therefore, Rubisco that has more carboxylation capacity would be advantageous in situations of excessive heat (Zhu et al. 2010). Regardless, at moderately high temperatures, the low activation state of Rubisco would be the main cause of the decreased photosynthetic rate (Kim and Portis 2005) due to the suppression of Rubisco activase activity (Salvucci and Crafts-Brandner 2004). The lower regeneration capacity of ribulose-1,5-bisphosphate (RuBP) is another limiting factor for the photosynthetic rate at moderately high temperatures. The decrease in the Rubisco activation state may be a regulatory response to the limitation of one of the processes restricting the regeneration of RuBP, including damage to the thylakoid reactions (Sharkey 2005). When overexpressed, enzymes involved in RuBP regeneration, such as sedoheptulose-1,7-bisphosphatase, reduce the photosynthesis inhibition caused by heat (Feng et al. 2007). In this context, an improved response of photosynthesis to warmer temperatures by manipulating RuBP regeneration seems to be a promising way to increase thermal tolerance. The accumulation of compatible osmolytes is an important adaptive mechanism to heat stress. Under heat, different plant species can accumulate a variety of osmolytes, such as sugars, sugar alcohols, proline, and betaines, as a way to increase tolerance to heat stress, such as in water and salt stresses. Due to the pivotal role of osmolytes in response to environmental stresses, the selection of materials with a greater potential for the accumulation of these osmolytes, whether through traditional genetic breeding, marker-assisted selection or genetic engineering, is of special interest (Ashraf and Foolad 2007). In summary, the measurement of membrane thermal stability is a good indicator of high temperature tolerance. In this context, the electrolyte leakage method, which is used to check the loss of cell compartmentalization, seems very sensitive for the quantification of temperature tolerance, as demonstrated in rice (Tripathy et al. 2000) and beans (Thiaw and Hall 2004). The carbon isotope discrimination method in cotton was also positively linked with the selection of more heat-resistant materials (Lu et al. 1996). In addition to these tools, the chlorophyll a fluorescence and the SPAD (Soil–Plant Analysis Development) index have been used as good predictors of genotypes that are tolerant to heat stress. 3 The Physiology of Abiotic Stresses 33 3.4.2 Strategies to Increase Heat Tolerance To increase heat tolerance, crop breeding has been conducted by biotechnological methods, such as the control of membrane composition (Murakami et al. 2000) or the production of cultivars with the constitutive expression or overexpression of HSPs (Wang and Luthe 2003). Studies have indicated that plant tolerance to heat is a multigenic characteristic. Despite the genetic complexity and difficulties found, some heat-tolerant strains and hybrid cultivars have been developed in crops such as tomato (Scott et al. 1995). In beans, genes that increase heat tolerance cause a progressive effect on dwarfism due to shortening of the main stalk internodes (Ismail and Hall 1998). Similarly, in cotton, heat-tolerant strains have a substantial reduction in size (Lu and Zeiger 1994). These changes facilitate the planting density and may lead to lower leaf temperatures. High planting density can lead to changes in canopy architecture that can help to reduce thermal stresses with minimal effects on water consumption. 3.5 Nitrogen (N) Deficiency The availability of N in soils, especially in tropical soils, is usually far below that required for crop species to achieve satisfactory yields. This inadequacy is due to the fundamental importance of N for plant metabolism, as this element is a constituent of chlorophyll, proteins, and nucleic acids, among other molecules, and N is accumulated in large quantities in most species. The major way to overcome N deficiency is through nitrogenous fertilizers, which is applied in large quantities due to low N use efficiency (NUE, i.e., the mass of the harvested product per amount of N applied) of crops, usually approximately 30–40 % (Raun and Johnson 1999). This low NUE implies significant economic and environmental losses. Therefore, NUE optimization in crop species is essential to more sustainable agricultural production. The search for more efficient cultivars with respect to N use has been the goal of many breeding programs, especially those for cereals because 65 % of the N fertilizer production in the world is used for cereal crops (Garnett et al. 2009). The greatest efforts have been concentrated in rice, maize, and wheat crops (Hirel et al. 2007), for which there is already extensive knowledge about the genetic and physiological determinants of NUE. In this chapter, NUE physiological components, which are the product of the N uptake efficiency (NUpE, i.e., total N in the plant per amount of N applied), and the N utilization efficiency (NUtE, i.e., mass of the harvested product for the total N in the plant), are emphasized. 34 P. C. Cavatte et al. 3.5.1 Physiological Strategies for Increasing NUE 3.5.1.1 Physiological Strategies for Increasing NUpE The NUpE comprises the uptake of NO3- and NH4+ ions from the soil, which are held by a series of transporters located in the root epidermis. At the whole plant level, NUpE also depends on the cell storage capacity of these ions and their translocation to the uptake site, which may be in the roots or shoots, depending on the species (Chardon et al. 2010). NO3- uptake is performed by high-affinity transporter systems (HATS) or low-affinity transporter systems (LATS), which operate at micromolar and millimolar concentration ranges, respectively. Two gene families encode these transporters: NRT1, responsible for HATS, and NRT2, for LATS. NH4+ uptake also involves high and low-affinity transporters, which are encoded by the aminomethyltransferase (AMT) gene family. The absorption process is regulated by the ion concentration in the soil and by the N status in the plant. Typically, the level of glutamine acts as a negative signal for absorption. Once absorbed, NO3- can be stored in vacuoles, reduced in the root cortex cells, transported to the xylem and then to the shoots, or undergo efflux to the soil. NH4+ is almost entirely assimilated in the roots, with very little transported to the aerial parts, but recent studies have suggested that many species can transport considerable amounts of NH4+ in the xylem, when the assimilative capacity of the roots has been exceeded (Garnett et al. 2009). The most direct strategy for increasing NUpE would be an increase in the rootto-shoot ratio, enabling a greater investment in roots and consequently higher nutrient uptake. This strategy, however, also implies carbon deviation from the shoots to the roots, which can limit the carbon fixation ability of the plant as a whole and thus its productivity, as suggested by studies that have identified quantitative trait loci (QTLs) in which there is usually a negative correlation between the size of the root system and the productivity of the plant (Coque and Gallais 2007). Increased root length density (higher number of roots of a smaller diameter per volume) has been suggested to be a way to increase the specific surface of the root system without altering the proportion of carbon invested. Because N transport occurs primarily through mass flow, however, the root surface may have a limited importance for an increase in N uptake. These aspects are highly dependent on soil type and NO3- and NH4+ prevalence in the soil. Thus, having a larger root length density may be advantageous in soils with high leaching potential and/or where NH4+ (less mobile than NO3-) is the predominant form to be absorbed. The ability of post-anthesis N uptake capacity and its importance in grain filling also plays a key role in crop yield. In maize, 50 % of N is absorbed in the grains during the post-anthesis phase, whereas in rice and wheat, 70–90 % of N is absorbed prior to anthesis (Hirel et al. 2007). Therefore, the selection of genotypes that are able to extend N uptake during the crop cycle can be an important strategy to increase productivity and/or grain quality. In maize, the largest genotypic 3 The Physiology of Abiotic Stresses 35 variation found in protein content breeding was associated with higher N uptake after anthesis (Coque and Gallais 2007). A difficulty often found by crop breeders resides in the negative correlation between productivity and N concentration in grains. In winter wheat, post-anthesis N absorption was shown to be highly correlated with the relationship between protein concentration and productivity; whether this correlation occurs in other crops remains to be demonstrated (Bogard et al. 2010). At the physiological level, the increase in the number of transporters present in the root would also be a potential alternative for increasing N uptake. Roots usually have a high capacity for NO3- absorption, but overexpression of the involved transporters, especially HATS, did not translate into increases in NUE (Good et al. 2004). Alternatively, promising laboratory results have not been validated under field conditions (Quaggiotti et al. 2003). These HATS carriers are subjected to strong transcriptional and post-translational control, which would minimize N uptake during certain stages of the plant cycle, even when the concentration of N in the soil is abundant. Therefore, methods to ‘‘circumvent’’ the regulatory mechanisms of N uptake must be identified. Currently, the overexpression of alanine aminotransferase in the roots has resulted in increased NUpE in canola by reducing the levels of glutamine uptake and increasing the levels of alanine, a metabolite that would be ‘‘invisible’’ to the regulatory systems, allowing increased NO3- influx (Good et al. 2007). In addition to the increase in the NO3- influx, an increase in its storage capacity in the cells is also desirable. Although there is some genetic variability for this trait, the extent to which the NUE would be affected by an increased NO3- influx remains unclear (Hirel et al. 2007). An increase in N storage capacity should be coupled to a greater ability to use the N excess in the harvested product, if such an increase is a breeding program goal (Triboi and Triboi-Blondel 2002). 3.5.1.2 Physiological Strategies for Increasing NUtE NUtE is the optimum between two components, the N remobilization efficiency (NRE) and the N assimilation efficiency (NAE). NAE is the assimilation of inorganic N into organic N through a series of enzymatic reactions, which begin in the cytoplasm via the action of nitrate reductase (NR), which reduces NO3- to NO2-. The NO2- is then transported to plastids, where it is rapidly reduced to NH4+ by nitrite reductase (NiR), and the NH4+ is then assimilated by the glutamine synthetase/2-oxoglutarate aminotransferase (GS/GOGAT) pathway, where GS fixes NH4+ into a molecule of glutamate to form glutamine, which further reacts with a molecule of 2-oxoglutarate to form two molecules of glutamate via GOGAT catalysis. NRE is the result of a set of enzymes active during leaf senescence that are responsible for recycling NH4+ and amino acids from proteolysis events, and NRE also involves the efficiency of phloem transport of newly synthesized amino acids. The key enzymes involved in N remobilization are cytosolic glutamine synthetase (GS1), glutamate dehydrogenase (GDH), asparagine synthetase (AS), and other transaminases. The importance of these enzymes is due to their products, 36 P. C. Cavatte et al. asparagine and glutamine, which are the main amino acids transported in the phloem during senescence because of their high N/C ratios. NUtE is dependent on the efficiency of N in biomass formation, the effect of N on carbohydrate partition, the nitrate reduction efficiency, and the NRE of senescent tissues (Foulkes et al. 2009). Therefore, approaches to increase NUtE concern both carbon and N metabolism, mainly because the enzymes involved in these routes are co-regulated (Nunes-Nesi et al. 2010), and the existence of overlap in QTLs associated with N assimilation and starch synthesis (Zhang et al. 2010). For a long time, nitrate reduction was considered a limiting factor in N uptake, leading to great efforts to select cultivars with higher NR activities. In general, this approach resulted in small NUE increments due to the overexpression of NR in transgenic plants (Good et al. 2004). Recent evidence suggests that the limiting factor in nitrate reduction would be providing reducing power in the form of NADH, which makes mitochondrial metabolism a new target to improve NUE (Foyer et al. 2011). The most promising efforts have been related to the role of enzymes involved in N remobilization due to the uptake of N during grain filling being generally insufficient to meet the demand of the reproductive organs. Among these enzymes, GS1 has received greater attention because of its role in metabolism, in which it participates in all processes involving N uptake and recycling, mainly of NH4+ released in the photorespiration process, which can be ten times larger than the NH4+ derived from NO3- assimilation (Masclaux-Daubresse et al. 2010). The activity of GS1 is considered an excellent marker for an increase in physiological NUE, regardless of the developmental stage, and N nutritional conditions in the plant. The transgenic approaches related to GS1 overexpression are largely positive, but there are insufficient details about plant behavior under field conditions. In rice transgenic lineages overexpressing GS1, for example, there were NUtE increases but not NUE increases under N limiting field conditions with respect to the control plants (Brauer et al. 2011). The manipulation of photosynthesis at both the leaf and canopy levels is aimed at increasing the photosynthetic N use efficiency (PNUE), i.e., the amount of CO2 assimilated per unit of N in the leaf. PNUE is also an important strategy for increasing NUtE, especially in species in which assimilated CO2 has been little explored using breeding strategies. The PNUE depends on nitrogenous compound investment not directly related to the photosynthetic process, such as some secondary metabolic compounds. Reducing the content of N per area unit in parallel to the maintenance of the maximum photosynthetic rate is an important strategy to increase PNUE. In addition, increased solar radiation use efficiency (RUE, i.e., the amount of biomass produced per amount of absorbed solar radiation), through changes in plant architecture that lead to more erect leaves, allows a better irradiance distribution along the canopy, decreasing the canopy extinction coefficient, and increasing the RUE of the whole plant. Recently, emphasis has been placed on cultivars with a reduced rate of senescence in the field, i.e., cultivars with the ‘‘stay-green’’ characteristic. The functional stay-green phenotypes are those with delayed senescence combined 3 The Physiology of Abiotic Stresses 37 with extended photosynthetic activity, which translates into a greater capacity for the synthesis of photoassimilates throughout the crop cycle and a greater capacity for N uptake post-anthesis. A simple physiological marker for assessing these phenotypes involves the determination of the chlorophyll concentration in leaves, as green leaves generally have higher levels of N. In addition, spectral reflectance methods allow large population screening to identify stay-green phenotypes. Determining whether there is extended photosynthetic activity in these stay-green phenotypes in which the delayed senescence is not always followed by increased photosynthetic activity is, thus, essential. The stay-green phenotypes generally have lower RUEs that may impair the protein content in the grains. Hence, the feasibility of using these phenotypes depends on the characteristics desired in the final product. A point to be considered in this approach is which plant organ contributes the most to the N that is remobilized to the grain. In wheat, the true stalk can account for up to 30 % of remobilized N, and thus, an efficient strategy for wheat breeding would be to increase the stalk capacity to store N coupled to a high efficiency of stalk N remobilization in the seeds, allowing the leaves to remain green longer, contributing a higher supply of assimilates, and leading to a greater extent of N absorption during post-anthesis. Moreover, knowledge of crop physiology per se is crucial in determining the strategy to be adopted in breeding programs aiming to improve the quality of the product harvested because certain features are incompatible with others. For example, when a high content of starch (e.g., maize) or oil (e.g., canola) is desired in the harvestable product, the increased N concentration in seeds varies inversely with the starch and oil levels. Conversely, in rice and wheat, if higher grain protein content is desired, higher N concentration in seeds (or vegetative parts in the case of maize used for silage) becomes the target of the breeder, based on positive correlations between N and protein content in these grains (Chardon et al. 2012). Given the diversity of strategies aimed at NUE improvement, which may include molecular to macroscopic characteristics, multidisciplinary approaches, including agronomic, genetic, biochemical, and physiological aspects are of special interest. In the short term, the use of appropriate and sustainable agronomic practices is the best approach to increase NUE. One of the factors responsible for low NUE in crops lies in the screening assays conducted under excess N conditions, selecting plants that are less efficient in the use of N. In addition, soil conditions and the environment can be limiting factors for NUE during the crop cycle. Notably, the strategies that plants use to increase NUE under conditions of high N availability may be different from those used during low availability and may also vary between different species. Improving biological N fixation (BNF) in legumes and the introduction of this mechanism in non-legume crops has been the focus of several studies in recent years, particularly with respect to obtaining satisfactory production and using more sustainable technologies. The main BNF limitations are the high susceptibility of some rhizobia strains to environmental stresses and the low capacity of inoculant strains to compete with the native soil microbial population. One way to overcome these problems is the more careful selection and production of inoculants to be 38 P. C. Cavatte et al. used, isolating specific strains of interest for the species best adapted to the prevailing soil and climate conditions in the local culture (Cummings 2005). The use of transgenic rhizobia strains has also been tested and may be promising way to increase the plant-rhizobia interaction. Attempts to introduce the BNF in nonlegumes have been performed using N-fixing endophytic and plant growthpromoting bacteria, especially the diazotrophic bacterium Gluconacetobacter diazotrophicus, which colonizes the roots of some tropical grasses, particularly sugarcane but also sweet potatoes, coffee, pineapple, tea, mango, and rice (Deb Roy et al. 2010). Recently, Cocking et al. (2006), using a new inoculation technique, were able to successfully promote the colonization of maize, rice, wheat, and tomato roots with G. diazotrophicus, but whether these colonized plants have a better N nutrition under field conditions remains to be determined. 3.6 Phosphorus (P) Deficiency P is one of 17 essential elements needed for plant growth, playing a central role in a variety of processes, including energy generation, nucleic acid synthesis, membrane synthesis and stability, enzymatic activation and inactivation, redox reactions, signaling, carbohydrate metabolism, and N fixation. Although the total content of P in tropical soils may be relatively high, typically ranging from 500 to 2,000 ppm, the total bioavailable P may be only a few parts per million (Sanyal and Dedatta 1991) due to the adsorption of this element by iron and aluminum (Al) oxyhydroxides, the main constituents of the clay fraction of most tropical soils. Thus, low P availability is a major factor limiting the development and growth of plants. Hereafter, physiological components of P use efficiency (PUE; i.e., mass of harvested product per amount of P applied), which is the product of the P acquisition or uptake efficiency (PUpE; i.e., the total plant P per amount of P applied) and the P utilization efficiency (PUtE; i.e., the mass of harvested product per total amount of P in the plant), are emphasized. 3.6.1 Physiological Strategies for Increasing PUpE Processes that lead to greater PUpE include changes in the morphology and architecture of the root system, increased production and secretion of phosphatases, increased exudation of organic acids, and increased expression of inorganic phosphate (Pi) transporters (Lynch and Brown 2001). Although outside the scope of this chapter, the most prevalent evolutionary adaptation for terrestrial plants to acquire P occurs through mycorrhizal symbiosis (Tibbett and Sanders 2002). Adjustments that improve P acquisition in soil are key because of the relative immobility of this element, which has concentrations that are typically high in even the most superficial soil profiles. Root architecture refers to the spatial 3 The Physiology of Abiotic Stresses 39 configuration complexity of the root system, which arises in response to soil conditions. Given the uneven availability of P throughout the soil, root system architecture can improve soil exploitation, especially where mineral elements are more available. Changes in the root system architecture associated with improved P acquisition include basal roots with more horizontal growth, which results in shallower roots, the increased formation of adventitious and lateral roots and the increased density and length of root hairs. Overall, these characteristics were positively related to PUpE in beans (Lynch and Brown 2001; Wang et al. 2010), soybeans (Zhao et al. 2004), and rice (Wissuwa 2003). Under normal growth and development, plant roots exude a large variety of organic compounds, including simple sugars, organic acids, amino acids, phenolic compounds, quinones, flavonoids, hormones, proteins, and polysaccharides (Marschner 1995). Among these compounds, the organic acids, which favor P solubilization, and strigolactones, which are signaling compounds to mycorrhizal colonization, are key for adaptation to environments with low P availability. Recent studies on the mechanisms of P acquisition show that the plants grown in the presence of low P availability have higher root synthesis of low molecular weight organic acids. In some species, there has been an increase in acid phosphatase activity in roots and in its exudation in response to P deficiency, demonstrating their importance for PUpE increase (Vance et al. 2003). Increased phosphatase activity in the shoots has, however, been found, revealing that the main function of phosphatase appears to be associated with the remobilization of P in the plant as a strategy to keep the level of cytoplasmic Pi adequate to maintaining the metabolism and contributing to the PUtE increase. 3.6.2 Physiological Strategies for Increasing PUtE Processes that occur using less P and thus promote greater PUtE involve decreased growth rates, greater remobilization of internal Pi, changes in carbohydrate metabolism, avoidance of pathways that require a high supply of P, and the use of alternative respiratory systems (Uhde-Stone et al. 2003). In fact, the greater PUtE is mainly attributed to the efficient reuse and retranslocation of P stored in plants. The highest activity of acid phosphatase contributes to the increased efficiency of P utilization in beans through the remobilization of P from old leaves (Kouas et al. 2009). Studies on sorghum, tomatoes, wheat, and soybeans showed that the remobilization of P is not directly correlated only with senescence, as occurs in young tissues (Akhtar et al. 2008). Another important aspect is the release of Pi from the vacuole when P is limited (Akhtar et al. 2008). Pi is stored in vacuoles when P is abundant and is released under conditions of P deficiency, which helps the plants meet the P demands involved in maintaining Pi homeostasis. The mechanism of transporting P to the vacuole by Pi transporters in the tonoplast is still unclear, and high affinity Pi transporters could play an important role in P uptake (Ai et al. 2009). 40 P. C. Cavatte et al. The conservation of Pi internal reserves is considered an important adaptation for growth under conditions of low P availability, leading to greater PUtE. Because several enzymes in the glycolytic pathway depend on Pi or adenosines (ATP/ADP) as co-substrates, metabolism could be impaired by reduced Pi availability, as occurs in plants grown in soils with limited P. The energy generation and carbon skeleton production continue under these conditions, as shown by the exudation of substantial amounts of organic acids in the rhizosphere. In response to P limitation, many plants show remarkable metabolic flexibility, using alternative metabolic pathways instead of reactions that are Pi- or ATP-dependent. Although ATP and ADP concentrations are reduced, the pyrophosphate (PPi) concentration appears to be buffered (Duff et al. 1989; Dancer et al. 1990) under Pi deficiency. Considered a by-product of secondary metabolism, PPi can be used as an energy donor, enabling ATP availability for other limiting metabolic processes (Theodorou and Plaxton 1996; Plaxton and Carswell 1999). An example is a well-documented reaction that generates fructose-1,6-bisphosphate, which under P-deficient conditions is catalyzed by the enzyme PPi-dependent phosphofructokinase instead of ATP-dependent phosphofructokinase. Regarding the flexibility of the adaptive response to P deficiency, which is present in the glycolytic pathway, adjustments to maintain mitochondrial electron transport must be considered. The significant reduction of ADP and Pi concentration in plants subjected to P limitation inhibits the cytochrome c pathway because low concentrations of ADP and Pi can result in a high ATP/ADP ratio, which is responsible for the inhibition of oxidative phosphorylation. Thus, the induction of the alternative oxidase pathway during P deficiency has been proposed to be responsible for maintaining mitochondrial electron transport and for metabolic continuity (Vance et al. 2003). P deficiency usually results in the accumulation of secondary metabolites, such as flavonoids and indole alkaloids. The increased synthesis of anthocyanins is a frequent response of plants subjected to P deficiency, protecting the plant against photo-inhibitory damages (Takahashi et al. 1991). In addition to the involvement of senescence, phenolic compounds may be exudated in the rhizosphere in response to P deficiency, acting as chelating or reducing agents, and increasing the absorption of P. Sclerophylly is another common adaptation concerning the increase of phenolic metabolism in many species in low P availability environments. Sclerophillous species usually have hard and rigid leaves, with heavily lignified secondary walls, which assume a secondary metabolic relationship. Loveless (1962) postulated that the sclerophylly is an adaptation for nutrient storage, particularly of P and N. Unlike primary metabolism, secondary metabolism in general does not consume large amounts of Pi but may recycle significant amounts of Pi from phosphate esters. Secondary metabolism, however, produces excessive reducing equivalents, leading to the acidification of the cytosol, which can lead to the activation of alternative oxidase and other pathways to consume the excess reducing equivalents (Sakano 2001). 3 The Physiology of Abiotic Stresses 41 3.7 Aluminum (Al) Toxicity Al is the most abundant metal and the third most common element in the Earth’s surface, occurring mainly in the form of stable Al silicate complexes, which is non-toxic to plants (Ma and Ryan 2010). Under acidic conditions, however, the Al 3+ solubilizes and forms octahedron hexahydrate ((Al(H2O)3+ 6 ), also known as Al . This form of Al is toxic to plants, even at micromolar concentrations (Kochian et al. 2005). Globally, approximately 30 % of all land area consists of acid soils, and 50 % of the world’s cultivable lands are potentially acidic; thus, Al toxicity is one of the most important limitations to agricultural production (Piñeros et al. 2005). In Brazil, for example, more than 500 million hectares are formed of acid soils, especially those covered by savannah (Cerrado biome) vegetation (Vitorello et al. 2005). The soils of these areas have high acidity (average pH 4.6), a high concentration of Al and manganese and deficiencies of Ca2+, Mg2+, and P. These limitations, if not corrected, can lead to remarkable drops in crop productivity. The application of limestone (CaCO3 ? MgCO3) is an effective way to correct soil acidity but is often not an economically viable option for less capitalized producers. In addition, limestone corrects only the surface layers and is not effective in correcting subsoil acidity due to the low mobility of the limestone soluble components. The development of genotypes tolerant to soil Al has gained high importance. In the last decades, great efforts have been made to identify and characterize the mechanisms of plant tolerance to toxic levels of Al (Samac and Tesfaye 2003). Understanding these mechanisms is key for the development of procedures to rapidly select Al-resistant plants with good performance in acid soils (Barceló and Poschenrieder 2002). There is great variability in Al tolerance between species and even between genotypes within a species (Huang et al. 2009). The mechanisms of Al tolerance can basically be summarized into two classes: (i) those that eliminate absorbed Al or prevent/reduce its uptake by the roots (Al exclusion) and (ii) detoxification mechanisms, which usually act by Al complexation, followed by the transfer, and storage of these complexes in vacuoles (internal tolerance) (Hartwig et al. 2007). 3.7.1 Morpho-Physiological Responses and Alterations to Al Toxicity Al3+ has a high load/atomic radius, which allows it to form highly stable electrostatic bonds with negatively charged compounds, such as phosphates and carboxylic groups (Berthon 1996). Thus, many cellular structures, such as the cell wall, plasma membrane, cytoskeleton, and nucleus, are targets of Al3+ toxicity. 42 P. C. Cavatte et al. The primary site of Al accumulation and toxicity is the root meristem, specifically the distal part of the transition zone. The rapid root growth inhibition after exposure indicates that the Al quickly stops cell expansion and elongation before inhibiting cell division (Kochian et al. 2005). After more prolonged exposure of the root system to Al, its toxicity is manifested through a set of symptoms expressed in its continuous and increasing effect on the morphology and physiology of the roots, involving reductions in the following: biomass; the number and length of the roots, often combined with an increase in the mean radius and root volume; and the uptake of water and mineral nutrients, resulting in severe losses of root elongation and ultimately productivity. Recent studies have shown that the binding of Al to cell wall components alters the cation exchange capacity (Panda et al. 2009), viscoelasticity (Ma et al. 2004), and other properties of the cell wall, causing changes that block growth. Al can reduce the elasticity of the cell wall and stimulate the synthesis and accumulation of lignin (Peixoto et al. 2007) via the activation of a peroxidase (POD) linked to the cell wall, which is involved in the improvement of hydroxyproline-rich glycoprotein binding to phenolic acids. This improvement then contributes to secondary wall thickening, which results in lower root growth and elongation. Other enzymes activated by Al include NADH oxidase, phenylalanine ammonialyase (PAL), and lipoxygenase (LOX). NADH oxidases are responsible for the synthesis of H2O2, which is necessary for rapid polymer binding catalyzed by cell wall POD. PAL is a key enzyme in the biosynthesis of phenylpropanoids, and LOX is responsible for the peroxidation of membrane polyunsaturated fatty acids, leading to the formation of hydroperoxides. These compounds are highly reactive and are quickly degraded into compounds that, by the octadecanoic pathway, lead to the production of jasmonic acid, which acts in the lignin synthesis signaling pathway (Xue et al. 2008). Furthermore, Al can disrupt the cytoskeletal dynamics, interacting with microtubules and actin filaments (Kochian et al. 2004). Al can interfere with signal transduction, particularly in the Ca2+ signaling pathway (Rengel and Zhang 2003). Al may also increase callose synthesis, blocking the plasmodesmata (Sivaguru et al. 2000) and preventing cell wall loosening, thus limiting the expansion of cells (Jones et al. 2006). The plasma membrane has a negatively charged surface, making it a sensitive target for Al toxicity. Al strongly binds to phospholipids, which alters the lipid composition (Peixoto et al. 2001), reduces membrane fluidity, and increases the folding density of lipids (Chen et al. 1991). Moreover, Al can inhibit the H+-ATPase in the plasma membrane, preventing the formation, and maintenance of the H+ gradient (Ahn et al. 2001). Therefore, Al interferes with the secondary transport of ions, indirectly causing an alteration of ion homeostasis in root cells. Al also rapidly and effectively inhibits the influx of Ca2+ into cells by modulating the activity of transporters by changing the membrane potential (Kochian et al. 2005). Although most of the Al associated with the root system is found in the apoplast (Xue et al. 2008), a significant portion of this cation can penetrate rapidly and interact with molecules and subcellular structures of the symplast (Taylor et al. 2000), such as the nucleus of cells in the meristematic regions of the root apex. 3 The Physiology of Abiotic Stresses 43 Due to its affinity for phosphate groups, Al binds DNA, negatively affecting its template activity and chromatin structure (Silva et al. 2000) and modifying the cell division process (Barceló and Poschenriede 2002; Kochian et al. 2005). 3.7.2 Physiological Mechanisms of Al Tolerance Al tolerance or internal detoxification can be accomplished by its complexation in the symplast with different organic compounds and/or by compartmentalization of Al or its complexes in vacuoles (Hartwig et al. 2007). In this context, Al would change little or nothing in plant metabolism, allowing growth and development even after Al input into the symplast. This tolerance mechanism is found mainly in endemic species of regions with acidic soils, where the ability to address Al toxicity is a prerequisite for survival (Ryan and Delhaize 2010). There are, however, few species that accumulate high concentrations of Al in their shoots without suffering from its toxicity (Jansen et al. 2002). The main tolerance mechanisms that promote Al exclusion or prevent its absorption by the roots include Al immobilization in the cell wall, Al selective permeability in the plasma membrane, pH increases in the rhizosphere or the root apoplast and exudation of organic acids (e.g., citrate, oxalate and malate), and phenolic compounds by the roots. The synthesis and exudation of organic acids is perhaps the major mechanism of Al tolerance. Several evidences support this statement, as discussed by Kochian et al. (2004): (i) a strong correlation exists between Al tolerance and the exudation of organic acids in many species; (ii) the addition of organic acids in the nutritive medium reduces Al toxicity; (iii) Al/ organic acid complexes (di-and tri-carboxylic) do not cross the membrane and are not significantly absorbed by the roots; (iv) the exudation of organic acids, activated by Al, occurs at the root apex, the location of the primary effect of Al toxicity; (v) in general, the activation of the exudation mechanism is triggered specifically by Al3+; and (vi) there are, in the plasma membrane, anionic channels activated by Al that facilitate the efflux of organic acids. Two temporal patterns of organic acid exudation have been identified and characterized in plants (Ma et al. 2001). In Pattern I, the plants are characterized by having an almost immediate response to the release of organic acids by the roots when exposed to Al. This process appears to involve the activation of preexisting proteins, as found in wheat, tobacco, and barley. In plants with Pattern II (e.g., sorghum), the existence of a lag-phase between Al exposure and organic acid release is found, and this process is assumed to involve the induction of gene expression (Magalhães et al. 2007). Several studies have shown that excess Al induces ROS production, triggering oxidative stress (Boscolo et al. 2003). Genotypes with more robust antioxidant systems are usually more tolerant to excess Al, but the mechanism by which Al exacerbates the formation of ROS is still not fully understood (Darkó et al. 2004). Although how Al3+ acts in the cell to induce ROS formation is not exactly known, 44 P. C. Cavatte et al. its direct involvement in redox reactions seems unlikely because it is not a transition metal. Possibly, Al3+, due to its high affinity for biomembranes, can cause changes in membrane structure, thereby promoting the formation of ROS (Cakmark and Horst 1991). Moreover, the affinity of Al3+ for biomembranes can cause rigidity and facilitate chain reactions mediated by Fe2+ ions, which increases lipid peroxidation (Yamamoto et al. 2001). As Al accumulates preferentially in the roots, its most important effects occur in this region, and indirect effects are thought to occur in the shoots, mainly by affecting the translocation of nutrients (Lindon et al. 1999). In shoots, however, Al3+ can also cause oxidative stress, which can be indirectly monitored by the assessment of fluorescence parameters, particularly those monitoring the maximum quantum yield of photosystem II. 3.7.3 Identification of Tolerant Plants to High Al The use of Al-tolerant cultivars stands out as the most effective strategy for the production of economically important crops in acid soils. The development of plants resistant to mineral stresses is more profitable than correcting a soil nutrient deficiency or toxicity (Miyasaka et al. 2006). The most used method to evaluate Al toxicity is the comparison of the root growth of plants cultivated in a nutrient solution with an acid pH and Al with the root growth of control plants cultivated in the absence of Al. Therefore, Al differential tolerance may be assessed by comparing the root elongation of different genotypes in solutions with increasing concentrations of Al and is expressed as relative root growth, i.e., the root elongation ratio of the plants from the treatment with Al and those from the treatment without Al. Furthermore, the root growth potential after Al treatment can be evaluated. In this case, the seedlings are grown in a nutrient solution without Al. After approximately 48 h, they are transferred to a nutrient solution containing Al, where they remain for another 48 h, and are then returned to the initial solution for another 72 h. At that point, the seedlings are evaluated for the main root growth after the damage caused by Al3+ (Boscolo et al. 2003). 3.8 Conclusion Over the past years, our understanding of plant adaptation to abiotic stresses has increased enormously. However, there are still many gaps in our knowledge on the physiological mechanisms associated with successful crop growth and production in stressful environments. For instance, drought-stress signaling, which is essential for rational genetic engineering programs aimed at durable stress tolerance, is still very poorly understood (Chinnusamy et al. 2005b). 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