**4. Aspects of mineral nutrition in the relationship between water deficits and plant physiology**

Under water stress, plants develop various physiological and molecular mechanisms to maintain productivity. Among these mechanisms, Nepomuceno et al. (2001) highlight the activation of genes induced by drought to promote cell tolerance to dehydration and osmot‐ ic adjustment to maintain the water potential and turgor close to optimum levels. Addition‐ ally, to minimize the oxidative damage to cells generated by reactive oxygen species (ROS), plants develop antioxidant systems (Apel & Hirt, 2004).

In addition to the internal mechanisms in plants, the negative effects of water stress can be minimized through a balanced supply of nutrients (Waraich et al., 2011). Among the nu‐ trients classified as essential (Dechen & Nachtigall, 2006), potassium (K), phosphorus (P) and calcium (Ca) are the most studied in relation to their roles in reducing the effects of wa‐ ter stress on the physiology of soybean (Waraich et al, 2011).

### **4.1. Effects of potassium**

early water stress treatments, while for the cultivars 'MG/BR-46' (Conquista) and 'BR 16' (Stolf-Moreira et al., 2011), Ci increased as the water deficit progressed, indicating differ‐ ent physiological responses for different soybean cultivars. Furthermore, an increase in the intrinsic efficiency of water use was observed when the cultivars 'CD 202', 'CD 226RR', BR 16 and EMBRAPA 48 were subject to episodes of water restriction (Catuchi et al., 2011 and 2012). According to Manavalan et al. (2009), this increase may indicate bet‐ ter control of water loss via transpiration, contributing to the productivity of soybean. However, it is important to take into account that the studies discussed herein used dif‐ ferent methods of water stress induction, which could interfere with making more relia‐

A Comprehensive Survey of International Soybean Research - Genetics, Physiology, Agronomy and Nitrogen

Reduction of net photosynthesis in soybean plants can be induced by both stomatal and non-stomatal factors (of both biochemical and photochemical origin). When a crop is sub‐ jected to a water deficit, the plants can reduce their stomatal conductance (gs), limiting the entry of CO2 into the substomatal chambers and thus reducing the diffusion of carbon to the site of carboxylation, resulting in significant decreases in carbon assimilation (Yu et al., 2004). Furthermore, Flexas et al. (2006a) report that the effects of water stress on the initial activity of Rubisco may be reproduced by induction of stomatal closure, independent of the reduction in the relative water content in the leaves of soybean plants. Thus, we can expect a lower regulation of photochemical and biochemical processes when the availability of CO2 is the most limiting component for photosynthesis in plants under severe water stress (Flexas

Although Pankovic et al. (1999) report that the content of Rubisco in soybean leaves increas‐ es as plants acclimate to water stress, other authors have observed that under the imposition of water deficits in pine, tobacco and soybean, there is reduced transcription of the subunits of this enzyme (Pelloux et al., 2001, Kawaguchi, et al., 2003, Majumdar et al., 1991, respec‐ tively). Reduction of the Rubisco activity in soybean plants under drought stress can be in‐ duced by reducing the content of the enzyme itself and possibly through increases in strongly binding inhibitors, as reported by Flexas et al. (2006a). When stomatal closure oc‐ curs for a period of several days, another mechanism involving gene expression can operate, resulting in a decrease in the total amount of Rubisco and/or an increase in the content of

Moreover, when the availability of CO2 and biochemical activity are reduced due to wa‐ ter deficits, the excess reductants in the photochemical apparatus must be dissipated as heat or drained through alternative electron sinks (Miyake et al., 2009, 2010) to reduce photoinhibition and the production of reactive oxygen species (ROS). Studies have re‐ vealed that PSII of soybean plants is resistant to moderate water stress (Kirova et al., 2008), and the potential quantum efficiency of PSII (Fv / Fm) and electron transport rate (ETR) are not altered by the imposition of water stress (Ohashi et al., 2006). Bertolli et al. (2012) reported that the decrease in the ETR was more sensitive than the decrease of Fv / Fm when the relative water content declined in soybean plants (cv. CD202), supporting the idea that the potential photochemical efficiency would not be readily affected by wa‐ ter deficiency. However, the reduction of the ETR could be due to a lower energy (ATP /

ble and suitable comparisons among cultivars (Bertolli et al., 2012).

inhibitors that bind strongly to this enzyme (Flexas et al., 2006a).

et al. 2006b).

Relationships

282

Potassium is considered to be the second most extracted element in soybean crops (Malavol‐ ta, 2006). From 1,000 kg of seeds produced by a soybean crop, 20 kg of K2O can be extracted (Mascarenhas, 2004). In this context, in some agricultural production systems, particularly tropical systems, K application is required to ensure soil productivity. More than 60 en‐ zymes involved in cell metabolism are K dependent for normal activity because this nutrient

is an important enzymatic activator (Prado, 2008). Moreover, K plays an important role in cell expansion, which involves the formation of a large central vacuole occupying 80% to 90% of cell volume.

Under water deficit conditions, stomatal conductance is reduced (Oliveira et al, 2005) conse‐ quently decreasing the intercellular CO2 concentration (Kaiser, 1987, Lawlor & Tezara, 2009). Thus, the light energy used for the fixation of CO2 is diverted to O2, generating high accu‐ mulation of ROS in the chloroplast (Pitzschke et al, 2006). According to Cakmak (2005), when plants are grown under low K availability, the production of free radicals may be in‐ creased because the lack of this nutrient disturbs the opening and closing mechanism of sto‐ mata, causing a reduction of photosynthesis, and consequently, the excess electrons are diverted to the production of ROS. Therefore, under conditions of water stress, the plant ex‐ hibits an increased demand for K to maintain photosynthesis and protect the chloroplasts from oxidative damage. This author also stresses the importance of K in the translocation of assimilates. Under K deprivation, there is reduced exportation of the products of photosyn‐ thesis to the drain region of the plant. Thus, the accumulation of photoassimilates in the chloroplast can decrease the fixation of CO2 through down-regulation, thus increasing the generation of ROS.

According to Prado (2008), K promotes maintenance of the turgor of guard cells, allow‐ ing better opening and closing dynamics of the stomatal pores. Sangakkara et al. (2000) evaluated the effect of moisture and K fertilization on the physiology of two common bean cultivars and observed that the addition of K to the system via a nutrient solution promoted an increased photosynthetic rate under conditions of water stress in both culti‐ vars. Catuchi et al. (2012) evaluated the net CO2 assimilation rate (A) in two soybean cul‐ tivars under water deficit conditions as well as 12 hours after rehydration and following supplementation with 0, 90 and 180 mg dm-3 K. The authors concluded that in general, the A values (Figure 3) in both cultivars decreased by 50% under water deficit, irrespec‐ tive of the K level. In contrast, after rehydration, the cultivar BR 16 showed A values that were 27% higher in plants without the addition of K and 42% higher in plants sup‐ plied with 90 mg dm-3 K compared to the values in plants under drought. However, the higher dose of K did not allow the recovery of A after rehydration. Moreover, Embrapa 48 responded positively to supplementation with two doses of K in terms of the recov‐ ery of A. While in plants without added K, there was no recovery of A observed. Plants that received doses of 90 and 180 mg dm-3 K showed A values that were 57 and 38% higher, respectively, than those in plants under water stress. These responses indicate that K may promote greater recovery of photosynthesis in soybean after a period of wa‐ ter restriction. According to Flexas et al. (2004), the intensity and duration of water re‐ striction are key factors that define the speed and rate of recovery of plants after rehydration. In general, plants subjected to severe drought stress exhibit recovery of only 40-60% of the maximum photosynthetic rate on the next day. In a study performed by Catuchi et al. (2012), these values were only achieved in plants that were supplemented with K. The response of the recovery of plants supplied with K via fertilization may be related to the influence of this nutrient on the repair of oxidative damage to cells under conditions of water stress (Soleimanzadeh et al., 2010). The higher photosynthetic rates of plants supplied with K after recovery could provide greater restoration of plant growth, minimizing productivity losses.

**Figure 3.** Average of net photosynthesis (Pn) of cultivars BR-16 (A) and Embrapa 48 (B) grown under water stress (40%) and 12 hours after rehydration. The letters above the bars indicate the statistical difference (p < 0,005) between the water levels in each dose of potassium (adapted from Catuchi et al, 2012).

#### **4.2. Effects of phosphorus**

is an important enzymatic activator (Prado, 2008). Moreover, K plays an important role in cell expansion, which involves the formation of a large central vacuole occupying 80% to

A Comprehensive Survey of International Soybean Research - Genetics, Physiology, Agronomy and Nitrogen

Under water deficit conditions, stomatal conductance is reduced (Oliveira et al, 2005) conse‐ quently decreasing the intercellular CO2 concentration (Kaiser, 1987, Lawlor & Tezara, 2009). Thus, the light energy used for the fixation of CO2 is diverted to O2, generating high accu‐ mulation of ROS in the chloroplast (Pitzschke et al, 2006). According to Cakmak (2005), when plants are grown under low K availability, the production of free radicals may be in‐ creased because the lack of this nutrient disturbs the opening and closing mechanism of sto‐ mata, causing a reduction of photosynthesis, and consequently, the excess electrons are diverted to the production of ROS. Therefore, under conditions of water stress, the plant ex‐ hibits an increased demand for K to maintain photosynthesis and protect the chloroplasts from oxidative damage. This author also stresses the importance of K in the translocation of assimilates. Under K deprivation, there is reduced exportation of the products of photosyn‐ thesis to the drain region of the plant. Thus, the accumulation of photoassimilates in the chloroplast can decrease the fixation of CO2 through down-regulation, thus increasing the

According to Prado (2008), K promotes maintenance of the turgor of guard cells, allow‐ ing better opening and closing dynamics of the stomatal pores. Sangakkara et al. (2000) evaluated the effect of moisture and K fertilization on the physiology of two common bean cultivars and observed that the addition of K to the system via a nutrient solution promoted an increased photosynthetic rate under conditions of water stress in both culti‐ vars. Catuchi et al. (2012) evaluated the net CO2 assimilation rate (A) in two soybean cul‐ tivars under water deficit conditions as well as 12 hours after rehydration and following supplementation with 0, 90 and 180 mg dm-3 K. The authors concluded that in general, the A values (Figure 3) in both cultivars decreased by 50% under water deficit, irrespec‐ tive of the K level. In contrast, after rehydration, the cultivar BR 16 showed A values that were 27% higher in plants without the addition of K and 42% higher in plants sup‐ plied with 90 mg dm-3 K compared to the values in plants under drought. However, the higher dose of K did not allow the recovery of A after rehydration. Moreover, Embrapa 48 responded positively to supplementation with two doses of K in terms of the recov‐ ery of A. While in plants without added K, there was no recovery of A observed. Plants that received doses of 90 and 180 mg dm-3 K showed A values that were 57 and 38% higher, respectively, than those in plants under water stress. These responses indicate that K may promote greater recovery of photosynthesis in soybean after a period of wa‐ ter restriction. According to Flexas et al. (2004), the intensity and duration of water re‐ striction are key factors that define the speed and rate of recovery of plants after rehydration. In general, plants subjected to severe drought stress exhibit recovery of only 40-60% of the maximum photosynthetic rate on the next day. In a study performed by Catuchi et al. (2012), these values were only achieved in plants that were supplemented with K. The response of the recovery of plants supplied with K via fertilization may be related to the influence of this nutrient on the repair of oxidative damage to cells under

90% of cell volume.

Relationships

284

generation of ROS.

Because of its role in the formation of adenosine triphosphate (ATP), phosphorus (P) plays key roles in the production of energy necessary for photosynthesis, the translocation of as‐ similates and many other metabolic processes. In its inorganic form, P is the substrate or end product in many enzymatic reactions, including photosynthesis and carbohydrate metabo‐

lism, and it is essential for the regulation of metabolic pathways in the cytoplasm and chlor‐ oplast, sucrose and starch synthesis, triose phosphate transport, translocation of sucrose and hexose synthesis (Araújo & Machado, 2006).

According to Lantmann & Castro (2004), for each ton of soybeans produced, the plant con‐ sumes 15 kg of P2O5 on average. Under conditions of low soil water availability, there is a marked reduction in P uptake by plants (Santos et al., 2006). When there is a lack of inorgan‐ ic P (Pi) in the chloroplast, decreases in the production of ATP and NADPH may occur, re‐ sulting in a decrease in the regeneration of ribulose-1,5-biphosphate, which is crucial in the photosynthetic assimilation of CO2 (Lawlor & Cornic, 2002).

The decrease in ATP synthesis in the chloroplast may be caused by low availability of free cytoplasmic Pi, which is exchanged for triose phosphate in the chloroplast by phosphate transporters that use Pi as a substrate (Flügge et al., 2003). The carbon partitioning between starch and sucrose is dependent on the concentration of cytoplasmic Pi, which regulates the export of triose-P from the chloroplast to the cytosol, and a decrease in the recycling of P between the cytoplasm and chloroplasts can generate inhibition of photosynthesis via carbo‐ hydrate accumulation (Foyer, 1988). During drought periods lasting approximately ten days, the diffusive flux of P from the soil to plants stops almost completely, causing a signif‐ icant loss of productivity (Novais & Smyth, 1999). Thus, there is a need for a stock of P un‐ der optimal conditions of water availability to reduce the effects of the lack of P during water stress (Prado, 2008). Furthermore, after rehydration, the absorption and uptake of P should begin rapidly to restore the diffusion flow.

The direct role of P in the maintenance of plant productivity under low water availability is also related to the maintenance of stomatal conductance (Waraich et al., 2011). This function of P is associated with the osmotic regulation of stomatal guard cells because P supplemen‐ tation can be related to the accumulation of proline, which is an important regulator of cell osmolarity (Al-Karaki et al., 1996). Firmano et al. (2009) evaluated the effects of P on photo‐ synthesis in soybean plants grown under a water deficit and observed (Figure 4) that fertili‐ zation with 200 kg ha-1 P maintained net photosynthesis under water stress better in comparison to what was observed in plants that were not supplemented. According to these authors, these results were due to increased stomatal conductance promoted by P under conditions of water restriction.

Santos et al. (2006) evaluated the effect of foliar supplementation with inorganic phosphate (Pi) in two common bean genotypes, A320 and Ouro Negro, under water deficit conditions for 7 days. They observed that the rates of net photosynthesis and stomatal conductance were not affected by supplementation of Pi during dehydration in either genotype. Howev‐ er, after rehydration, stomatal conductance and photosynthesis were increased associated with foliar Pi being supplied in relation to the plants without Pi supplementation. Likewise, as noted by Firmano et al. (2009) in soybean, the role of Pi in the regulation of photosynthe‐ sis recovery after a water deficit appears to be important in reducing the deleterious effects of a temporary lack of water.

**Figure 4.** Average of net photosynthesis (Pn) and stomatal conductance (Gs) of soybean plants grown with and with‐ out water restriction. The lowercase letters above the bars indicates the statistical difference (p < 0,05) between the P doses in each water level (adapted from Firmano et al, 2009).

#### **4.3. Effects of calcium**

lism, and it is essential for the regulation of metabolic pathways in the cytoplasm and chlor‐ oplast, sucrose and starch synthesis, triose phosphate transport, translocation of sucrose and

A Comprehensive Survey of International Soybean Research - Genetics, Physiology, Agronomy and Nitrogen

According to Lantmann & Castro (2004), for each ton of soybeans produced, the plant con‐ sumes 15 kg of P2O5 on average. Under conditions of low soil water availability, there is a marked reduction in P uptake by plants (Santos et al., 2006). When there is a lack of inorgan‐ ic P (Pi) in the chloroplast, decreases in the production of ATP and NADPH may occur, re‐ sulting in a decrease in the regeneration of ribulose-1,5-biphosphate, which is crucial in the

The decrease in ATP synthesis in the chloroplast may be caused by low availability of free cytoplasmic Pi, which is exchanged for triose phosphate in the chloroplast by phosphate transporters that use Pi as a substrate (Flügge et al., 2003). The carbon partitioning between starch and sucrose is dependent on the concentration of cytoplasmic Pi, which regulates the export of triose-P from the chloroplast to the cytosol, and a decrease in the recycling of P between the cytoplasm and chloroplasts can generate inhibition of photosynthesis via carbo‐ hydrate accumulation (Foyer, 1988). During drought periods lasting approximately ten days, the diffusive flux of P from the soil to plants stops almost completely, causing a signif‐ icant loss of productivity (Novais & Smyth, 1999). Thus, there is a need for a stock of P un‐ der optimal conditions of water availability to reduce the effects of the lack of P during water stress (Prado, 2008). Furthermore, after rehydration, the absorption and uptake of P

The direct role of P in the maintenance of plant productivity under low water availability is also related to the maintenance of stomatal conductance (Waraich et al., 2011). This function of P is associated with the osmotic regulation of stomatal guard cells because P supplemen‐ tation can be related to the accumulation of proline, which is an important regulator of cell osmolarity (Al-Karaki et al., 1996). Firmano et al. (2009) evaluated the effects of P on photo‐ synthesis in soybean plants grown under a water deficit and observed (Figure 4) that fertili‐ zation with 200 kg ha-1 P maintained net photosynthesis under water stress better in comparison to what was observed in plants that were not supplemented. According to these authors, these results were due to increased stomatal conductance promoted by P under

Santos et al. (2006) evaluated the effect of foliar supplementation with inorganic phosphate (Pi) in two common bean genotypes, A320 and Ouro Negro, under water deficit conditions for 7 days. They observed that the rates of net photosynthesis and stomatal conductance were not affected by supplementation of Pi during dehydration in either genotype. Howev‐ er, after rehydration, stomatal conductance and photosynthesis were increased associated with foliar Pi being supplied in relation to the plants without Pi supplementation. Likewise, as noted by Firmano et al. (2009) in soybean, the role of Pi in the regulation of photosynthe‐ sis recovery after a water deficit appears to be important in reducing the deleterious effects

hexose synthesis (Araújo & Machado, 2006).

Relationships

286

photosynthetic assimilation of CO2 (Lawlor & Cornic, 2002).

should begin rapidly to restore the diffusion flow.

conditions of water restriction.

of a temporary lack of water.

The main functions of Ca in plants are acting as a component of the cell wall and as a second messenger in signaling associated with different processes in the cell. This nutrient plays an important role in ion uptake, root development and the germination of pollen grains (Vitti et al., 2006). During stress, Ca plays an important role in the regulation of plant metabolism, along with the calmodulin protein, which can promote the maintenance of cellular metabo‐ lism under water deficit conditions (Waraich et al, 2011). Ca assists the plant in its recovery after a water shortage because this nutrient functions in the activation of the ATPase en‐ zyme in the cell membrane, promoting the pumping back to the cell of electrolytes that were lost because of membrane damage caused by water deficit (Palta, 1990, Waraich et al., 2011).

In addition to the direct effects of K, P, and Ca on the maintenance of plant metabolism un‐ der water deficit conditions, balanced nutrition regarding all essential elements (both mac‐ ro- and micronutrients) can support plant development under limiting conditions by improving the initial steps of vegetative growth, such as leaf area expansion. This improved growth will allow the achievement of high photosynthetic rates and, hence, good root devel‐ opment, thereby improving the absorption of water into deeper layers and allowing the plants to survive water deficit periods.

### **4.4. Future directions**

Breeders and geneticists involved in soybean breeding are interested in consolidating the current knowledge about physiology and functional genomics to improve crop breeding programs (Manavalan et al., 2009), especially based on studies aimed at providing the infor‐ mation needed to improve the resistance / tolerance of cultivars to a multitude of stress fac‐ tors (Kulcheski et al., 2011, Makbul et al., 2011). Through proteomic analysis, 145 genes that are differentially expressed according to the imposition of water stress were identified in two soybean cultivars, MG/BR46 [Conquista] and BR 16, that are considered tolerant and sensitive to water deficits, respectively (Stolf-Moreira et al., 2011). These genes were classi‐ fied into nine functional categories: energy, transcription factors, metabolism, stress re‐ sponses, protein synthesis, cell communication, the cell cycle, cellular transport, and other unknown functions. Additionally, 11 micro-RNAs that show different expression patterns during the imposition of biotic and abiotic stress were identified in the cultivars 'Embrapa 48' (tolerant to drought stress) and 'BR 16' (sensitive to water stress) (Kulcheski et al., 2011), and the transcription of several other proteins related to oxidative damage, isoflavonoids and lignin synthesis was detected in soybean under water stress (Yamaguchi et al., 2010). Furthermore, Alam et al. (2010) reported that there are two enzymes involved in carbohy‐ drate metabolism (UDP-glucose pyrophosphorylase and 2.3-biphosphoglycerate independ‐ ent phosphoglyceratemutase) that are suppressed after exposure to a water deficit. The levels of these enzymes tended to revert to the basal level after rehydration of the plants, suggesting that the change in the allocation of carbon in soybean plants under drought may indicate an adaptive response. According to the authors of this report, the metabolism of carbohydrates is one of the processes that are most susceptible to water stress, after photo‐ synthesis. Other studies have identified several soybean wildtypes that can be specifically adapted to adverse conditions, such as wind, water logging, salinity and water deficits, and may be useful for identifying genes related to tolerance / resistance to a variety of biotic and abiotic stresses (Lee et al., 2010). Such studies are required because genetic diversity has been lost in the process of domestication of *G. max* (Hyten et al., 2006), and wildtype soy‐ bean have been useful for contributing new and unique genes to increase yields under dif‐ ferent worldwide crop conditions (Wang et al., 2004).

Moreover, as discussed in previous sections of this chapter, to improve soybean growth un‐ der water deficit conditions, the application of additional strategies is necessary, such as constant development of new soil management techniques, allowing the development of root systems to increase the water intake capacity as well as provide balanced nutrition to the crop, supporting adequate development of the plants throughout their life cycle.
