**Tolerance to Drought in Leguminous Plants Mediated by** *Rhizobium and Bradyrhizobium*

Allan Klynger da Silva Lobato, Joaquim Albenísio Gomes da Silveira, Roberto Cezar Lobo da Costa and Cândido Ferreira de Oliveira Neto

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/54094

**1. Introduction**

The water availability is considered the climatic factor with large effect on agricultural productivity, being responsible to determine species distribution in different climate zones around the globe [1]. Effects of drought depend of plant development stage, inten‐ sity, and duration of the water restriction. In other hand, plant adaptive strategies will determine the tolerance level, and consequently your survival on these conditions of in‐ adequate water supply [2].

Water deficit is an abiotic factor that affects the agricultural production with high frequency and intensity, influencing aspects related to plant development, such as decrease in photo‐ synthesis rate, reduction in leaf area [3], and stomata closing [4]. Crops normally present performance affected by water deficiency, which can cause lower growth and development (Figure 1), with progressive reduction in leaf dry matter [5] and consequent repercussion on production parameters, such as number of grains and pods per plant.

Root system presents complex strategy aiming to maintain water supply in conditions of water deficit, by increasing the root elongation rate and completely inhibiting the shoot [6]. On the other hand, plants growing in low water potentials normally present root thinner [7], and this morphological modification is an adaptation to increase water absorption efficien‐ cy. Therefore, a combination of changes in morphological, physiological and biochemical levels are necessary to plant survival in environments affected by drought.

The persistence of rhizobial strains, and their symbiotic performance in current and subse‐ quent seasons are affected by numerous biotic and abiotic factors [10], with drought stress and nitrogen deprivation, being among the most significant in many parts of the world [11]. Other important factor is the root exudation ability, which it will determine plant microbe

Tolerance to Drought in Leguminous Plants Mediated by *Rhizobium and Bradyrhizobium*

http://dx.doi.org/10.5772/54094

Molybdenum is an essential element for soil microorganisms, since it serves as a cofactor for different enzymes involved in the metabolism of nitrogen, carbon and sulfur. Before the synthesis of molybdoenzymes, uptake of molybdate, which is the more stable form of mo‐ lybdenum), its activation to an appropriate form, and its incorporation into the organic frac‐

The presence of molybdenum is necessary during formation of several proteins, including the nitrogenase, the molybdoenzyme that reduces atmospheric dinitrogen (N2) into ammo‐

nitrous oxide (N2O) to N2, when the cells are cultured under oxygen-limiting conditions [14]. The first reaction of denitrification, is carried out by the periplasmic Mo-containing nitrate reductase [15]. In addition, the reaction under normal conditions is described as N2 + 8 e–

The enzyme mechanism requires reduction of the Fe protein by electron donors such as fer‐ redoxin and flavodoxin, transfer of single electrons from the Fe protein to the MoFe protein (which is dependent on MgATP hydrolysis) and, finally, internal electron transfer in the MoFe protein by the P cluster to the FeMo cofactor substrate-binding site. Each electrontransfer step requires an obligatory cycle of association of the Fe and MoFe proteins to form

**Figure 3.** Schematic representation of the nitrogenase Fe protein cycle. The Fe protein dimer is shown in light blue with the cube representing the [4Fe–4S] cluster coloured black to indicate the reduced form and red to represent the oxidized form. The α and β subunits of the MoFe protein are depicted as orange and pink, respectively, the yellow squares represent the P cluster and the black diamond represents the FeMo cofactor. Changes in the oxidation state

) [13]. This bacterium is also capable of denitrification, via nitric oxide (NO) and

+ 8

51

associations so that the survival and tolerance of rhizobia during water restriction.

tion of the molybdenum-cofactors, are required [12].

+ 16 MgATP → 2 NH3 + H2 + 16 MgADP + 16Pi.

a complex (Figure 3), after which the two components dissociate [16].

nia (NH4

H+

+

of the MoFe protein are not shown [16].

**Figure 1.** Visual aspect of shoot in *Phaseolus vulgaris* plants exposed to drought by four days.

The biological fixation of nitrogen is the capacity of an organism to divide the molecule of nitrogen (N2) and to combine hydrogen atoms (H+ ), forming ammonium (NH4 + ) [8], being carried out by a distinct group of microorganisms, singly or under symbiosis. The *Bradyrhi‐ zobium* and *Rhizobium* genders are described as soil bacteria that have ability to infect root hair of leguminous plants, and it to induce nodule formation (Figure 2), with subsequent fix‐ ation of nitrogen [9].

**Figure 2.** Visual aspect of root system of *Vigna unguiculata* plants inoculated with *Bradyrhizobium*. The red arrows indicate nodules formed after infection process.

The persistence of rhizobial strains, and their symbiotic performance in current and subse‐ quent seasons are affected by numerous biotic and abiotic factors [10], with drought stress and nitrogen deprivation, being among the most significant in many parts of the world [11]. Other important factor is the root exudation ability, which it will determine plant microbe associations so that the survival and tolerance of rhizobia during water restriction.

Molybdenum is an essential element for soil microorganisms, since it serves as a cofactor for different enzymes involved in the metabolism of nitrogen, carbon and sulfur. Before the synthesis of molybdoenzymes, uptake of molybdate, which is the more stable form of mo‐ lybdenum), its activation to an appropriate form, and its incorporation into the organic frac‐ tion of the molybdenum-cofactors, are required [12].

The presence of molybdenum is necessary during formation of several proteins, including the nitrogenase, the molybdoenzyme that reduces atmospheric dinitrogen (N2) into ammo‐ nia (NH4 + ) [13]. This bacterium is also capable of denitrification, via nitric oxide (NO) and nitrous oxide (N2O) to N2, when the cells are cultured under oxygen-limiting conditions [14]. The first reaction of denitrification, is carried out by the periplasmic Mo-containing nitrate reductase [15]. In addition, the reaction under normal conditions is described as N2 + 8 e– + 8 H+ + 16 MgATP → 2 NH3 + H2 + 16 MgADP + 16Pi.

**control drought**

The biological fixation of nitrogen is the capacity of an organism to divide the molecule of

carried out by a distinct group of microorganisms, singly or under symbiosis. The *Bradyrhi‐ zobium* and *Rhizobium* genders are described as soil bacteria that have ability to infect root hair of leguminous plants, and it to induce nodule formation (Figure 2), with subsequent fix‐

**Figure 2.** Visual aspect of root system of *Vigna unguiculata* plants inoculated with *Bradyrhizobium*. The red arrows

), forming ammonium (NH4

+

) [8], being

**Figure 1.** Visual aspect of shoot in *Phaseolus vulgaris* plants exposed to drought by four days.

nitrogen (N2) and to combine hydrogen atoms (H+

ation of nitrogen [9].

50 Responses of Organisms to Water Stress

indicate nodules formed after infection process.

The enzyme mechanism requires reduction of the Fe protein by electron donors such as fer‐ redoxin and flavodoxin, transfer of single electrons from the Fe protein to the MoFe protein (which is dependent on MgATP hydrolysis) and, finally, internal electron transfer in the MoFe protein by the P cluster to the FeMo cofactor substrate-binding site. Each electrontransfer step requires an obligatory cycle of association of the Fe and MoFe proteins to form a complex (Figure 3), after which the two components dissociate [16].

**Figure 3.** Schematic representation of the nitrogenase Fe protein cycle. The Fe protein dimer is shown in light blue with the cube representing the [4Fe–4S] cluster coloured black to indicate the reduced form and red to represent the oxidized form. The α and β subunits of the MoFe protein are depicted as orange and pink, respectively, the yellow squares represent the P cluster and the black diamond represents the FeMo cofactor. Changes in the oxidation state of the MoFe protein are not shown [16].

Several leguminous such as *Vigna unguiculata* and *Cicer arietinum* are considered tolerant to water deficit, and important mechanisms were developed by this species to tolerate inade‐ quate water supply. For example, biochemical modifications in carbon metabolism, such as increase in sucrose [17], as well as significant interference in nitrogen metabolism, like re‐ duction of soluble proteins [5] and increase in total amino acids [18] contribute to osmotic adjustment of these plants (Figure 4).

**2. Objective**

water restriction.

Aims of this chapter is to define (i) water deficiency and biological fixation of nitrogen, to explain (ii) as this symbiotic process can promote beneficial repercussions to plant and microorganism, and to present (iii) the attenuation of negative impacts on nodule and plant, besides nitrogen compounds and morphological parameters of plants exposed to

Tolerance to Drought in Leguminous Plants Mediated by *Rhizobium and Bradyrhizobium*

http://dx.doi.org/10.5772/54094

53

Drought is environmental component that affect crop yields worldwide. In nature, this stress is multifaceted problems that are usually associated with other adverse circumstances, which limit plant performance such as water shortage and nutrient deficits. In order to as‐ sess the osmotic stress, Sassi et al. [19] monitored two *Phaseolus vulgaris* cultivars inoculated

Leaf osmotic potential (Ψo) decreased in stressed plants in both cultivars. A minimum value of −2.3 MPa was reached in Cv. 'Flamingo' plants under mannitol-induced osmotic stress (Figure 5 A). Ψo decreased in stressed nodules, reaching −1.3 MPa in Cv. 'Coco Blanc' and −1.7 MPa in Cv. 'Flamingo' (Figure 5 B). Therefore, Cv. 'Flamingo' showed a better osmotic

**Figure 5.** Variation of osmotic potential (Ψo) in response to osmotic stress in leaves (A) and nodules (B) mediated by

In control leaves of both cultivars, RWC remained close to 80% (Figure 6 A). After 15 days of osmotic treatment, RWC was 65%in mannitol-treated plants of Cv. 'Flamingo', and only

**3. Water maintenance in leaf and nodule produced by inoculation**

with *Rhizobium*, being cvs. 'Flamingo' (tolerant) and Cv. 'Coco Blanc' (sensitive).

adjustment response to osmotic stress both in leaves and nodules [19].

50 mM mannitol. Values represents mean ± SE (n=6) [19].

**Figure 4.** Glutamine synthetase activity (a), total soluble amino acids (b) and total soluble proteins (c) in Vigna ungui‐ *culata* plants cv. Vita 7 subjected to 4 days of water restriction and 2 days of rehydration. Means followed by the same letter are not significantly different by the Tukey test at 5% of probability. The bars represent the mean standard error and the arrow the arrow indicates the rehydration point [5].

## **2. Objective**

Several leguminous such as *Vigna unguiculata* and *Cicer arietinum* are considered tolerant to water deficit, and important mechanisms were developed by this species to tolerate inade‐ quate water supply. For example, biochemical modifications in carbon metabolism, such as increase in sucrose [17], as well as significant interference in nitrogen metabolism, like re‐ duction of soluble proteins [5] and increase in total amino acids [18] contribute to osmotic

**Figure 4.** Glutamine synthetase activity (a), total soluble amino acids (b) and total soluble proteins (c) in Vigna ungui‐ *culata* plants cv. Vita 7 subjected to 4 days of water restriction and 2 days of rehydration. Means followed by the same letter are not significantly different by the Tukey test at 5% of probability. The bars represent the mean standard error

and the arrow the arrow indicates the rehydration point [5].

adjustment of these plants (Figure 4).

52 Responses of Organisms to Water Stress

Aims of this chapter is to define (i) water deficiency and biological fixation of nitrogen, to explain (ii) as this symbiotic process can promote beneficial repercussions to plant and microorganism, and to present (iii) the attenuation of negative impacts on nodule and plant, besides nitrogen compounds and morphological parameters of plants exposed to water restriction.

## **3. Water maintenance in leaf and nodule produced by inoculation**

Drought is environmental component that affect crop yields worldwide. In nature, this stress is multifaceted problems that are usually associated with other adverse circumstances, which limit plant performance such as water shortage and nutrient deficits. In order to as‐ sess the osmotic stress, Sassi et al. [19] monitored two *Phaseolus vulgaris* cultivars inoculated with *Rhizobium*, being cvs. 'Flamingo' (tolerant) and Cv. 'Coco Blanc' (sensitive).

Leaf osmotic potential (Ψo) decreased in stressed plants in both cultivars. A minimum value of −2.3 MPa was reached in Cv. 'Flamingo' plants under mannitol-induced osmotic stress (Figure 5 A). Ψo decreased in stressed nodules, reaching −1.3 MPa in Cv. 'Coco Blanc' and −1.7 MPa in Cv. 'Flamingo' (Figure 5 B). Therefore, Cv. 'Flamingo' showed a better osmotic adjustment response to osmotic stress both in leaves and nodules [19].

**Figure 5.** Variation of osmotic potential (Ψo) in response to osmotic stress in leaves (A) and nodules (B) mediated by 50 mM mannitol. Values represents mean ± SE (n=6) [19].

In control leaves of both cultivars, RWC remained close to 80% (Figure 6 A). After 15 days of osmotic treatment, RWC was 65%in mannitol-treated plants of Cv. 'Flamingo', and only 45% in Cv. 'Coco Blanc'. These results indicate that osmotic stress caused an important re‐ duction in shoot water supply. The same trend was observed in nodules (Figure 6 B). In‐ deed, data showed decreased nodule RWC in both stressed cultivars. This decrease was higher in Cv. 'Coco Blanc' treated nodules [19].

ence in sensivity of N2 fixation in tropical legumes, under water stress, is not clearly under‐ stood [23]. Based in these problems reported, Figueiredo et al. [24] investigated Vigna unguiculata plants exposed to 3 inoculation forms (BR-2001, EI-6, and control) combined

Tolerance to Drought in Leguminous Plants Mediated by *Rhizobium and Bradyrhizobium*

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55

Water deficit response in cowpea appears to be directly related to a reduction in nodule mass (Table 1), which may (after a severe stress, S6) have affected nodule structural constitu‐ ents. However, in moderate stress (S3) the impact on nodule water content was higher than

(1)For S1 to S6 see Table 1. \*, \*\*Significant at the 0.05 and 0.01 probability level. In each column (lower letters) and in each line (capital letters), the means followed by the same letter do not differ statistically (*p<* 0.05) from each other,

**Table 1.** Nodule dry matter (NDM) and nodule water content (NWC) in cowpea with (BR-2001 and EI-6) and without

**5. Interference positive on nitrogen compounds of plants inoculated and**

Cowpea (*Vigna unguiculata* [L.] Walp.) is a leguminous with high protein content, large ca‐ pacity of fixation of the atmospheric nitrogen (N2) and low requirements to soil fertility [25], being frequently cultivated by farmers in Northern and Northeastern regions of the Brazil. This species constitutes the main subsistence culture, being the grain used as protein source in feeding [26]. Cowpea presents important agronomical characteristics, such as rusticity and precocity, besides being considered a plant adapted to conditions of limited or insuffi‐

(C) *Bradyrhizobium* spp. inoculation at different degrees of water stress [24].

with 6 different degrees of water stress (-1.5, -2.0, -4.0, -6.0, -8.0, and -10.0 kPa).

on the changes in nodule mass [24].

according to Tukey's test

**exposed to water deficit**

cient water availability [27].

**Figure 6.** Effect of mannitol-induced osmotic stress on relative water content (RWC) in Flamingo and Coco blanc bean cultivar leaves (A) and nodules (B). Values represents mean ± SE (n=6) [19].

Mannitol-induced water deficit produced substantial dehydration that led to decreasing Ψo (Figure 6). The decrease in Ψo is considered a potential mechanism of cellular drought re‐ sistance as it enables turgor maintenance and growth continuation [20]. Cv. 'Flamingo' ex‐ hibited lower Ψo under osmotic treatment. It was able to uptake more water and then grow more when exposed to decreased Ψo, thus it turned out to be a better drought tolerant culti‐ var than Cv. 'Coco Blanc' [21]. This may be attributed to maintenance of the leaf and nodule water status under stressed conditions (Figure 5). Several mechanisms could be involved in contributing to water retention.

## **4***. Bradyrhizobium* **ameliorates negative effects in plants exposed to drought**

The relationship between the water status in the plant and N2 fixation, mainly under water stress, and the changes in nodule morphology have been studied in some temper‐ ate legumes [22]. However, tropical legumes growing in arid regions, have not received adequate attention.

Even where information is available, the degree of water stress in the plants was not clearly defined, which makes it difficult to make comparisons. The structural basis for the differ‐ ence in sensivity of N2 fixation in tropical legumes, under water stress, is not clearly under‐ stood [23]. Based in these problems reported, Figueiredo et al. [24] investigated Vigna unguiculata plants exposed to 3 inoculation forms (BR-2001, EI-6, and control) combined with 6 different degrees of water stress (-1.5, -2.0, -4.0, -6.0, -8.0, and -10.0 kPa).

45% in Cv. 'Coco Blanc'. These results indicate that osmotic stress caused an important re‐ duction in shoot water supply. The same trend was observed in nodules (Figure 6 B). In‐ deed, data showed decreased nodule RWC in both stressed cultivars. This decrease was

**Figure 6.** Effect of mannitol-induced osmotic stress on relative water content (RWC) in Flamingo and Coco blanc bean

Mannitol-induced water deficit produced substantial dehydration that led to decreasing Ψo (Figure 6). The decrease in Ψo is considered a potential mechanism of cellular drought re‐ sistance as it enables turgor maintenance and growth continuation [20]. Cv. 'Flamingo' ex‐ hibited lower Ψo under osmotic treatment. It was able to uptake more water and then grow more when exposed to decreased Ψo, thus it turned out to be a better drought tolerant culti‐ var than Cv. 'Coco Blanc' [21]. This may be attributed to maintenance of the leaf and nodule water status under stressed conditions (Figure 5). Several mechanisms could be involved in

**4***. Bradyrhizobium* **ameliorates negative effects in plants exposed to**

The relationship between the water status in the plant and N2 fixation, mainly under water stress, and the changes in nodule morphology have been studied in some temper‐ ate legumes [22]. However, tropical legumes growing in arid regions, have not received

Even where information is available, the degree of water stress in the plants was not clearly defined, which makes it difficult to make comparisons. The structural basis for the differ‐

higher in Cv. 'Coco Blanc' treated nodules [19].

54 Responses of Organisms to Water Stress

cultivar leaves (A) and nodules (B). Values represents mean ± SE (n=6) [19].

contributing to water retention.

**drought**

adequate attention.

Water deficit response in cowpea appears to be directly related to a reduction in nodule mass (Table 1), which may (after a severe stress, S6) have affected nodule structural constitu‐ ents. However, in moderate stress (S3) the impact on nodule water content was higher than on the changes in nodule mass [24].


(1)For S1 to S6 see Table 1. \*, \*\*Significant at the 0.05 and 0.01 probability level. In each column (lower letters) and in each line (capital letters), the means followed by the same letter do not differ statistically (*p<* 0.05) from each other, according to Tukey's test

**Table 1.** Nodule dry matter (NDM) and nodule water content (NWC) in cowpea with (BR-2001 and EI-6) and without (C) *Bradyrhizobium* spp. inoculation at different degrees of water stress [24].

## **5. Interference positive on nitrogen compounds of plants inoculated and exposed to water deficit**

Cowpea (*Vigna unguiculata* [L.] Walp.) is a leguminous with high protein content, large ca‐ pacity of fixation of the atmospheric nitrogen (N2) and low requirements to soil fertility [25], being frequently cultivated by farmers in Northern and Northeastern regions of the Brazil. This species constitutes the main subsistence culture, being the grain used as protein source in feeding [26]. Cowpea presents important agronomical characteristics, such as rusticity and precocity, besides being considered a plant adapted to conditions of limited or insuffi‐ cient water availability [27].

Beneficial effects proportioned by the inoculation on growth parameters as leaf, stem and root are largely explored and well known in leguminous plants [28-30], but informations more specific of this symbiotic process on essential compounds such as amino acids and proteins are limited. Figueiredo et al. [24] report that inoculation using *Bradyrhizobium* can alleviate the negative consequences in *Vigna unguiculata* plants induced to water deficiency, but study conducted by Serraj and Sinclair [31] revealed that water supply presents reper‐ cussion on symbiotic efficiency.

Based on this overview, Barbosa et al. [32] carried out a study aiming to investigate if nitro‐ gen compounds exercise influence on accumulation of dry matter in *Vigna unguiculata* plants exposed to combined action of inoculation and water deficit.

The concentration of total soluble amino acids in plants subjected to inoculation was higher only in tolerant plants, if compared with same treatments of plants non-inoculat‐ ed (Figure 7 A). Water deficit promoted a significant increase in this variable to all treat‐ ments. The tolerant cultivar presented lower changes, in comparison with same treatments in sensitive cultivar.

Total soluble proteins of inoculated plants presented higher values (Figure 7 B), when com‐ pared to same treatments in non-inoculated plants. Water deficit caused a significant de‐ crease in both cultivars, presenting higher variation in sensitive plants.

For proline the inoculated plants presented higher values, comparing with same treatments in non-inoculated plants (Figure 7 C). The two cultivars demonstrated higher values in wa‐ ter deficit, when compared with respective controls. These results present a greater variation in tolerant plants, if compared with same treatments in sensitive plants.

Tolerant plants submitted to inoculation presented significant increase in amino acids, and these results are attributed to biological fixation of nitrogen. The nitrogenase enzyme pro‐ motes the nitrogen absorption in form of nitrogen gas (N2) and conversion to ammonium (NH4 + ). In addition, the higher formation of amino acids probably is linked to increase in ac‐ tivity of enzymes glutamine synthetase (GS), being your activity depending of ATP (adeno‐ sine-5'-triphosphate), and glutamate synthase (GOGAT). In addition, the increase in amino acids of plants exposed to inoculation is due to greater flux and better assimilation of nitro‐ gen in form of ammonium, concomitantly with higher activity of GS and GOGAT enzymes. Ramos et al. [33] evaluating the responses in Glycine max plants under water deficit and in‐ oculation of Bradyrhizobium japonicum observed also an increase in concentration of total soluble amino acids.

The concentration of total soluble amino acids in plants under water deficit increased in all treatments. This increment occurred probably due to increase in activity of protease enzymes, responsible by breakdown of proteins aiming to adjust osmotically the plant [34]. Similar results on increase in amino acids were obtained to Costa et al. [35] investi‐ gating *Vigna unguiculata* plants. Delfini et al. [36] evaluating the responses of two *Arachis hypogaea* cultivars submitted to inoculation of *Bradyrhizobium sp.* showed significant in‐ crease in amino acids.

3

**0**

**tolerant sensitive tolerant sensitive inoculated non-inoculated**

**Figure 7: Figure 7.** Total soluble amino acids (A), total soluble proteins (B), and proline (C) in two contrasting *Vigna unguiculata* plants under water deficit and subjected to inoculation. Means followed by the same letter are not significantly differ‐

The increase showed in total soluble proteins induced by inoculation suggests that bacte‐ ria action resulted in increase in nitrogen supply through secondary route, that is regu‐ lated by the nitrogenase [37], because in this study was not verified increase in activity of the nitrate reductase after inoculation. Hristozkova et al. [38] evaluating the responses

**inoculated non-inoculated**

**c**

**2**

**4**

**6**

**Proline**

**(µmol g DM-1)** **8**

**10**

**12**

**0**

**2**

**4**

**Total soluble**

 **proteins**

**(mg g DM-1)**

**6**

**8**

**10**

**0**

**12**

**50**

**Total soluble**

 **amino acids**

**(µmol g DM-1)**

**100**

**150**

**200**

**water deficit control**

**b**

**c**

**b b**

**d d**

**c**

**b**

**d d**

**<sup>b</sup> <sup>b</sup>**

Tolerance to Drought in Leguminous Plants Mediated by *Rhizobium and Bradyrhizobium*

**c**

**A**

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57

**a**

**B**

**c**

**C**

**b**

**e e**

**c**

**d**

**a**

**a**

**<sup>c</sup> <sup>c</sup>**

**a a**

**b**

**a**

**b**

**cd**

**a**

**a**

**b**

ent by the Scott-Knott test at 5% of probability. The bars represent the mean standard error [32].

**b**

**a**

**a**

Tolerance to Drought in Leguminous Plants Mediated by *Rhizobium and Bradyrhizobium* http://dx.doi.org/10.5772/54094 57

Beneficial effects proportioned by the inoculation on growth parameters as leaf, stem and root are largely explored and well known in leguminous plants [28-30], but informations more specific of this symbiotic process on essential compounds such as amino acids and proteins are limited. Figueiredo et al. [24] report that inoculation using *Bradyrhizobium* can alleviate the negative consequences in *Vigna unguiculata* plants induced to water deficiency, but study conducted by Serraj and Sinclair [31] revealed that water supply presents reper‐

Based on this overview, Barbosa et al. [32] carried out a study aiming to investigate if nitro‐ gen compounds exercise influence on accumulation of dry matter in *Vigna unguiculata* plants

The concentration of total soluble amino acids in plants subjected to inoculation was higher only in tolerant plants, if compared with same treatments of plants non-inoculat‐ ed (Figure 7 A). Water deficit promoted a significant increase in this variable to all treat‐ ments. The tolerant cultivar presented lower changes, in comparison with same

Total soluble proteins of inoculated plants presented higher values (Figure 7 B), when com‐ pared to same treatments in non-inoculated plants. Water deficit caused a significant de‐

For proline the inoculated plants presented higher values, comparing with same treatments in non-inoculated plants (Figure 7 C). The two cultivars demonstrated higher values in wa‐ ter deficit, when compared with respective controls. These results present a greater variation

Tolerant plants submitted to inoculation presented significant increase in amino acids, and these results are attributed to biological fixation of nitrogen. The nitrogenase enzyme pro‐ motes the nitrogen absorption in form of nitrogen gas (N2) and conversion to ammonium

The concentration of total soluble amino acids in plants under water deficit increased in all treatments. This increment occurred probably due to increase in activity of protease enzymes, responsible by breakdown of proteins aiming to adjust osmotically the plant [34]. Similar results on increase in amino acids were obtained to Costa et al. [35] investi‐ gating *Vigna unguiculata* plants. Delfini et al. [36] evaluating the responses of two *Arachis hypogaea* cultivars submitted to inoculation of *Bradyrhizobium sp.* showed significant in‐

). In addition, the higher formation of amino acids probably is linked to increase in ac‐ tivity of enzymes glutamine synthetase (GS), being your activity depending of ATP (adeno‐ sine-5'-triphosphate), and glutamate synthase (GOGAT). In addition, the increase in amino acids of plants exposed to inoculation is due to greater flux and better assimilation of nitro‐ gen in form of ammonium, concomitantly with higher activity of GS and GOGAT enzymes. Ramos et al. [33] evaluating the responses in Glycine max plants under water deficit and in‐ oculation of Bradyrhizobium japonicum observed also an increase in concentration of total

cussion on symbiotic efficiency.

56 Responses of Organisms to Water Stress

treatments in sensitive cultivar.

(NH4 +

soluble amino acids.

crease in amino acids.

exposed to combined action of inoculation and water deficit.

crease in both cultivars, presenting higher variation in sensitive plants.

in tolerant plants, if compared with same treatments in sensitive plants.

**Figure 7: Figure 7.** Total soluble amino acids (A), total soluble proteins (B), and proline (C) in two contrasting *Vigna unguiculata* plants under water deficit and subjected to inoculation. Means followed by the same letter are not significantly differ‐ ent by the Scott-Knott test at 5% of probability. The bars represent the mean standard error [32].

The increase showed in total soluble proteins induced by inoculation suggests that bacte‐ ria action resulted in increase in nitrogen supply through secondary route, that is regu‐ lated by the nitrogenase [37], because in this study was not verified increase in activity of the nitrate reductase after inoculation. Hristozkova et al. [38] evaluating the responses

3

in *Pisum sativum* plants under inoculation and molybdenum application also obtained in‐ crease in protein levels.

The decrease in protein levels promoted by the water deficit is associated to decrease of the protein synthesis combined with increase of proteolytic enzymes, responsible by breakdown of soluble proteins in plants [39]. Costa [40] obtained similar results studying *Vigna unguicu‐ lata* subjected to water deficit, corroborating these results.

The increase of proline levels provoked by the inoculation is probably linked to better amino acids utilization such as glutamic acid and arginine, being the glutamic acid the precursor of the proline, while arginine can suffer reaction mediated by enzyme called of pyrrolline-5 carboxylate reductase (P5CR) and consequently to liberate proline [33].

Kohl et al. [41] also observed higher amounts of proline in *Glycine max* plants inoculated with *Bradyrhizobium japonicum*, contributing with results of this study. The increase of pro‐ line in plants under water deficit is a response to loss of cell turgescence [42]. Nogueira et al. [43] describe that the proline accumulation has been related with drought tolerance in high‐ er plants, actuating as osmoregulator agent with the objective to keep water in plant tissue [44]. Similar behavior was described by González et al. [45] working with *Pisum sativum* plants under water restriction.

## **6***. Bradyrhizobium* **producing better performance on morphological parameters**

Beneficial effects proportioned by the inoculation on growth parameters are largely ex‐ plored in crops as *Phaseolus vulgaris* and *Glycine max*, but informations on dry matter ac‐ cumulation of *Vigna unguiculata* under water deficit is limited. Barbosa et al. [32] conduced an experiment with 2 cultivars (tolerant and sensitive) combined with 2 water regimes (water deficit and control), and 2 inoculation forms (inoculated and non-inocu‐ lated), totalizing 8 treatments.

In shoot dry matter the inoculation provoked increase, considering same treatments in plants non-inoculated (Figure 8 A). However, this increase only was significant in toler‐ ant cultivar under inoculation and irrigation. Water deficit occasioned a significant de‐ crease in shoot dry matter, with exception in tolerant cultivar non-inoculated. The tolerant plants presented better results, if compared with sensitive plants independently of treat‐ ment (Figure 8 A).

The inoculation provoked increase in plant dry matter, with exception in sensitive cultivar under irrigation (Figure 8 B), comparing to same treatments in plants non-inoculated. Water deficiency induced decrease in plant dry matter for all treatments, being these significant re‐ sults when compared with control plants. Independently of treatments was showed that sensitive cultivars presented lower values, if compared to tolerant cultivars.

4

**0**

**tolerant sensitive tolerant sensitive inoculated non-inoculated**

**Figure 8: Figure 8.** Shoot dry matter (A), plant dry matter (B), number of leaves (C) in two contrasting Vigna unguiculata plants under water deficit and subjected to inoculation. Means followed by the same letter are not significantly different by

For leaf number occurred increase after inoculation, with exception in sensitive plants under water deficiency (Figure 8 C). Water deficit proportioned decrease in values of leaf number, being significant in comparison with control plants. In tolerant cultivar were obtained high‐

**inoculated non-inoculated**

**10**

**20**

**Leaf (number)**

**30**

**40**

**50 0**

**5**

**10**

**Plant dry matter (g)**

**15**

**20**

**25**

**30 0**

**5**

**10**

**Shoot dry matter (g)**

**15**

**20**

**25**

**30**

**water deficit control**

**b**

**b <sup>b</sup> <sup>b</sup>**

Tolerance to Drought in Leguminous Plants Mediated by *Rhizobium and Bradyrhizobium*

**<sup>d</sup> <sup>d</sup>**

**c c**

**a**

**b**

**a**

**ddc<sup>c</sup>**

**<sup>b</sup> <sup>b</sup> c**

**c**

**de <sup>e</sup>**

**c**

**A**

http://dx.doi.org/10.5772/54094

59

**b**

**B**

**C**

**d**

**c**

**d**

**c**

**b**

**b**

**c**

**a**

**a**

**a**

**b**

the Scott-Knott test at 5% of probability. The bars represent the mean standard error [32].

er values of leaf number, if compared with sensitive cultivar.

**b**

**b**

Tolerance to Drought in Leguminous Plants Mediated by *Rhizobium and Bradyrhizobium* http://dx.doi.org/10.5772/54094 59

in *Pisum sativum* plants under inoculation and molybdenum application also obtained in‐

The decrease in protein levels promoted by the water deficit is associated to decrease of the protein synthesis combined with increase of proteolytic enzymes, responsible by breakdown of soluble proteins in plants [39]. Costa [40] obtained similar results studying *Vigna unguicu‐*

The increase of proline levels provoked by the inoculation is probably linked to better amino acids utilization such as glutamic acid and arginine, being the glutamic acid the precursor of the proline, while arginine can suffer reaction mediated by enzyme called of pyrrolline-5-

Kohl et al. [41] also observed higher amounts of proline in *Glycine max* plants inoculated with *Bradyrhizobium japonicum*, contributing with results of this study. The increase of pro‐ line in plants under water deficit is a response to loss of cell turgescence [42]. Nogueira et al. [43] describe that the proline accumulation has been related with drought tolerance in high‐ er plants, actuating as osmoregulator agent with the objective to keep water in plant tissue [44]. Similar behavior was described by González et al. [45] working with *Pisum sativum*

**6***. Bradyrhizobium* **producing better performance on morphological**

Beneficial effects proportioned by the inoculation on growth parameters are largely ex‐ plored in crops as *Phaseolus vulgaris* and *Glycine max*, but informations on dry matter ac‐ cumulation of *Vigna unguiculata* under water deficit is limited. Barbosa et al. [32] conduced an experiment with 2 cultivars (tolerant and sensitive) combined with 2 water regimes (water deficit and control), and 2 inoculation forms (inoculated and non-inocu‐

In shoot dry matter the inoculation provoked increase, considering same treatments in plants non-inoculated (Figure 8 A). However, this increase only was significant in toler‐ ant cultivar under inoculation and irrigation. Water deficit occasioned a significant de‐ crease in shoot dry matter, with exception in tolerant cultivar non-inoculated. The tolerant plants presented better results, if compared with sensitive plants independently of treat‐

The inoculation provoked increase in plant dry matter, with exception in sensitive cultivar under irrigation (Figure 8 B), comparing to same treatments in plants non-inoculated. Water deficiency induced decrease in plant dry matter for all treatments, being these significant re‐ sults when compared with control plants. Independently of treatments was showed that

sensitive cultivars presented lower values, if compared to tolerant cultivars.

crease in protein levels.

58 Responses of Organisms to Water Stress

plants under water restriction.

lated), totalizing 8 treatments.

ment (Figure 8 A).

**parameters**

*lata* subjected to water deficit, corroborating these results.

carboxylate reductase (P5CR) and consequently to liberate proline [33].

**Figure 8: Figure 8.** Shoot dry matter (A), plant dry matter (B), number of leaves (C) in two contrasting Vigna unguiculata plants under water deficit and subjected to inoculation. Means followed by the same letter are not significantly different by the Scott-Knott test at 5% of probability. The bars represent the mean standard error [32].

For leaf number occurred increase after inoculation, with exception in sensitive plants under water deficiency (Figure 8 C). Water deficit proportioned decrease in values of leaf number, being significant in comparison with control plants. In tolerant cultivar were obtained high‐ er values of leaf number, if compared with sensitive cultivar.

4

Shoot dry matter was maximized after the inoculation procedure, being this fact linked to probably increase in nodule number in root (data not shown), as well as it proportioned higher absorption and availability of nitrogen to plant [46-47]. Similar results linked to shoot dry matter were found by Figueiredo et al. [24] in research with *Vigna unguiculata* plants ex‐ posed to inoculation of *Bradyrhizobium*.

al possibilities linked to symbiosis between plant-microorganisms. Additionally, it present‐ ed essential compounds such as molybdenum and reactions during process of biological fixation of nitrogen. Also was demonstrated the water maintenance in leaf and nodule pro‐ duced after inoculation. Based in novel results, were related interference positives on nitro‐ gen compounds such as total soluble amino acids, proline, and total soluble proteins. Other results prove the beneficial repercussion produced by inoculation with *Bradyrhizobium* on

Tolerance to Drought in Leguminous Plants Mediated by *Rhizobium and Bradyrhizobium*

http://dx.doi.org/10.5772/54094

61

This chapter had financial support from Conselho Nacional de Pesquisa (CNPq/Brazil) and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES/Brazil) for Lobato

, Joaquim Albenísio Gomes da Silveira2

and Cândido Ferreira de Oliveira Neto1

1 Núcleo de Pesquisa Vegetal Básica e Aplicada, Universidade Federal Rural da Amazônia,

2 Laboratório de Metabolismo e Estresse de Plantas, Universidade Federal do Ceará, Fortale‐

[1] Rockström J, Falkenmark M. Semiarid crop production from a hydrological perspec‐ tive: Gap between potential and actual yield. Crit. Rev. Plant Sci 2000;19 319-326.

[2] Kramer PJ, Boyer JS. Water relations of plant and soils. Academic Press, New York.

[3] Fontana DC, Berlato MA, Bergamaschi H. Micrometeorological alterations in soy‐ beans grown under different water regimes. Pesquisa Agropecuária Brasileira

[4] Santos RF, Carlesso R. Water deficit and morphologic and physiologic behavior of plants. Revista Brasileira de Engenharia Agrícola e Ambiental 1998;2 287-294.

,

morphological parameters.

**Acknowledgements**

**Author details**

Paragominas, Brazil

za, Brazil

**References**

1995.

1992;27 661-669.

Allan Klynger da Silva Lobato1

Roberto Cezar Lobo da Costa1

AKS.

The water deficit reduced the production of shoot dry matter, with these effects associated to negative interference of water deficiency on biochemical processes as nitrate assimilation and biological fixation of nitrogen [35], modifying indirectly the partitioning of photo-assim‐ ilates in root and shoot, and consequent decrease in accumulation of shoot biomass [48]. Similar results were found by Mendes et al. [49] working with two *Vigna unguiculata* culti‐ vars submitted to drought during two stages.

The inoculation proportioned increase of total dry matter, and this result must be linked to better development and efficiency of root system, in which presents higher nitrogen absorp‐ tion using the nodulation process. I addition, normally the higher nitrogen fixation will pro‐ duce increase in amino acids and also proteins [36], and it exercises influence on photoassimilates availability.

Similar responses were described by Sassi et al. [50] investigating two *Phaseolus vulgaris* cul‐ tivars subjected to inoculation with bacteria of *Rhizobium* gender. Plants under water defi‐ ciency frequently have the production of dry matter reduced, being this decrease related to fact that water deficit affects several metabolic processes such as absorption of water and nutrients, which are fundamental to keep adequate growth and development rates.

Nascimento [51] also reported that water deprivation affects the osmotic mechanism, and by consequence reduces the CO2 supply, that is essential in photosynthetic process. Similar re‐ sults were found by Leite and Virgens Filho [52] studying *Vigna unguiculada* plants exposed to water deficit.

The increase in leaf number promoted by inoculation is occasioned by the higher num‐ ber of nodules in root, and consequently due to the better biological fixation of nitrogen [53]. Araújo et al. [54] studying *Vigna unguiculata* and *Leucaena leucocephala* plants also re‐ ported an increase of this variable, confirming the results obtained in this work. The lower leaf number after water deficiency is caused by the process of leaf abscission, and this fact occurs due to substrate not to present water and nutrient sufficient to supply the plant exigencies [4]. Correia and Nogueira [55] obtained similar results with *Arachis hypogaea* plants under water deficit.

## **7. Final considerations**

This chapter was structured with recent informations on capacity of *Bradyrhizobium* and *Rhi‐ zobium* to mediate tolerance in leguminous plants submitted to water deficit, which it can be used by students, teachers, researchers, scientists and farmers. It revealed concepts, effects, and results on water deficiency and your consequences on plants, as well as explored sever‐ al possibilities linked to symbiosis between plant-microorganisms. Additionally, it present‐ ed essential compounds such as molybdenum and reactions during process of biological fixation of nitrogen. Also was demonstrated the water maintenance in leaf and nodule pro‐ duced after inoculation. Based in novel results, were related interference positives on nitro‐ gen compounds such as total soluble amino acids, proline, and total soluble proteins. Other results prove the beneficial repercussion produced by inoculation with *Bradyrhizobium* on morphological parameters.

## **Acknowledgements**

Shoot dry matter was maximized after the inoculation procedure, being this fact linked to probably increase in nodule number in root (data not shown), as well as it proportioned higher absorption and availability of nitrogen to plant [46-47]. Similar results linked to shoot dry matter were found by Figueiredo et al. [24] in research with *Vigna unguiculata* plants ex‐

The water deficit reduced the production of shoot dry matter, with these effects associated to negative interference of water deficiency on biochemical processes as nitrate assimilation and biological fixation of nitrogen [35], modifying indirectly the partitioning of photo-assim‐ ilates in root and shoot, and consequent decrease in accumulation of shoot biomass [48]. Similar results were found by Mendes et al. [49] working with two *Vigna unguiculata* culti‐

The inoculation proportioned increase of total dry matter, and this result must be linked to better development and efficiency of root system, in which presents higher nitrogen absorp‐ tion using the nodulation process. I addition, normally the higher nitrogen fixation will pro‐ duce increase in amino acids and also proteins [36], and it exercises influence on

Similar responses were described by Sassi et al. [50] investigating two *Phaseolus vulgaris* cul‐ tivars subjected to inoculation with bacteria of *Rhizobium* gender. Plants under water defi‐ ciency frequently have the production of dry matter reduced, being this decrease related to fact that water deficit affects several metabolic processes such as absorption of water and

Nascimento [51] also reported that water deprivation affects the osmotic mechanism, and by consequence reduces the CO2 supply, that is essential in photosynthetic process. Similar re‐ sults were found by Leite and Virgens Filho [52] studying *Vigna unguiculada* plants exposed

The increase in leaf number promoted by inoculation is occasioned by the higher num‐ ber of nodules in root, and consequently due to the better biological fixation of nitrogen [53]. Araújo et al. [54] studying *Vigna unguiculata* and *Leucaena leucocephala* plants also re‐ ported an increase of this variable, confirming the results obtained in this work. The lower leaf number after water deficiency is caused by the process of leaf abscission, and this fact occurs due to substrate not to present water and nutrient sufficient to supply the plant exigencies [4]. Correia and Nogueira [55] obtained similar results with *Arachis*

This chapter was structured with recent informations on capacity of *Bradyrhizobium* and *Rhi‐ zobium* to mediate tolerance in leguminous plants submitted to water deficit, which it can be used by students, teachers, researchers, scientists and farmers. It revealed concepts, effects, and results on water deficiency and your consequences on plants, as well as explored sever‐

nutrients, which are fundamental to keep adequate growth and development rates.

posed to inoculation of *Bradyrhizobium*.

60 Responses of Organisms to Water Stress

vars submitted to drought during two stages.

photoassimilates availability.

*hypogaea* plants under water deficit.

**7. Final considerations**

to water deficit.

This chapter had financial support from Conselho Nacional de Pesquisa (CNPq/Brazil) and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES/Brazil) for Lobato AKS.

## **Author details**

Allan Klynger da Silva Lobato1 , Joaquim Albenísio Gomes da Silveira2 , Roberto Cezar Lobo da Costa1 and Cândido Ferreira de Oliveira Neto1

1 Núcleo de Pesquisa Vegetal Básica e Aplicada, Universidade Federal Rural da Amazônia, Paragominas, Brazil

2 Laboratório de Metabolismo e Estresse de Plantas, Universidade Federal do Ceará, Fortale‐ za, Brazil

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**Chapter 4**

**Comparison Between the Water and Salt Stress Effects**

Abiotic stress limits crop productivity [1], and plays a major role in determining the distri‐ bution of plant species across different types of environments. Abiotic stress and its effects on plants in both natural and agricultural settings is a topic that is receiving increasing at‐ tention because of the potential impacts of climate change on rainfall patterns and tempera‐ ture extremes, salinization of agricultural lands by irrigation, and the overall need to maintain or increase agricultural productivity on marginal lands. In the field, a plant may experience several distinct abiotic stresses either concurrently or at different times through

In reference [3] are showed some common examples of the abiotic stresses a plant may en‐ counter which include a decreased availability of water, extremes of temperature including freezing, decreased availability of essential nutrients from the soil (or conversely the buildup of toxic ions during salt stress), excess light (especially when photosynthesis is restricted)

In the face of a global scarcity of water resources and the increased salinization of soil and water, abiotic stress is already a major limiting factor in plant growth and will soon become even more severe as desertification covers more and more of the world's terrestrial area. Plants are often subjected to periods of soil and atmospheric water deficits during their life cycle. Moreover, the faster-than-predicted change in global climate [4] and the different available scenarios for climate change suggest an increase in aridity for the semiarid regions of the globe. Together with overpopulation, this will lead to an overexploitation of water re‐ sources for agriculture purposes and increased constraints on plant growth and survival aid,

> © 2013 de Oliveira et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© 2013 de Oliveira et al.; licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

**on Plant Growth and Development**

Nara Lídia Mendes Alencar and Enéas Gomes-Filho

Additional information is available at the end of the chapter

or increased hardness of the soil that restricts root growth.

Alexandre Bosco de Oliveira,

http://dx.doi.org/10.5772/54223

**1. Introduction**

the growing season [2].
