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 .
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 . Effects of drought depend of plant development stage, intensity, 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 inadequate water supply .
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 photosynthesis rate, reduction in leaf area , and stomata closing . Crops normally present performance affected by water deficiency, which can cause lower growth and development (Figure 1), with progressive reduction in leaf dry matter  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 . On the other hand, plants growing in low water potentials normally present root thinner , and this morphological modification is an adaptation to increase water absorption efficiency. Therefore, a combination of changes in morphological, physiological and biochemical levels are necessary to plant survival in environments affected by drought.
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+) , being carried out by a distinct group of microorganisms, singly or under symbiosis. The Bradyrhizobium 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 fixation of nitrogen .
The persistence of rhizobial strains, and their symbiotic performance in current and subsequent seasons are affected by numerous biotic and abiotic factors , with drought stress and nitrogen deprivation, being among the most significant in many parts of the world . 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 molybdenum), its activation to an appropriate form, and its incorporation into the organic fraction of the molybdenum-cofactors, are required .
The presence of molybdenum is necessary during formation of several proteins, including the nitrogenase, the molybdoenzyme that reduces atmospheric dinitrogen (N2) into ammonia (NH4+) . 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 . The first reaction of denitrification, is carried out by the periplasmic Mo-containing nitrate reductase . In addition, the reaction under normal conditions is described as N2 + 8 e– + 8 H+ + 16 MgATP → 2 NH3 + H2 + 16 MgADP + 16Pi.
The enzyme mechanism requires reduction of the Fe protein by electron donors such as ferredoxin 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 electron-transfer 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 .
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 inadequate water supply. For example, biochemical modifications in carbon metabolism, such as increase in sucrose , as well as significant interference in nitrogen metabolism, like reduction of soluble proteins  and increase in total amino acids  contribute to osmotic adjustment of these plants (Figure 4).
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 assess the osmotic stress, Sassi et al.  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 .
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 reduction in shoot water supply. The same trend was observed in nodules (Figure 6 B). Indeed, data showed decreased nodule RWC in both stressed cultivars. This decrease was higher in Cv. ‘Coco Blanc’ treated nodules .
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 resistance as it enables turgor maintenance and growth continuation . Cv. ‘Flamingo’ exhibited 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 cultivar than Cv. ‘Coco Blanc’ . 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 temperate legumes . 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 difference in sensivity of N2 fixation in tropical legumes, under water stress, is not clearly understood . Based in these problems reported, Figueiredo et al.  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).
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 constituents. However, in moderate stress (S3) the impact on nodule water content was higher than on the changes in nodule mass .
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 capacity of fixation of the atmospheric nitrogen (N2) and low requirements to soil fertility , 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 . Cowpea presents important agronomical characteristics, such as rusticity and precocity, besides being considered a plant adapted to conditions of limited or insufficient water availability .
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.  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  revealed that water supply presents repercussion on symbiotic efficiency.
Based on this overview, Barbosa et al.  carried out a study aiming to investigate if nitrogen 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-inoculated (Figure 7 A). Water deficit promoted a significant increase in this variable to all treatments. 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 compared to same treatments in non-inoculated plants. Water deficit caused a significant decrease 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 water 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 promotes 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 activity of enzymes glutamine synthetase (GS), being your activity depending of ATP (adenosine-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 nitrogen in form of ammonium, concomitantly with higher activity of GS and GOGAT enzymes. Ramos et al.  evaluating the responses in Glycine max plants under water deficit and inoculation 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 . Similar results on increase in amino acids were obtained to Costa et al.  investigating Vigna unguiculata plants. Delfini et al.  evaluating the responses of two Arachis hypogaea cultivars submitted to inoculation of Bradyrhizobium sp. showed significant increase in amino acids.
The increase showed in total soluble proteins induced by inoculation suggests that bacteria action resulted in increase in nitrogen supply through secondary route, that is regulated by the nitrogenase , because in this study was not verified increase in activity of the nitrate reductase after inoculation. Hristozkova et al.  evaluating the responses in Pisum sativum plants under inoculation and molybdenum application also obtained increase 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 . Costa  obtained similar results studying Vigna unguiculata 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 .
Kohl et al.  also observed higher amounts of proline in Glycine max plants inoculated with Bradyrhizobium japonicum, contributing with results of this study. The increase of proline in plants under water deficit is a response to loss of cell turgescence . Nogueira et al.  describe that the proline accumulation has been related with drought tolerance in higher plants, actuating as osmoregulator agent with the objective to keep water in plant tissue . Similar behavior was described by González et al.  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 explored in crops as Phaseolus vulgaris and Glycine max, but informations on dry matter accumulation of Vigna unguiculata under water deficit is limited. Barbosa et al.  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-inoculated), 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 tolerant cultivar under inoculation and irrigation. Water deficit occasioned a significant decrease in shoot dry matter, with exception in tolerant cultivar non-inoculated. The tolerant plants presented better results, if compared with sensitive plants independently of treatment (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 results when compared with control plants. Independently of treatments was showed that sensitive cultivars presented lower values, if compared to tolerant cultivars.
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 higher values of leaf number, if compared with sensitive cultivar.
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.  in research with Vigna unguiculata plants exposed to inoculation of Bradyrhizobium.
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 , modifying indirectly the partitioning of photo-assimilates in root and shoot, and consequent decrease in accumulation of shoot biomass . Similar results were found by Mendes et al.  working with two Vigna unguiculata cultivars 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 absorption using the nodulation process. I addition, normally the higher nitrogen fixation will produce increase in amino acids and also proteins , and it exercises influence on photoassimilates availability.
Similar responses were described by Sassi et al.  investigating two Phaseolus vulgaris cultivars subjected to inoculation with bacteria of Rhizobium gender. Plants under water deficiency 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  also reported that water deprivation affects the osmotic mechanism, and by consequence reduces the CO2 supply, that is essential in photosynthetic process. Similar results were found by Leite and Virgens Filho  studying Vigna unguiculada plants exposed to water deficit.
The increase in leaf number promoted by inoculation is occasioned by the higher number of nodules in root, and consequently due to the better biological fixation of nitrogen . Araújo et al.  studying Vigna unguiculata and Leucaena leucocephala plants also reported 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 . Correia and Nogueira  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 Rhizobium 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 several possibilities linked to symbiosis between plant-microorganisms. Additionally, it presented essential compounds such as molybdenum and reactions during process of biological fixation of nitrogen. Also was demonstrated the water maintenance in leaf and nodule produced after inoculation. Based in novel results, were related interference positives on nitrogen 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.