Open access peer-reviewed chapter

Role of Boron Nutrient in Nodules Growth and Nitrogen Fixation in Soybean Genotypes Under Water Stress Conditions

Written By

Nacer Bellaloui, Alemu Mengistu, My Abdelmajid Kassem, Craig A. Abel and L.H.S. Zobiole

Submitted: September 12th, 2013 Reviewed: September 20th, 2013 Published: January 29th, 2014

DOI: 10.5772/56994

Chapter metrics overview

2,539 Chapter Downloads

View Full Metrics

1. Introduction

Boron is an essential nutrient for plant growth, development, and seed quality [1-4]. Previous research indicated the involvement of B in cell wall structure [5,6]; cell membrane integrity [2,7]; sugar metabolism [2], especially sugar alcohols [3,8]; nitrogen assimilation and fixation [9,10]; nodules [11,12], nodullin protein (ENOD2) and malfunction of oxygen diffusion barrier [13]; phenolic metabolism [2,14,15]; ion uptake [2,16]; and plasma membrane-bount H+ ATPase [7,17,18].

Boron is required for nodules growth and nitrogen fixation [9,11-13], and boron deficiency can occur under certain environmental stress factors even when boron level in soil is adequate [19], leading to yield loss. Among the environmental stress factors that can lead to boron deficiency in plants is drought or water stress. Drought is a major environmental stress factor limiting crop yields worldwide [20], and maintaining boron levels within plants under drought conditions is critical. Boron has low mobility in the phloem [21], although boron mobility in the phloem depends on plant species [3, 22]. Under water stress conditions, plant increases abscisic acid (ABA) production [23], possibly affecting photosynthetic rate in drought-stressed plants, leading to stomata closure and transpiration rate reduction. Under these conditions boron uptake and translocation, and boron movement from leaves to seed is reduced, decreasing seed boron concentration [10]. Although soybean nodule growth and symbiotic N2 fixation are sensitive to drought [20,24,25], we hypothesized that drought can result in boron deficiency within the plant, impacting nodule growth, N2 fixation, and CO2 accumulation. The objective of this research was to investigate the effects of foliar boron on nodules growth and nitrogen fixation rates in several soybean genotypes under water stress. To avoid the confounding effects of multi-environmental factors in the field on boron application effects, the experiment was conducted under greenhouse conditions. In addition to the current research findings, the present chapter will also highlight previous and current major research findings in boron nutrition and the role of B in nodule growth and symbiotic nitrogen fixation in soybean.

Advertisement

2. Materials and methods

A repeated greenhouse experiment was conducted. Cultivars of maturity group (MG) III Pella, Williams 82, Hutcheson, and Forest, were used. Seeds of soybean cultivar were germinated in flat trays in vermiculite, and then uniform size seedlings at V1 stage were transplanted into 9.45 L size pots. Soil in pots was a Dundee silt loam (fine-silty, mixed, active, thermic Typic Endoqualfs) with pH 6.3, 1.1% organic matter, a cation exchange capacity of 15 cmol/kg, and soil textural fractions of 26% sand, 56% silt, and 18% clay, average B concentration was 0.72 mg kg-1. The soil contained an abundant native population of B. japonicum. Water stress was introduced as reported by Bellaloui [20,26]. Briefly, soil in pots were weighed and then saturated with deionized water and left to drain and weighed again to obtain the water field capacity using soil water sensors inserted in pots and measured by Soil Moisture Meter (WaterMark Company, Inc., Wisconsin, USA). Plants were divided into well watered (soil water potential between –15 to –20 kPa) (this was considered field capacity for the control plants), moderate water stress (soil water potential between -90 and -100 kPa), and severely water stressed (soil water potential between –150 to –200 kPa). Boron was foliar-applied as boric acid at a rate of 1.1 kg ha-1 once at flowering stage (R1-R2) and once at seed-fill stage (R5-R6). Combined treatments were well watered plants with no B (W-B), well watered plants with B (W+B); water stressed plants with no B (WS-B); water stressed plants with B (WS+B); severely water stressed plants with no B (SWS-B); severely water stressed plants with B (SWS+B). Samples were taken five days after the second B application for nitrate reductase assay to measure the rate nitrate reductase activity (NRA), nitrogenase, and leaf B. Mature seed were weighed at R8 (harvest maturity stage). Plants were considered fully matured when they reached R8 according to [27]. Greenhouse conditions were about 34°C ± 9°C during the day and about 28°C ± 8°C at night with a photosynthetic photon flux density (PPFD) of about 800 - 2300 μmol m-2 s-1, as measured by Quantum Meter (Spectrum Technology, Inc., Illinois, USA). The big range of light intensity reflects a bright, sunny, or cloudy day. The source of lighting was a mixture of natural light, bulb light (60 W), cool white (250 W). To avoid differences in the day-length between the two experiments, the two experiments were conducted simultaneously at the same time and during the normal growing season (from April to September) for the Early Soybean Production System in the midsouth USA, and this is to be consistent with the normal photoperiod for soybean growth [10].

2.1. Nitrate reductase assay

The rate of nitrate reductase activity (NRA) was determined according to [28, 29]. Briefly, NRA was measured in the fully expanded leaves and nodules. Nodules were gently and carefully separated from roots and placed in NRA assay buffer solution. A fresh leaf sample of about 0.3 g was placed in 10 mL of potassium phosphate buffer at a concentration of 100 mM, pH 7.5, containing 1% (v/v) 1-propanol, in the flask. The incubation solution was vacuum-filtered for 1 min, and then flashed with nitrogen gas for 30 s, and then incubated at 30°C. A samples of 0.5 mL was taken at regular intervals (0, 60, 120, 180, and 300 min) for nitrite measurement. Samples were extracted with 5 mL of deionized water and reacted with 1.0 mL of 1% (w/v) sulfanilamide in 10% v/v HCl and 1.0 mL of N-naphthyl-(1)-ethylenediamine dihydro- chloride (0.1%). Nitrite concentration in samples was measured by reading the absorbance at 540 nm after 30 minutes using a Beckman Coulter DU 800 spectro- photometer (Fullerton, CA). A standard curve was produced using KNO2 as a source of NO2 in the tested samples according to (Bellaloui et al., 2006). To measure the enzyme activity where there was no limiting concentration of NO3 in the incubation culture solution (potential nitrate reductae activity, PNRA), exogenous NO3 was added at a concentration of 10 mM as KNO3.

2.2. Acetylene reduction assay

Destructive method for acetylene reduction assay was used. Three plants from each replicate were harvested five days after the second B application (at seed-fill stage). Nitrogenase activity was assayed using the acetylene reduction assay as described elsewhere [29-31]. Roots with nodules intact were excised and incubated in 60 mL plastic syringes. Roots from each replicate and from each treatment were placed in the syringes in the Mason jars and sealed. A 10% volume of air was then removed and replaced with an equal volume of acetylene. After 1 h of incubation at room temperature, duplicate 1.0 mL gas samples were removed and analyzed by gas chromatography for ethylene formation and carbon dioxide evolution. The gas chromatography (Agilent HP6960, Agilent Technologies, Wilmington, DE) was equipped with manual injector, injector loop, and sample splitter. A flame ionization detector (FID) and a thermal conductivity detector (TCD) were used. Using the sample loop and splitter, 0.25 mL of gas was directed into a 30 m length × 0.53 mm i.d. alumina megabore column (115-3532) connected to the FID, and 0.25 mL of sample was injected into a HP-PLOT D column (30 m length × 0.53 mm i.d. megabore with 40 µm film; 1905D-Q04) connected to the TCD using helium as a carrier gas. Chromatographs were integrated using Chem Station software. Standard curves for ethylene and carbon dioxide were updated and produced for each day. Samples having <9% acetylene were not used in the analysis.. Nodules were carefully removed and counted, and then oven-dried at 60°C for 4 - 5 days.

2.3. Boron determination

The concentration of total B was measured in the fully expanded leaves after the second foliar B application, and in seeds at harvest maturity stage. Boron concentration was determined according to Azomethine—H method [19,32-34]. Briefly, 1 g of dry sample was placed in a porcelain crucible for ashing at 500°C for 8 hr. Samples then were extracted with 20 mL of 2 M HCl at 90°C for 10 min and azomethine-H solution containing 0.45% before the analysis (John et al., 1975) with a buffer solution contained 25% ammonium acetate, 1.5% EDTA, and 12.5% acetic acid. The concentration of B in the samples were determined spectrophotometrically by using color development after 45 minutes. Samples were read at 420 nm using a Beckman Coulter DU 800 spectrophotometer (Fullerton, California). Boron analysis in soil was conducted using Inductively Coupled Plasma spectrometry (ICP) using Thermo Elemental, Thermo Jarrell-Ash model 61E ICP, USA [10].

2.4. Analysis of δ15N (15N/14N ratio) and δ13C (13C/12C ratio) using natural abundance

Natural abundance of δ 15N and 13C isotopes was determined using about 0.9 mg of ground seeds. Isotopic analysis was conducted using a Thermo FinniGlyn Delta Plus Advantage Mass Spectrometer with a FinniGlyn ConFlo III, and Isomass Elemetal Analyzer (Bremen, Germany) according to [26, 35,36]. Isodat software version 2.38 was used to obtain Delta values [26]. The elemental combustion system was Costech ECS 4010 with an autosampler (Bremen, Germany).

2.5. Determination of seed sucrose

Seed sucrose concentration was measured in mature seeds. Sucrose concentration was measured according to [37,38] using an AD 7200 diode array feed analyzer (Perten, Springfield, IL). Briefly, about 25 g of seed were ground using a Laboratory Mill 3600 (Perten, Springfield, IL). Initial calibration equations were developed by the Department of Agronomy and Plant Genetics, University of Minnesota St Paul, MN using Thermo Galactic Grams PLS IQ software, developed by Perten company (Perten, Springfield, IL). Analyses of sugars were performed based on a seed dry matter basis [26, 37, 39].

2.6. Seed glucose determination

Glucose concentration in mature seeds was measured enzymatically using Glucose (HK) Assay Kit from Sigma, USA, Product Code GAHK-20, 2012) [40]. In this reaction, glucose is phosphorylated by adenosine triphosphate (ATP) in a reaction catalyzed by hexokinase. Glucose-6-phosphate (G6P) produced is then oxidized to form 6-phosphogluconate in the presence of oxidized nicotinamide adenine dinucleotide (NAD) in a reaction catalyzed by glucose-6-phosphate dehydrogenase (G6PDH). During this oxidation, an equimolar amount of NAD is reduced to NADH, and the increase in absorbance at 340 nm is directly proportional to glucose concentration in the sample. Mature seed samples were ground using a Laboratory Mill 3600 (Perten, Springfield, IL). A dry, ground sample of 0.1 mg was extracted with deionized water. The extraction procedure of glucose from seeds was conducted according to [26], and as instructed by Glucose (HK) Assay Kit from Sigma. The concentration of glucose was measured spectrophotometrically by reading the samples at 340nm using a Beckman Coulter DU 800 spectrophotometer (Fullerton, CA). The concentration of glucose was expressed as mg g dwt-1.

2.7. Seed fructose determination

Fructose concentration is mature seeds was determined enzymatically according to Fructose Assay Kit from Sigma, USA, Product Code FA-20, 2012 [41]. In this reaction, fructose is phosphorylated by ATP in a reaction catalyzed by hexokinase, and the produced fructose 6-phosphate is then converted to G6P by phosphoglucose isomerase (PGI). The oxidation of G6P to 6-phosphogluconate takes place in the presence of NAD in the reaction catalyzed by glucose-6-phosphate dehydrogenase (G6PDH). An equimolar amount of NAD is reduced to NADH, and the consequent increase in absorbance at 340 nm is directly proportional to fructose concentration in a sample. A sample of 0.1 mg was extracted according to Fructose Assay Kit from Sigma, and is detailed in Bellaloui et al (2013) as instructed by Fructose Assay Kit from Sigma. The concentration of fructose in samples were measured spectrophotometrically by reading the samples at absorbance of 340nm using a Beckman Coulter DU 800 spectrophotometer (Fullerton, CA). The concentration of fructose was expressed as mg g dwt-1.

2.8. Experimental design and statistical analysis

Treatments were arranged in a split plot design with irrigation as a main block and B treatment as sub-plot. Four replicates were used, and each replicate consisted of a pot containing three plants. Proc Mixed was used for data analysis of variance in SAS [42]. Means were separated by Fisher’s least significant difference test at the 5% level of probability using Proc GLM analysis in SAS [42]. Since there were no interactions between the two experiments, the data were pooled and combined.

Advertisement

3. Results and discussion

Analysis of variance showed that boron application (T) and irrigation (IR) were significant for grain weight, nodule mass, nitrogen fixation (NF), nitrate reductase activity (NRA), N, B, and natural abundance of δ15N and δ13C (Table 1), and sugars (Table 2). Cultivar (CV) was significant for some parameters and not significant for others, indicating differences in cultivar responses to the B application and water stress. Both B application and IR significantly interacted (T×IR and E×T×IR interactions) for these parameters, indicating that B effects on these parameters depended on IR (watered or water stressed conditions). There were no significant interactions between Experiment (E) and T or IR, indicating that the effect of B application or IR had similar effect in each experiment (Table 1). Therefore, the data were pooled and combined [26].

3.1. Effect of B and water stress on grain weight, nodule mass and nodule number

Foliar B application to watered plants (W+B) resulted in significant increase (P≤0.05) in grain weight, nodule mass, and nodule number (Table 3). For example, in Pella cultivar the increase of grain weight, nodule mass, and nodule number was 18.7%, 38.5%, and 33.3 %, respectively. These parameters were different between cultivars and each cultivar responded differently to foliar B (Table 3). Previous research indicated that B plays a major role for plant growth and development [1, 2] and crop quality [4,19]. It was shown that foliar B improved seed set, seed yield, and seed quality of alfalfa [19] and sugar beet [4], and altered seed composition in soybean [10]. Previous research showed that B is an essential nutrient for the development of nitrogen-fixing root nodules in pea (Pisum sativum) [9]. A lower level of infection of the host plants with Rhizobiumwas noticed in plants grown in B-deficient medium compared to plants supplied with adequate B [9]. It was shown that that little or no ability to fix N2 under B-deficient plants [11]. Recently, it was found that FB increased nodule weight under irrigated greenhouse conditions [43]. The current results showed that, even though B concentration in leaves was above the critical level (20 mg B kg-1, critical level of B in leaves for normal plant growth) [44], FB resulted in a positive effect on seed and nodule weights, agreeing with previous research of those of [7, 43, 45]. Our results showed that foliar B increased grain weight, nodule mass and number due to the stimulatory effects of B on growth and development [9,11] and nodule improvement.

3.2. Effect of B and water stress on nitrogen fixation and nitrogen assimilation

Foliar application of B resulted in higher rates of nitrogen fixation (increase of nitrogenase), root respiration, and nitrogen assimilation (increase of nitrate reductase activity, NRA), (Table 4). Foliar B application to moderately water stressed plants (Table 4) increased grain weight, nodule number and mass, nitrogen fixation and assimilation. However, foliar B application to severely water stress plants did not result in an increase in these parameters because of the damaging effects of water stress to nitrogen metabolism enzymes, especially nitorgenase and nitrate reductase (Table 5). Researchers reported that B is an essential micronutrient for the development of nitrogen-fixing root nodules [11], and plants grown in B-deficient medium showed lower infection of the host plants with Rhizobium compared to plants supplied with adequate B [11]. It was reported that nodules showed little or no ability to fix N2 in B-deficient plants, leading to N deficiency and necrosis of nodulated pea plants [9]. This indicated that nitrogen fixation in soybean was sensitive to B deficiency [9,12], and B deficiency can result in the reduction in early nodullin protein (ENOD2) in nodule parenchyma cells and malfunction of oxygen diffusion barrier [13]. It was hypothesized that B protects nitrogenase against oxygen damage by influencing membrane integrity and function [13] and may interact with membrane glycoproteins and glycolipids to maintain the proper conformation in nitrogen-fixing cells [5]. Although B has been shown to be essential for nodule growth and development, there is no convincing evidence that there is a direct effect of B on nitrogen metabolism [2,13,46,]. Nitrate assimilation, reflected by the key enzyme in nitrogen assimilation, was higher in W+B plants, indicating that B enhanced nitrate assimilation. The stimulatory effects of nitrogen assimilation by B was also reported by others (Bellaloui et al., 2011 AJPS. This is because nitrogen metabolism in legumes is both a result of both symbiotic N2 fixation and mineral N assimilation processes. During this process, atmospheric N2 is fixed by the enzyme nitrogenase in the bacteroids of nodules [47], and nitrate reduction (assimilation) is catalyzed by the enzyme nitrate reductase (NR). Both NR and nitrogenase enzymes coexist in nodules competing for reductant [48]. It appears that B may stimulate de novo synthesis and making nitrate (enzyme substrate) available for the enzyme nitrate reductase. Adding 0.5 mM B to the buffer solution increased NRA by 30% in WS+B compared with WS-B, and adding 10 mM NO3 to the buffer solution increased NRA by 55% and 40% in leaves and nodules, respectively (data not shown). In SWS plants, adding B or NO3 did not enhance NRA (data not shown). This indicated that both B and NO3 stimulated NR enzyme somehow, maybe by facilitating nitrate availability in the cytoplasm to NR for reduction or enhancing nitrate translocation from the vacuoles to the cytoplasm, leading to higher NRA activity. This hypothesis was supported by the effect of B ion uptake [2,16] and the direct or indirect effects of B on the plasma membrane bound H+ ATPase (plasmalemma H+-ATPase activity) [7,17], cell wall structure and membrane integrity [2,7]. Our results are supported by [2] in that B may have an indirect influence on nitrate uptake and assimilation, and enhance NRA by inducing nitrate availability and increasing protein de novo synthesis as a result of nitrate absorption [49]. This observation is supported by [13] who found that adequate level of B increased NRA and decreased nitrate in xylem sap compared to deficiency level. The relationship between nitrogen fixation and nitrogen assimilation and how this relationship is influenced by foliar B and its impact on seed protein and oil and sugars is still not well established.

Source of variabilitySeed weightNodule massNodule numberARA(NF)Leaf NRANoduleNRAB in leavesN inleavesδ15Nδ 13C
Experiment (E)NSNSNSNSNSNSNSNSNSNS
Treatment (T)*********************
Water stress (WS)**********************
Cultivar (CV)***NSNSNSNSNSNSNS
E×TNSNSNSNSNSNSNSNSNSNS
E×WSNSNSNSNSNSNSNSNSNSNS
E×CVNSNSNSNSNSNSNSNSNSNS
T×WS*************
T×CV***********
CV×WS**************
E×T×WS×CV****************

Table 1.

Analysis of variance of the effects of foliar boron on seed weight (100 seed weight, g), nodule mass (mg plant-1), nodule number plant-1, [nitrogen fixation (acetylene reduction assay (ARA), μmol of C2H4 plant-1 h-1)], leaf nitrate reductase activity (NRA, µmol NO2 g-1h-1), and nodule NRA (µmol NO2 g-1h-1), boron (B, mg kg-1)) and nitrogen (N, %) in leaves and seeds, and in δ15N and in δ 13C isotope values in seeds in genotypes of maturity group III (Pella and William 82) and MG V (Hutcheson and Forest) under well watered and water stressed conditions (WS) with and without foliar boron (B) treatments (T) under greenhouse conditions a.

* Significance at P≤ 0.05; ** Significance at P≤ 0.01; *** Significance at P≤ 0.001.


Source of variabilityGlucoseFructoseSucroseRaffinoseStachyose
Experiment (E)NSNSNSNSNS
Treatment (T)********
Irrigation (IR)*********
Cultivar (CV)***NSNS
E×TNSNSNSNSNS
E×IRNSNSNSNSNS
E×CVNSNSNSNSNS
T×IR******
T×CV******
CV×IR******
E×T×IR×CV******

Table 2.

Analysis of variance of the effects of foliar boron on sugars (mg g-1 dwt) in genotypes of maturity group III (Pella and William 82) and MG V (Hutcheson and Forest) under well watered and water stressed conditions (IR) with and without foliar boron (B) treatments (T) under greenhouse conditions a.

* Significance at P≤ 0.05; ** Significance at P≤ 0.01; *** Significance at P≤ 0.001.


Watered soybean
VarietyBoronGrain weight (100 seed weight, g)Nodule mass(mg dwt plant-1)Nodule number plant-1ARA(NF)(μmol of C2H4plant-1 h-1)Root respiration(mmol of CO2 evolved/g of root/h)Leaf NRA (µmol NO2 g-1 h-1)NoduleNRA(µmol NO2g-1h-1)
PellaW-B16 a65 a33 a11.6 a7.5 a5.6 ab4.7 ab
W 8215 a68 a28 b10.5 a7.8 a4.9 b5.2 a
Hutcheson14 b61 b25 b10.1 a6.8 b5.7 ab3.8 b
Forrest14 b64 a17 c11.4 a6.4 b6.3 a4.2 ab
Pella19 a90 a44 a15.4 a9.7 b7.5 ab6.4 a
W 82W+B18 a86 ab38 abc14.7 ab10.5 ab6.8 b7.8 a
Hutcheson16 b84 b36 c13.2 b10.6 ab7.4 ab5.2 b
Forrest17 b86 ab38 abc13.6 b11.5 a8.3 a5.8 b

Table 3.

Effect of foliar boron on soybean seed weight (100 seed weight, g), nodule mass (mg plant-1), nodule number plant-1, ARA (μmol of C2H4 plant-1 h-1), root respiration (mmol of CO2 evolved/g of root/h), leaf NRA (µmol NO2 g-1h-1), and nodule NRA (µmol NO2 g-1h-1) in genotypes of maturity group III (Pella and William 82) and MG V (Hutcheson and Forest) under well watered conditions without boron (W-B) and with boron (W+B) under greenhouse conditions a.

aSoybean plants were grown at field capacity at -15 to -20 kPa [10]. Soybeans were grown under greenhouse conditions similar to those in Bellaloui et al. (2011). Values within columns and within each B treatment sharing a letter are not significantly different (P>0.05) using Fishers’ test. W 82=Williams 82.


Watered stressed soybean
VarietyBoronGrain weight (100 seed weight, g)Nodule mass (mg dwt plant-1)Nodule number plant-1ARA(NF)Root respirationLeaf NRA(µmol NO2g-1h-1)NoduleNRA(µmol NO2g-1h-1)
PellaWS-B9.4 a45.3 b22.4 a7.6 a4.3 b3.6 a2.4 b
W 8210.3 a51.6 a24.1 a8.7 a4.9 b2.7 b3.6 a
Hutcheson9.7 a42.7 b21.5 a6.5 b5.4 b3.3 a2.7 b
Forrest10.1 a46.3 b14.5 b5.7 c6.3 a4.7 a2.5 b
Pella11.1 a56.7 b28.6 a10.5 a5.4 c5.4 b4.7 ab
W 82WS+B12.5 a66.4 a31.4 a9.7 a7.6 b7.5 a5.3 a
Hutcheson12.6 a57.3 b30.7 a10.4a8.7 a5.8 b4.7 ab
Forrest11.5 a63.2 a19.6 b7.4 b9.8 a6.3 a3.9 b

Table 4.

Effect of foliar boron on soybean seed weight (g), nodule mass (mg plant-1), nodule number plant-1, ARA ((μmol of C2H4 plant-1 h-1), root respiration (mmol of CO2 evolved/g of root/h), leaf NRA (µmol NO2 g-1h-1), and nodule NRA (µmol NO2 g-1h-1) in genotypes of maturity group III (Pella and William 82) and MG V (Hutcheson and Forest) under water stress conditions without boron (WS-B) and with boron (WS+B) under greenhouse conditions a.

a Soybean plants were grown under water stress (WS) (-90 to -100 kPa soil water potential). Soybeans were grown under greenhouse conditions similar to those previously reported [10]. Values within columns and within each B treatment sharing a letter are not significantly different (P>0.05) using Fishers’ test. W 82=Williams 82.


Severe watered stressed soybean
VarietyBoronGrain weight(100 seed weight, g)Nodule mass(mg dwt plant-1)Nodule number plant-1ARA(NF)Root respirationLeaf NRA(µmol NO2 g-1h-1)NoduleNRA(µmol NO2 g-1h-1)
PellaSWS-B4.4 a21.5 b20.7 a4.6 a2.1 c2.1 a0.7 a
W 825.5 a28.5 a21.6 a5.3 a3.3 b1.5 b0.9 a
Hutcheson5.3 a27.5 a19.6 a4.7 a3.6 b2.4 a0.8 a
Forrest4.7 b22.6 b21.5 a3.2 b4.7 a1.7 b0.8 a
Pella4.3 c25.4 a22.6 b5.1 a2.6 b2.4 a0.8 a
W 82SWS+B6.0 a30.6 a26.5 a4.8 a2.4 b0.9 b0.6 a
Hutcheson5.5 b26.5 a29.7 a3.4 b3.8 a2.1 a0.8 a
Forrest4.1 c21.6 b19.6 b3.2 b4.9 a1.2 b0.7 a

Table 5.

Effect of foliar boron on soybean seed weight (g), nodule mass (mg plant-1), nodule number plant-1, nitrogen fixation ( ARA, μmol of C2H4 plant-1 h-1), root respiration (mmol of CO2 evolved/g of root/h), leaf NRA (µmol NO2 g-1h-1), and nodule NRA (µmol NO2 g-1h-1) in genotypes of maturity group III (Pella and William 82) and MG V (Hutcheson and Forest) under severe water stress conditions without boron (SWS-B) and with boron (SWS+B) under greenhouse conditions a.

a Soybean plants were grown under severe water stress (soil water potential between –150 to –200 kPa). Soybeans were grown under greenhouse conditions similar to those previously reported [10]. Values within columns and within each B treatment sharing a letter are not significantly different (P>0.05) using Fishers’ test. W 82=Williams 82.


Foliar boron application increased B in leaves and seed in watered plants (Figure 1). No significant B concentration differences were observed between leaves and seeds B in each watered treatment in each cultivar, indicating that B movement from leaves to seeds was not limited. In severely water-stressed plants, application of foliar B did not significantly increase B in leaves, and B movement from leaves to seed was limited, indicated by the large accumulation of B in leaves and small accumulation of B in seeds. Similar trend of N in leaves and seed was noticed (Figure 2), indicating a close relationship between B and N.

3.3. Effects of B and water stress on seed sugars

Since B plays an important role in carbohydrate mobility and since carbohydrates are a source of reducing power in nitrogen assimilation, sugar profiling was also investigated. Our research demonstrated that foliar B application resulted in higher sucrose, glucose, and fructose under irrigated conditions, but under severe water stress these mono and disaccharides sugars decreased, but stachyose and raffinose increased (Table 6,7,8).

This indicated that there was a redistribution of sugars under severe water stress, and this shift may provide plants with an adaptive mechanism to tolerate the stress.

Foliar boron resulted in higher seed sucrose, glucose, and fructose concentrations in W+B plants, indicating B involvement in sugar metabolism and synthesis. The involvement of B in sugar synthesis and distribution is not understood, but B involvement in sugar movement and metabolism was previously reported [2,3,50]. The decrease of sucrose, glucose, and fructose in SWS-B and SWS+B plants compared with W+B and W-B indicated that B enhanced accumulation of sucrose, glucose, and fructose concentrations may be due to B role in sugar movement within the plants, and that severe water stress limited sugars movement due reduction of B uptake and stomatal conductance. The increase of seed stachyose concentration in seeds of SWS-B and SWS+B plants indicated that severe water stress affects the distribution of sugar fractions, in our case the increase of stachyose and raffinose and decrease of sucrose, glucose, and fructose concentrations. The increase of stachyose under water stress may indicate the role of stachyose in plant tolerance to biotic and abiotic stress [14,15]. It was also reported that raffinose and galactinol levels may play an important role in plant tolerance to biotic and abiotic stress [14,15], and the accumulation of galactinol and raffinose may protect the plant from drought [51], and the activity of sucrose synthase, the main enzyme involved in sucrose hydrolysis in nodules, was significantly inhibited under drought conditions [52,53]. The biological functions of raffinose and stachyose are not clear [54], but previous research reported that oligosaccharides (sucrose, raffinose, and stachyose) are related to seed quality [55] and the acquisition of desiccation tolerance during seed development and maturation.

Soybean seed sugars are important to soybean seed industry because they determine the quality of seeds beside protein and oil. This is because soybean seed with high raffinose and stachyose concentrations are undesirable and have negative effects on the nutritive value of soymeal and seed consumed by human. Stachyose and raffinose are indigestible by humans and animals, especially monogastric animal such as chicken and pigs, causing flatulence or diarrhea [56]. On the other hand, low raffinose and stachyose levels in soybean seed are desirable [57]. High level of seed sucrose, glucose, and fructose are desirable because it improves taste and flavor of tofu, soymilk, and natto [58]. Currently, soybean cultivars with improved sugar profiles have been released to the market [58], and breeding for desirable sugars in soybean or agricultural practices to improve seed sugars are needed.

Watered soybean
VarietyBoronGlucose(mg g-1)Fructose(mg g-1)Sucrose(mg g-1)Raffinose(mg g-1)Stachyose(mg g-1)
PellaW-B1.5 b0.73 a35.4 b5.4 a32.6 b
W 822.1 a0.65 b42.4 a4.8 b43.6 a
Hutcheson1.5 b0.64 b32.7 b5.3 ab42.8 a
Forrest2.2 a0.61 b30.8 b5.7 a41.6 a
Pella2.3 b0.93 b57.4 b5.4 ab35.4 b
W 82W+B2.8 a1.22 a63.2 a5.1 b44.6 a
Hutcheson2.1 b0.89 b53.8 b5.7 a42.6 a
Forrest2.9 a0.94 b59.7 b5.2 b46.5 a

Table 6.

Effect of foliar boron on soybean seed sugars in genotypes of maturity group III (Pella and William 82) and MG V (Hutcheson and Forest) under well watered conditions without boron (W-B) and with boron (W+B) under greenhouse conditions a.

a Soybean plants were grown at field capacity at -15 to -20 kPa according to Bellaloui et al., (2011). Soybeans were grown under greenhouse conditions similar to those previously reported [10]. Values within columns and within in each B treatment sharing a letter are not significantly different (P>0.05) using Fishers’ test. W 82=Williams 82.


VarietyBoronGlucose(mg g-1)Fructose(mg g-1)Sucrose(mg g-1)Raffinose(mg g-1)Stachyose(mg g-1)
PellaWS-B0.8 b0.74 a19.3 a6.5 b45.5 b
W 821.1 b0.65 b16.4 b7.3 a52.5 a
Hutcheson1.5 a0.42 c21.3 a7.4 a47.3 b
Forrest0.9 b0.69 b15.4 b6.4 b50.3 a
Pella1.5 b0.76 a27.5 ab7.1 a41.6 b
W 82WS+B1.5 b0.75 a25.4 b6.2 b48.7 a
Hutcheson1.9 a0.64 b31.2 a7.4 a50.3 a
Forrest1.6 b0.72 a30.5 a6.3 b48.5 a

Table 7.

Effect of foliar boron on soybean seed sugars in genotypes of maturity group III (Pella and William 82) and MG V (Hutcheson and Forest) under water stress conditions without boron (WS-B) and with boron (WS+B) under greenhouse conditions a.

a Soybean plants were grown under water stress (WS) (-90 to -100 kPa soil water potential). Soybeans were grown under greenhouse conditions similar to those previously reported [10]. Values within columns and within each B treatment sharing a letter are not significantly different (P>0.05) using Fishers’ test. W 82=Williams 82.


VarietyBoronGlucose(mg g-1)Fructose(mg g-1)Sucrose(mg g-1)Raffinose(mg g-1)Stachyose(mg g-1)
PellaSWS-B0.65 a0.54 a17.5 a8.5 b73.5 a
W 820.53 b0.43 b14.3 b9.5 a65.4 b
Hutcheson0.51 b0.47 b15.3 ab8.3 b73.7 a
Forrest0.48 c0.52 a11.6 c9.5 a65.7 b
Pella0.59 a0.53 a19.5 a8.5 b62.1 b
W 82SWS+B0.48 c0.47 b13.2 b9.5 a73.6 a
Hutcheson0.52 b0.51 a17.6 a8.7 b68.5 ab
Forrest0.51 b0.48 b14.2 b7.2 c68.5 ab

Table 8.

Effect of foliar boron on soybean seed sugars in genotypes of maturity group III (Pella and William 82) and MG V (Hutcheson and Forest) under severe water stress conditions without boron (SWS-B) and with boron (SWS+B) under greenhouse conditions a.

a Soybean plants were grown under severe water stress (soil water potential between –150 to –200 kPa). Soybeans were grown under greenhouse conditions similar to those previously reported [10]. Values within columns and within each B treatment sharing a letter are not significantly different (P>0.05) using Fishers’ test. W 82=Williams 82.


Figure 1.

Effects of foliar boron application (1.1 kg B ha-1) on boron concentration in leaves and seed in soybean genotypes in watered (A,B) and severe water stressed (C,D) soybean genotypes. Soybean plants were grown under severe water stress (soil water potential between –150 to –200 kPa). Soybeans were grown under greenhouse conditions similar to those previously reported [10].

Figure 2.

Effects of foliar boron application (1.1 kg B ha-1) on nitrogen percentage in leaves and seed in soybean genotypes in watered (A,B) and severe water stressed (C,D) soybean genotypes. Soybean plants were grown under severe water stress (soil water potential between –150 to –200 kPa). Soybeans were grown under greenhouse conditions similar to those previously reported [10].

3.4. Effect of B and water stress on δ15N (15N/14N ratio) and δ13C (13C/12C ratio)

Foliar B did not result in changes in 15N/14N or 13C/12C ratios, but significant differences in these ratios were observed between irrigated and non-irrigated soybean with or without foliar B (Figure 3). The alteration of 15N/14N by increasing 15N (derived from soil nitrogen that is used for nitrate assimilation) and decreasing 14N (derived from atmospheric nitrogen that is used for nitrogen fixation) indicated that the source of nitrogen use changed, and plants favored N from soil over atmospheric nitrogen, indicating that nitrogenase is more sensitive than nitrate reductase under water stress. The mechanisms of this shift are not understood, but one possible explanation is that the shift in 15N/14N may reflect a possible mechanism to compensate for the inhibition of nitrogen fixation under water stress conditions. Previous research indicated that δ15N values in the xylem and plant tissues were associated with acquired N, and changed with N metabolism [59]. The increase in δ13C or higher 13C/12C ratio (less negative) in seed under severe water stress conditions indicated that the source of carbon fixation used was shifted. Previous research reported that that the δ13C value in plant tissues can be affected by water supply [60], plant physiology [61], and mycorrhizal infection [62]. The level of δ13C was dependent on the environmental factors and their association with plant gas exchange, stomatal conductance, and CO2 fixation [63]. It was found that drought stress leads to stomatal closure and 13C fixation increase, resulting in less discrimination against δ13C [64,65]. Previous research indicated that the the shift in 13C/12C ratio was a result of a shift in carbon fixation metabolism from ribulose bisphosphate (RuBP) carboxylase pathway to phosphoenolpyruvate carboxylase (PEP). This shift resulted in δ13C enrichment [60]. It was found that in C3 species, to which soybean belongs, carbon isotope composition changes among and between genotypes correlated with water use efficiency, and the stable isotope 13C would be discriminated against during photosynthesis, leading to a smaller 13C to 12C ratio [66]. The enrichment of 13C may be due closure of stomatal conductance under severe water stress, leading to 13C fixation increase and less 13C discrimination [67,68]. Our current results are in agreement with previous reports that environmental stresses, including drought, can alter δ13C due to the drought effects on the balance between stomatal conductance and carboxylation [67,68,69].

During carbon fixation by photosynthesis, the naturally occurring stable isotope 13C is discriminated against, and plants would have a smaller 13C to 12C ratio than 13C to 12C ratio in fixed CO2 of the air, suggesting a possible use of this technique to select for water use efficiency (Farquhar et al., 1989). Our results demonstrated that δ 15N and δ13C values changed and enrichment occurred under water stress conditions, suggesting that both nitrogen and carbon metabolism pathways were affected during water stress, impacting seed production and seed quality.

Figure 3.

Effects of foliar boron application (1.1 kg B ha-1) on seed δ15N (15N/14N Ratio) (A-C) and δ13C (13C/12C Ratio) (D-H) in soybean genotypes in watered plants with B (A) and without B (B); in water stressed plants with B (C) and without B (D); in soybean genotypes in watered plants with B (E) and without B (F); in water stressed plants with B (G) and without B (H); Soybean plants were grown under severe water stress conditions (soil water potential between –150 to –200 kPa). Soybeans were grown under greenhouse conditions similar to those previously reported [10].

Advertisement

4. Conclusions

Foliar boron application resulted in nodule growth by increasing the number and mass of nodules under well watered or moderate water stress conditions. Also, foliar boron resulted in higher nitrogen fixation and nitrogen assimilation under well watered or moderate water stress conditions. Foliar B application under severe water stress did not enhance nodule number or mass, nitrogen fixation and nitrogen assimilation. This is because severe water stress altered nitrogen and carbon fixation as indicated by changes in the values of 15N and 13C natural isotopes. Nitrogen fixation is more sensitive than nitrogen assimilation under severe water stress conditions. Foliar B enhanced seed sugars under well watered conditions, but sever water stress resulted in redistribution of sugar fractions by increasing monosaccharides such as glucose and fructose, decreasing sucrose as a result, but increasing both raffinose and stachyose due to their possible roles in drought and the acquisition of desiccation tolerance during seed development and maturation. Increasing sugars by foliar B is desirable as high glucose, fructose, and sucrose contribute to soybean seed quality by improving the taste and flavor of soymeal based products such as tofu, soymilk, and natto.

Although our research showed that B has beneficial effects on nodules and nitrogen fixation and seed quality, further research is needed to test these findings under field and drought conditions in multi-year and multi-location experiments so that recommendations can be made.

Advertisement

Acknowledgments

We thank Sandra Mosley for lab assistance and greenhouse management. Also, we thank Leslie Price for his technical assistance on nitrogen and carbon isotopes measurements. The U.S. Department of Agriculture (USDA) prohibits discrimination in all its programs and activities on the basis of race, color, national origin, age, disability, and where applicable, sex, marital status, familial information, political beliefs, reprisal, or because all or part of an individual’s income is derived from any public assistance program. (Not all prohibited bases apply to all programs.) Persons with disabilities who require alternative means for communication of program information (Braille, large print, audiotape, etc.) should contact USDA’s TARGET Center at (202) 720-2600 (voice and TDD). To file a complaint of discrimination, write to USDA, Director, Office of Civil Rights, 1400 Independence Avenue, S.W., Washington, D.C. 20250-9410, or call (800) 795-3272 (voice) or (202) 720-6382 (TDD). USDA is an equal opportunity provider and employer.

References

  1. 1. Pilbeam DJ, Kirkby EA. The Physiological Role of Boron in Plants. Journal of Plant Nutrition 1983; 6, 563–582.
  2. 2. Marschner H. Mineral Nutrition of Higher Plants. San Diego, CA (Academic Press) 1995; p379-396.
  3. 3. Brown PH, Bellaloui N, Dandekar AM, Hu H. Transgenically Enhanced Sorbitol Synthesis Facilitates Phloem Boron Transport and Increases Tolerance of Tobacco to Boron Deficiency. Plant Physiology 1999; 119, 17-20.
  4. 4. Dordas C, Apostolides G, Goundra O. Boron Application Affects Seed Yield and Seed Quality of Sugar Beets. Journal of Agricultural Sciences 2007; 145, 377–384.
  5. 5. Brown PH, Bellaloui N, Wimmer MA, Bassil ES, Ruiz J, Hu H, Pfeffer H, Dannel F, Romheld V. Boron in Plant Biology. Plant Biology 2002; 4, 205–223.
  6. 6. Hu H, Brown PH. Localization of Boron in Cell Walls of Squash and Tobacco and its Association with Pectin: Evidence for a Structural Role of Boron in the Cell Wall. Plant Physiology 1994; 105, 681–689.
  7. 7. Schon MK, Blevins DG. Foliar Boron Applications Increase the Final Number of Branches and Pods on Branches of Field-Grown Soybeans. Plant Physiology 1990; 92, 602-607.
  8. 8. Bellaloui N, Brown PH, Dandekar AM. Manipulation ofin vivoSorbitol Production Alters Boron Uptake and Transport in Tobacco. Plant Physiology 1999; 119, 735-742.
  9. 9. Bolaños L, Brewin NJ, Bonilla I. Effects of Boron on Rhizobium-Legume Cell--Surface Interactions and Node Development. Plant Physiology 1996; 110, 1249–1256.
  10. 10. Bellaloui N, Abbas HK, Gillen AM, Abel CA. Effect of Glyphosate-Boron Application on Seed Composition and Nitrogen Metabolism in Glyphosate-Resistant Soybean. Journal of Agriculture and Food Chemistry 2009a; 57, 9050–9056.
  11. 11. Bolaños L, Esteban E, de Lorenzo C, Fernández-Pascual M, deFelipe MR, Garate A, Bonilla I. Essentiality of Boron for Symbiotic Dinitrogen Fixation in Pea (Pisum sativum) Rhizobium Nodules. Plant Physiology 1994; 104, 85–90.
  12. 12. Carpena RO, Esteban E, Sarro MJ, Peñalosa J, Gárate A, Lucena JJ, Zornoza P. Boron and Calcium Distribution in Nitrogen-Fixing Pea Plants. Plant Sciences 2000; 151, 163–170.
  13. 13. Bonilla I, Mergold-Villasenor C, Campos ME, Sanchez N, Perez H, Lopez I, Castrejon L, Sanchez F, Cassab, GI. The Aberrant Cell Walls of Boron-Deficient Bean Root Nodules have no Covalently Bound Hydroxyproline-/Proline-Rich Protein. Plant Physiology 1997; 115, 1329–1340.
  14. 14. Bellaloui N, Mengistu A, Fisher DK, Abel CA. Soybean Seed Composition as Affected by Drought andPhomopsisin Phomopsis Susceptible and Resistant Genotypes. Journal of Crop Improvement2012a;26, 428-453.
  15. 15. Bellaloui N, Mengistu A, Zobiole LHS, Shier WT. Resistance to Toxin-Mediated Fungal Infection: Role of Lignins, Isoflavones, other Seed Phenolics, Sugars, and Boron in the Mechanism of Resistance to Charcoal Rot Disease in Soybean. Toxin Reviews 2012b; 31, 16-26.
  16. 16. Goldbach H. Influence of Boron Nutrition on Net Uptake and Efflux of 32P and 14C-Glucose inHelianthus annuusRoots and Cell Cultures ofDaucus carota. Journal of Plant Physiology 1985; 118, 431–438.
  17. 17. Camacho-Cristóbal JJ, González-Fontes A. Boron Deficiency Decreases Plasmalemma H+-Atpase Expression and Nitrate Uptake, and Promotes Ammonium Assimilation into Asparagine in Tobacco Roots. Planta 2007; 226, 443-451.
  18. 18. Camacho-Cristóbal JJ, Rexach J, González-Fontes A. Boron in Plants: Deficiency and Toxicity. Journal Integrative Plant Biology 2008; 50, 1247-1255.
  19. 19. Dordas C. Foliar Boron Application Improves Seed Set, Seed Yield, and Seed Quality of Alfalfa. Agronomy Journal 2006; 98, 907–913.
  20. 20. Sinclair TR, Purcell LC, King CA, Sneller CH, Chen P, Vadez V. Drought Tolerance and Yield Increase of Soybean Resulting from Improved Symbiotic N2 Fixation. Field Crops Research 2007; 101, 68–71.
  21. 21. Malavolta E, Vitti GC, Oliveira AS. Avaliação do Estado Nutricional das Plantas: Princípioseaplicações. Piracicaba: Potafos; 1997.
  22. 22. Brown P.H, Hu H. Phloem Mobility of Boron is Species Dependent: Evidence for Phloem Mobility in Sorbitol-rich Species. Annals of Botany 1996; 77, 497-505.
  23. 23. Planchet E, Rannou O, Ricoult C, Boutet-Mercey S, Maia-Grondard A, Limami AM. Nitrogen Metabolism Responses to Water Deficit Act Through both Abscisic Acid (ABA)-Dependent and Independent Pathways inMedicago truncatulaDuring Post-Germination. Journal of Experimental Botany 2011; 62, 2, 605–615.
  24. 24. Sinclair TR, Serraj R. Legume Nitrogen Fixation and Drought. Nature 1995; 378, 344.
  25. 25. Serraj R, Sinclair TR, Purcell LC. Symbiotic N2 Fixation Response to Drought. Journal of Experimental Botany 1999; 50, 143–155.
  26. 26. Bellaloui N, Hu Y, Mengistu A, Kassem MA, Abel, CA. Effects of Foliar Boron Application on Seed Composition, Cell Wall Boron, and Seed δ15N and δ 13C isotopes in Water-stressed Soybean Plants. Frontiers in Plant Nutrition 2013; 4, 1-12.
  27. 27. Fehr WR, Caviness CE. Stages of Soybean Development. Iowa Agriculture and Home Economics Experiment Station. Iowa State University, Ames, Iowa; 1977.
  28. 28. Klepper L, Hageman RH. The Occurrence of Nitrate Reductase in Apple Leaves. Plant Physiology 1969; 44, 1, 110-114.
  29. 29. Bellaloui N, Reddy KN, Zablotowicz RM, Mengistu A. Simulated Glyphosate Drift Influences Nitrate Assimilation and Nitrogen Fixation in Non-Glyphosate- Resistant Soybean, Journal of Agriculture and Food Chemistry 2006; 54, 9, 3357-3364.
  30. 30. Hardy RWF, Holsten D, Jackson EK, Burns RC. The Acetylene-Ethylene Assay for Nitrogen Fixation: Laboratory and Field Evaluation. Plant Physiology 1968; 43, 1185-1205.
  31. 31. Zablotowicz RM, Focht DD, Cannell GH. Nodulation and N Fixation of Field Grown California Cowpeas as Influenced by Irrigated and Drought Conditions. Agronomy Journal 1981; 73, 9-12.
  32. 32. Lohse G. Microanalytical Azomethine-H Method for Boron Determination in Plant Tissue. Communications in Soil Science and Plant Analysis 1982; 13, 2.
  33. 33. Bellaloui N, Reddy KN, Gillen AM, Abel CA. Nitrogen Metabolism and Seed Composition as Influenced by Foliar Boron Application in Soybean. Plant and Soil 2010a; 336, 143–155.
  34. 34. Bellaloui N, Smith JR, Gillen AM, Ray JD. Effect of Maturity on Seed Sugar in the Early Soybean Production System as Measured on Near-Isogenic Soybean Lines. Crop Science 2010b; 49, 608–620.
  35. 35. Bellaloui N, Mengistu A. Seed Composition Is Influenced by Irrigation Regimes and Cultivar Differences in Soybean. Irrigation Science 2008; 26, 261-268.
  36. 36. Bellaloui N, Mengistu A, Paris RL. Soybean Seed Composition in Cultivars Differing in Resistance to Charcoal Rot (Macrophomina phaseolina). Journal of Agricultural Sciences 2008; 146, 667-675.
  37. 37. Wilcox JR, Shibles RM. Interrelationships Among Seed Quality Attributes in Soybean. Crop Science 2001; 41, 11–14.
  38. 38. Bellaloui N, Mengistu A, Fisher DK, Zobiole L HS, Abbas HK. Irrigation Management: Effects of Soybean Diseases on Seed Composition in Genotypes Differing in their Disease Resistance under Irrigated and Non-irrigated Conditions. In: Irrigation Management, Technologies, Ali MH (ed.), Chapter 8, Nova Publishers, 2012; p193-228.
  39. 39. Boydak E, Alpaslan M, Hayta M, Gercek S, Simsek M. Seed Composition of Soybeans Grown in the Harran Region of Turkey as Affected by Row Spacing and Irrigation. Journal of Agricultural and Food Chemistry 2002; 50, 718-720.
  40. 40. Sigma, USA. Glucose (HK) Assay Kit Product Code GAHK-20. ProducIninformation, 2012.http://www.sigmaaldrich.com/etc/medialib/docs/Sigma/Bulletin/gahk20bul.Par.0001.File.tmp/gahk20bul.pdf (accessed July 29 2013).
  41. 41. Sigma, USA. Fructose Assay Kit, Product Code FA-20. ProductIninformation, 2012. http://www.sigmaaldrich.com/etc/medialib/docs/Sigma/Bulletin/fa20bul.Par.0001.File.tmp/fa20bul.pdf (accessed July 29 2013).
  42. 42. SAS. SAS 9.1 TS LeVel 1M3, Windows Version 5.1.2600, SAS Institute: Cary; 2001.
  43. 43. Yamagishi M, Yamamoto Y. Effects of Boron on Nodule Development and Symbiotic Nitrogen Fixation in Soybean Plants. Soil Science and Plant Nutrition 1994; 40, 265-274.
  44. 44. Mills HA, Jr. Jones JB. Plant Analysis Handbook II. Micromacro Publisher: Athens; 1996.
  45. 45. Reinbott TM, Blevins DG. Response of Soybean to Foliar-Applied Boron and Magnesium and Soil-Applied Boron. Journal of Plant Nutrition 1995; 18, 179-200.
  46. 46. Shelp BJ. Physiology and Biochemistry of Boron in Plants. In: Gupta UC (ed.) Boron and its Role in Crop Production, USA, Boca Raton, FL. CRC Press; 1993; p 53–85.
  47. 47. Kanayama Y, Kimura K, Nakamura Y, Ike T. “Purification and Characterization of Nitrate Reductase from Nodule Cytosol of Soybean Plants. Physiologia Plantarum 1999; 105, 3, 396-401.
  48. 48. Caba JM, Lluch C, Ligero F. Distribution of Nitrate Reductase Activity inVicia faba: Effect of Nitrate and Plant Genotype. Physiologia Plantarum 1995; 93, 667-672.
  49. 49. Ruiz JM, Baghour M, Bretones G, Belakbir A, Romero L. Nitrogen Metabolism in Tobacco Plants (Nicotiana TabacumL.): Role of Boron as a Possible Regulatory Factor. International Journal of Plant Sciences 1998; 159, 121–126.
  50. 50. Perica S, Bellaloui N, Greve C, Hu H, Brown PH. Boron Transport and Sugar Metabolism in Olive. Journal of the American Society for Horticultural Science 2001; 126, 291-296.
  51. 51. Taji T, Ohsumi C, Iuchi S, Seki M, Kasuga M, Kobayashi M, Yamaguchi-Shinozaki K, Shinozaki K. Important. Roles of Droughtand Cold inducible Genes for Galactinol Synthase in Stress Tolerance inArabidopsis thaliana.Plant Journal 2002; 29, 417–426.
  52. 52. Streeter JG. Effects of Drought on Nitrogen Fixation in Soybean Root Nodules. Plant, Cell & Environment 2003; 26, 1199–1204.
  53. 53. González EM, Gordon AJ, James CL, Arrese-Igor C. The Role of Sucrose Synthase in the Response of Soybean Nodules to Drought. Journal of Experimental Botany 1995; 46, 1515-1523.
  54. 54. Ren C, Bilyeu KD, Beuselinck PR. Composition, Vigor, and Proteome of Mature Soybean Seeds Developed Under High Temperature. Crop Science 2009; 49, 1010–1022.
  55. 55. Wilson RF. Seed Composition. In Boerma H, Specht JE (eds), Soybeans: Improvement, Production, and Uses, Madison, WI. American Society of Agronomy, Crop Science Society of America, and Soil Science Society of America: 2004; p621–668.
  56. 56. Liu K. Soybeans Chemistry, Technology, and Utilization.New York, NY 1997: Chapman & Hall.
  57. 57. Obendorf RL, Horbowicz M, Dickerman AM, Brenac P, Smith ME. Soluble Oligosaccharides and Galactosyl Cyclitols in Maturing Soybean Seeds in Planta andIn Vitro. Crop Science 1998; 38, 78–84.
  58. 58. Hou A, Chen P, Alloatti J, Li D, Mozzoni L, Zhang B, Shi A. Genetic Variability of Seed Sugar Content in Worldwide Soybean Germplasm Collections. Crop Science 2009; 49, 903-912.
  59. 59. Yoneyama T, Handley LL, Schrimgeour CM, Fisher DB, Raven JA. Variations of the Natural Abundances of Nitrogen and Carbon Isotopes inTriticum aestivumwith Special Reference to Phloem and Xylem Exudates. New Phytologist 1997; 137, 205-213.
  60. 60. O'Leary M.H. Environmental Effects on Carbon Isotope Fractionation in Terrestrial Plants. In: Wada E, Yoneyama T, Minagawa M, Ando T, Fry BD (eds), Kyoto, Kyoto University Press, 1995; p517-530.
  61. 61. Kumarashinghe KS, Kirda C, Arag M, Zapata F, Danso KSA. 13C isotope Discrimination Correlated with Biological Nitrogen Fixation in Soybean (Glycine maxL. Merrill). Plant and Soil 1992; 139, 145–147.
  62. 62. Högberg P. 15N Natural Abundance as a Possible Marker of the Ectomycorrhizal Habit of Trees in Mixed African Wood Land. New Phytology 1990; 115, 483-486.
  63. 63. Livingston NJ, Guy RD, Sun ZJ, Ethier GJ. The Effects of Nitrogen Stress on the Stable Carbon Isotope Composition, Productivity and Water Use Efficiency of White Spruce (Piceaglauca(Moench) Voss) Seedlings. Plant Cell & Environment. 1999; 22, 281–289.
  64. 64. Sun ZJ, Livingston, NJ, Guy RD, Ethier GJ. Stable Carbon Isotopes as Indicators of Increased Water Use Efficiency and Productivity in White Spruce (Picea glauca(Moench) Voss) Seedlings. Plant Cell & Environment. 1996; 19, 887–894.
  65. 65. Matsushima M. (2007). Nitrogen and Water Availabilities and Competitiveness of Bluejoint: Spruce Growth and Foliar Carbon-13 and Nitrogen-15 Abundance.S.S.S.A. J.71, 2007; p1547-1556.
  66. 66. Farquhar GD, Richards RA. Isotopic Composition of Plant Carbon Correlates with Water-Use Efficiency of Wheat Genotypes. Australian Journal of Plant Physiology 1984; 11, 6, 539-552.
  67. 67. Fotelli MN, Rennenberg H, Holst T, Mayer H, Geßler A. Carbon Isotope Composition of Various Tissues of Beech (Fagus sylvatica) Regeneration Is Indicative of Recent Environmental Conditions within the Forest Understory. New Phytologist 2003; 159, 1, 229-244.
  68. 68. Kume A, Satomura T, Tsuboi N, Chiwa M, Hanba YT, Nakane K, Horikoshi T, Sakugawa H. Effects of Understory Vegetation on the Ecophysiological Characteristics of an Overstory Pine, Pinus Densiflora. Forest Ecology and Management 2003; 176, 195-203.
  69. 69. Robinson D, Handley LL, Scrimgeour CM, Gordon DC, Forster BP, Ellis RP. Using Stable Isotope Natural Abundances (δ15N and δ13C) to Integrate the Stress Responses of Wild Barley (Hordeum spontaneumC. Koch.) Genotypes. Journal of Experimental Botany 2000; 51, 342, 41-50.

Written By

Nacer Bellaloui, Alemu Mengistu, My Abdelmajid Kassem, Craig A. Abel and L.H.S. Zobiole

Submitted: September 12th, 2013 Reviewed: September 20th, 2013 Published: January 29th, 2014