American Journal of Plant Sciences, 2011, 2, 692-701
doi:10.4236/ajps.2011.25084 Published Online November 2011 (http://www.SciRP.org/journal/ajps)
Effect of Water Stress and Foliar Boron
Application on Seed Protein, Oil, Fatty Acids, and
Nitrogen Metabolism in Soybean*
Nacer Bellaloui
Crop Genetics and Production Research Unit, USDA-ARS, Stoneville, MS, USA.
Received August 17th, 2011; revised September 6th, 2011; accepted October 15th, 2011.
ABSTRACT
Effects of water stress and foliar boron (FB) application on soybean (Glycine max (L) Merr.) seed composition and
nitrogen metabolism have not been well investigated. Therefore, the objective of this study was to investigate the effects
of water stress and FB on seed protein, oil, fatty acids, nitrate reductase activity (NRA), and nitrogenase activity (NA).
A repeated greenhouse experiment was conducted where one set of soybean plants were subjected to water stress (WS),
and the other set was watered (W). Foliar boron (B) was applied at rate of 0.45 kg·ha−1. Treatments were watered-
plants with no FB (W), watered-plants with FB (WB), water-stress plants with no FB (WS), and water-stress plants with
FB (WSB). The results showed that seed protein and oil percentage were significantly (P < 0.05) higher in WB than
other treatments. Oleic acid increased and linolenic acid decreased in WB and WSB. Significant (P < 0.05) increase in
NRA in leaves and roots and NA occurred in WB compared to W. In WSB, NRA in leaves and roots or nitrogenase ac-
tivities were higher than those in WS. Nitrate reductase activity in nodules was greater in WB than in W, and was
higher in WSB than in WS. The concentration of B in leaves and seed were significantly (P < 0.05) higher in W than in
WS. Seed 15N/ 14N and 13C/12C natural abundance were altered between watered- and watered-stressed plants. These
results suggest that water stress and FB can influence seed composition, and nitrogen metabolism, and 15N/14N and
13C/12C ratios, reflecting environmental and metabolic changes in carbon and nitrogen fixation pathways. Lack of B
translocation from leaves to seed under water stress may suggest a possible mechanism of limited B translocation under
water stress. These findings may be beneficial to breeders to select for B translocation efficiency under drought condi-
tions. Altered 15N/14N and 13C/12C under water stress can be used as a tool to select for drought tolerance using N and C
isotopes in the breeding programs.
Keywords: Boron Nutrition, Nitrate Reductase, Nitrogenase, Nitrogen Assimilation, Nitrogen Fixation, Nitrogen
Metabolism, Seed Composition, Nitrogen and Carbon Isotopes
1. Introduction
Soybean is a major crop in the world, and soybean seed
is a major source of protein and oil. The quality of soy-
bean seed is determined by the content and composition
of protein, oil, and fatty acids. Soybean seed protein
ranges from 34 to 57% of total seed weight, with a mean
of 42%, and oil content ranges from 8.3 to 28%, with a
mean of 19.5% [1]. The concentration of saturated fatty
acids in soybean seed oil ranges from 10% to 12% palmitic
acid (C16:0), and 2.2 to 7.2% stearic acid (C18:0) [2].
The mean concentration of unsaturated fatty acids in
soybean seed oil is 24% oleic acid (C18:1), 54% linoleic
acid (C18:2), and 8.0% linolenic acid (C18:3) [3]. Higher
oleic acid and lower linolenic acid are desirable traits for
oil stability and long-term shelf storage for industrial and
processing purposes, but higher linolenic acids (essential
polyunsaturated) are desirable for human nutrition. Hy-
drogenation of polyunsaturated fatty acids such as lino-
lenic acid leads to trans-isomers, which are associated
with increased incidence of heart disease [4]. However,
monounsaturated fatty acids such as oleic acid are less
susceptible to oxidative changes during refining, storage,
and frying. Consequently, the food industry is becoming
increasingly interested in producing soybean seed with a
*Mention of trade names or commercial products in this publication is
solely for the purpose of providing specific information and does no
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imply recommendation or endorsement by the U.S. Department o
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Effect of Water Stress and Foliar Boron Application on Seed Protein, Oil, Fatty Acids, 693
and Nitrogen Metabolism in Soybean
high content of oleic acid and low linoleic and linolenic
acids [5]
Boron is an essential micronutrient for plant growth
and development [6-8]. Boron was reported to have an
important role in nodule development of Vicia faba [9],
and was found to be an essential micronutrient for the
development of nodules in pea (Pisum sativum) [10]. It
was reported that Rhizobia inside nodules exhibited little
or no ability to fix N2 in B-deficient plants, resulting in
N deficiency and necrosis of nodulated pea plants [11].
Nitrogenase fixes atmospheric N2 in the bacteroids of
nodules [12], and nitrate reduction (assimilation) is cata-
lyzed by the enzyme nitrate reductase, a limiting step in
nitrate assimilation. Both nitrate reductase and nitro-
genase enzymes coexist in nodules and compete for a
reducing agent (reductant) [13].
The role of B in flower set, fruit set, seed set, and seed
quality was reported in other species, and it was found
that floral and fruiting organs are sensitive to B defi-
ciency [14,15]. Higher B requirements during flowering
and seed set were shown even where B levels in leaves
are in the adequate range [16]. Recently, it was found
that foliar B application improved seed protein and seed
oleic fatty acid [17], and seed yield and seed quality of
alfalfa [16], and increased fruit set [18].
In spite of B role in structure, metabolism, and im-
proving yield and seed quality in plants, its effect on seed
composition and yield in soybean has not been yet estab-
lished. Therefore, the objective of this research was to in-
vestigate the effects of foliar B application on seed com-
position (protein, oil, and fatty acids), nitrogen assimila-
tion, and nitrogen fixation in soybean. Since changes in
plant physiology and environment resulting from water
stress or drought may alter natural abundances of carbon
isotopes (δ13C) and nitrogen isotopes (δ15N) in higher
plants [19-21], seed nitrogen (δ15N) and carbon (δ13C)
isotopes were also investigated.
2. Materials and Methods
A greenhouse experiment was repeated twice. Seeds of
soybean cultivar AG4903RR were germinated in flat
trays in vermiculite. Uniform size seedlings at about V1
stage were transplanted into 9.45 L size pots filled with
field soil. The soil 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,
and it contained an abundant native population of B.
japonicum. To induce water stress, soil in pots were
weighed and then saturated with dionized water and left
to drain and weighed again to obtain the water field
capacity as measured by soil water sensors inserted in
pots. Soil water potential was measured daily using Soil
Moisture Meter (WaterMark Company, Inc., Wisconsin,
USA). Water stressed plants were kept between –90 and
–100 kPa. This represented a moderate to severe water
stress level for soybean under greenhouse conditions.
Watered plants were kept between –15 to –20 kPa (this
was considered field capacity for the control plants) [17].
Half of the plants from each B treatment was watered
(W), and the other half was water stressed (WS). Treat-
ments were watered plants with no foliar B (W), watered
plants with foliar B (WB), water stress plants with no
foliar B (WS), and water stress plants with foliar B
(WSB). Boron, as boric acid, of a rate of 0.45 kg·ha−1
was foliar applied using hand sprayer, and measures to
avoid boron drift to the control plants were taken [17].
Boron was applied at R1-R2 (flowering stage) and
R5-R6 (seed-fill stage) [22], or not applied (control).
Samples were taken five days after each foliar B
application for NRA, nitrogenase, and leaf B. Mature
seed were weighed at R8 (physiological maturity stage).
Plants were considered fully matured when they reached
R8 according to [22]. Treatments were arranged in a split
plot design with irrigation as a main block and B
treatment as sub-plot. Four replicates were used for each
treatment and for each sampling time for each expe-
riment. Each pot with four individual plants was con-
sidered one replicate Greenhouse conditions were about
34˚C ± 9˚C during the day and about 28˚C ± 7˚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
range of light intensity reflects a bright, sunny, or cloudy
day. The source of lighting in the greenhouse was a
mixture of natural light, bulb light (60 W), cool white
(250 W). To be consistent with the normal photoperiod
for soybean growth and to avoid differences in the
day-length between the two experiments, the two expe-
riments 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.
2.1. Nitrate Reductase Assay
Nitrate reductase activity (NRA) was determined ac-
cording to [23,24]. Briefly, NRA was measured in leaves
(fully expanded leaves), stems, roots, and nodules. Nod-
ules were gently and carefully separated from roots and
placed in NRA assay buffer solution. Approximately 0.3
g of plant tissue 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 incu-

Effect of Water Stress and Foliar Boron Application on Seed Protein, Oil, Fatty Acids, 694
and Nitrogen Metabolism in Soybean
bation solution was vacuum filtered for 1 min, flashed
with nitrogen gas for 30 s, and then incubated at 30˚C.
Samples of 0.5 mL were 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%). The samples were read at 540 nm after
30 minutes using a Beckman Coulter DU 800 spectro-
photometer (Fullerton, CA). A standard curve made of
nitrite concentrations was prepared and used to measure
nitrite concentrations in the plant tissue (Bellaloui et al.,
2006). Potential NRA (PNRA) was measured by adding
exogenous nitrate to the incubation solution at a concen-
tration of 10 mM as KNO3.
2.2. Acetylene Reduction Assay
Nitrogenase activity (NA) was assayed using the acety-
lene reduction assay as described elsewhere [25,26]. Briefly,
roots with nodules intact were excised and incubated in 1
L Mason jars. Four roots were placed 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 chromato-
graphy for ethylene formation. An Agilent HP6960 (Agi-
lent Technologies, Wilmington, DE) gas chromatograph
was equipped with manual injector, injector loop, sample
splitter, flame ionization detector (FID), and thermal
conductivity detector (TCD). A sample of 0.25 mL of
gas was directed into a 30 m length × 0.53 mm i.d. alu-
mina 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 in-
tegrated using Chem Station software. Standard curves
for ethylene were developed for each day of analysis and
used to determine ethylene. Nodules were obtained by
carefully removing the nodules from the roots, and the
dry weight was determined following oven-drying at
60˚C for 4 - 5 days.
2.3. Boron Determination
Boron in fully expanded leaves and seed was determined
according to Azomethine—H method [17,27]. Briefly, 1
g of dry samples was placed in a porcelain crucible for
ashing at 500˚C for 8 hr. Then, samples were extracted
with 20 mL of 2 M HCl at 90˚C for 10 min. After filtra-
tion, the samples were transferred to plastic vials and 2
mL of the solution was added to 4 mL of buffer solution
and 4 mL of azomethine-H solution containing 0.45%
azomethine-H and 1% of ascorbic acid prepared right
before the analysis [28]. The buffer solution contained
25% ammonium acetate, 1.5% EDTA, and 12.5% acetic
acid. Boron concentration measurement was performed
after 45 minutes of color development. Samples were
read using a Beckman Coulter DU 800 spectrophoto-
meter (Fullerton, California) at 420 nm. Boron analysis
in soil was conducted at The University of Georgia, Soil,
Plant, and Water Laboratory, Athens, GA. Briefly, a soil
sample of 5.0 g: 20 mL Mehlich 1 solution was used.
Boron concentration was analyzed using Inductively
Coupled Plasma spectrometry (ICP) using Thermo Ele-
mental, Thermo Jarrell-Ash model 61E ICP, USA.
2.4. Analysis of δ15N (15N/14N Ratio) and δ13C
(13C/12C Ratio) Using Natural Abundance
Delta 15N and 13C natural abundance was determined from
nitrogen isotope, 15N/14N ratio, and carbon isotope, 13C/
12C ratio, 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, Ger-
many). Isodat software version 2.38 was used to obtain
Delta values [29,30]. The elemental combustion system
was Costech ECS 4010 with an autosampler (Bremen,
Germany).
2.5. Analysis of Seed Protein, Oil, and Fatty
Acids
Seed protein, oil, and fatty acids were analyzed using
near-infrared (NIR) reflectance [24,31], diode array feed
analyzer, Perten. The calibration was developed by the
University of Wisconsin, USA using Perten’s Thermo
Galactic Grams PLS IQ software. The calibration was
developed for unique samples using AOAC methods [32,
33]. The analysis was performed on the basis of percent
dry matter [31,34].
2.6. Experimental Design and Statistical Analysis
Treatments were arranged in a split plot design with irri-
gation as a main block and B treatment as sub-plot. The
data were subjected to analysis of variance using Proc
GLM in SAS [35]. Means were separated by Fisher’s
least significant difference test at the 5% level of pro-
bability. Since there were no interactions between the
two experiments for the measured variables, the data
were pulled and combined.
3. Results and Discussion
3.1. Seed and Nodule Weights
Seed (Figure 1(a)) and nodule (Figure 1(b)) weights

Effect of Water Stress and Foliar Boron Application on Seed Protein, Oil, Fatty Acids, 695
and Nitrogen Metabolism in Soybean
were higher in W and WB, with the highest weight ob-
served in WB treatment. This was observed at both
R1-R2 and R5-R6 stages (Figures 1(b) and (c)). This
indicates the significance positive effects of foliar B on
seed and nodule weights. The lowest seed and nodule
weight were observed in WS and WSB. The increase of
soybean seed weight by foliar B was previously reported
by other researchers. For example, it was found that B
application increased soybean seed yield [36,37]. In gen-
eral, B application in the field had negative, positive, or
no yield responses from direct applications of B fertilizer
[36-39]. The increase of nodule weight by FB indicates
the positive response of nodules to B. It was reported that
B is an essential micronutrient for the development of
nitrogenfixing root nodules in pea (Pisum sativum) [40].
This may indicate that B induces nodule formation and
development, as suggested by [40], or lowers infection of
the host plants with Rhizobium in plants grown in
B-deficient medium compared with plants supplied with
adequate B [40]. Recently, it was found that FB in-
creased nodule weight under irrigated greenhouse condi-
tions [17]. Since B concentrations in leaves of WS plants
fall below 20 mg·B·kg–1, critical level of B in leaves for
normal plant growth [41], a positive response to FB was
expected. However, the non-response of WS or WSB to
FB may be due to water stress effects. The current results
showed that, even though B concentration in leaves was
above the critical level, FB had a positive effect on seed
and nodule weights. Therefore, the current results agreed
with previous research of those of [17,36,37] in that FB
can increase seed and nodule weights in soybean.
3.2. Boron Concentrations in Leaves and Seed
Boron concentration in leaves was significantly higher in
W and WB than in WS and WSB, with leaf B in WB
being the highest (Figure 1(d)). This was noticed at
R1-R2 and R5-R6 stages. Seed B concentration was the
highest in WB (Figure 1(d)). The increase in B con-
centration in leaves of WSB did not result in higher B
concentration in seed. The higher B concentrations in
leaves of WSB than in WS indicate that FB leads to
higher B concentrations in leaves under water stress. The
lower level of B concentration in leaves of WS plants
indicates that B was not taken up under water stress even
when B concentration in soil was adequate. Although
soil B in the used soil was considered adequate, B con-
centration in leaves of WS was significantly lower than
those of WSB due to water stress. The lower B concen-
tration in seed in spite of transferrable B from leaves to
seed in in WS and WSB plants indicated that B translo-
cation from leaves to seed was limited under water stress,
suggesting that the positive or beneficial effect of FB on
Figure 1. Effect of foliar boron (B) application on seed
weight (a), nodule weight at R1-R2 stage (b), nodule weight
at R5-R6 stage (c), boron in leaf and seed (d). Foliar B
treatments were: W = plants were watered with no foliar B;
WB = Plants were watered and received foliar B; WS =
plants were water-stressed with no foliar B; WSB = plants
were water-stressed and received foliar B. Foliar B was
applied at R1-R2 (flowering stage and at R5-R6 (seed-fill
stage). Bar Values are means ± SE.
soybean may depend on B translocation from leaves
(source) to seed (sink). Limited translocation of B from
leaves to seed under water stress condition could under-
line one of the mechanisms of how water stress affects B
nutrition. If this is the case, then FB application under
water stress or drought conditions in the field may in-

Effect of Water Stress and Foliar Boron Application on Seed Protein, Oil, Fatty Acids, 696
and Nitrogen Metabolism in Soybean
crease B concentrations in leaves and seed. This may ex-
plain the controversial literature on the effect of B on
soybean yield. For example, during dry growing season,
application of FB may increase seed yield, but under nor-
mal growing season with normal rainfall, application of
FB may not increase seed yield. The higher cell wall B
percentage in WS (91%) and WSB (86%) compared with
that of W (71%) or WB (61%) plants (data not shown),
emphasized the structural role of B in cell wall, as su-
ggested by previous researchers [17,42].
3.3. In Vivo Nitrate Reductase Activity
Five days after FB, NRA at R1-R2 in leaves and roots of
W and WB was significantly higher than in those of WS
or WSB (Figure 2(a)). Leaves of WSB showed higher
Figure 2. Effect of foliar boron (B) application on nitrate
reductase activity (NRA) in soybean leaves, roots, stems,
and nodules. Foliar B treatments were: W = plants were
watered with no foliar B; WB = Plants were watered and
received foliar B; WS = plants were water-stressed with no
foliar B; WSB = plants were water-stressed and received
foliar B. Foliar B was applied at R1-R2 (flowering stage (a)
and at R5-R6 (seed-fill stage) (b). Bar Values are means ±
SE.
NRA than those of WS. Same pattern of NRA was ob-
served at R5-R6 growth stage (Figure 2(b)). Adding ni-
trate to the incubation solution (potential nitrate reduc-
tase activity, PNRA) to either leaves or roots resulted in
a significant increase of PNRA in WS and WSB plants
compared with W and WB (data not shown). The per-
centage increase of potential NRA in WS was 47% in
leaves and 41% in roots, and in WSB was 35% in leaves
and 31% in roots compared with their equivalent NRA
without adding nitrate to the incubation solution (data
not shown). Nitrate reductase activity per organ (leaves,
stems, or roots) and per plant showed the same pattern as
those of NRA per g fwt (Figure 3(a)). Similar pattern
was observed five days after FB at R5-R5 (Figure 3(b)).
Total NRA (µmol·nitrite·plant–1·h–1) showed that W
plants had the highest NRA followed by W, WSB, and
finally WS, following similar pattern of those in NRA
per g fwt or per organ (Figure 4(a)). The higher NRA in
leaves and roots in W and WB indicates the nega-
Figure 3. Effect of foliar boron (B) application on nitrate
reductase activity (NRA) per soybean organ (leaves, roots,
stems). Foliar B treatments were: W = plants were watered
with no foliar B; WB = Plants were watered and received
foliar B; WS = plants were water-stressed with no foliar B;
WSB = plants were water-stressed and received foliar B.
Foliar B was applied at R1-R2 (flowering stage (a) and at
R5-R6 (seed-fill stage) (b). Bar Values are means ± SE.

Effect of Water Stress and Foliar Boron Application on Seed Protein, Oil, Fatty Acids, 697
and Nitrogen Metabolism in Soybean
tive effect of water stress on nitrogen assimilation, lead-
ing to NRA inhibition. The increase of leaf NRA with
FB indicates that B may stimulate NRA by, possibly,
increasing nitrate uptake or translocation of nitrate from
vacuoles (inaccessible nitrate for reduction) to cytoplasm
(accessible nitrate for reduction). The higher increase in
PNRA in roots in WS and WSB than those of W or WB
indicates that water stress limited nitrate (substrate)
availability to nitrate reductase, and this was reflected by
the striking increase of PNRA when nitrate was added to
the incubation solution. This observation also suggests
that nitrate reductase was inactive under water stress
conditions, and can be active when the substrate, nitrate,
becomes available. Severe water stress (soil water poten-
tial between –150 to –195 kPa) led to complete irrever-
sible NRA (data not shown).
The increase of NRA by FB may be due to an induc-
ing, indirect effect [7] of B on nitrate assimilation [17].
The exact mechanism of B effects on NRA is not yet
understood, but it was suggested that B may induce ni-
trate uptake and nitrate availability to nitrate reductase,
enhance de novo synthesis of the enzyme and its effects
on cell membrane. The influence of B on ion uptake was
previously reported [7,43], and was suggested to be me-
diated by direct or indirect effects of B on the plasma
membrane-bount H+ ATPase [36,44,45], cell wall struc-
ture and membrane integrity [7,36]. The current results
support those of [7] in that B has an indirect effects by
inducing nitrate uptake and assimilation [17], increasing
nitrate availability to NR, and contributes to membrane
cell membrane cell wall integrity.
3.4. δ15N (15N/14N ratio) and δ13C (13C/12C Ratio)
Natural Abundance
There was no difference in 15N/14N or 13C/12C between
irrigated foliar applied and non-foliar applied soybean.
However, there were significant changes in 15N/14N
and 13C/12C ratios between irrigated and non-irrigated
soybean with or without FB (Figures 4(b) and (c)). Water
stress altered 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) (Figure 4(a)). This indi-
cates that water stress inhibited nitrogen fixation, may be
due to that nitrogenase is more sensitive than nitrate re-
ductase. This shift in 15N/14N may reflect a possible
mechanism to compensate for the inhibition of nitrogen
fixation under water stress conditions. It has been re-
ported that the δ15N values in the xylem and plant tissues
are associated with acquired N, and the value can be
altered because of N metabolism [21]. There was also a
change in 13C/12C ratio between irrigated and non-
irrigated soybean with or without FB (Figure 4(c)). The
Figure 4. Effect of foliar boron (B) application on nitrate
reductase activity (NRA) per whole plant (a) and on seed δ
15N (15N/14N ratio) (b), and on seed δ 13C (13C/12C ratio) (c).
Foliar B treatments were: W = plants were watered with no
foliar B; WB = Plants were watered and received foliar B;
WS = plants were water-stressed with no foliar B; WSB =
plants were water-stressed and received foliar B. Bar Va-
lues are means ± SE.
increase in δ13C (higher 13C/12C ratio = less negative) in
seed of water stressed soybean indicates that water stress
altered the source of carbon fixation. It was reported that
the δ13C value in plant tissues can be influenced by water
supply and temperature [46], plant physiology [47], and
mycorrhizal infection [48]. There are two systems for
carbon fixation in N2-fixing plants, depending on where
the carbon is fixed. In nodules carbon fixation is cata-
lyzed by phosphoenolpyruvate (PEP) carboxylase; in
leaves carbon is fixation is catalyzed by ribulose bispho-
sphate (RuBP) carboxylase. Therefore, the incorporation
of fixed carbon by the nodules may change the δ13C
value in the nodulated plants. The δ13C values of carbon
fixed by PEP carboxylase are less negative than that of
CO2 fixed by RuBP carboxylase [46]. It was found that

Effect of Water Stress and Foliar Boron Application on Seed Protein, Oil, Fatty Acids, 698
and Nitrogen Metabolism in Soybean
for C3 species, the variability of carbon isotope composi-
tion among and between genotypes correlated with water
use efficiency, and during carbon fixation by photosyn-
thesis, the naturally occurring stable isotope 13C is dis-
criminated against, and plants would have a smaller 13C
to 12C ratio than 13C to 12C ratio in fixed CO2 of the air
[49]. The altered 13C to 12C ratio could be due closure of
stomatal conductance under drought, resulting in an in-
crease of 13C fixation, leading to less 13C discrimination
[50,51]. The current results support previous research
that environmental stresses, including drought, can alter
δ13C as a result of effects on the balance between sto-
matal conductance and carboxylation [50-52].
3.5. Acetylene Reduction Assay
Five days after FB at R1-R2, FB resulted in higher
nitrogen fixation in WB and WSB plants, with nitrogen
fixation being the highest in WB plants. This pattern was
shown at R1-R2 and R5-R6 (Figures 5(a) and (b)). The
relationship between B and nitrogen fixation is not yet clear.
Figure 5. Effect of foliar boron (B) application on nitroge-
nase activity in soybean at R1-R2 stage (a) and at R5-R6
stage (b). Foliar B treatments were: W = plants were wa-
tered with no foliar B; WB = Plants were watered and re-
ceived foliar B; WS = plants were water-stressed with no
foliar B; WSB = plants were water-stressed and received
foliar B. Foliar B was applied at R1-R2 (flowering stage (a)
and at R5-R6 (seed-fill stage) (b). Bar Values are means ± SE.
However, previous research suggested that nodules under
B deficiency had little or no ability to fix N2, leading to
N deficiency and necrosis of nodulated pea plants [11].
Recently, it was shown that FB increased nitrogen
fixation under irrigated greenhouse conditions [17]. In
spite of the possible explanation that limited B can
reduce early nodullin protein (ENOD2) in nodule pa-
renchyma cells and malfunction of oxygen diffusion
barrier, especially under B deficiency [53]. There is no
convincing evidence that there is a direct effect of B on
nitrogen metabolism [7,17,53,54]. The current results
showed that FB increased nitrogenase activity under both
water stress and irrigated conditions, agreeing with pre-
vious studies [11,17,40]. It was suggested that B may
protect nitrogenase against the oxygen damage to mem-
brane integrity and function [53] or may maintain the
proper conformation in nitrogen-fixing cells [14]. Further
research is needed to elucidate the relationship between
B and nitrogenase.
3.6. Seed Protein, Oil, and Fatty Acids
In seed of WB plants, protein and oil percentage were
significantly (P < 0.05) higher than those of W, WS, or
WSB (Table 1). Compared to seed protein in W plants,
the increase in seed protein was 13.8% in WB, 3% in WS,
and 8% in WSB. Seed oil increased 11% in WB, but
decreased 9.8% in WS and 14% in WSB compared to
those of W plants. The most noticeable change was in
Table 1. Effect of foliar boron (B) application on seed pro-
tein, oil, monounsaturated fatty acid (oleic), and polyun-
saturated fatty acid (linolenic). Foliar B treatments were:
W = plants were watered with no foliar B; WB = Plants were
watered and received foliar B; WS = plants were water-
stressed with no foliar B; WSB = plants were water-
stressed and received foliar B(*).
Concentration
Treatments Protein
(g·kg–1)
Oil
(g·kg–1)
Oleic
(g·kg–1)
Linolenic
(g·kg–1)
W 398 d 204 b 200 d 83 a
WB 453 a 226 a 299 a 59 b
WS 410 c 1844 c 220 c 85 a
WSB 431 b 175 b 274 b 58 b
Content
Treatments Protein
(g·plant-1)
Oil
(g·plant-1)
Oleic
(g·plant-1)
Linolenic
(g·plant-1)
W 6.67 b 3.42 b 3.37 b 1.39 a
WB 9.81 a 4.90 a 6.51 a 1.28 b
WS 5.96 c 2.62 c 3.17 c 1.23 c
WSB 6.14 c 2.49 c 3.90 c 0.83 d
*Means within a column for each water treatment separately followed by the
same letter are not significantly different at the 5% level.

Effect of Water Stress and Foliar Boron Application on Seed Protein, Oil, Fatty Acids, 699
and Nitrogen Metabolism in Soybean
oleic acid. Compared to those of W plants, oleic acid
increased 49% in WB, 10% in WS, and 36.9% in WSB.
On the other hand, linolenic acid was decreased by FB,
opposing the pattern of oleic acid. Compared with those
of W plants, the decrease in linolenic acid was 28.6% in
WB and 29.8% in WSB. The increase of seed protein
was not accompanied with a decrease in oil in WB com-
pared with those of W plants. The inverse relationship
between protein and oil in soybean is well established
[55]. However, this inverse relationship was shown in W,
WS, and WSB. The increase in oleic acid and decrease in
linolenic acid with foliar B application in watered and
water-stressed plants indicated that both FB and water
stress can result in seed constituent altering. Expressing
the seed constituents on seed weight per plant, water
stressed plants with or without foliar B (WS or WSB)
had lower protein, oil, and linolenic acid compared to
watered plants (W), and this was expected based on
previous research. Previous research showed that there
was a positive relationship between soil B and seed
protein and oleic acid, suggesting an indirect role of B in
seed composition [56]. Other research showed that foliar
B application increased soybean seed protein and oleic
acid concentrations [17,57]. Recently, it was found that
foliar B application under irrigated conditions increased
seed protein and oleic acid, and decreased linolenic acid.
Although the effect of fertilizers on seed composition is
still inconsistent, the current results support previous
research that foliar B alters seed composition.
4. Conclusions
Foliar B application can alter seed composition by in-
creasing seed protein and oleic acid and decreasing lino-
lenic acids. Foliar B application at both flowering and
seed fill stages induced nitrogen assimilation and nitro-
gen fixation, suggesting a close relationship between B
nutrition and nitrogen metabolism. The nature of this
relationship is still unclear (Bellaloui et al., 2010). Water
stress inhibited nitrogen assimilation and nitrogen fixa-
tion, and altered 15N/14N and 13C/12C ratios. Boron trans-
location from leaves to seed was limited under water
stress, suggesting a possible mechanism of B transloca-
tion within soybean plants. The current research suggests
that soybean selection for B translocation efficiency is an
important trait for soybean B nutrition under drought
conditions such as in ESPS. Alteration in 15N/14N and
13C/12C ratios under water stress indicates that nitrogen
and carbon isotopes could be used as a tool in selecting
for drought tolerant soybean lines.
5. Acknowledgements
We thank Sandra Mosley and Earl Gordon for field and
lab technical assistance, and Albert Tidwell for field
management. Also, we thank Leslie Price for his tech-
nical assistance on nitrogen isotope measurements.
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