American Journal of Plant Sciences, 2011, 2, 702-715
doi:10.4236/ajps.2011.25085 Published Online November 2011 (http://www.SciRP.org/journal/ajps)
Influence of Planting Date on Seed Protein, Oil,
Sugars, Minerals, and Nitrogen Metabolism in
Soybean under Irrigated and Non-Irrigated
Environments*
Nacer Bellaloui1, Krishna N. Reddy2, Anne M. Gillen1, Daniel K. Fisher2, Alemu Mengistu3
1Crop Genetics Research Unit, USDA-ARS, Stoneville, MS, USA; 2Crop Production Systems Research Unit, USDA-ARS, Stone-
ville, USA; 3 Crop Genetics Research Unit, USDA-ARS, Jackson, TN, USA.
Received August 17th, 2011; revised September 21st, 2011; accepted October 15th, 2011.
ABSTRACT
Information on the effect of planting date and irrigation on soybean [Glycine max (L.) Merr.] seed composition in the
Early Soybean Production System (ESPS) is deficient, and what is available is inconclusive. The objective of this re-
search was to investigate the effects of planting date on seed protein, oil, fatty acids, sugars, and minerals in soybean
grown under irrigated (I) and non-irrigated (NI) co ndition s. A 2-yr field experiment was conducted in Stoneville, MS in
2007 and 2008. Soybean was planted during second week of April (early planting) and second week of May (late plant-
ing) each year. Results showed that under irrigated condition, early planting increased seed oil (up to 16% increase)
and oleic acid (up to 22.8% increase), but decreased protein (up to 6.6% decrease), linoleic (up to 10.9% decrease)
and linolenic acids (up to 27.7% decrease) compared to late planting. Under I conditions, late planting resulted in
higher sucrose and raffinose and lower stachyose compared with early planting. Under NI conditions, seed of early
planting had higher protein (up to 4% increase) and oleic acid (up to 25% increase) and lower oil (up to10.8% de-
crease) and linolenic acids (up to 13% decrease) than those of late planting. Under NI, stachyose concentration was
higher than sucrose or raffinos e, especially in early planting. Under I, early planting resulted in lower lea f and seed B,
Fe, and P concentrations compared with those of late planting. Under NI, however, early planting resulted in higher
accumulation of leaf B and P, but lower seed B and P compared with those of late planting. This research demonstrated
that both irrigation and planting date have a significant influence on seed protein, oil, unsaturated fatty acids, and
sugars. Our results suggest that seed of late planting accumulate more B, P, and Fe than those of early planting, and
this could be a beneficial gain. Limited translocation of nutrients from leaves to seed under NI is undesirable. Soybean
producers may u se this information to maintain yield and seed quality , and soybean breeders to select for seed quality
traits and mineral translocation efficiency in stress environments.
Keywords: Mineral Nutrition, Oligosaccharides, Raffinose, Stachyose, Seed Composition, Sucrose
1. Introduction
Soybean is a major crop in the word and a source of pro-
tein, oil, carbohydrates, and other nutrients for humans
and animals [1]. Seed contains about 40% protein, 20%
oil, and 33% carbohydrates [2]. Soybean seed soluble
carbohydrates, including disaccharides (sucrose) and oli-
gosaccharides (raffinose and stachyose), contribute to
seed quality [3]. The average soybean seed contains 9%
to 12% total soluble carbohydrates, of which 4% to 5% are
sucrose (C12H22O11), 1 to 2% are raffinose (C18H32O16),
and 3.5% to 4.5% are stachyose (C24H42O21) [4]. Raffinose
and stachyose are undesirable seed quality traits because
they have detrimental effects on food and feed quality,
causing flatulence or diarrhea in nonruminants [3], and
soybean with low raffinose and stachyose is desirable
because of increased feed energy efficiency, mineral
uptake, and reduced flatulence for nonruminant animals.
Soybean seed with high sucrose is desirable because it
*Mention of trade names or commercial products in this publication is
solely for the purpose of providing specific information and does no
t
imply recommendation or endorsement by the U.S. Department o
f
A
g
riculture.

Influence of Planting Date on Seed Protein, Oil, Sugars, Minerals, and Nitrogen Metabolism in Soybean under 703
Irrigated and Non-Irrigated Environments
improves taste and flavor in tofu, soymilk, and nato [1].
Although the Early Soybean Production System (ESPS)
showed yield benefit under irrigated and non-irrigated
conditions [5], lower seed quality and substandard ger-
mination of seed [6,7] and variability of seed composi-
tion constituents [8-10] are still a challenge. Planting date
was used as a management strategy to optimize yield and
seed quality. This is because changing of the planting
date would lead to a change in environmental factors,
including temperature and rainfall. Although it is well
established that a late planting date results in higher seed
quality (seed germination), in ESPS for maturity group
IV and V, effects of planting date on seed composition
constituents (protein, oil, fatty acids, and sugars) have
not been well investigated.
Previous research showed that oil concentration in-
creased with early planting, but this increase pattern was
not consistent across locations [11-13]. On the other hand,
it was found that protein concentration increased and oil
concentration decreased with late planting [12,13]. This
variability in seed composition constituents across envi-
ronment and location is still a challenge for normal and
novel modified seed constituent lines. For example, it was
reported that the instability of high oleate germplasm lines
across environments was due to mainly to the effect of
temperature on the enzymes controlling biosynthesis of
soybean seed fatty acids, especially at a seed-fill (R5-R6)
stage [14-16]. It was suggested that palmitic and lino-
lenic acids may decrease with later planting date, but
stearic acid may increase, and this may be due to tem-
perature changes during seed maturation at later planting
[15]. On the other hand, oleic acid levels increased and
linoleic and linolenic acid levels decreased when soy-
beans were grown in warmer environments [17]. Higher
oleic acid and lower linoleic and linolenic acids in soy-
bean seed oil are desirable because of their contribution
to the stability of the oil. The effect of temperature on
oleic and linolenic acid was explained previously in that
temperature may affect oleate and linoleate desaturases
[18], decrease oleyl and linoleyl desaturase activities at
35˚C [19], decrease ω-6 desaturase enzyme, encoded by
the FAD2-1A gene, and desaturases degraded at high
growth temperatures of 30˚C [20].
Based on the above literature, effects of planting date
on seed composition are still inconclusive. Therefore, the
objective of this research was to further investigate effects
of planting date and irrigation on seed protein, oil, fatty
acids, sugars, and mineral nutrition. Since nitrogen is an
essential nutrient for seed protein, and nitrogen metabo-
lism in legumes is a result of both N2 fixation and as-
similation [21,22], the effect of planting date and irriga-
tion on nitrogen fixation and assimilation were also in-
vestigated.
2. Materials and Methods
2.1. Field and Growth Conditions
A 2-yr field study was conducted during 2007 and 2008
at the USDA-ARS Crop Production Systems Research
farm, Stoneville, MS (33˚26'N latitude), The soil was a
Dundee silt loam (fine-silty, mixed, active, thermic Typic
Endoqualf) with pH 6.7, 1.1% organic matter, a cation
exchange capacity of 15 cmol/kg, and soil textural fra-
ctions of 26% sand, 55% silt, and 19% clay. The experi-
mental area was disked, subsoiled, disked, and bedded in
the fall of the previous year. Prior to planting, the raised
beds were smoothed as needed. Soybean was planted in
102-cm wide rows using a MaxEmerge 2 planter (Deere
and Co., Moline, IL) at 285,000 seeds/ha. Soybean cul-
tivar “AG4604RR/S” was planted April 9 and May 10 in
2007 and April 8 and May 12 in 2008. Pendimethalin at
1.12 kg·ai/ha plus paraquat at 1.12 kg·ai/ha were applied
to the entire experimental area immediately after each
planting. Paraquat was applied to kill existing weeds at
planting, Pendimethalin was used to provide early-season
weed control. Glyphosate at 0.84 kg·ae/ha was applied at
4 - 5 weeks after planting soybean to the entire experi-
mental area for postemergence weed control. Herbicides
were applied with a tractor-mounted sprayer with TeeJet
8004 standard flat spray nozzles (TeeJet Spraying Sy-
stems Co., Wheaton, IL), delivering 187 L/ha water at
179 kPa. All plots were hand weeded periodically through-
out the season to keep weed-free. No fertilizer nitrogen
was applied and the crop was irrigated on an as-needed
basis each year. Each treatment plot consisted of twelve
rows spaced 102-cm apart and 15.2 m long. At harvest
about 200 soybean pods were randomly sampled from
the middle four rows for seed. Soybean from middle
eight rows in each plot were harvested using a combine,
and grain yield was adjusted to 13% moisture.
2.2. Seed Analysis for Protein, Oil, and Fatty
Acids
Mature seed collected at harvest were analyzed for pro-
tein, oil, and fatty acids. Approximately 25 g of seed from
each plot were ground using a Laboratory Mill 3600
(Perten, Springfield, IL). Analyses were conducted by
near infrared reflectance [23] using a diode array feed
analyzer AD 7200 (Perten, Springfield, IL). Calibrations
were developed by the University of Minnesota, using
Perten’s Thermo Galactic Grams PLS IQ software. The
calibration curve has been regularly updated for unique
samples according to AOAC methods [24,25]. Analyses
of protein and oil were performed based on a seed dry
matter basis [23,26].

Influence of Planting Date on Seed Protein, Oil, Sugars, Minerals, and Nitrogen Metabolism in Soybean under
704
Irrigated and Non-Irrigated Environments
2.3. Seed Analysis for Sucrose, Raffinose, and
Stachyose
Matured seed collected at harvest from each planting
date were analyzed for sucrose, raffinose, and stachyose
concentrations [10]. About 25 g of seed from each plot
were ground using a Laboratory Mill 3600 (Perten, Spring-
field, IL). Analyses were conducted by near infrared
reflectance [9,23] using an AD 7200 array feed analyzer
(Perten, Springfield, IL). Calibrations were developed by
the Department of Agronomy and Plant Genetics, Uni-
versity of Minnesota St Paul, MN, using Thermo Galac-
tic Grams PLS IQ software, developed by Perten com-
pany (Perten, Springfield, IL). Analyses of sugars were
performed based on a seed dry matter basis [23,26].
2.4. Seed N, S, and Mineral Composition
Mature seed collected at harvest were analyzed for N, S,
Ca, Mg, and Zn concentrations at The University of
Georgia’s Soil, Plant, and Water Laboratory, Athens, GA.
Seed Ca, Mg, and Zn concentrations were analyzed by
digesting 0.5 g of dried ground seed in HNO3 in a mi-
crowave digestion system. Values were then determined
using inductively coupled plasma spectrometry. Nitrogen
and S were measured in a 0.25-g sample using an ele-
mental analyzer (LECO CNS-2000, LECO Corporation,
MI). For seed B, Fe, and P, concentrations were deter-
mined as indicated in the following sections.
2.5. Nitrate Reductase Activity
Nitrate reductase activity (NRA) was measured in the
fully expanded leaves at R1-R2 from each plot. The mea-
surement of NRA was made according to the method of
[27] and was described for soybean in detail by others
[28]. To determine potential NRA (PNRA) where nitrate
availability is not limited, nitrate at a concentration of 10
mM as KNO3, was added to the incubation solution. Ni-
trate reductase activity was expressed as µmol 2
NO

·g
fwt–1·hour–1).
2.6. Acetylene Reduction Assay
Ten soybean plants were randomly sampled from each
plot at R1-R2 for nitrogenase activity (nitrogen fixation
activity, NFA) measurement. Plants were excavated with
roots and shoot and transported to the laboratory for NA.
Nitrogenase activity was assayed within 30 min of colle-
ction using the acetylene reduction assay to measure NA
[29,30]. Roots with nodules intact were excised and in-
cubated in 1 L Mason jars (two jars per plot). Six roots
were placed in the Mason jars and sealed, and a 10%
volume of acetylene was added. After 1 h of incubation
at room temperature, gas samples were removed and
analyzed by gas chromatography using a flame ioniza-
tion detector (FID) and a thermal conductivity detector
(TCD) for determination of ethylene.
2.7. Boron Measurement
Boron concentration was measured in seed from each
plot using the Azomethine-H method [31]. Calcium car-
bonate powder was added to 1.0 g seed samples before
ashing at 500˚C for 8 hours to prevent losses of volatile
B compounds. Ashed samples, then, were extracted with
20 ml of 2 M HCl at 90˚C for 10 min, filtered and trans-
ferred to plastic vials. A 2 ml sample of the solution was
added to 4 ml of buffer solution (containing 25% ammo-
nium acetate, 1.5% EDTA, and 12.5% acetic acid) and 4
ml of freshly prepared azomethine-H solution (0.45%
azomethine-H and 1% of ascorbic acid) [32]. Samples
were left at room temperature for at least 45 min for
color development, and B concentration was determined
using a Beckman Coulter DU 800 spectrophotometer
(Fullerton, CA, USA) at 420 nm.
2.8. Iron Measurement
Seed iron from each plot was measured after acid wet
digestion, extraction, and reaction of the reduced ferrous
Fe with 1, 10-phenanthroline [10,33,34]. A 2 g sample of
dried ground seed was digested in nitric acid (70% m/m
HNO3). After the acids were removed by volatilization,
the soluble constituents were dissolved in 2 M HCl.
Standard solutions of iron were prepared in 0.4 M HCl
and ranged from 0.0 to 4 μg·ml−1 Fe. Phenanthroline so-
lution of 0.25% m/v was prepared in 25% v/v ethanol. A
fresh quinol solution (1% m/v) reagent was prepared on
the day of use. An aliquot of approximately 4 ml was
pipetted into a 25 ml volumetric flask. A concentration
of 0.4 M HCl solution was used to dilute the aliquot to 5
ml. A volume of quinol solution was added and mixed,
and then 3 ml of phenanthroline solution and 5 ml of tri-
sodium citrate solution (8% m/v) were added. The mix-
ture solution containing the aliquot, HCl, phenanthroline,
tri-sodium citrate, was diluted to 25 ml. The mixture
stood for 4 h and the absorbance of the samples was read
at 510 nm using a Beckman Coulter DU 800 spectro-
photometer.
2.9. Phosphorus Measurement
Seed phosphorus was measured spectrophotometrically
as the yellow phospho-vanado-molybdate complex [35,
36]. A dry seed sample of 2 g was ashed, then 10 ml of 6
M HCl was added. Samples were placed in a water bath
at 70˚C to evaporate the solution. After drying, the sam-
ples were kept under heat, and 2 ml of 36% m/m HCl
was added, and gently boiled. Then, 10 ml of water was

Influence of Planting Date on Seed Protein, Oil, Sugars, Minerals, and Nitrogen Metabolism in Soybean under
Irrigated and Non-Irrigated Environments
705
added and the solution was carefully boiled for about 1
min. The samples were transferred and diluted to 50 ml
in a volumetric flask. After the first 2 ml were discarded,
the sample solution was then filtered and kept for P
analysis. A volume of 5 ml of the sample was taken, and
5 ml of 5 M HCl and 5 ml of ammonium molybdate-
ammonium metavanadate (a solution of ammonium mo-
lybdate, (NH4)2MoO4 (25 g/500 ml water), and ammo-
nium metavanadate, NH4VO3) (1.25 g/500 ml water) rea-
gent were added, diluted to 50 ml, and allowed to stand
for 30 min at ambient temperature before measurement.
Phosphorus standard solution (0 - 50 μg/ml of phospho-
rus) was prepared using dihydrogen orthophosphate dis-
solved in both water and 36% m/m HCl. Phosphorus
concentration was measured using a Beckman Coulter
DU 800 spectrophotometer at 400 nm.
2.10. Statistical Analysis
The experiment was conducted in a split plot arrange-
ment of treatments in a randomized complete block de-
sign with irrigation as the main plot and planting date as
the sub-plot with six replications. The experiment (exp)
was repeated twice. Each sub-plot consisted of twelve
rows of 1 - 2-cm apart and 15.2 m long. The data were
subjected to analysis of variance using Proc Mixed using
SAS [37]. Means were separated by Fisher’s least signi-
ficant difference test at the 5% level of probability. Data
were averaged across years (as main effect means) if the
year by treatment interactions were not significant and
data were presented separately for each year when inter-
actions were significant.
3. Results and Discussion
Analysis of variance indicated that irrigation and plant-
ing date were the major sources of seed composition
changes (Tables 1 and 2). Seed constituents responded
differently to the interactions between year, irrigation,
and planting date, indicating that effect of irrigation and
planting date on some seed constituents were different in
each year (Tables 1 and 2).
3.1. Seed Yield
Seed yield of early planting (April) was greater than
yield from late planting (May) under I conditions (Table
3), but no consistent difference in yield between early
and late planting was observed under non-irrigated con-
ditions. It was demonstrated that early planting showed
higher yield under irrigated and non-irrigated plants
[5,38]. Our yield results under irrigated conditions sup-
port previous research. The observation that yield under
NI was higher in one year only could be due to differ-
ences in growing season environmental factors of tem-
perature and rainfall. Weather data (Figure 1) showed
different patterns of rainfall and temperature, and these
differences could be a source of inconsistency of yield
across years under NI [39]. In 2007, the rainfall pattern
in June and July was favorable to Early planting and in
2008, rainfall pattern in July and August was favorable
to late planting. Overall, rainfall was higher in 2008
compared to 2007 (Check the rainfall data for accuracy
of this statement). In 2008, both early and late planted
soybean under NI produced relatively high yields with a
narrow difference.
Table 1. Analysis of variance (F-value and level of significance) of the effect of year, planting date (Planting), irrigation (Irri),
and their interactions for seed protein, oil, and fatty acids (g·kg–1) and seed sugars (mg·g–1)*.
Source of variability Protein Oil Oleic
(C18:1)
Linoleic
(C18:2)
Linolenic
(C18:3) Sucrose Raffinose Stachyose
Year 16*** NS NS NS 4.37* 12** NS NS
Exp NS NS NS NS NS NS NS NS
Irri 89*** 3.8* 121*** 64*** 18** 150*** 68*** 18**
Planting 52*** 150*** 148*** 84*** 35*** 188*** 11** 9.2**
Year × exp NS NS NS NS NS NS NS NS
Year × irri 13*** NS 4.3* NS NS NS NS NS
Year × planting 4.8* NS 11** NS NS NS NS 4.1*
Exp × irri 4.8* NS NS NS NS NS NS NS
Exp × planting NS NS NS NS NS NS NS NS
Irri × planting NS NS NS 30*** NS 11** NS 3.9**
Year × irri × planting NS NS NS NS 5.4* NS NS 8.8**
Year × block × irri × planting NS NS NS NS 2.4* 2.9* NS 2.7**
*Significant at P < 0.05; **Significant at P < 0.01; ***Significant at P < 0.001. NS = non-significant at the P < 0.05.

Influence of Planting Date on Seed Protein, Oil, Sugars, Minerals, and Nitrogen Metabolism in Soybean under
706
Irrigated and Non-Irrigated Environments
Table 2. Analysis of variance (F-value and level of significance) of the effect of year, planting date (Planting), irrigation (Irri),
and their interactions for boron (B), iron (Fe) in leaves and seed (mg·kg–1), phosphorus (P) in leaves and seed (%), weight (wt)
(kg·ha–1), nitrogen fixation activity (NFA, µmol C2H2·plant–1·h–1), nitrate reductase activity(NRA, µmol ·g·fwt–1·h–1)*
2
NO 
Source of variability B leaves B seed Fe leaves Fe seed P leaves P seed Wt NFA NRA
Year NS 9.9* 12** 24** NS NS 67*** NS 7*
Exp NS NS NS NS NS NS NS NS NS
Irri 244*** 96*** 143*** 284*** 180*** 61*** 88*** 114*** 134***
Planting 120*** 40*** 51*** 24*** 15** NS NS 21*** 12**
Year × exp NS NS NS 15** NS NS NS NS NS
Year × irri NS NS 4.3* 5.0* NS NS 7.0** NS 4.08*
Year × planting NS NS NS NS NS NS 7.2** NS NS
Exp × irri NS NS NS 7.3* 4.2* NS NS NS 4.1*
Exp × planting NS NS NS 5.5* NS NS NS NS NS
Irri × planting NS NS NS NS 41*** NS 16.1*** 9.2** 7.6**
Year × irri × planting NS 5.2* NS NS NS NS 28.9** NS NS
Year × exp × irri × planting NS NS NS NS NS NS 6.29** NS NS
*Significant at P < 0.05; **Significant at P < 0.01; ***Significant at P < 0.001. NS = non-significant at the P < 0.05.
Table 3. Effect of planting date on soybean yield, seed protei n, oil, and fatty acids (oleic, linoleic, and linolenic under irrigated
(IR) and non-irrigated (NI) conditions. The experiment was conducted in 2007 and 2008 at Stoneville, MS, USA. *
2007
Irrigation Planting Yield
(kg·ha–1)
Protein
(g·kg–1)
Oil
(g·kg–1)
Oleic
(g·kg–1)
Linoleic
(g·kg–1)
Linolenic
(g·kg–1)
I April 4705 a 409 b 243 a 270 a 531 b 60 b
May 3801 b 413 a 214 b 220 b 585 a 83 a
NI April 4255 a 432 a 249 a 308 a 523 b 62 b
May 3123 b 415 b 222 b 246 b 544 a 69 a
2008
I April 5832 a 405 b 243 a 259 a 525 b 74 b
May 4479 b 429 a 209 b 221 b 589 a 83 a
NI April 4043 b 453 a 245 a 301 a 512 b 61 b
May 4256 a 437 b 213 b 275 b 521 a 73 a
*Means within a column and within each irrigation treatment (I or NI) followed by the same letter are not significantly different at the 5% level as determined
by Fishers’ LSD test.
Figure 1. Rainfall (cm) and maximum temperature (Tmax)
and minimum temperature (Tmin) in ºC in 2007 and 2008.
3.2. Seed Composition
Under I conditions, early planting resulted in higher oil
and oleic acid, but lower protein, linoleic, and linolenic
acids (Table 3). Late planting resulted in higher protein,
linoleic and linolenic acids, but lower oil and oleic acid.
Late planting resulted in higher sucrose and raffinose
concentrations and lower stachyose concentration com-
pared with early planting under irrigation (Figure 2).
Combined sugar (sucrose + stachyose + raffinose) con-
centration was not consistent across years (Figure 3).
Results from previous research on the effect of planting
date on seed composition were inconsistent. For example,
it was found that early planting resulted in higher oil
(1.54 g·kg–1 increase) concentration at Arlington, WI,
USA than late planting, but planting date did not affect
protein concentration at Hancock, WI, USA [40]. On the
other hand, planting date did not affect oil or protein con-
tent at Hancock [40]. Other researchers found the oppo-

Influence of Planting Date on Seed Protein, Oil, Sugars, Minerals, and Nitrogen Metabolism in Soybean under 707
Irrigated and Non-Irrigated Environments
Figure 2. Effect of early planting (April planting) and late planting (May planting) and irrigated and non-irrigated environ-
ments in 2007 (a, b, c) and in 2008 (d, e, f) on seed concentrations of sucrose (a, d), raffinose (b, e), and stachyose (c, f). Bar
values are means ± SE.

Influence of Planting Date on Seed Protein, Oil, Sugars, Minerals, and Nitrogen Metabolism in Soybean under
708
Irrigated and Non-Irrigated Environments
Figure 3. Effect of early planting (April planting) and late
planting (May planting) and irrigated and non-irrigated en-
vironments on the seed concentrations of combined sugars
(sucrose + raffinose + stachyose) in 2007 (a) and in 2008 (b).
Bar values are means ± SE.
site in that delayed planting increased protein concentra-
tion, but decreased oil concentration [12].
In our experiment, the increase of oil and oleic acid in
the early planting may be due to higher temperature dur-
ing the seed-fill stage, especially from July to August.
The higher increase in protein, linoleic, and linolenic
acids at late planting may be due to lower temperature
coinciding with the seed-fill stage from August to Sep-
tember. The effect of temperature on seed composition
was previously reported [9,41-43]. It was reported that
the inconsistency of seed composition constituents in
previous research could be due to the range of tempera-
tures under which soybean grow. This was explained by
[43], who suggested that differences in protein levels in a
given genotype could depend on the range of tempera-
ture during the seed-fill. They proposed that later matur-
ing genotypes may accumulate more protein than early
maturing, and this is because the late maturing soybean
may have developed its seed under mean daily tempera-
ture of less than 20˚C. Since maximum temperature in
Mississippi Delta can exceed 36˚C during flowering and
seed fill, it is possible that the inconsistency of the results
in the literature could be due to environment, genotype,
and their interactions.
The variability in the relationship between seed com-
position constituents and temperature may depend on the
range of temperature. For example, it was found that the
range of maximum temperature during filling period for
Harosoy early isolines was from 31.6˚C to 33.6˚C in 2004
and from 33.5˚C to 35.5˚C in 2005 [9]. However, for Clark
late isolines the maximum temperature was from 31.8˚C to
33.5˚C in 2004 and from 33.2˚C to 36˚C in 2005. The dif-
ferences in temperature ranges between the two years
resulted in protein decrease as temperature increased in
2004, but protein increased as temperature increased
further in 2005 [9]. This observation was also found by
other researchers. Piper and Boote found that protein
was high between 20˚C and 25˚C, but higher when tem-
perature was lower than 20˚C or greater than 25˚C [42].
Oil concentration increased as temperature increased up
to a point, then oil concentration decreased as tempera-
ture increased [44-46]. The increase of oil with maxi-
mum temperature was also observed in by others [8,14].
The increase in sucrose and raffinose in late planting
under irrigated and non-irrigated conditions (Figure 1)
could be due to lower temperature at the seed-fill stage
that coincided with late planting. Since planting date
leads to temperature changes during the critical stages of
growth, seed sugar concentrations can be discussed in the
context of planting date and temperature. Generally, late
planting moves the seed fill period to a cooler tempera-
tures compared with early planting for maturity group IV
and V, and this can be observed from the weather data
for maximum and minimum temperatures (Figure 1). Li-
terature indicated that the effect of temperature on sugars
was not consistent. For example, it was found that low
and high temperatures (from 18˚C/13˚C - 33˚C/28˚C)
had no effect on raffinose levels, slightly decreased sta-
chyose at the highest temperature 33˚C/28˚C, and sig-
nificantly decreased sucrose content as the temperature
increased [47]. In a growth chamber experiment on
mid-high oleic acid breeding line N98-4445A (MG III),
combined sugars (sucrose, raffinose, and starchyose) in
mature seed grown under high temperature (37˚C/30˚C)
did not change compared with those grown under 27˚C/
18˚C [48]. Recently, Bellaloui et al. conducted a field
experiment on Clark and Harosoy isolines and found that

Influence of Planting Date on Seed Protein, Oil, Sugars, Minerals, and Nitrogen Metabolism in Soybean under 709
Irrigated and Non-Irrigated Environments
sucrose, stachyose, and combined sugars had a signify-
cant positive linear relationship with maximum tempera-
ture in 2004, but negative relationship in 2005 [10]. The
inconsistency was suggested to be due to differences in
temperatures between years since the range of maximum
temperature during the last 20 d before maturity (filling
period) for the Clark isoline set ranged from 31.7˚C to
33.4˚C in 2004 and from 33.3˚C to 36.12˚C in 2005 [10].
It was concluded that the increase in galactinol synthase
activity and galactinol content, and the decrease in myo-
inositol during sugar partitioning during late seed devel-
opment may be associated with sugar metabolism in
soybean seeds [49]. In addition, the regulation of raffi-
nose oligosaccharide accumulation may also depend on
galactosyl transferase activity [49]. Further research is
needed to reconcile the controversial literature results on
the effects of temperature on seed sugars.
3.3. Mineral Seed Composition
Early planting under irrigation resulted in a decrease in
leaf and seed B, Fe, and P concentration compared with
late planting (Figures 4 and 5). However, early planting
under NI resulted in significant accumulation of leaf B
and P, but less seed B and seed P compared with late
planting (Figures 4 and 5). Non-irrigation conditions
resulted in lower leaf and seed B and P, but there was no
consistency for leaf and seed Fe. The lower concentra-
tion of B, P, and Fe in leaves and seed in early planting
may indicate that the uptake and translocation of these
nutrients during seed-fill stages may be affected by hi-
gher temperature. The effect of temperature on uptake
and translocation of mineral nutrients were previously
reported [50-52].
3.4. Nitrogen Metabolism
Both nitrogen fixation (nitrogenase activity, µmol C2H2
plant–1·h–1) and nitrogen assimilation (nitrate reductase
activity, µmol 2 NO

·gfwt·h–1) were higher in early plant-
ing than in late planting in 2007 and 2008 (Figure 6),
reflecting that nitrogen metabolism activity is associated
with yield [53]. Non-irrigation resulted in lower nitrogen
fixation and assimilation, indicating the sensitivity of ni-
trogen metabolism to drought or water stress [54-56].
Adding nitrate to the assay medium of leaves of plants
grown under NI resulted in a striking increase in NRA,
indicating that there was limitation of nitrate availability
in leaf cells (data not shown). The increase of NRA by
adding nitrate to the assay medium indicates that water
stress inhibited nitrate uptake and translocation to leaves.
Our results show that nitrogen metabolism activity may
explain the yield benefits of early planting in the ESPS.
4. Conclusions
Planting date altered seed composition and mineral nutri-
tion in soybean. Late planting tends to increase oil and
oleic acid, but decrease protein, linoleic and linolenic
acid under irrigated conditions. Under non-irrigation,
protein and oleic acid tend to increase, but linoleic and
linolenic acids tend to decrease to decrease. Total seed
constituents were higher at early planting than late plant-
ing because of the higher yield at early planting (Table
4). Late planting under irrigated conditions also tends to
increase sucrose and raffinose, but decrease stachyose.
Under non-irrigated conditions sucrose tends to decrease.
Planting date effects on seed composition may be associ-
ated with the shift in temperatures. Early planting is as-
Table 4. Effect of planting date on total seed constituents (protein, oil, and fatty acids) under irrigated (IR) and non-irrigated
(NI) conditions. The experiment was conducted in 2007 and 2008 at Stoneville, MS, USA.*
2007
Irrigation Planting
Protein
(kg·ha–1)
Oil
(kg·ha–1)
Oleic
(kg·ha–1)
Linoleic
(kg·ha–1)
Linolenic
(kg·ha–1)
I April 1924 a 1142 a 1269 a 2496 a 282 b
May 1571 b 813 b 835 b 2222 b 314 a
NI April 1841 a 1059 a 1310 a 2225 a 265 a
May 1297 b 694 b 767 b 1698 b 214 b
2008
I April 2361 a 1415 a 1512 a 3060 a 434 a
May 1920 b 938 b 991 b 2638 b 373 b
NI April 1833 a 990 a 1209 a 2078 b 245 b
May 1858 a 904 b 1169 a 2217 a 311 a
*Means within a column and within each irrigation treatment (I or NI) followed by the same letter are not significantly different at the 5% level as determined
by Fishers’ LSD test.

Influence of Planting Date on Seed Protein, Oil, Sugars, Minerals, and Nitrogen Metabolism in Soybean under
710
Irrigated and Non-Irrigated Environments
Figure 4. Effect of early planting (April planting) and late planting (May planting) and irrigated (a, b, c) and non-irrigated
(d, e, f) environments on seed concentrations of boron (B), iron (Fe), and phosphorus (P) in 2007. Bar values are means ±
SE.

Influence of Planting Date on Seed Protein, Oil, Sugars, Minerals, and Nitrogen Metabolism in Soybean under 711
Irrigated and Non-Irrigated Environments
Figure 5. Effect of early planting (April planting) and late planting (May planting) and irrigated (a, b, c) and non-irrigated
(d, e, f) environments on seed concentrations of boron (B), iron (Fe), and phosphorus (P) in 2008. Bar values are means ±
SE.

Influence of Planting Date on Seed Protein, Oil, Sugars, Minerals, and Nitrogen Metabolism in Soybean under
Irrigated and Non-Irrigated Environments
712
Figure 6. Effect of early planting (April planting) and late planting (May planting) and irrigated and non-irrigated environ-
ments on nitrogen fixation activity/nitrogenase activity (NFA), NA (a, c) and nitrate assimilation (nitrate reductase activity,
NRA) (b, d) in 2007 and 2008. Bar values are means ± SE.
sociated with cooler temperature and Late planting is
associated with hotter temperature during flowering and
seed fill periods. Lack of translocation of B, Fe, and P
from leaves to seed under non-irrigated conditions may
suggest foliar application of these nutrients may be need-
ed, especially under deficiencies of these nutrients in
soil.
5. Acknowledgements
We thank Albert Tidwell field technical assistance, San-
dra Mosley for lab technical assistance, and Debbie Boy-
kin for statistical assistance. This research was funded by
United States Department of Agriculture, Agricultural
Research Service, project number 6402-21000-034-000.
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