Soybean seed protein, oil, fatty acids, N, and S partitioning as affected by node position and cultivar differences

doi:10.4236/as.2010.13014

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Vol.1, No.3, 110-118 (2010) Agricultural Sciences

doi:10.4236/as.2010.13014

Soybean seed protein, oil, fatty acids, N, and S

partitioning as affected by node position and cultivar

differences1

Nacer Bellaloui*, Anne M. Gillen

Crop Genetics Research Unit, USDA-ARS, Stoneville, USA; * Corresponding Author: nacer.bellaloui@ars.usda.gov

Received 6 July 2010; revised 21 August 2010; accepted 24 August 2010.

ABSTRACT

The mechanisms controlling the partitioning of

seed composition constituents along the main

stem in soybean are still controversial. There-

fore, the objective of this study was to investi-

gate seed protein, oil, and fatty acids partition-

ing in soybean cultivars along the main stem.

The cultivars were DT97-4290, maturity group

(MG) IV; Stressland, MG IV; Hutcheson, MG V;

TracyM, MG VI. Seeds were harvested based on

position on the plant (top nodes, middle nodes,

and bottom nodes). At R8 (physiological matur-

ity stage), DT97-4290, Hutcheson, and Stress-

land had higher percentage of protein and oleic

acid and lower percentage of oil and linolenic

acid in top node seed compared with bottom

node seed. The increase of protein in top node

compared with the bottom node across the two

experiments ranged from 15.5 to 19.5%, 7.0 to

10.5%, 14.2 to 15.8%, 11.2 to 16.5%, respectively

for DT97 - 4290, Hutcheson, Stressland, and

TracyM. Except for TracyM, the increase of oleic

acid in the top node ranged from 45.4 to 93%,

depending on the cultivar. Conversely, the de-

crease in the top node seed ranged from 14.4 to

26.8% for oil and from 5.7 to 34.4% for linolenic

acid, depending on the cultivar. The partitioning

trend of seed composition constituents at R6

(seed - fill stage) was inconsistent. Except for

Stressland, seed oleic acid was higher at R6

than at R8. The higher protein and oleic acid

concentrations in the top node seed was accom-

panied by higher activity of nitrate reductase

activity, higher chlorophyll concentration, higher

nitrogen (N) and sulfur (S) percentages in the

fully expanded leaves at R5-R6 growth stage,

and higher seed nitrogen (N) and sulfur (S)

percentages in DT 97-4290 and Stressland. The

current research suggests that the partitioning

of seed protein, oil, and fatty acids in nodes

along the plant depended on the position of

node on the main stem, cultivar differences,

seed N and S status, and tissue N and S parti-

tioning. The higher nitrate reductase activity at

the top nodes, accompanied higher protein and

oleic acid, and the changes of oleic acid at R6

and R8 along the stem, were not previously re-

ported, and need further investigation. The cur-

rent knowledge is useful for soybean germplasm

selection for desirable traits such protein and

oleic acid, and for accurate measurements of

seed composition constituents in breeding lines.

Keywords:

Seed Composition; Nitrogen Assimilation;

Soybean; Nitrogen; Sulfur

1. INTRODUCTION

Soybean seed is a source of protein and oil for human

nutrition and a source of soybean meal for livestock feed.

Soybean protein meal and soybean oil accounted for

69% and 30%, respectively in 2006 and 2007, of the

world's supply of protein meal and edible oil [1]. Many

international and domestic soybean processors prefer

soybean with at least 340 g kg-1 protein and 190 g kg-1

oil, assuming 130 g kg-1 seed moisture [2]. Soybean seed

quality is determined by the quantity and quality of pro-

tein and oil content (seed composition).

Previous studies showed that soybean seed protein

and oil qualities and quantities significantly vary as

function of node position [3,4]. However, the source of

this variability is not yet understood, and the literature

about seed protein and oil partitioning along the main

1Mention of trade names or commercial products in this publication is

solely for the purpose of providing specific information and does not

imply recommendation or endorsement by the U.S. Department o

Agriculture.

N. Bellaloui et al. / Agricultural Sciences 1 (2010) 110-118

111

stem is still controversial. Understanding the mecha-

nisms of this variability would allow for efficient breed-

ing and selection for higher seed composition qualities

and for more accurate seed composition measurements

in breeding lines. It was reported that seed oil content on

lower nodes in indeterminate cultivars was higher than

the seed on the upper nodes [3]. For determinate culti-

vars, however, the highest oil content in seed was re-

corded in seeds from terminal nodes [3]. It was found a

nearly linear increase in protein content of seeds from

bottom to the top nodes in indeterminate and high pro-

tein breeding lines [5]. Bennett et al. [6] showed higher

oil concentration in soybean seed from basal plant nodes

compared with upper plant nodes. On the other hand,

seed from the middle nodes on determinate cultivars had

higher oil and lower protein than seeds from either the

top or bottom of the plants [3]. Other research indicated

that the lowest seed protein concentration occurred at

lowest nodes in both determinate and indeterminate

types, and protein concentration increased linearly from

397 g kg-1 at the lowest node to 442 g kg-1 at the highest

node in determinate and increased from the lowest (398

g kg-1) and increased progressively through 14 in inde-

terminate, with no significant differences between nodes

12 and 16 [4]. Huskey et al. [7] found that protein con-

centration of 241 individual seeds from different posi-

tions in ‘Forrest’ soybean did not differ between the top

third (415 g kg-1) and the bottom third (410 g kg-1) of

plants. Both these concentrations were higher than the

middle third of plants (392 g kg-1).

Nitrogen (N) and sulfur (S) mobilization from vegeta-

tive tissues to seed, their status in tissue and seed, and

their distribution along the main stem could be a limiting

factor for seed composition constituents, especially for

protein production and amino acid profile. This is be-

cause high mobilization of N from vegetative tissue to

seed is required for seed composition [8,9] and S [10].

Seed N concentration is correlated with N availability

within plants, and the contribution of N remobilization

to seed N accounted for about 80% to 90% in soybean

(Glycine max) [11], 43% to 94% in rain-fed grown lentil

(Lens culinaris) [12], 84% in bean plants (Phaseolus

vulgaris) [13], and 80% in Vicia faba [14]. It was shown

that seed N content significantly different between R5,

R6, and R7 growth stages. Mean N contents were 6.28%

at R5, 6.35% at R6, and 6.68% at R7 [15].

Seed N is approximately one-half mobilized N under

normal field conditions [16], and soybean mobilizes 66

to 79% of its vegetative N [17], Nitrogen and protein in

vegetative tissues are important sources of re-mobiliz-

able S [18]. It was found that soybeans mobilize 61 to

82% and 66 to 79%, respectively, for vegetative N and S

to seed [17,19]. Egli and Bruening [20] suggested that

total plant N supply to the seed may be the primary lim-

iting factor for seed protein and yield accrual. It was

concluded that the proportion of mobilized N found in

the seed at harvest is more dependent on the amount of

N stored in the vegetative tissues than on the amount of

N either taken up from soil or fixed during seed filling

[21]. On the other hand, N acquisition by soybean plants

involves two interdependent systems, uptake and reduc-

tion of soil NO3

- by nitrate reductase enzyme, and N2

fixation by Bradyrhizobium japonicum in the nodules

using nitrogenase, both of which are dependent on en-

ergy supplied by photosynthesis [22]. Reduction in pho-

tosynthesis during the seed-fill period would limit both

C and N assimilation, reducing seed yield and seed pro-

tein and oil. For example, when photosynthesis was re-

duced by shading, a decrease in yield was associated

with a decrease in seed size, oil concentration, and in-

creased protein concentration [23]. The rate of these

processes would limit N and C sources for seed growth.

Naeve and Shibles [24] found S mobilization to seed

dependent on the quantity of S stored in leaf tissue.

Soybean leaf S concentration declines during seed-fill

period [25,26] as does N/S ratio [25].

Based on the above discussion, the mechanisms con-

trolling the variability of seed composition constituents

as function of nodal position are still not well known,

and available results are still not consistent. Therefore,

the objective of this experiment was to further investi-

gate the partitioning of seed protein, oil, fatty acid at R6

and R8 [27] in determinate and indeterminate soybean

with maturity group ranged from IV to VI. To provide

more sources of variability in the measured variables, we

included different cultivars, with different MG and dif-

ferent stem architecture (terminate and indeterminate).

Possible relationships between nodal seed composition

and seed N and S partitioning on different nodes, N as-

similation and chlorophyll concentration in leaves from

different nodes were also investigated.

2. MATERIALS AND METHODS

Two greenhouse experiments were conducted at Delta

States Research Center in Stoneville, MS. Since seed

samples were the main plant organ in this study, the two

experiments were conducted in different greenhouse bays

and in different years. Therefore, variability between the

two experiments was expected. Soybean seed were ger-

minated in flat trays in vermiculite. Four uniform size

seedlings at V1 stage were transplanted into each 9.45 L

size pot filled with field soil (sandy loam, fine-loamy,

mixed thermic Molic Hapludalfs) with pH 6.3, 1.1% or-

ganic matter, and soil textural fractions of 26% sand, 56%

silt, and 18% clay, and it contained an abundant native

population of B. japonicum. Soil was irrigated as needed

N. Bellaloui et al. / Agricultural Sciences 1 (2010) 110-118

112

based on the soil water potential, monitored by tensiome-

ters. Soil water potential was measured every other day

using Soil Moisture Meter (WaterMark Company, Inc.,

Wisconsin, U.S.A.). The soil water potential was kept

between −15 to −20 kPa (this was considered field capac-

ity for the soil used in this experiment). Greenhouse con-

ditions were: temperature was about 32oC ± 8oC during

the day and about 20oC ± 5oC at night with a photosyn-

thetic photon flux density (PPFD) of about 500-2100

µmol m-2 s

-1, measured by Quantum Meter (Spectrum

Technology, Inc., Illinois, U.S.A.). The range of light in-

tensity reflects a bright and sunny or cloudy day, respec-

tively. The source of lighting in the greenhouse was a

mixture of natural light, standard incandescent bulb light

(60 W), and cool white incandescent bulb (250 W). Cul-

tivars grown were maturity group (MG) IV DT97-4290,

indeterminate; MG IV Stressland, indeterminate; MG V

Hutcheson, determinate; MG VI TracyM, determinate.

Selection of cultivars was based to include different MG

(MG IV and V are maturities often used in the region by

growers), different stem architecture (determinate and

indeterminate), different stress tolerance level (Stressland

is adapted to harsh stress environment compared with

others). The selection would create enough variability for

the measured the variables (protein, oil, fatty acids, and N,

S) to investigate the distribution pattern.

The nodes on the main stem were divided into Top,

Middle, and Bottom. For example, if there were 15 nodes

on the main stem, the nodes would have been divided

such that the upper five nodes were considered top third

(Top); the following five nodes were the middle third

(Middle); the lowest five nodes were the bottom third

(Bottom). The number of nodes ranged from 10 to 15.

Seed samples were harvested from R6 and R8 plants. R6

seed samples were dried under room temperature. Each

cultivar was harvested based on its maturity time, and

each cultivar was considered mature when 95% of seed

pods on the main stem had been fully matured.

2.1. In Vivo Nitrate Reductase Assay and

Seed Nutrient Analysis

In all experiments, fully expanded leaves at seed-fill

stage (R5-R6) were sampled for nitrate reductase activity

(NRA). NRA was measured in the fully expanded leaf

according to Hunter [28] and Bellaloui et al. [29]. Briefly,

approximately 0.3 g of fully expanded leaf at R5-R6 was

placed in 10 mL of potassium phosphate buffer at a con-

centration of 100 mM, pH 7.5, containing 1% (v/v) 1-

propanol, in the flask. The incubation solution was va-

cuum filtered for 1 min, and the flask and contents were

flashed with nitrogen gas for 30 s and then incubated at

30°C. A sample of 0.5 mL was taken at regular intervals

(0, 60, 120, 180, and 300 min) for nitrite determination.

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 dihy-

drochloride (0.1%). After 30 min, the samples were read

at 540 nm using a Beckman Coulter DU 800 spectropho-

tometer (Fullerton, CA). The concentration of nitrite was

calculated from a calibration curve made of potassium

nitrite (KNO2). To determine potential NRA (PNRA) un-

der conditions when nitrate concentration would not be a

limiting factor, exogenous nitrate in form of KNO3 was

added to the incubation solution at a concentration of 10

mM. The seed samples were analyzed at The University

of Georgia’s Soil, Plant, and Water Laboratory, Athens,

GA, to determine N and S concentration. Measurements

of N an S were conducted on a 0.25 g sample of soil using

an elemental analyzer.

2.2. Chlorophyll Concentrations

Chlorophyll measurements were made on fully ex-

panded leaf at R5-R6. Leaves were sampled from four

randomly selected soybean plants that were additionally

planted for chlorophyll and NRA. Chlorophyll was ex-

tracted according to Hiscox and Israelstam [30]. Chloro-

phyll concentration was determined spectrophotometri-

cally using a Beckman Coulter DU 800 spectrophotome-

ter (Fullerton, California, U.S.A.). Chlorophyll concentra-

tion was calculated using the equation of Arnon [31] and

expressed as milligrams of chlorophyll per gram of leaf

fresh weight.

2.3. Protein, Oil, and Fatty Acids Analysis

Seeds from each replicate and from each treatment

were sampled and analyzed for seed composition using

near-infrared (NIR) reflectance diode array feed analyzer

(Perten, Spring Field, IL, U.S.A.) for protein, oil, fatty

acids [29,32]. Calibrations were developed by Perten us-

ing Thermo Galactic Grams PLS IQ. The calibration

curve has been regularly updated from six months to one

year for unique samples according to AOAC methods

[33,34]. The analysis was performed on the basis of per-

cent dry matter [29].

2.4. Statistical Analysis

Treatments were arranged in a split plot design with

four replications. Cultivar was a main plot, stage (R6 and

R8) was a sub-plot, and position (Top, Middle, and Bot-

tom) was a sub-sub-plot. Analysis of variance using Proc

Mixed was conducted using a split plot model using SAS

[35]. Level of significance was at P ≤ 5%. Since the two

experiments were significantly different in a preliminary

analysis (data not shown), as expected, each experiment

N. Bellaloui et al. / Agricultural Sciences 1 (2010) 110-118

113

was analyzed separately.

3. RESULTS

3.1. Seed Composition Constituents

Since each experiment was conducted in a different

greenhouse bay and in different years, it was expected that

interactions between experiment (Exp) and other variables

(cultivar, stage, and position) will be significantly (P ≤ 0.5)

different for seed composition constituents (protein, oil,

oleic and linolenic acids, N, and S) (data not shown).

Therefore, results were presented separately by experi-

ment (Exp 1 and Exp 2) and stage (R6 and R8). In Exp 1,

mean values at R6 (Table 1) showed that seed protein

concentration was the highest and oil was the lowest in

top node seed in DT97-4290, Hutcheson, Stressland. Tra-

cyM had no differences in oil concentration among the

nodes, but it showed the same relationship of protein

concentration among nodes as the other cultivars. All cul-

tivars had the highest oleic acid and protein in the top

node and the lowest in the bottom node. Linolenic acid

was expected to be inversely related to oleic acid based on

prior experience with R8 growth stage seed composition

values, but this was not always the case at R6 for Stress-

land and TracyM. The lowest values for linolenic acid

were in the top and middle node for DT97-4290 and

Hutcheson. At R8, the concentration of seed protein and

oleic acid was greater in the top node seed and middle

node seed, and lower in the bottom seed in all cultivars,

except Hutcheson. Oil concentration was generally lowest

in the top node and highest in the bottom node, which is

the inverse of protein and oleic acid concentration, except

for Hutcheson. Hutcheson did not fit the pattern of the

other cultivars for oleic acid partitioning at R8 because it

had the highest oleic acid in the middle node, rather than

the top node. Compared to R6, the R8 values for linolenic

acid were more consistent with the lower value in the top

node and the higher value in the bottom node for all cul-

tivars.

In Exp 2, the pattern of seed constituents partitioning at

R6 growth stage were similar for all variables for DT97-

4290, but the patterns were different from those in Exp1

for the other cultivars (Table 2). Stressland and TracyM

showed the opposite trend of those in DT97-4290, i.e.,

lower protein concentration and oleic acid in the top seed

nodes, and oil and linolenic acid were higher in the bot-

tom seed than the top seed nodes at R6 (Table 2). In Exp

1, oleic acid was highest in the top node for all cultivars,

but in the Exp 2 it was lower in the top node for Stress-

land and TracyM.

At R8, protein was consistently higher in top node and

oil was consistently lower in the top node in the two ex-

periments and among all the cultivars. For oleic acid,

DT97-4290, Hutcheson, and Stressland consistently had

higher oleic acid in the top nodes and lower in the bottom

nodes. TracyM did not follow the same pattern as other

Table 1. Experiment 1, mean values of protein, oil, and fatty acids percentages (%) as affected by node position (Top = T, Middle = M,

Bottom = B) and cultivar at R6 (seed-fill stage) and R8 (physiological maturity stage).

R6 R8

Cultivar Position Protein Oil Oleic Linolenic Protein Oil Oleic Linolenic

T 42.93 a 15.52 b 32.55 a 5.69 b 47.33 a 18.82 c 30.45 a 6.99 b

M 42.47 a 15.57 b 31.78 a 5.78 b 45.56 b 20.91 b 25.06 b 7.21 a

B 39.64 b 20.59 a 27.80 b 6.73 a 39.61 c 23.53 a 20.73 c 10.19 a

DT 97-4290

LSD = 0.627 0.444 3.20 0.40 0.464 0.404 3.258 0.397

T 43.47 a 16.80 b 23.15 a 4.63 b 47.68 a 19.85 c 27.013 b 6.15 c

M 42.75 a 16.25 c 23.73 a 4.20 c 45.06 b 20.53 b 28.910 a 7.18 b

B 40.77 b 21.33 a 19.05 b 5.65 a 43.15 c 23.66 a 18.575 c 8.95 a

Hutcheson

LSD = 0.722 0.507 1.086 0.244 0.309 0.414 0.811 0.415

T 44.88 a 15.88 c 35.45 a 4.43 ab 48.55 a 19.46 c 23.83 a 6.63 b

M 45.07 a 18.51 b 33.43 b 5.00 a 47.28 b 20.59 b 22.55 b 6.88 ab

B 42.22 b 21.81 a 23.03 c 4.75 b 41.91 c 24.36 a 12.34 c 7.13 a

Stressland

LSD = 0.958 0.928 1.126 0.238 0.520 0.562 0.916 0.281

T 43.02 a 20.65 a 26.10 a 5.08 a 46.68 a 20.69 b 23.63 a 5.52 b

M 42.76 a 20.99 a 23.65 b 4.95 a 44.49 b 20.85 b 20.90 b 5.30 b

B 41.54 b 20.75 a 19.23 c 4.47 b 40.08 c 24.50 a 11.12 c 8.42 a

TracyM

LSD = 0.372 0.437 0.998 0.382 0.600 0.585 1.128 0.68

Notes: means within a column in each cultivar followed by the same letter are not significantly different at P ≤ 5%. Four replicates were used.

N. Bellaloui et al. / Agricultural Sciences 1 (2010) 110-118

114

Table 2. Experiment 2, mean values of protein, oil, and fatty acids percentages (%) as affected by node position (Top = T, Middle = M,

Bottom = B) and cultivar at R6 (seed-fill stage) and R8 (physiological maturity stage).

R6 R8

Cultivar Position Protein Oil Oleic Linolenic Protein Oil Oleic Linolenic

T 44.24 a 16.24 b 31.50 a 5.48 b 47.89 a 19.39 c 27.58 a 5.92 b

M 42.67 b 16.30 b 23.91 b 5.63 b 46.30 b 21.72 b 23.66 b 7.46 a

B 40.00 c 22.73 a 18.67 c 6.95 a 41.48 c 23.17 a 15.28 c 6.28 b

DT 97-4290

LSD = 0.574 0.557 1.33 0.596 0.408 0.522 0.768 1.017

T 43.43 a 15.58 b 23.94 a 5.16 c 47.89 a 17.31 c 20.42 a 7.10 b

M 41.81 b 16.52 b 21.58 b 5.82 b 47.48 a 21.91 b 18.50 b 6.55 b

B 38.96 c 21.88 a 16.14 c 7.04 a 44.75 b 23.66 a 13.08 c 8.31 a

Hutcheson

LSD = 0.549 1.346 1.086 0.285 1.123 0.619 1.060 0.588

T 39.91 c 23.58 a 17.19 c 6.32 b 48.60 a 18.81 c 22.30 a 6.37 c

M 46.48 a 16.83 c 38.10 a 7.02 a 48.17 b 21.99 b 22.04 a 6.83 b

B 44.60 b 17.58 b 31.55 b 6.59 b 42.55 c 24.69 a 11.87 b 7.67 a

Stressland

LSD = 0.452 0.700 0.992 0.302 0.426 0.414 1.192 0.424

T 39.66 b 22.03 a 21.10 b 8.05 a 47.56 a 20.45 c 22.10 b 6.17 b

M 42.21 a 20.68 ab 22.61 a 6.36 b 44.97 b 22.27 b 21.63 b 6.00 b

B 42.47 a 21.40 b 22.89 a 6.10 b 42.77 c 23.89 a 23.73 a 7.84 a

Tracy

LSD = 0.4679 0.8614 3.273 1.1433 0.715 0.780 1.377 0.569

Notes: means within a column in each cultivar followed by the same letter are not significantly different at P ≤ 5%. Four replicates were used.

Table 3. Mean values of nitrate reductase activity (NRA, µmol nitrite/g fwt/hour) and chlorophyll concentrations (mg/g fwt) in the fully

expanded leaf at R5-R6 (seed fill-stage) in Experiment 1 and experiment 2 as affected by node position (Top = T, Middle = M, Bottom

= B) and cultivar.

Experiment 1 Experiment 2

Cultivar Position NRA Chlorophyll NRA Chlorophyll

T 4.82 a 2.43 a 5.76 a 1.98 a

M 4.98 a 2.51 a 5.32 b 2.29 b

B 3.31 b 1.69 b 3.45 c 1.53 c

DT 97-4290

LSD = 0.3945 0.4404 0.166 0.2726

T 5.47 a 2.86 b 6.01 a 2.86 a

M 5.60 a 3.63 a 5.18 b 2.17 b

B 3.62 b 1.33 c 3.37 c 1.18 c

Hutcheson

LSD = 0.3908 0.2293 0.3157 0.2398

T 5.95 a 3.43 a 5.43 a 1.64 c

M 5.18 b 3.51 a 5.18 a 3.16 a

B 5.43 b 1.81 b 5.43 a 2.53 b

Stressland

LSD = 0.3408 0.1893 0.3845 0.2395

T 5.55 a 3.25 a 4.62 b 2.16 b

M 5.85 a 3.05 a 5.39 a 3.27 a

B 3.98 b 1.83 b 5.19 a 2.92 a

Tracy

LSD = 0.3693 0.2735 0.4662 0.4293

Notes: means within a column in each cultivar followed by the same letter are not significantly different at P ≤ 5%. Four replicates were used.

N. Bellaloui et al. / Agricultural Sciences 1 (2010) 110-118

115

cultivars. Linolenic acid was also consistently lower in the

top nodes in all cultivars and in both experiments.

3.2. Partitioning of in Vivo Nitrate Reductase

Activity, Chlorophyll, and N and S

Both nitrate reductase activity (NRA) and chlorophyll

concentration were lower in lower leaves than those of

upper leaves (Table 3). This pattern was consistent for all

cultivar, except for Stressland. Seed N concentrations (Ta-

ble 4) showed a pattern of increase from bottom to top

node seed in DT97-4290, Hutcheson, and Stressland at R8.

This pattern was not shown in Tracy (Table 4). For seed S,

only DT 97-4290 showed a pattern of increase from bot-

tom to top node seed (Table 4). At R6, the pattern of in-

crease from bottom to top node seed was consistent only

in DT 97-4290 (Table 4).

4. DISCUSSION

Our research showed that two cultivars (DT97-4290,

indeterminate, and Hutcheson, determinate) had consis-

tent trend in that protein and oleic acid percentage were

higher and oil and linolenic acid percentage were lower in

upper nodes (Top and Middle) compared with those of

lower nodes (Bottom). These results support those found

by Escalante and Wilcox [4] for both determinate and

indeterminate, and support those found by Collins and

Cartter [3] for determinate type only. Stressland, indeter-

minate, and TracyM, determinate, showed inconsistency.

Patterns of distribution of these constituents are contro-

versial, non-consistent. For example, it was reported that

seed oil in the lower nodes was higher than those of upper

nodes in indeterminate cultivars, and the opposite trend

was noticed in determinate cultivars [3]. Escalante and

Wilcox [4] found an increase in seed protein from bottom

to the top nodes in indeterminate lines, and that the middle

nodes had higher seed oil and lower protein on determi-

nate cultivars than seeds from either the top or bottom

nodes. Escalante and Wilcox [4] also found that the lowest

seed protein concentration occurred at lowest nodes in

both determinate and indeterminate types. Other research-

ers did not show the same observation in that protein

concentration of 241 individual seeds from different posi-

tions in ‘Forrest’ soybean did not differ between the top

third (415 g kg-1) and the bottom third (410 g kg-1) of

plants, and these concentrations were higher than the

middle third of plants (392 g kg-1) [7]. The seed composi-

tion constituent partitioning along the stem may be con-

trolled by stem architecture, type of growth (determinate

or indeterminate), maturity, and genotype.

Since our objective was to understand some of the

physiological mechanisms controlling the partitioning of

seed composition constituents along the whole plant, it

was thought that temperature and light distribution (gra-

dient) from top to bottom canopy could be involved. In

our experiment, light intensity in the lower canopy (lower

Table 4. Mean values of seed N and S percentages in Experiment 1 (Exp 1) and Experiment 2 (Exp 2) as affected by node position (Top

= T, Middle = M, Bottom = B) and cultivar at R6 (seed-fill stage) and R8 (physiological maturity stage).

Exp1 at R6 Exp 2 at R6 Exp1 at R8 Exp 2 at R8

Cultivar Position N S N S N S N S

T 5.28 a 0.55 a 5.82 a 0.64 a 6.91 a 0.57 a 6.34 a 0.50 a

DT 97-4290 M 5.45 a 0.50 a 5.45 b 0.60 b 6.83 a 0.47 b 6.63 a 0.55 b

B 4.83 b 0.43 b 4.78 c 0.48 c 5.86 b 0.41 c 5.80 b 0.42 c

LSD = 0.3222 0.0517 0.2386 0.0359 0.1682 0.026 0.3461 0.0453

T 4.25 c 0.68 a 5.78 a 0.60 b 6.79 a 0.51 a 6.96 a 0.52 b

Hutcheson M 4.98 a 0.63 b 5.85 a 2.00 a 6.83 a 0.47 b 6.53 b 0.56 a

B 4.68 b 0.65 ab 4.63 b 0.59 b 6.43 b 0.47 b 6.08 c 0.54 ab

LSD = 0.1818 0.0351 0.2412 0.70272 0.2448 0.0246 0.3387 0.0337

T 6.05 a 0.50 a 6.12 b 1.89 a 6.70 a 0.48 a 6.77 a 0.56 a

Stressland M 5.35 b 0.45 b 7.02 a 2.06 a 6.88 a 0.46 b 6.56 b 0.56 a

B 4.53 c 0.35 c 6.86 b 2.04 a 5.81 b 0.35 c 5.20 c 0.54 a

LSD = 0.2675 0.0468 0.3188 0.70272 0.2432 0.0174 0.1468 0.0228

T 5.83 a 0.35 a 4.93 a 0.62 a 6.33 b 0.55 b 5.49 a 0.58 a

Tracy M 5.95 a 0.30 b 4.61 b 0.63 a 6.69 a 0.56 a 5.17 b 0.58 a

B 5.85 a 0.30 b 4.88 a 0.57 b 6.85 a 0.57 a 5.24 b 0.53 b

LSD = 0.2725 0.037 0.1491 0.0495 0.1617 0.0146 0.2057 0.021

Notes: means within a column in each cultivar followed by the same letter are not significantly different at P ≤ 5%. Four replicates were used.

N. Bellaloui et al. / Agricultural Sciences 1 (2010) 110-118

116

nodes) was about 500 µmol m-2 s

-1 compared with about

2100 µmol m-2 s

-1 (about 76% less) of those of upper

nodes. Both nitrate reductase activity (NRA) and chloro-

phyll concentration were lower than those of upper leaves

(Table 3). This pattern was consistent for all cultivar ex-

cept for Stressland. Since Stressland was developed for

stress environment [36], its response to light intensity due

to shade effect could be different. Total seed amino acids,

proline, cysteine, and methionine amino acids were 20, 40,

30, 25% higher, respectively (data not shown). Shade ef-

fect was reported by other researchers. For example,

Burkley et al. [37] found that highly shaded soybean

leaves in the high plant density treatment accumulated

triacylglycerol up to 25% of total leaf lipid compared to

leaves in the upper canopy. They also found that shade did

not affect leaf chlorophyll content, but reduced linolenic

acid content, which was a companied by a proportional

increase in oleic and linoleic acids. They concluded that

triacylglycerol accumulation was a result of altered carbon

metabolism and not a senescence response. Our results

disagreed with Burkley et al. [37] in that chlorophyll con-

centration decreased in three cultivars, but not in Stress-

land. Recently, Proulx and Naeve [23] showed that shade

and pod removal resulted in preferential accumulation of

protein over oil, and the altered protein accumulation over

oil became more pronounced under shade conditions. It

was explained that these effects appeared to be a result of

more limited seed oil accrual under shade conditions.

Shade treatments have shown different effects on seed

size, protein, and oil concentration, and this is because

photosynthesis during the seed fill period is the primary

source of energy for both seed growth [38,39] and N as-

similation via NO3

– uptake and N2 fixation [22]. Therefore,

inhibition of photosynthesis rate during seed-fill period

can limit C and N assimilation, leading to reduced seed

yield and accrual seed protein and oil. Other reports

showed that shade reduced yield and seed size with no

effect on protein [40], reduced yield with no effect on seed

size, protein, or oil [41], or reduced yield with variable

effects on seed size, protein, and oil [42].

Our results showed that the activity of nitrate reductase

consistently increased from bottom to top nodes in all

cultivars, except in Stressland. This indicates that nitrate

assimilation is lower in the bottom of the canopy, and this

may be due to low photosynthesis rate promoted by lower

light intensity in the lower canopy due to shade. Adding

exogenous nitrate to the incubation solution resulted in a

significant increase of NRA, especially in lower leaves

(increase of 35% comparing to the upper node leaves).

The higher increase of NRA in lower leaves, when ex-

ogenous nitrate was added, than the upper leaves indicated

lower nitrate availability for maximum NRA as indicated

by others [43,44]. The lower activity of nitrate reductase

(NR) in lower node leaves could be a result of inhibition

of de novo synthesis of NR molecules due to less light

intensity as light is a source of the reducing power

(NADPH). Therefore, the partitioning of seed composi-

tion constituents along the main stem could be partially

due to nitrogen assimilation, lower chlorophyll concentra-

tion, and different maturity times of individual pods along

the main stem. More work is needed to identify the rela-

tionships between these factors and seed composition.

Seed N and S concentrations pattern at R8 appear to be

more indicative for final N and S concentration status in

seed than at R6. Therefore, seed N or S at R6 may not

reflect the total seed N and S at R8. The increase of N and

S from bottom to top nodes was accompanied by the in-

crease of protein and oleic acid, especially in DT97-4290

and Stressland. Previous studies reported that N and S

mobilization from vegetative tissues to seed could be a

limiting factor for seed constituent accumulation. This is

because seed protein requires a high demand of mobiliza-

tion of stored N in vegetative tissues [7,8] and S [9]. Also

it was found that mobilization of S to seed depended on

the quantity of S stored in leaf tissue [23]. The increase in

total seed amino acids, proline amino acid, and cysteine

and methionine (Sulfur amino acids) (data not shown) is

significant observation that may contribute to the inter-

pretation of seed composition constituent distribution

along the main stem, but further research is needed.

Therefore, NRA and chlorophyll concentrations could

explain the partitioning of seed constituents along the

main stem. However, seed constituents partitioning could

be explained by seed N at R8 only in some cultivars such

as DT97-4290 and Hutcheson, and by seed S in only

DT97-4290, indicating that using seed N and S at R6 or

R8 to explain constituents partitioning cannot be general-

ized.

5. CONCLUSIONS

The current research suggests that the partitioning of

protein, oil, and fatty acids concentration along the main

stem depended on the position of nodes on the main stem,

cultivar differences, and light intensity. Seed protein and

oleic acid concentrations were higher in the top nodes, but

oil and linolenic acid concentrations were lower in the

bottom nodes. This pattern was consistent at R8 stage

compared with R6, indicating that R8 is more indicative

for final seed composition status, and the full period from

R6-R8 is important for achieving maximum accumulation

of seed composition constituents. Cultivar Stressland did

not follow the same trend as other cultivars, especially at

R6, indicating that stress tolerance cultivars such as

Stressland may pose a different mechanism for seed con-

stituent partitioning. Higher protein and oil are obtained at

N. Bellaloui et al. / Agricultural Sciences 1 (2010) 110-118

117

R8, but oleic acid accumulation was greater at R6, except

for Hutcheson, reflecting the effect of cultivar differences

and stage on oleic acid accumulation. Pattern differences

in N and S along the main stem among cultivars may de-

pend on N and S requirements of each cultivar, which is

genetically controlled. Further studies are needed to in-

vestigate the relationships between N and S supply and

uptake, and seed composition constituent accumulation,

especially amino acids. This knowledge provides with

further understanding of the dynamics of nutrient distribu-

tion and seed composition constituents. Also, the current

research is useful for soybean germplasm and pod selec-

tion for desirable traits such protein and oleic acid and for

accurate measurements of seed composition constituents

in breeding lines.

6. ACKNOWLEDGEMENTS

The authors are thankful to Sandra Mosley for seed

composition analysis. We are also thankful to Debbie

Boykin for statistical assistance. This research was funded

by United States Department of Agriculture, Agricultural

Research Service, project number is 6402-21000-034-000.

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