Article pubs.acs.org/JAFC
Environmental Stability of Seed Carbohydrate Profiles in Soybeans Containing Different Alleles of the Raffinose Synthase 2 (RS2) Gene Kristin D. Bilyeu*,† and William J. Wiebold§ †
Plant Genetics Research Unit, Agricultural Research Service, U.S. Department of Agriculture, 110 Waters Hall, Columbia, Missouri 65211, United States § Division of Plant Sciences, University of Missouri, Columbia, Missouri 65211, United States ABSTRACT: Soybean [Glycine max (L.) Merr.] is important for the high protein meal used for livestock feed formulations. Carbohydrates contribute positively or negatively to the potential metabolizable energy in soybean meal. The positive carbohydrate present in soybean meal consists primarily of sucrose, whereas the negative carbohydrate components are the raffinose family of oligosaccharides (RFOs), raffinose and stachyose. Increasing sucrose and decreasing raffinose and stachyose are critical targets to improve soybean. In three recently characterized lines, variant alleles of the soybean raffinose synthase 2 (RS2) gene were associated with increased sucrose and decreased RFOs. The objective of this research was to compare the environmental stability of seed carbohydrates in soybean lines containing wild-type or variant alleles of RS2 utilizing a field location study and a date of planting study. The results define the carbohydrate variation in distinct regional and temporal environments using soybean lines with different alleles of the RS2 gene. KEYWORDS: soybean, [Glycine max (L.) Merr], sucrose, stachyose, carbohydrate, environment
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INTRODUCTION The value in soybean comes from the extracted vegetable oil and the high protein meal. Soybean meal is a major source of protein for livestock feeds, and >75% of the soybean meal utilized in the United States is consumed by poultry and swine. Soluble carbohydrates in soybean meal are potential contributors to livestock metabolizable energy, and understanding the stability of altered carbohydrate traits is an important step toward improving soybean meal. The predominant soluble carbohydrates present in soybean seeds are sucrose and the raffinose family of oligosaccharides (RFOs), raffinose and stachyose.1 Poultry and swine are both in the monogastric livestock class, and neither produce the appropriate enzymes in their digestive systems to convert RFOs into metabolizable energy.2 RFOs are therefore considered antinutritional factors that have a negative impact on metabolizable energy.3 Sucrose is a disaccharide that is an important positive source of metabolizable energy for monogastric livestock. Sucrose and the galactosyl cyclitol galactinol are the substrates for RFO biosynthesis. The role for galactinol in animal nutrition has not been characterized. The enzyme raffinose synthase (RS; EC 2.4.1.82) is a galactinol-sucrose galactosyltransferase that creates raffinose and the free cyclitol myo-inositol. In addition to potential sources of metabolizable energy in soybean seed meal, soluble carbohydrates such as sucrose, galactinol, and RFOs have multiple critical roles in plant development. Carbohydrates produced from photosynthesis in general are used in plants as carbon building blocks, energy currency, signaling molecules, and stress responders.1,4,5 Although the accumulation of sucrose and RFOs was thought to be essential for desiccation aspects of seed development and energy reserves utilized upon seed imbibition, recent results have demonstrated no significant reduction in seed quality © XXXX American Chemical Society
(germination/emergence) for soybean seeds with altered carbohydrate profiles.6−10 Our previous research and collaborations have led to the development of molecular tools for selection and new soybean lines with altered seed carbohydrate profiles. We identified the soybean raffinose synthase 2 (RS2) gene encoded by Glyma06g18890 in Glycine max v1.1 to have a major role in soybean seed carbohydrate profile. Two variant alleles of RS2 were identified that caused increases in seed sucrose along with decreases in raffinose and stachyose compared to lines with functional versions of RS2.11,12 From the PI 200508, the RS2 allele contained a three-nucleotide deletion that resulted in the absence of a conserved amino acid (RS2 p.Try331del), whereas the induced mutant line named 397 contained a SNP in the RS2 allele that resulted in a missense amino acid change (RS2 p.Thr107Ile). In addition, we characterized novel sources of soybean lines that contained the RS2 p.Try331del mutation plus additional genetic factors that resulted in a unique carbohydrate profile.10,13 We have reported a comparison of the carbohydrate profiles from a single field environment of four classes of soybean lines with different RS2 alleles in similar genetic backgrounds.13 Each class produced a characteristic carbohydrate signature, with stachyose content being the most distinguishing feature. When two planting dates were compared for the same lines, no consistent overall trends were observed in carbohydrate components, nor were there significant differences identified when early- and late-maturing lines within a class were compared.13 Received: October 1, 2015 Revised: January 15, 2016 Accepted: January 22, 2016
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DOI: 10.1021/acs.jafc.5b04779 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
Article
Journal of Agricultural and Food Chemistry Table 1. Soybean Lines Used in both the Location Study and the Planting Date Study and Their Characteristics
a
line
source
category
phenotype
RS2 allelea
ref
W82 534545 397 KB0715 SGUL
Williams 82 cultivar food grade tofu Williams 82 mutagenesis PI 200508 (W82 background) breeding/selection
wild-type tofu low RFOs-1 low RFOs-2 ultralow RFOs
reference high sucrose sucrose/low RFO sucrose/low RFO sucrose/ultralow RFO
RS2 RS2 rs2 (p.Thr107Ile) rs2 (p.Try331del) rs2 plus (p.Try331del)
15 14 11 12 10
RS2 is defined here as Glyma06g18890 in Glycine max v1.1.
Table 2. Location Descriptions Used in the Location Study dates code
location
NEMO NWMO COMO SWMO SEMO
LaGrange Grand Pass Columbia Nevada Portageville
latitude 40.0422° 39.2053° 38.9483° 37.8408° 36.4297°
longitude N N N N N
91.5006° 93.4433° 92.3339° 94.3556° 89.7011°
W W W W W
soil type
planting
harvest
Westerville silt loam Haynie silt loam Mexico silt loam Parsons silt loam Tiptonville silt loam
May 12 June 10 May 4 June 7 May 10
Oct Oct Oct Oct Oct
10 14 7 24 10
Table 3. Historical and 2011 Average Monthly Temperatures (°C) in the Location Study NEMO April May June July Aug Sept a
NWMO
2011
norm
11.7 16.7 22.2 27.2 24.4 17.8
11.7 17.2 22.2 24.4 23.9 19.4
a
COMO
SWMO
SEMO
2011
norm
2011
norm
2011
norm
2011
norm
12.2 16.1 24.4 28.3 24.4 17.8
12.2 17.8 22.8 25.0 24.4 19.4
14.4 16.7 23.9 27.8 25.0 17.8
12.8 17.8 22.8 25.0 24.4 20.0
14.4 17.8 26.1 30.0 27.2 20.0
13.9 18.9 23.9 26.7 26.1 21.1
16.1 18.9 27.2 28.3 26.1 20.6
15.0 20.6 25.0 26.7 26.1 22.2
Norm = 30-year average; source: Midwestern Regional Climate Center. ultralow stachyose levels.10,13 All of the lines used are classified as latematurity group III. Planting Date Study. This experiment was conducted in 2011 at the University of Missouri Bradford Research and Extension Center near Columbia, MO, USA (38.9483° N, 92.3339° W). The predominant soil type at this location is Mexico silt loam (fine, smectitic, mesic, Aeric Vertic Epiaqualfs). The experiment design was a split plot with whole plots arranged in randomized complete block and three replications. Whole plots were five planting dates (April 13, May 4, June 3, June 16, and July 1, 2011), and split plots were the five cultivars (Table 1). Plots were planted without tillage using a four-row Kinze planter with a planting depth of 3.8 cm. The previous crop was corn (Zea mays L.). Seeding rate was 430 000 seeds/ha, and it was not adjusted for possible emergence differences among planting dates. Plot size was four 0.76 m rows wide and 7.6 m long. Before planting, a broadcast application of 23 kg P/ha and 57 kg K/ha was made. Plots were monitored daily for two stages of development, R5 and R7.17 Seed-filling period was the number of days between these two stages. Daily minimum and maximum air temperatures were recorded with a weather station located about 800 m from the experiment site. These temperature data were averaged for the days involved in the seed-filling period. At harvest maturity, plots were harvested with a plot combine. A grain sample from each plot was captured and stored for further processing. Analyses of variances were calculated using the Proc Mixed routine of SAS. Replications were considered random, whereas planting date and cultivar and their interaction were considered fixed. Means were compared using Fisher’s protected LSD with a type 1 error of 0.05. Pearson’s simple correlation coefficients were calculated between each of the recovered carbohydrate fractions of each cultivar and the average maximum and minimum temperatures during the seed-filling period of that cultivar.
We have observed an environmental effect on the accumulation of sucrose and RFOs in selected food grade lines.14 The results suggested that cooler temperatures during seed maturation correlated with an increase in sucrose and a decrease in raffinose and stachyose for the food grade line. We are now poised with a collection of soybean lines that are capable of producing distinct and genetically characterized RFO profiles, albeit in a limited range of similar maturity groups. The objective of this research was to evaluate the environmental stability of the carbohydrate profile of five distinct classes of soybean lines. The major hypothesis is that the environment will have a measurable impact on the accumulation of galactinol, sucrose, and RFOs. The experiment was set up with two components: a location study utilizing five locations and a planting date study using five planting dates at one location to capture different field environments and conditions as well as successively later maturity due to planting date differences.
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MATERIALS AND METHODS
The soybean lines used in this study are listed in Table 1 with their characteristics. ‘Williams 82’15 is a reference conventional maturity group (MG) III cultivar containing wild-type alleles of the RS2 gene, and it is referred to here as W82. Line 534545 is a large seed tofu type soybean variety also containing wild-type alleles of the RS2 gene provided by the Missouri Soybean Programs office.14 Line 397 is a mutant of Williams 82 containing p.Thr107Ile alleles of the RS2 gene.11,16 KB0715 is an experimental soybean line with a pedigree of Williams 82 (Williams 82 × PI 200508) that was selected for homozygous alleles of RS2 p.Try331del.12 SGUL, which contains homozygous alleles of RS2 p.Try331del, is an experimental highprotein soybean line that was developed by a private company for its B
DOI: 10.1021/acs.jafc.5b04779 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
Article
Journal of Agricultural and Food Chemistry Table 4. Summary of Carbohydrate Content as Percent of Seed for Soybean Lines in the Location Study line
NEMO
NWMO
COMO Galactinol 0.0 0.0 0.5 0.5 1.1
W82 534545 397 KB0715 SGUL
0.0 0.0 0.5 0.7 1.1
0.0 0.0 0.4 0.6 0.8
mean
0.5 ab
0.4 b
SWMO
0.4 a Sucrose 5.8 8.8 8.5 7.6 8.8
W82 534545 397 KB0715 SGUL
8.0 12.1 12.3 9.1 9.8
8.7 11.1 11.7 11.3 11.3
mean
10.3 a
10.8 a
7.9 b
W82 534545 397 KB0715 SGUL
0.8 0.9 0.1 0.0 0.0
1.4 1.4 0.2 0.0 0.0
Raffinose 0.7 0.6 0.1 0.1 0.0
mean
0.4 d
0.6 a
W82 534545 397 KB0715 SGUL
5.4 5.0 2.7 1.8 0.1
5.2 4.9 2.2 0.9 0.1
mean
3.0 c
2.6 d
Stachyose 5.5 5.5 2.9 1.7 0.3 3.2 b
W82 534545 397 KB0715 SGUL
14.2 18.0 15.6 11.6 11.0
15.3 17.4 14.5 12.8 12.2
Sum 12.0 14.9 12.0 9.9 10.2
mean
14.1 ab
14.4 a
11.8 cd
a
0.0 0.0 0.5 0.5 1.2
0.0 0.0 0.3 0.3 0.6
0.4 a
0.2 c
5.9 10.3 9.4 7.2 9.8
0.3 e
SEMO
5.1 7.7 6.6 6.4 8.0
8.5 b
6.8 c
1.0 0.9 0.1 0.1 0.0
1.1 0.8 0.3 0.3 0.0
0.4 c
0.5 b
5.9 6.2 3.2 2.0 0.5
6.0 5.7 3.6 2.7 0.7
3.6 a
3.7 a
a b c d e
12.8 17.4 13.2 9.8 11.5
12.2 14.2 10.8 9.7 9.3
b a b c c
12.9 bc
11.2 d
mean 0.0 0.0 0.5 0.5 0.9
da d c b a
6.7 10.0 9.7 8.3 9.5
c a a b a
1.0 0.9 0.2 0.1 0.0
a b c d e
5.6 5.5 2.9 1.8 0.3
a a b c d
13.3 16.4 13.2 10.8 10.8
b a b c c
b
Means in the same column with different letters are significantly different (LSD 0.05). Means in the same row with different letters are significantly different (LSD 0.05). Location Study. This experiment was conducted in 2011 at five locations (Table 2). The experiment design was a randomized complete block with four replications. Treatments were the five cultivars. Planting procedures were similar to those of the planting date study. Grain samples were collected during harvest and stored for processing. Analyses of variances were calculated using the Proc Mixed routine of SAS. Replications were considered random, whereas location and cultivar and their interaction were considered fixed. Means were compared using Fisher’s protected LSD with a type 1 error of 0.05. Monthly average temperatures were recorded with a weather station located within the same county as each of the experiment sites (Table 3). The 30-year average for each site was obtained from the Midwestern Regional Climate Center.
Carbohydrate Analysis. A subset of the total soluble carbohydrates (galactinol, sucrose, raffinose, and stachyose) in soybean was determined by high-performance ion chromatography with pulsed amperometric detection (PAD) employing a Dionex ICS-5000 with an electrochemical detector (Thermo Scientific Dionex, Waltham, MA, USA). The method was similar to that in previous studies, but our instrument system changed from an Agilent HPLC to a Dionex chromatography system.13 On the basis of our standard curves, the limit of detection for each of the four carbohydrates measured was approximately 0.1 μg/μL injected sample. A random three-seed subsample was lyophilized and ground to a fine powder in liquid N2. The 12.5 mg samples were combined with 1 mL of extraction buffer (50% (v/v) ethanol) in 2 mL tubes and incubated for 30 min at 70 °C with intermittent shaking (three times). Samples were centrifuged for 10 min at 16000g. The supernatants were transferred to a deep-well C
DOI: 10.1021/acs.jafc.5b04779 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
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Journal of Agricultural and Food Chemistry Table 5. Summary of Carbohydrate Content as Percent of Seed for Soybean Lines in the Date of Planting Study line
date 1
date 2
date 3 Galactinol 0.0 0.0 0.5 0.8 1.2
W82 534545 397 KB0715 SGUL
0.0 0.0 0.5 0.4 1.2
0.0 0.0 0.5 0.5 1.1
mean
0.4 ab
0.4 a
W82 534545 397 KB0715 SGUL
6.9 8.4 8.6 7.7 8.9
5.8 8.7 8.5 7.5 8.8
mean
8.1 b
7.9 b
W82 534545 397 KB0715 SGUL
0.8 0.6 0.1 0.1 0.0
0.7 0.6 0.1 0.1 0.0
mean
0.3 b
0.3 b
W82 534545 397 KB0715 SGUL
5.8 5.4 2.8 1.6 0.2
5.5 5.5 2.9 1.7 0.3
mean
3.2 ab
3.2 a
0.5 a Sucrose 7.6 11.6 11.4 8.6 9.2
0.3 a Stachyose 5.4 5.0 2.7 1.2 0.3 2.9 ab
12.0 14.8 12.0 9.8 10.2
Sum 13.8 17.4 14.7 10.6 10.7
mean
12.0 b
11.8 b
13.4 a
a
mean
0.0 0.1 0.4 0.8 1.1
0.0 0.1 0.5 0.5 0.7
0.0 0.0 0.5 0.6 1.1
da d c b a
0.5 a
0.4 b
7.3 10.0 10.7 8.9 9.2
d ab a c bc
0.9 0.6 0.1 0.0 0.0
a b c d d
5.4 5.1 2.8 1.3 0.2
a a b c d
13.5 15.8 14.1 10.9 10.5
b a b c c
9.9 a
Raffinose 0.8 0.8 0.1 0.0 0.0
13.5 14.4 12.0 9.8 10.3
date 5
8.3 10.1 12.4 9.7 9.1
9.7 a
W82 534545 397 KB0715 SGUL
date 4
7.6 11.0 12.7 11.1 10.1 10.5 a
0.9 0.8 0.2 0.0 0.0
1.1 0.5 0.1 0.0 0.0
0.4 a
0.3 ab
5.3 5.1 2.7 1.3 0.0
4.9 4.6 2.7 0.8 0.1
2.9 bc
2.6 c
14.5 16.1 15.7 11.8 10.2
13.6 16.2 16.0 12.4 10.9
13.7 a
13.8 a
b
Means in the same column with different letters are significantly different (LSD 0.05). Means in the same row with different letters are significantly different (LSD 0.05). 96-well plate. For each sample, 50 μL was removed to a well of a fresh 96-well plate, dried under vacuum, and resuspended to 250 μL (5× dilution) with water. Soluble carbohydrates were separated after a 10 μL injection on a Dionex Carbo Pac PA 10 analytical column (250 mm × 4 mm, 10 μm) connected to a Carbo Pac PA 10 guard column (50 nm × 4 nm). The mobile phase was 90 mM NaOH (blanketed with helium) with a flow rate of 1.5 mL min−1. A gold electrode was used in the electrochemical cell of the detector, and the settings were (time in seconds/volts) 0/0.1, 0.2/0.1, 0.4/0.1, 0.41/−2.0, 0.42/−2.0, 0.43/0.6, 0.44/−0.1, and 0.5/−0.1. Run time was a total of 48 min, with the first 18 min for sample separation followed by a 15 min washing step with 200 mM NaOH and a 15 min re-equilibration step with 90 mM NaOH. Peak areas were integrated for galactinol, sucrose, raffinose, and stachyose. Carbohydrates were quantified on the basis of standard curves generated for each carbohydrate. We report here the
content of galactinol, sucrose, raffinose, and stachyose as the percent of dry seed weight. Other Compositional Analyses. Nitrogen content of the seeds was measured according to the manufacturer’s recommendations (LECO, St. Joseph, MI, USA) using a weighed, approximately 100 mg, subsample of lyophilized seed powder via the Dumas method with a LECO truSpec model FP-428 nitrogen analyzer instrument. Protein content of the seeds was inferred from nitrogen content using a protein correction factor of 6.25. For sulfur (S) measurement, 10 mL of concentrated nitric acid was added to 0.5 g of dried ground sample and digested for 30 min using a MARSXpress microwave digestion unit (CEM, Matthews, NC, USA). Once the samples were digested (30 min), they were diluted with distilled water to 50 mL, and S concentration was determined using VISTA-MPX simultaneous ICPOES spectroscopy (Varian, Inc., Palo Alto, CA, USA). D
DOI: 10.1021/acs.jafc.5b04779 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
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Journal of Agricultural and Food Chemistry
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RESULTS Location Study. The five Missouri locations used in this study were LaGrange (NEMO), Grand Pass (NWMO), Columbia (COMO), Nevada (SWMO), and Portageville (SEMO). Higher sucrose contents and lower stachyose contents are desirable for the production of improved quality soybean meal, although an equation to assign a positive value to sucrose and a negative value to RFOs has not yet been established. The mean sucrose contents across locations were significantly different and ranked highest for the group consisting of 534545, 397, and SGUL, followed by KB0715 and W82 (Table 4). At each location, the W82 line had the least sucrose, whereas the ranking for the highest sucrose content line was not consistent across locations. The differences between the highest and lowest sucrose contents for the lines between locations was 3.3% of the seed. Overall, the highest mean sucrose contents were achieved at the NEMO and NWMO locations, followed by COMO and SWMO, and the SEMO location produced significantly lower mean sucrose contents across lines. The largest difference across locations was 4.0% of the seed. The mean stachyose contents across locations were significantly different for the lines except for W82 and 534545, which were not different from each other but ranked the highest (Table 4). Significantly different decreasing stachyose contents were measured for 397, KB0715, and SGUL. The relative ranking for stachyose content was consistent for the lines at every location, with no significant difference observed between W82 and 534545. The difference between the highest and lowest overall mean stachyose content was between W82/534545 and SGUL, and it represents 5.0% of the seed and an 18-fold reduction in stachyose. Overall, the lowest mean stachyose contents were achieved at the NWMO location, but the difference between the lowest stachyose location and the two highest stachyose locations (SWMO and SEMO) was only 1.0% of the seed. Galactinol and raffinose represent intermediates in RFO biosynthesis, with galactinol a substrate for both raffinose synthase and stachyose synthase; raffinose is a substrate for stachyose synthase. Both components were present at low or undetectable levels in soybean seeds in the location study (Table 4). Galactinol was below the limit of detection in soybean lines with functional RS2 alleles (W82 and 534545) in all locations. There were significant differences for galactinol content among the remaining mutant RS2 soybean lines across locations, with SGUL having the highest galactinol, followed by KB0715 and then 397. There were small but significant differences for galactinol across locations, with NWMO and SEMO producing the lowest mean galactinol contents. Raffinose content was significantly different for each soybean line across all locations (Table 4). Raffinose was below detection limits for line SGUL at every location, whereas KB0715 raffinose was undetectable for NEMO and NWMO locations. The highest raffinose content was from W82, which was significantly higher than the next highest raffinose content from 534545. Each location produced a significantly different mean raffinose content (in descending order: NWMO > SEMO > SWMO > NEMO > COMO). There were significant differences in the sum of the four measured carbohydrates (galactinol, sucrose, raffinose, and stachyose) accumulated in seeds from the location study for the
different lines (Table 4). 534545 had the highest sum, W82 and 397 were not significantly different from each other but lower than 534545, and KB0715 and SGUL had the lowest sum carbohydrate values. The locations generating sum mean carbohydrates in overlapping order from the highest to the lowest were NWMO, NEMO, SWMO, COMO, and SEMO. The difference in the means between the highest and lowest sum carbohydrate locations was 3.2% of the seed. Date of Planting Study. The soybean lines chosen for this experiment had appropriate similar maturity group designations (approximately maturity group late III) for the locations selected to allow pod fill and maturation under similar environmental conditions. Because we did not have soybean lines of contrasting maturity group designations that contained our array of RFO-controlling genes, we elected to synchronously vary the environment during pod fill by establishing plots during successively later planting dates. Five planting dates were used, and this strategy successfully affected the timing of seed development to span a period of approximately 3 weeks for any variety to reach physiological maturity from the first planting date to the fifth planting date. The mean sucrose contents across dates separated into two groups with dates 1 and 2 producing lower sucrose contents than dates 3, 4, and 5, and the greatest difference between dates was 2.6% of the seed (Table 5). Ranking for sucrose content was as follows: Lines 397 and 534545 produced the highest sucrose contents, then SGUL and KB0715, and the lowest sucrose content was in W82. The top four lines clustered away from W82, with a range of sucrose contents of 1.8% of the seed and then a gap of 1.6% of the seed to the W82 level of sucrose. There were small significant differences across planting dates for mean stachyose content. The three earlier planting dates had higher stachyose content means compared to the two latest planting dates, with the largest difference in mean stachyose contents between dates 2 and 5 at only 0.6% of the seed. There were major significant differences for the mean stachyose content among lines across planting dates. Increasing mean stachyose contents were produced with lines SGUL, KB0715, 397, and 534545 and W82. The difference between W82 and SGUL was 5.2% of the seed, representing a 27-fold reduction in mean stachyose content for line SGUL compared to W82. As in the location study, both galactinol and raffinose were present in low or undetectable levels in soybean seeds for the date of planting study (Table 5). Galactinol was below the limit of detection in soybean lines with functional RS2 alleles (W82 and 534545) for all planting dates except dates 4 and 5 for 534545. There were significant differences for galactinol content among the remaining mutant RS2 soybean lines across planting dates, with the rank order for galactinol identical to the order in the location study, but there was no obvious trend for galactinol content for the planting dates. The mean raffinose content across planting dates was undetectable in lines KB0715 and SGUL (Table 5). W82 had the highest mean raffinose content, followed by 534545 and 397. There was no obvious trend for raffinose content for the planting dates. There were significant differences in the sum of the four carbohydrates accumulated in seeds from the date of planting study for the different lines. 534545 had the highest sum; W82 and 397 were not significantly different from each other but lower than 534545, and KB0715, and SGUL had the lowest sum carbohydrate values, the same ranking as the location E
DOI: 10.1021/acs.jafc.5b04779 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
Article
Journal of Agricultural and Food Chemistry
locations (NEMO and NWMO) had lower temperatures during the pod-filling period (August) for the lines than for the most southern locations (SWMO and SEMO) (Table 3). It was notable that the SEMO location produced the lowest sucrose contents and the highest stachyose contents overall. No significant correlations were observed between carbohydrate components. The simple correlation coefficients between seed protein content and the carbohydrate components were moderate for protein and stachyose (−0.51), protein and galactinol (0.59), and protein and raffinose (−0.45), but weak for protein and sucrose (−0.24). Seed sulfur content was not significantly correlated with any of the carbohydrate components for the lines in the location study (data not shown).
study. Dates 3, 4, and 5 produced the highest mean sum carbohydrates, whereas dates 1 and 2 were significantly lower in sum carbohydrates. Significant Correlations. We hypothesized that temperature during pod fill may play a role in carbohydrate partitioning. The most significant correlation identified in this study was a putative environmental effect discovered in the date of planting study. Significant simple correlation coefficients were observed for all four carbohydrate components (when present in the lines) with the mean minimum/maximum daily temperature during pod filling (from maturity stage R5 to R7) (Table 6). In general, sucrose was negatively correlated with
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Table 6. Pearson Simple Correlation Coefficients between Average Daily Minimum and Maximum Temperatures during Seed Filling and the Various Carbohydrate Components
DISCUSSION Previous research has investigated genotype and environment effects on soybean seed carbohydrates. For commodity and food-type soybean lines, several studies have reported relatively small ranges for sucrose, raffinose, and stachyose contents, when directly measured, for different soybean cultivars, indicating little genotypic variation.14,18−21 Sucrose was the most variable carbohydrate component.22 It is becoming apparent that there are significant environmental effects on sucrose and RFOs for both commodity-type soybeans and lines with altered carbohydrate profiles. Although not exhaustively studied, generally, lower temperatures during pod filling or late planting correlated with more desirable carbohydrate profiles (increased sucrose and decreased RFOs).13,14,22,23 Because we previously investigated the variation in seed carbohydrate profiles for a number of experimental soybean lines with contrasting alleles of the RS2 gene, the present research intended to extend our understanding of the variation in galactinol, sucrose, raffinose, and stachyose for a small number of inbred soybean lines with defined contrasting alleles of the RS2 gene across a set of Missouri locations and staggered planting dates.13 Indeed, we observed significant variation for different carbohydrate components. Our results are distinct from previous research because the utilized altered RS2 alleles are currently the simplest genetic approach to a “high” sucrose and “low” RFO trait that may increase metabolizable energy in soybean meal. In addition, we utilized different environments and staggered planting dates to better understand the stability of the altered carbohydrate profile in soybean seeds. In sum, we evaluated five distinct locations in addition to five successive planting dates at one location for a total of nine environments. The weather data for our locations indicated that temperatures were similar to 30-year averages for the pod-filling period. Our results demonstrate that altered carbohydrate soybeans can produce a high sucrose (>8% dry) and low RFOs (
