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CROP PHYSIOLOGY & METABOLISM

Genotypic and Environmental Variation in Soybean Seed Cell Wall Polysaccharides

^a Dep. of Agronomy and Plant Genetics, 1991 Upper Buford Circle, Univ. of Minnesota, St. Paul, MN 55108 USA
^b USDA-ARS, Plant Sciences Res. Unit, St. Paul, MN 55108 USA

somers{at}biosci.cbs.umn.edu

	ABSTRACT

TOP NOTES ABSTRACT INTRODUCTION Materials and methods Results Discussion REFERENCES

 
Soybean [Glycine max (L.) Merr.] seed cell wall polysaccharides (CWP) have been characterized, but little is known about their genotypic variation. This information would be beneficial for determining genetic strategies for manipulating CWP content and composition. Seed CWP was determined by the Uppsala total dietary fiber method to quantify monosaccharides of CWP in 14 soybean genotypes from Maturity Groups 00 to I grown at four Minnesota locations. CWP concentration of mature whole seed varied from 158 to 176 g/kg dry matter (DM). Genotypic but not environmental effects were significant for total CWP concentration. For individual monosaccharide concentrations, both genotypic and environmental effects were present. Seed of five genotypes were separated into cotyledon and seed coat for CWP analysis. Genotypic variation for CWP concentration was mostly in cotyledon and not seed coat. Pectin was mostly in cotyledon rather than seed coat with 80.8 and 14.6 g pectin/kg of whole seed DM in cotyledon and seed coat, respectively. Only xylose, glucose, galactose, and uronic acid concentrations were significantly different among genotypes in cotyledon. The correlation between CWP concentration and protein plus oil concentration among the 14 genotypes was r = -0.724, which suggests that an increase in protein plus oil content is associated with a reduction of CWP concentration. The genotypic variation observed suggests that it is possible to breed for reduced CWP. However, genotypic variation for some monosaccharides was limited, suggesting that other methods of genetic manipulation may be more efficient in reducing these monosaccharides.

Abbreviations: Ara, arabinose • CWP, cell wall polysaccharide • DM, dry matter • Fuc, fucose • Gal, galactose • Glc, glucose • Man, mannose • Rha, rhamnose • UA, uronic acid • Xyl, xylose

	INTRODUCTION

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CELL WALL POLYSACCHARIDES account for about 12% of dry matter in soybean cotyledons (Daveby and Aman, 1993) and represent a substantial amount of dry matter deposited during seed development. While CWP composition has been quantified, the genotypic and environmental variation in soybean seed CWP concentration have not been examined. Although cellulose content, quantified as insoluble crude fiber, has been analyzed in some genotypes (Youssef et al., 1980; Kapoor et al., 1975), pectin and hemicellulose have not. Monosaccharides of soybean CWP were quantified (Daveby and Aman, 1993) in a study that did not examine genotypic and environmental influences on polysaccharide concentration. Quantifying the genotypic and environmental variation in soybean seed CWP would provide information on the feasibility of breeding for altered CWP concentration.

Cell wall polysaccharides can be separated into cellulose, pectin, and hemicellulose on the basis of solubility (Hatfield, 1989). Cellulose and hemicellulose are structural polysaccharides that are hydrophobic and have no or limited solubility in water. Cellulose is a straight chain polymer of ß-1–4-linked glucose units whose close packing and hydrogen bonding with other cellulose microfibrils form a strong insoluble fiber (Hatfield, 1989). Hemicellulose is comprised of xylans, ß-1–4-linked polymers of xylose, and xyloglucans, ß-1–4-linked polymers of glucose with xylose side chains. The side chains produce various amounts of branching which prevent the close packing of fibers seen in cellulose but do allow some hydrogen bonding to occur. Pectin is the most highly branched polysaccharide. Its galacturonic acid residues form ionic bonds with Ca²⁺ providing sites for interpolysaccharide chain binding. Pectin is water soluble, although its extractability varies because of associations formed with other polymers and ions (Monro, 1991). It has been postulated that the branching and association of pectin with hemicellulose and cellulose determine the porosity of plant cell walls (Carpita and Gibeaut, 1993).

Cell wall polysaccharide composition of soybean seed has been determined by isolating polysaccharides on the basis of their solubility and then degrading the polysaccharides into their monosaccharide sugars (Aspinall and Cottrell, 1971). Some monosaccharide sugars that comprise cell wall polysaccharides tend to be predominately in either pectin or hemicellulose. However, pectin and hemicellulose are defined by their solubilities rather than by their constituent monosaccharide subunits, such that some monosaccharides like xylose and arabinose are found to a small degree in both polysaccharides. Hot water soluble polysaccharides isolated from mature soybean cotyledons are comprised of arabinans, arabinogalactans, and an acidic polysaccharide complex containing galacturonic acid, galactose, arabinose, xylose, fucose, and rhamnose (Aspinall and Cottrell, 1971). Hemicellulose isolated from soybean hulls consisted mainly of xylose with some glucose, galactose, and arabinose (Sannella and Whistler, 1962). Galactomannans and several other water soluble acidic polysaccharides have been isolated from soybean hulls as well (Aspinall et al., 1967). While the composition of soybean seed CWP has been characterized, the variation of CWP content has not been examined among genotypes. The objective of this study was to investigate genotypic and environmental variation in CWP and its monosaccharide subunits in soybean genotypes grown in Minnesota.

	Materials and methods

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Fourteen soybean genotypes of Maturity Groups 00 to I (`Bert', `Council', `Faribault', `Glacier', `Granite', `Hendricks', `Kato', `Lambert', `McCall', `Minnatto', `Ozzie', `Parker', `Proto', `Toyopro') were grown at Rosemount, Becker, Waseca, and Lamberton, MN, during 1995 and 1996. The genotypes were grown in standard yield plots and managed for uniform yield evaluations. Seed samples were taken from the yield trials for analysis. Whole seed was ground in a Stein mill and defatted by Soxlet extraction with petroleum ether. Oil concentration was calculated as the difference in weight before and after defatting and was corrected for water content. Protein was quantified by microkjeldahl (N x 6.25). Ash was determined as the residue remaining after combustion in a muffle furnace for 6 h. All data were expressed on a dry matter (DM) basis.

Cell wall polysaccharides were quantified after hydrolysis to their monosaccharide subunits by the Uppsala total dietary fiber method (Theander et al., 1995). Starch was removed and protein reduced by mixing 100 mg defatted flour in 5 mL of 0.1 M sodium acetate (pH 5.0) and treating it with -amylase (Sigma, St. Louis, MO)¹ at 95°C for 1 h followed by amyloglucosidase (Boehringer Manneheim, Indianapolis, IN) at 60°C for 3 h. Twenty-eight milliliters of 950 mL/L ethanol were added to reach a final concentration of 800 mL/L ethanol and the samples left at 4°C overnight. Samples were centrifuged for 15 min. at 1650 x g and the pellet rinsed and centrifuged twice with 15 mL 800 mL/L ethanol and once with acetone. The pellet was dried over N₂ and desiccated before acid hydrolysis. Samples were hydrolyzed in 1.5 mL of 12 M H₂SO₄ at 30°C for 1 h, diluted to 0.4 M H₂SO₄, and autoclaved for 1 h. Neutral sugars were acetylated and derivatized sugars were quantified by GC-FID. Uronic acids were quantified colorimetrically (Ahmed and Labavitch, 1977). Pectin concentration was estimated as the sum of galactose, arabinose, rhamnose, fucose, and uronic acids. Cell wall polysaccharides were defined as the sum of all of the sugars.

Five genotypes, `Danatto', Faribault, Lambert, Minnatto, and Toyopro, grown at Becker and Rosemount, MN, in 1996 were used for analysis of seed coat and cotyledon cell wall polysaccharides. Mature dry seeds were broken into pieces with a mortar and pestle. Seed coats were isolated by vacuum and any residual cotyledon or embryonic axis material was removed from the seed coats with forceps. Pieces of cotyledon free of seed coat and the embryonic axis were collected with forceps. Seed tissues used in this study were analyzed as above except they were ground in a cyclone mill to pass a 1-mm screen.

Samples were analyzed in duplicate and mean values were used in analysis of variance and correlations (Analytical Software, 1992). Data for whole seed were analyzed with genotype, location, and year being the main effects. Genotype was considered a fixed effect and locations and years random effects. Data from the seed coat, cotyledon, and whole seed study were analyzed separately with genotype and location as the main effects.

	Results

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Whole Seed Cell Wall Polysaccharide
To evaluate genotypic and environmental variation in CWP concentration, whole seed of 14 soybean cultivars grown in four locations over 2 yr were analyzed for CWP, oil, protein, and seed weight (Table 1) . The genotypes studied were chosen because they exhibited significant variation in mean oil content ranging from 202 g/kg for Minnatto to 244 g/kg for Lambert. The cultivars varied for protein, seed weight, and CWP (Table 1). The effect of genotype on these components was highly significant (data not shown). Year effects and location x year effects on oil content were significant at the 0.05 level (data not shown). Seed weight exhibited highly significant genotype x location and location x year interactions (data not shown).

Whole Seed Partitioning
Comparing Year 1 and Year 2, pectin was 4.9 g/kg DM higher in the second year than in the first year with each of the pectin sugars contributing to the increase (data not shown). This was accompanied by glucose and mannose combined being 5.6 g/kg DM lower in Year 2 than Year 1. Total cell wall polysaccharide was 165.4 and 164.4 g/kg DM for Years 1 and 2, respectively. This indicates that there was more change in the composition of the CWP than there was in CWP concentration. As a result, year effects were observed for pectin and most of the monosaccharides while year effects were not observed for CWP.

Cell Wall Polysaccharide Correlations
Correlations were calculated to examine the relationships between the concentrations of CWP, monosaccharides, protein, and oil and seed weight (Table 4) . CWP concentration was not significantly correlated with either protein or oil content. However, CWP concentration was negatively correlated with the sum of protein plus oil (Table 4). Of the monosaccharides, only glucose was negatively correlated with protein plus oil. Oil and protein exhibited a negative correlation (r = -0.575) that has been previously reported (Hymowitz et al., 1972; Openshaw and Hadley, 1981).

Seed Coat and Cotyledon Cell Wall Polysaccharide
Because Minnatto is a small-seeded genotype and some correlations were significant only because of this genotype, we hypothesized these results may be related to seed coat representing a larger portion of the total mass for small seed. Therefore, to investigate sources of variation in CWP content and because control of biosynthetic pathways can vary by tissue type, seed of five soybean genotypes, Danatto, Faribault, Lambert, Minnatto, and Toyopro, grown at two locations were separated into seed coat and cotyledon and assayed for CWP content. Danatto was included because it is a small-seeded genotype of about equal size to Minnatto. On average, seed coat comprised 79 g/kg seed DM (Table 5) . Minnatto was the exception with a seed coat content of 100 g/kg seed DM. Danatto and the other lines examined exhibited similar seed coat content regardless of seed size.

	Discussion

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Seed CWP from 14 soybean genotypes grown in Minnesota exhibited genotypic variation indicating that it is possible to breed for reduced polysaccharides. Of pectin component sugars, galactose had the most genotypic variation. Pectin, and specifically galactose, were present mostly within the cotyledon (Table 5). In addition, genotypic variation in CWP concentration and its constituent sugars was more prevalent in the cotyledon than the seed coat (Table 5). While there didn't appear to be genotypic variation for uronic acids in whole seed, there was genotypic variation in the cotyledon. This suggests that whole seed analysis for uronic acids conceals the genotypic variation present in the cotyledon. Since most of the genotypic variation for the monosaccharides was present in the cotyledon, this tissue should be the primary target for studies to alter CWP levels. Seed coat had less pectin than cotyledon, but was high in glucose and xylose making seed coat high in cellulose and hemicellulose. While there was genetic variability for seed coat DM per seed, the lack of variability in seed coat CWP concentration may make altering CWP in the seed coat difficult without changing the amount of seed coat per seed as well.

In previous studies, soybean seed crude fiber, which is an estimate of cellulose content, averaged 4 to 5% of dry weight by proximate analysis (Youssef et al., 1980; Kapoor et al., 1975) and was related to seed weight and percentage of hull in the whole seed (Youssef et al., 1980). Larger seeds contained less crude fiber and had lower percentage hull. In the Minnesota genotypes, glucose was negatively correlated with seed weight (Table 4) and was in the same concentration range as reported for crude fiber by Youssef et al. (1980). Because about two-thirds of the glucose was in the seed coat (Table 5), the correlation between seed weight and glucose is most likely related to the percentage of seed coat in whole seed.

There is some variation between the current data and those from Daveby and Aman (1993). While the cotyledon CWP content they observed is similar to our data, Daveby and Aman (1993) reported lower seed coat levels of xylose, mannose, and galactose and a higher amount of UA. This may be related to the soybean genotypes sampled. Daveby and Aman (1993) used 12 genotypes for cotyledon analysis, while seed coat determination was limited to only one genotype (Daveby and Aman, 1993). They also used soybeans from a wider geographical area. The differences between the seed coat CWP content in the Minnesota genotypes and those previously reported may indicate that genotypes with more divergent CWP concentration in the seed coat can be found than was demonstrated in these Minnesota genotypes.

The lowest CWP level, 158 g/kg DM, was found in Toyopro, which contained the highest protein plus oil content, 644 g/kg DM (Table 1). Council contained one of the highest CWP contents, 172 g/kg DM, and had a significantly less protein plus oil content, 606 g/kg DM, than Toyopro. Council and Toyopro had similar seed sizes of 173 mg and 167 mg, respectively. This demonstrates that protein plus oil can be increased and CWP reduced without the change being due to increased seed size. The low CWP in Toyopro was due to the major constituents of CWP—galactose, arabinose, uronic acids, and glucose—being lower than the other genotypes (Table 3).

CWP and protein plus oil concentrations were negatively correlated (Table 4). Since the genotypes varied by 18 g/kg dry matter in cell wall polysaccharide, this implies that some genotypes contained up to 18 g/kg more dry matter in protein plus oil than in cell wall polysaccharides. However, the range in protein plus oil was 56 g/kg indicating that the variation in CWP does not account for all of the variation in protein plus oil. Soluble sugars are another component not determined in this study that may account for this difference.

There was significant genotypic variation for most of the CWP monosaccharides with most of the genotypic variation for these sugars localized within the cotyledon. Galactose was found almost completely within the cotyledon (Table 5). While glucose and uronic acids were divided between cotyledon and seed coat, genetic variation for these was located in the cotyledon. This suggests that the correlation between CWP and protein plus oil was based on genetic variation in the cotyledon more than in the seed coat. This is further supported by the observation that neither CWP or protein plus oil were correlated with seed weight. As a result, a decrease in cotyledon CWP may increase carbon available for protein and oil deposition.

	ACKNOWLEDGMENTS

 
The authors thank R.T. Jeo for his excellent technical assistance and instruction in the application of the total fiber method.

	NOTES

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Contribution no. 991130116 from the Minnesota Agric. Exp. Stn. This research supported in part by Minnesota Soybean Research and Promotion Council.

¹ Mention of a trade name, proprietary products, or specific equipment does not constitute a guarantee of the product by the University of Minnesota or the USDA, and also does not imply its approval to the exclusion of other products that may be suitable.

Received for publication March 22, 1999.

	REFERENCES

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• Aspinall, G.O., R. Begbie, and J.E. McKay. 1967. Polysaccharide components of soybeans. Cereal Sci. Today 12:223–228, 260–261.
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