Plant Physiol. (1985) 77, 532-5350032-0889/85/77/0532/04/$0 1.00/0

A Developmentally Regulated Hydroxyproline-Rich Glycoproteinfrom the Cell Walls of Soybean Seed Coats'

Received for publication September 17, 1984

GLADYS I. CASSAB, JORGE NIETO-SOTELO, JAMES B. COOPER, GERRIT-JAN VAN HOLST, ANDJOSEPH E. VARNER*Plant Biology Program, Department ofBiology,

ABSTRACT

In soybean seeds the level of hydroxyproline is regulated in a devel-opmental and tissue-specific manner. The seed coat contains approxi-mately 77% of the total hydroxyproline in the seed at all stages ofdevelopment. We determined the ratio of hydroxyproline to dry weightin a number of tissues within the seed; however, only the seed coat showsan increase in this ratio during development. Within the many cell layersof the seed coat, hydroxyproline is most abundant in the external layer.The hydroxyproline is present as an hydroxyproline-rich cell wall gly-coprotein. The protein is rich in hydroxyproline (36%), lysine (11%),proline (10%), histidine (9%), tyrosine (9%), and seine (8%). Thecarbohydrate portion is 90 mole % arabinose and 10 mole % plactose.The arabinose residues are attached to hydroxyproline mostly in the formof trisaccharides. The apparent molecular weight of this glycoprotein is100,000 daltons.

Hydroxyproline-rich glycoproteins of the cell wall are presentin a wide variety of plants (16). The level of the HRGPCWs2 isusually low in plants; but, in some instances, a rise above normallevels has been observed upon wounding (3, 27) and infection(8, 1 1), and the level is usually higher in cells in culture (15).Van Etten et al. (28) reported that seed coats and pericarps of

many plant species contain high levels ofthe amino acid hydrox-yproline. The testa is usually a hard coat whose physiologicalimportance arises from the presence ofan outer and inner cuticle,and one or more layers of thickened protective cells. Thesefeatures confer upon the testa mechanical strength and somedegree of impermeability to water and/or gases including 02, SOas to exert a regulatory influence on the metabolism, growth,and development of the inner tissues and organs of the seed (23).Indeed, the seed coats may determine seed size (5, 26).

Here, we report that during soybean seed development thelevel of hydroxyproline in the testa increases dramatically, andthe hydroxyproline to cell wall dry weight ratio is highest in theexternal layer of the seed coat. Moreover, this hydroxyproline ispresent as a cell wall glycoprotein which differs in its amino acidcomposition and arabinosylation pattern from the carrot cellwall glycoprotein previously characterized (29). This HRGPCWmay play a structural role in the seed coat (28) and could becharacteristic of this tissue.

'Supported by grants from the National Science Foundation (PCM7923550 and PCM 8104516), and the United States Department ofAgriculture (83-CRCR-I-12 17).

2Abbreviation: HRGPCW, hydroxyproline-rich glycoprotein fromcell wall.

Washington University, St. Louis, Missouri 63130

MATERIALS AND METHODS

Plant Material. Seeds of Glycine max (var Provar) were ob-tained from six stages of development: 8, 18, 19, 20, 21, and 26d after anthesis from plants grown in the greenhouse. At thesestages of development, the palisade and hour glass cell layerswere easily dissected from the rest ofthe seed coat. Integrity andpurity of the separated layers was verified by inspection by lightmicroscopy.Hydroxyproline Determination. Hydroxyproline content was

determined colorimetrically by the method of Drozdz et al. (6)after proteins or tissues were hydrolyzed in 6 N HCl at 120°C for3 h.

Cell Wall Isolation. Fresh seed coats (about 5 g) were groundin a glass homogenizer in 0.1% K-acetate buffer (pH 5.0) with 4mM Na2S205 and 1% insoluble PVP. The cell walls were washedwith 0.5% Nonidet P-40 (Sigma), 2 mm Na2S2O5, and thenresuspended and centrifuged 10 times with 50 ml of cold 2 mMNa2S205. Finally, the cell wall pellet was extracted with 10 ml of0.2 M CaC12, 4 mm Na2S205 for 12 h at 50C.

Isolation of Hydroxyproline-Rich Cell Wall Glycoprotein. TheCaCl2 extract was loaded onto a CM-Sepharose CL-6B (Sigma)column (30 x 2.5 cm) previously equilibrated with 0.02 M Tris-HCI (pH 8.0). The column was washed with two volumes of0.02 M Tris-HCl (pH 8.0), and the attached material was elutedwith a linear gradient of 0.02 to 0.5 M Tris-HCl (pH 8.0). Thehydroxyproline containing fractions (salt concentration, 0.3 MTris-HCl) were pooled and solid CsCl was added to a final densityof 1.4 g cc-'. Samples were centrifuged for 72 h at 250,000g in aBeckman SW-65 Ti rotor. The resulting CsCl gradient wasfractionated. The hydroxyproline containing fractions (densityof 1.435 g cc-') were pooled and dialyzed against 0.1 M NaClovernight and against deionized H20 for 2 d. The entire proce-dure was carried out at 50C. In all the fractionation procedures,the salt concentration of the samples was determined using arefractometer, the protein content by UV absorption at 280 nm,and the hydroxyproline content as described above.Chemical Analyses. Acid hydrolysis for the amino acid anal-

yses was performed in constant boiling HCI (Pierce) with 0.05%(v/v) mercaptoethanol under N2 for 24 h at 1 10°C. The resultingamino acids were then analyzed on an automatic amino acidanalyzer. Sugar composition was determined by GC of alditolacetate derivatives of the hydrolyzed polysaccharides (1 h at120°C in 2 M TFA [2]) on a 1% (w/v) OV-275 Gas Chrom AColumns (180 x 0.2 cm). Hydroxyproline arabinosides wereseparated by gel filtration (13).Gel Electrophoresis. SDS-PAGE was done according to La-

emmli (14) with 5 and 10% acrylamide. The gels were fixed andstained with periodic acid-silver stain (7).

Precipitation with ,-Galactosyl-Yariv Reagent. The precipita-tion of arabinogalactan-protein with ,-galactosyl-Yariv reagentwas carried out according to Jermyn and Yeow (12).

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HRGPCW IN SOYBEAN SEED COATS

18 20 26Days after Anthesis

01)a

0)

..

0x0

-C

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FIG. 1. Pattern of total hydroxyproline distri-bution during soybean seed development. A, Inthe entire seed; B, in the seed coat. Each valuerepresents the mean of three replicates.

Days after Anthesis

Table I. Ratios ofHydroxyproline to Dry Weight in Different Parts ofthe Developing Soybean Seed

Hydroxyproline: Dry Wt Ratio(pg/mg) at Following Days

Tissue after Anthesis

8 19 21 26Seed coat 0.3 2.1 3.1 4.6

External layer a 2.2 4.0 6.4Internal layer 1.6 1.6 1.8Hilum 3.0 4.5 4.6

Cotyledon 0.01 0.5 0.4 0.2Axis 1.0 0.2 0.4

a Not determined.

Scanning Electron Microscopy. Seeds were dissected, and thenfixed for 2 h in 3% (v/v) glutaraldehyde containing 100 mmphosphate buffer (pH 7.2). After two 15-min rinses in phosphatebuffer, tissue samples were slowly dehydrated in graded ethanolseries, and samples in absolute ethanol were dried using thecritical point dryer, fractured, attached to specimen studs, andcoated with gold-palladium prior to viewing with a Hitachi S-450 scanning electron microscope.

RESULTS

Distribution of Hydroxyproline in Soybeans. The seed coat hasthe highest ratio of hydroxyproline to dry weight (5.2 gg Hyp/mg dry weight) of the soybean plant compared with roots (0.2jig/mg), leaves (0.3 ug/mg), entire seed (0.9 sg/mg), stems (1.3ag/mg), and flowers (1.5 Ag/mg).The hydroxyproline content increases in the entire seed and

in all parts of the seed during its development (Fig. 1A). Theseed coat has about 77% of the total hydroxyproline in the seedat all stages. The ratio of hydroxyproline to dry weight increasesduring development only in the seed coat and not in the cotyle-dons and axis (Table I). To determine the distribution of hy-droxyproline in the various tissues of the seed coat, we dissectedit into the external and the internal layers and the hilum region.The external layer consists of two cell layers: epidermal palisadecells and hour glass cells. The internal layer has thin-walledparenchymatous tissue, vascular cells, and compressed cells (Fig.2). The total hydroxyproline content in the different sections ofthe seed coat was determined and is shown in Figure lB. Themaximum accumulation of hydroxyproline occurs in the exter-nal layer. At 26 d after anthesis, the external layer contains 73%of the total hydroxyproline of the seed coat. Moreover, the ratioof hydroxyproline to dry weight is greater than in any other part

ofthe seed (Table I). We also determined the amount of hydrox-yproline in isolated cell walls of different parts of the seed. Thehighest ratio ofhydroxyproline to cell wall dry weight is localizedin the outermost layer of the seed coat (data not shown).

Isolation of Hydroxyproline-Rich Glycoprotein from the CellWalls of Soybean Seed Coats. The HRGPCW of soybean seedcoat was isolated from seeds at stage 'N' (25 d after anthesis[2 1]). This particular stage was chosen because 60 to 80% of thepeptidyl-hydroxyproline is solubilized with high salt concentra-tion indicating that it is not covalently linked to the cell wall.We followed the same procedure reported by Stuart and Varner(27) with minor modifications. The density of the major proteineluted from the cation exchange chromatography was 1.435 gcc-l in a CsCl gradient. This is virtually the same density as thatof the HRGPCW from carrots (27).The HRGPCW was purified to homogeneity as indicated by

the appearance ofa single band in SDS-PAGE after staining withperiodic acid-silver stain in 10% and 5% polyacrylamide gels. In5% polyacrylamide gels, the HRGPCW shows an apparent molwt of 100,000 D. With this technique, however, the mol wt ofglycoproteins can be overestimated because they usually show alarger hydrodynamic radii than proteins with similar mol wt (1).The HRGPCW is rich in Hyp, Ser, Pro, Tyr, His, and Lys,

which make up 83% of the residues (Table II).The seed coat HRGPCW has arabinose and galactose as major

carbohydrates (Table II). The sugars were identified by compar-ison of retention times of known sugar standards as well as byco-elution of sample peaks coinjected with the standards on thegas chromatograph. The arabinosylation pattern as determinedby gel filtration (13) showed that arabinose is primarily attachedto hydroxyproline in oligosaccharide chains ofthree residues (seeTable II).The purified seed coat cell wall glycoprotein shows no reaction

with the ,8-galactosyl-Yariv reagent, which precipitates the ara-binogalactan proteins, another class of hydroxyproline-rich pro-teoglycans (9). Cell wall extracts from seed coats show littlereaction with this reagent. However, in the case of cell wallextracts from soybean flowers, 33% of the total hydroxyprolinereacts with the Yariv reagent.

DISCUSSION

The presence of hydroxyproline in the seed coat of severalplant species has been reported earlier (28). From the presentstudy, it is clear that the accumulation of hydroxyproline isdevelopmentally regulated in the soybean seed and is greater at26 d after anthesis than at the other stages analyzed. Moreover,the highest level of hydroxyproline is always found in the testaat the different stages of seed development, and interestingly,

100a)

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501

A Axis

El CotyedonEl Seed coat

nfl~0-E !

533

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Plant Physiol. Vol. 77, 1985

FIG. 2. Anatomy of soybean seed coat. PC, palisade cells; HG, hour glass cells; P, parenchyma; C, compressed cells; Cot, cotyledon. A, SEM ofan entire seed coat section; it is marked the external and internal layers used in this work. B, SEM showing the external layer as it is peeled awayfrom the internal layer during dissection. Also shown is a fragment of the cuticle (Cut) which covers the epidermal layer of the seed coat. C, SEMof the isolated external layer of the seed coat. The highest level of hydroxyproline is found in this layer (see Table I).

within the many cell layers of the coat, hydroxyproline is mostabundant in the outermost layer.The peptidyl-hydroxyproline isolated from soybean seed coats

is an hydroxyproline-rich glycoprotein. This glycoprotein is ap-proximately 2% of the total dry weight of the seed coat. Sixamino acids (Hyp, Ser, Pro, Tyr, His, and Lys) constitute 83%of the residues. This amino acid composition is similar to theamino acid composition of the well characterized HRGP from:

(a) aerated carrot slices (29); (b) potato tubers (20); (c) tobaccocallus (22); and (d) tomato cell suspension cultures (25). Therelatively high content of the basic amino acids histidine andlysine and the low content of acidic residues indicate that theprotein is a basic molecule, being positively charged at neutralpH values. This charge could be important for the interaction ofthe protein with other structural components, particularly polyu-ronates of the cell wall. The seed coat HRGPCW has a ratio of

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534 CASSAB ET AL.

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HRGPCW IN SOYBEAN SEED COATS

Table II. Composition ofHydroxyproline-Rich Glycoprotein from CellWalls ofSoybean Seed Coats

Amino Acids Sugars

mol % mol %Hyp 36.2 Ara 90.3Asx 2.1 Galb 9.2Thre 1.3 Glcc 0.5Sera 8.2Glx 2.4 HydroxyprolinePro 9.9 arabinosidesGly 4.0Ala 1.9 mol % total HypVal 2.5 Hyp-Ara 4 10Cys 1.0 Hyp-Ara 3 62Met 2.2 Hyp-Ara 2 7Ile 0.9 Hyp-Ara 1 10Leu 1.3 Hyp 11Tyr 8.5Phe 0.5His 8.8Lys 10.8Arg 1.0

a Not corrected for losses during hydrolysis. b Galactose is presum-ably linked to serine (Lamport et al., 1977). c Glucose is thought tobe a contamination.

Hyp to Pro (4: 1) that is similar to those ofthe bacterial agglutininsof potato tubers and tobacco callus (20, 22), but different fromthe carrot cell wall glycoprotein (46:1) (29).The carbohydrate content ofthe seed coat HRGPCW is similar

to that from other plant HRGPCWs (18, 29, 30). However, inthe seed coat HRGPCW, the arabinose is mainly bound tohydroxyproline in short side chains of three residues (HypAra3rather than HypAra4).The seed coat HRGPCW migrates as a single broad band after

electrophoresis in 5% polyacrylamide gels containing 10% SDS(data not shown), comparable to the bacterial agglutinins ofpotato tubers and tobacco callus (20, 22). This broad bandingpattern is fairly characteristic of glycoproteins and presumably isa consequence of the heterogeneity of the carbohydrate chains(18).

Finally, one distinguishing characteristic of the seed coatHRGPCW is that it accumulates during soybean seed develop-ment. Moreover, in the seed coat, the ratios of hydroxyprolineto dry weight is highest compared with any other part of thesoybean plant. This is of special interest in relation to a possiblerole in structure (28) and the protective function of the testa. Ithas been suggested that the cell wall glycoprotein is secreted assoluble monomer which becomes insolubilized in the wall byformation of isodytyrosine cross-links (4, 10). We are now inter-ested in determining whether these isodytyrosine cross-links existin the seed coat HRGPCW and are related to its presumedstructural role.

Acknowledgments-The authors are grateful to Meryl Weinstein (Fine ArtsMajor) for technical assistance in the first hydroxyproline assays, Terry Takehirofor technical assistance in the arabinosylation profile, and Mike Veith for excellentassistance in the scanning electron micrographs.

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