Plant Physiol. (1983) 71, 519-523 0032-0889/83/71/05 19/05/$00.50/0

Isolation and Characterization of Oat Aleurone and Starchy Endosperm Protein Bodies' Received for publication August 18, 1982 and in revised form November 11, 1982 ERIK T. DONHOWE2 AND DAVID M. PETERSON3 Department ofAgronomy, University of Wisconsin, and United States Department ofAgriculture, Agricultural Research Service, Madison, Wisconsin 53706

ABSTRACT MATERIALS AND METHODS To compare oat (Aena saltiwa L cv Froker) aleurone protein bodies Tissue Separations. Embryos of mature oat (Avena sativa L. cv with those of the starchy endosperm, methods were developed to isolate Froker) groats (caryopses) were removed, and the remaining seed these tissues from mature seeds. Aleurone protoplasts were prepared by was sliced 1-mm thick with a razor blade. For starchy endosperm euzymic digestion and filtration of groat (caryopsis) slices, and starchy preparation, slices were imbibed 16 h at 4VC, and then rolled as endosperm tissue was separated from the aleurone layer by squeezing described by Phillips and Paleg (18). The resulting slurry was slices of imbibed groats followed by filtration. Protein bodies were isolated filtered through a 750-.un nylon screen, and the filtrate was stored from each tissue by sucrose density gradient centrifigation. Ultrastructure on ice. This procedure effectively separated starchy endosperm of the isolated protein bodies was not Identical to that of the intact from the more resistant aleurone tissue, which was retained on the organelles, suggesting modification during Isolation or fixation. Both aleu- screen. Microscopic examination of the filtrate confirmed the rone and starchy endosperm protein bodies contained globulin and prolamin absence of aleurone tissue. storage protein, but minor dMerences in the protein-banding pattern by Aleurone protoplasts were prepared by incubating 25 g groat sodium dodecyl sulfate-polyacrylamide gel electrophoresis were evident. slices in 86 ml 0.7 M mannitol containing 1% (w/v) Cellulysin and The amino acid compositions of the protein body fractions were similar 0.25% (w/v) Macerase (Calbiochem-Behring),4 pH 5.5, at 260C and resembled that of oat g lo l The aleurone protein bodies contained with gentle agitation. The thin walls of the starchy endosperm phytic acid and protease activity, which were absent in starchy endosperm cells were digested more quickly than the thicker aleurone cell protein bodies. walls. At 1-h intervals, starchy endosperm cell contents were removed by filtration through a 750-pm nylon screen, and tissue retained by the screen was rinsed with 0.7 M mannitol and then incubated with fresh enzyme solution. After 4 h, only aleurone cells remained, as shown by the absence of any starch-containing cells adhering to the aleurone layer tissue on the nylon screen. The enzyme concentrations were then doubled, and after a 1-h Seed storage protein occurs primarily in protein bodies, which incubation, aleurone protoplasts were collected in the filtrate, are dense deposits of protein surrounded by a membrane (14). centrifuged at 75g for 3 min, washed in 0.7 M mannitol, and Within protein bodies, the proteinaceous matrix may contain resuspended in 2 to 5 ml of 13% (w/w) sucrose in gradient buffer. globoids and/or crystalloids. In the Gramineae, protein bodies Protein Body Isolation. Starchy endosperm filtrate or aleurone occur in the aleurone layer, starchy endosperm, and scutellum protoplasts were disrupted with three strokes of a Potter-Elvehjem (10). Aleurone protein bodies contain globoids and/or crystalloids, homogenizer and 0.5- to 1.0-ml aliquots were layered on 12-ml whereas those of the starchy endosperm do not (16). In mature linear 17 to 66% (w/w) sucrose density gradients in 10 mM Tris- caryopses of oats, aleurone protein bodies contain both globoids HC1, 1 mM EDTA, and 0.1 mM MgCl2, pH 7.5. Gradients were and crystalloids (3). The starchy endosperm protein bodies, by centrifuged 4 h at 230,000g~n.1 at 4°C in a Beckman SW 40 rotor. contrast, contain neither of these inclusions, but do show unchar- Alternatively, 2- to 3-ml aliquots were layered on 35-ml linear 36 acterized electron-lucent regions. to 66% (w/w) sucrose density gradients for centrifugation 4 h at In oats, the primary storage protein is a globulin, although 140,000g,., in a Beckman SW 27 rotor. Gradients were fraction- prolamin, the major storage protein ofmost cereals, is also present ated through an ISCO model 184 density gradient fractionator or (17). We considered it important to determine whether both the visible protein bands were aspirated with a Pasteur pipet. globulin and prolamin occurred within protein bodies ofoats, and Protein body fractions were frozen or stored on ice. whether protein bodies of the aleurone differed from those of the Electron Microscopy. Glutaraldehyde was added to protein starchy endosperm in protein contents or other characteristics. body fractions to a concentration of 6% (v/v), and the suspended Our approach was to devise methods to obtain preparations of protein bodies were fixed on ice for 1 h. Fixed samples were aleurone tissue uncontaminated by starchy endosperm and vice diluted with 2 volumes distilled H2O and centrifuged at 1700g for versa. From these tissues, protein bodies were isolated and char- 30 min. The pellets were resuspended in 2% (w/v) agar at 50°C acterized. and drops were solidified on chilled glass microscope slides. Cubes 1-mm square were placed in 80 mm cacodylate buffer, pH 7.4, ' Research supported by the United States Department of Agriculture, Agricultural Research Service, and the College of Agricultural and Life 4Mention of a trademark or proprietary product does not constitute a Sciences, University of Wisconsin, Madison. guarantee or warranty of the product by the United States Department of 2Present address: Adolph Coors Co., Golden, CO 80401. Agriculture and does not imply its approval to the exclusion of other 3To whom reprint requests should be addressed. products that may also be suitable. 519 520 DONHOWE AND PETERSON Plant Physiol. Vol. 71, 1983 postfixed in 2% (w/v)OS04 16 h at4VC, and dehydrated in an acetone series. They were stained with uranyl acetate and polym- erized in Spurr's resin (20). Silver sections were cut with glass knives, and these sections were stained with lead citrate and viewed with a transmission electron microscope. Assays. Sucrose density gradient fractions were assayed for protein by dye-binding (5). Fractions were diluted with 2 volumes distilled H20, and protein bodies were pelleted by centrifugation at 12,100g for analysis of N, P, phytic acid, amino acids, and protease. For total N,pellets were washed three times with 10%o (w/v) TCA, four times with acetone, resuspended in distilled H20, and assayed by digestion andcolorimetric analysis (7). For total P,1- ml aliquots of resuspended pellets were digested with 2.2 ml HC104 by boiling 30 min. Two drops 30% H202 were added, followed by an additional 15-min digestion. Neutralized digests were analyzed for P according to Fogg and Wilkinson (9). For phyfic acid, protein body pellets were resuspended in1 ml 0.5 N HCl and incubated with shaking1 h at 600C. Samples were centrifuged 30mi at 12,lOOg and supernatants were diluted to 10 ml. Phytic acid was determined as described by Latta and Eskin (13). For amino acid analysis, phytic acid was solubilized from protein body pellets as described above and protein was precipi- tated with 10% (w/v) TCA and washed with 80% (v/v) acetone. Aliquots were hydrolyzed in vacuo with 6 N HC1 containing 0.2% (v/v) phenol at110C for 20 h. The HCl was evaporated, and amino acids were dissolved in 0.2M lithium citrate buffer, pH 2.2, and analyzed on a Durrum D-500 amino acid analyzer. Protein-body pellets resuspended in10 ml 50 mm sodium citrate buffer, pH 6.0, were assayed for protease activity against casein. Following incubation, protein was precipitated with 10%o (w/v) TCA and soluble amino-N was assayed with ninhydrin (15). FIG. 1. Aggregated aleurone cells after 4 h of incubation in 0.7M Phytase activity was assayed by the method of Adams and Nov- mannitol containing 1% (w/v) Cellulysin and 0.25% (w/v) Macerase. Bar ellie (1). = 25,tm. SDS-Polyacrylmde Gel Electrophoresis. Protein body pellets from gradient fractions were washed with 10%o (w/v) TCA and three times with 80o (v/v) acetone, and then were denatured by 20 boiling 3 min in 1% (w/v) SDS. These samples were electropho- resed by the procedure of Laemmli (12) except that the stacking gel was 3.5% (w/v) acrylamide with 2.5% cross-linking and the separating gel was 12% (w/v) acrylamide with 1.7% cross-linking. Gels were stained with Coomassie Bfilliant Blue R-250. RESULTS E CD U) Incubation ofgroat slices with Cellulysin and Macerase initially degradedthe thin walls of the starchy endosperm tissue, leaving C31%.. the thick walled aleurone and seed coat intact. Four h of incuba- tion were required before all contaminating starch was removed from the aleurone layers, as determined by observation of iodine- stained tissue through a light microscope. Further incubation for 0~ 3 h with doubled enzyme concentrationsyielded aleurone proto- to plasts and small aggregates of cells (Fig. I) free of resistant seed coat tissue. The Phillips and Paleg procedure (18) for separating aleurone layers frpm starchy endosperm of wheat seeds, as modi- fwed for use with sliced oat seeds, yielded ample quantities of starchy endosperm tissue free of aleurone and seed coat. Sucr densitygradient centrifugation of disrupted aleurone or starchy endosperm tissues resulted in a large peak of protein Fraction equilibrating at a density of 1.23 g/cm3 (Fig. 2). This peak from FIG. 2. Protein distribution in 12-ml linear sucrose density gradients each tissue contained protein- bodies, as confirmed by electron following centrifugation for 4 h at 230,000g. of extracts from aleurone microscopy (see below). Smaller amounts of protein in the upper or starchy endosperm. Gradients contained 10 mm Tris-HCI, I mm EDTA, portion of the gradient represent soluble protein or protein solu- and 0.1 mM MgC12 at pH 7.5. bilized from disrupted organelles. A higher ionic strength gradient buffer (4) that we used initially solubilized most of the protein, contain spherical protein bodies and amorphous granular material yielding much smaller protein body peaks. that appeared to be disrupted protein bodies. Aleurone protein The high density protein peaks from both tissues were shown to bodies (Fig. 3) were 0.14 to 1.33 um in diameter and contained OAT PROTEIN BODIES 521 Table I. Chemical Composition ofOat Aleurone and Starchy Endosperm Protein Bodies Starchy Component Aleurone Endosperm % dy wt 12.56 15.73 P 0.82 0.69 Phytic acid 2.56 0.00

FIG. 3. Electron micrograph of aleurone protein body fraction isolated from a sucrose density gradient. Bar - I nm. * n^ .A..._H _

FIG. 5. SD-polyacrylamide gel electrophoresis patterns ofoat protein solubility fractions ared to aleurone and starchy endosperm protein body fractions. A, Albumin (buffer-soluble fraction); G, globulin (I M NaCl-soluble fraction); AL, aleurone protein body fraction; E, starchy endosperm protein body fraction; P. prolamin (70% ethanol-soluble frac- tion); GT, glutelin (1% SDS-soluble fraction). Analysis of isolated protein body fractions revealed that those from the starchy endosperm were almost entirely protein (assum- ing the protein contains 16% N), whereas those from the aleurone were about 80% protein (Table I). Phytic acid was detected in the from FIG. 4. Electron microgrph of frac- aleurone protein bodies, but was absent in those starchy starchy endosperm protein body endosperm. tion isolated from a sucrose density gradient. Bar = jnm. The amino acid compositions of the two protein body fractions electron-dense centers with light staining peripheral regions. Pro- were similar. Differences did not exceed 0.2 mol% for each amino tein bodies extracted from starchy endosperm were ofsimilar size acid residue except for greater leucine and isoleucine, and less and appearance (Fig. 4). The electron micrographs indicated that glutamine/glutamate, for the aleurone samples. The amino acid contaminating organelles were absent, and this was confirmed by compositions were quite similar to that of oat globulin (6). assaying gradient firtions for the marker enzymes Cyt c reductase Protein extracted from aleurone and from starchy endosperm (ER), Cyt oxidase (mitochondria), catalase (microbodies), and protein body fractions contained polypeptides which comigrated fumarase (mitochondria). Al these enzyme activities were found with globulin and prolamin polyeptides when electrophoresed in fractions with lower densities than the protein body peak (data on SDS-polyacrylamide gels (Fig. 5). The major polypeptide not shown). groups of the starchy endosperm preparation corresponded to the 522 DONHOWE AND PETERSON Plant Physiol. Vol. 71, 1983 protein bodies was highly modified. By reducing the Tris concen- tration to 10 mm and omitting the 20 mm KCI, the majority of protein was obtained in the protein body peak. The pH of the

- L- V gradient buffer had little effect, except that the peak equilibrated c * STARCHY ENDOSPERM at a slightly lower density at pH 4.5 to 5.5 than at pH 7.5 (data not shown). 0 I.- 0.6 , Electron micrographs of the isolated aleurone protein bodies show them to be free of other organelles (Fig. 3). The smaller average size in comparison to those of intact tissues (3) and the presence of granular amorphous material reveal that the larger 4 protein bodies were disrupted or were contracted during isolation or fixation. Globoid inclusions or their empty cavities, character- ) 0.4 istic offixed, sectioned intact aleurone protein bodies, were absent. o0 We suggest that this modification occurred upon fixation, because E/ phytic acid, the primary component ofthe globoid inclusions (16), was found in these gradient fractions by chemical analysis (Table I I). Lott (14) described the difficulties of preserving globoids with E 10.4 commonly used fixatives, and Wada and Maeda (23) found that globoid material was extracted with aqueous buffers during fixa- tion and dehydration. The lightly stained peripheral regions ofthe 0.2 protein bodies suggest some solubilization of protein may have occurred. The starchy endosperm protein body fraction was also free of other organelles, although some membranous material was present (Fig. 4). These protein bodies were also smaller than the average Time (H) size of those of intact tissue, and they contained lightly stained FIG. 6. Time course of casein hydrolysis by protein body fractions at peripheral regions. Again, the implication is that the larger protein pH 5.4. (0) Aleurone protein body fraction; (@) starchy endosperm protein bodies were disrupted and some solubilization ofprotein occurred. body fraction. Protein bodies extracted from whole grain have been reported a- and P-globulin polypeptides as described by Brinegar and to contain 70 to 80%o protein on a dry weight basis (16, 24). These Peterson (6). The aleurone preparation also contained these poly- percentages represent an average for combined aleurone and peptides, but the larger a polypeptide group was reduced in starchy endosperm protein bodies. Our data show that starchy staining intensity, and there were additional protein bands of endosperm protein bodies ofoats are higher in protein concentra- slightly greater mobility. The major prolamin polypeptides, inter- tion than aleurone protein bodies (Table I). mediate in mobility between the globulin polypeptides, were Total P concentration of oat protein body fractions was typical present in both protein body preparations. The starchy endosperm of reports from other cereal species (24) which range from 0.3 to protein bodies contained bands of low mobility not present in 2% by dry weight. Aleurone protein bodies were higher in P than aleurone protein bodies. The glutelin fraction contains previously those from starchy endosperm, reflecting the presence of phytic unextracted globulin, and thus shows band similarity. acid in the aleurone. Phytic acid was localized in the aleurone Protein body fractions from aleurone had protease activity over protein bodies, as has been reported for other cereals (22). a broad pH range with an optimum between pH 5.2 and 5.7. Evidence from amino acid analysis and SDS-polyacrylamide Activity at pH 5.4 was linear with time for at least 4 h, and 0.171 gel electrophoresis (Fig. 5) indicates the similarity between aleu- pmol of amino acids was released per h/mg protein (Fig. 6). rone and starchy endosperm protein bodies in protein composi- Starchy endosperm protein body fractions had no statistically tion. The major (globulin) and minor (prolamin) storage protein significant protease activity. There was no phytase activity (release bands were found in both preparations. Some differences in of P from sodium phytate) in either protein body extract. banding pattern and quantities of certain amino acids were ob- served, but most protein bands were similar. Thus, we conclude that there is a similarity between aleurone and starchy endosperm DISCUSSION protein bodies in their content of globulin and prolamin protein. Eastwood (8) isolated oat aleurone layers by incubation of We are aware of no comparable study in oats or other cereals, endosperm slices in a 2% solution of Meicelase P for 4 h. Further although Pernollet (16) suggests (without supporting evidence) incubation ofaleurone layers for 20 h in Meicelase P and pectinase that protein composition of these two types of protein bodies is yielded aleurone spheroplasts (cells). By using a combination of different. The only study of a similar nature is in rice, in which Cellulysin and Macerase, we obtqined isolated aleurone layers in some differences in electrophoretic behavior ofpolypeptides were 4 h, but aleurone protoplasts were obtained in 1 additional h, a found between two types of protein bodies, both found in starchy considerable improvement in time. Although our technique pro- endosperm (21). It is conceivable that within the oat aleurone, or vided clean preparations, yield was low because the aleurone cells within the oat starchy endosperm, there may also be a mixed were already coming apart before the last starchy endosperm cells population ofprotein bodies which differ in their relative concen- had been dismpted. After our work was completed, a report trations ofglobulin and prolamin storage protein. However, Saigo appeared (11) in which conditions of imbibition, enzyme concen- et al. (19) found no evidence for separate origins of two popula- tration, and osmoticum were optimized for isolation of aleurone tions of protein bodies in the starchy endosperm, and morpholog- protoplasts from Avenafatua in 91 to 94% yield. Further experi- ical appearance was uniform. Likewise, aleurone protein bodies mentation with various combinations of enzymes and conditions appeared morphologically similar (D. M. Peterson, R. H. Saigo, might better optimize yield of aleurone cells from A. sativa. and J. Holy, unpublished data). Saigo et al. (19) suggested that A low ionic strength gradient buffer was important to maintain the electron-lucent regions of the starchy endosperm protein bod- protein body integrity. The buffer of Bollini and Chrispeels (4) ies may contain prolamin, and the darker matrix the globulin, but solubilized the majority of the protein, and ultrastructure of the there is no direct evidence for this. OAT PROTEIN BODIES 523 Protease activity associated with protein bodies from ungermi- 9. FOGG DN, NT WILKINSON 1958 The colorimetric determination of phosphorus. Analyst 83: 406-414 nated seeds has been found in other cereals (2). The localization 10. FULCHER RG, TP O'BRIEN, DH SIMMONDS 1972 Localization of arginine-rich of protease activity in oat aleurone protein bodies may be typical. proteins in mature seeds of some members of the Gramineae. Aust J Biol Sci Adams and Noveilie (1) reported higher protease activity in 25: 187-197 sorghum aleurone protein bodies than in those from whole grain. 11. HooLEY R 1982 Protoplasts isolated from aleurone layers of wild oat (Avena fatua L.) exhibit the classic response to gibberellic acid. Planta 154: 29-40 Phytase activity was high in barley and maize, low in sorghum 12. LAEMMLI UK 1970 Cleavage of structural proteins during the assembly of the and babala, and absent in wheat and millet (2). We could not head of bacteriophage T4. Nature (Lond) 227: 680-685 detect phytase activity in oats. 13. LATrA M, M ESKIN 1980 A simple and rapid colorimetric method for phytate determination. J Agric Food Chem 28: 1313-1315 14. LoTT JNA 1980 Protein bodies. In NE Tolbert, ed, The Biochemistry of Plants, LITERATURE CITED Vol 1. Academic Press, New York, pp 589-623 15. MooRE S 1968 Amino acid analysis: aqueous dimethyl sulfoxide as solvent for 1. 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