Isolation and Characterization of Oat Aleurone and Starchy

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 precipitated 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 incubation 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.

Load more