Composition and Structure Protein Bodies Spherosomes ... · Protein bodies fromwholegrain are...

Plant Physiol. (1975) 55, 1-6

Composition and Structure of Protein Bodies and SpherosomesIsolated from Ungerminated Seeds ofSorghum bicolor (Linn.) Moench

Received for publication March 22, 1974 and in revised form July 10, 1974

CLIFFORD A. ADAMS AND LAWRENCE NOVELLIENational Chemical Research Laboratory, Councilfor Scientific and Industrial Research, Pretoria, South Africa

ABSTRACT

Protein bodies and spherosomes have been isolated frommature seeds of Sorghum bicolor (Linn.) Moench by a pro-cedure which successfully disrupts the protein starch complexin the grain. Protein bodies from whole grain are 68% proteinand have a distinct border and a monolithic appearance. Thosefrom embryo are 95% protein and have diffuse borders,vacuoles, and appear very granular. Aleurone tissue proteinbodies are 46% protein with a structure similar to those fromembryo, but possibly are composed of a protein carbohydratemixture. Spherosomes from all sources are quite similar incomposition and structure. They have an average compositionof 27% protein, 12% phosphorus, and 8.6% metals. Micro-scopically, they appear as small vesicles bounded by- a wallwhich is probably composed of protein and the potassium.magnesium salt of phytic acid.

Subcellular particles have been isolated from a variety ofungerminated seeds as reviewed by Ory (19). In cereals, mostwork has been done on the particles observed in the aleuronecells, although protein bodies in the endosperm have elicitedsome attention.Wheat aleurone cells are filled largely with two types of

particles (3, 21). One is the so-called aleurone grain whichconsists of a membrane bounded particle containing electrondense and electron transparent inclusions. Surrounding thesealeurone grains, are smaller ovoid bodies, called unidentifiedbodies by Buttrose (3) and spherosomes by Paleg and Hyde(21). The aleurone cells of barley have a similar microscopicstructure of aleurone grain surrounded by spherosomes (11,16).Using cytochemical methods, Jacobsen et al. (11) concluded

that the inclusions in barley aleurone grain consisted either ofphytin or of a protein-carbohydrate complex. However, theydid not isolate any of the particles to identify, unequivocally,these substances. A somewhat different particle was isolatedfrom aleurone cells of ungerminated barley by Ory andHenningsen (20). This was described as a protein body and, un-

like aleurone grains, it did not contain inclusions but con-sisted of protein and possibly carbohydrate.

Cytochemical observation of wheat aleurone cell contentssuggest they are not proteinaceous (24) although the aleuronelayer has traditionally been thought of as protein rich (10).Furthermore, subcellular particles isolated from rice aleurone

tissue were only 12% protein (25). These particles were com-posed primarily of phytic acid and metals.

Protein-rich particles known as protein bodies were studiedin developing wheat endosperm (2, 12), in maize (17), and insorghum (23). Wheat protein bodies comprising 72% protein(9) and rice protein bodies comprising 60% protein (18) havebeen prepared. These particles apparently constitute the re-serve storage protein in the endosperm. Isolated maize pro-tein bodies consist of zein, the major reserve protein of maize(4).Although there have been several microscopic and cyto-

chemical studies of various subcellular particles in cerealgrain, their chemical composition and structure are not un-equivocally resolved. We present data on the composition andstructure of two subcellular particles isolated from sorghum.One is a protein body and the other a spherosome. Theseparticles also have enzymatic activity which is described in asubsequent paper (1).

MATERIALS AND METHODS

Grain Preparation. Barnard's Red sorghum (Sorghumbicolor [Linn.] Moench) was used throughout the study. Whole,air-dried, viable grain was milled through a 1-mm sieve on anEBC mill (Casella Company, London) and then defatted inacetone at room temperature with a grain-acetone ratio of 1: 2.The defatted powder was air-dried and kept refrigerated untilused.

Aleurone tissue was prepared in a manner similar to thatdescribed by Tanaka (25). Whole grain was abraded in a ricepearling machine until 2.5% of the grain weight was removed.This weight represents outer husk material and was discarded.The next 7% of grain weight contained most of the aleuronetissue and was collected. This tissue was defatted in acetonebefore use. Pearling was continued, removing a further 4%of the grain weight and the final pearled grains, with very littlepigmented material left, were used as a source of embryos.Embryos were prepared from coarsely milled pearled grain

by flotation on carbon tetrachloride-benzene mixtures accord-ing to Johnston and Stern (15). The embryos were free fromouter pigmented husk materials and of endosperm. Wholeembryos were finely ground and de-fatted in acetone beforeuse.

Isolation of Particles. The same procedure was followed withall sources of plant material. Acetone powder was suspendedin 1% (w/v) sodium metabisulphite solution with a powderto solution ratio of 1:5, and stirred for 30 min at room tem-perature. The slurry was passed twice through a Fryma (Leu-mann and Uhlmann, Western Germany) mill and thenthrough a nylon cloth. The filtrate from the cloth was pumped

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ADAMS AND NOVELLIE

through a Sharples super centrifuge operating at low speed,with a flow rate of 900 ml/min. Starch granules were retainedin the barrel of the centrifuge and were discarded. The super-natant liquid was pumped once again through the centrifugeoperating at high speed with a flow rate of 300 ml/min.Particles designated as protein bodies were deposited in thebarrel of the centrifuge. These were removed, washed oncewith water, and either dehydrated in acetone for chemicalanalysis or resuspended in 0.1 M citrate buffer, pH 6.2, forelectron microscopy.The final supernatant from the Sharples super centrifuge

contain the particles designated as spherosomes. Thesespherosomes were coagulated by adding 1 volume of acetoneto the supernatant solution. Spherosomes settle under gravityand were washed once with 50% (w/v) acetone, and theneither dehydrated in acetone for chemical analysis or stored in50% (w/v) acetone for electron microscopy.

Chemical Analysis of Particles. Total nitrogen and phos-phorus was determined according to the procedure of Thomaset al. (26). Metals were estimated by atomic absorptionspectroscopy on the same extracts as used for nitrogen andphosphorus analysis. Soluble sugars and polysaccharide weredetermined as glucose equivalents according to Clegg (5). Amodification of the method of Jerumanis (13) was used toestimate polyphenol content. The air-dried particles were ex-tracted in 75% (v/v) aqueous dimethylformamide. A 0.1-mlaliquot of the centrifuged extract was diluted with 75% (v/v)aqueous dimethylformamide to about 4.5 ml; 0.1 ml of 3.5%(w/v) ferric ammonium citrate was added, followed by 0.1 mlof ammonium hydroxide (S.G. 0.91)-water (0.5 v/v). The mix-ture was adjusted to a final volume of 5 ml and after 10 min,the absorbance was determined at 525 nm. Polyphenol contentis expressed as tannic acid equivalents.

Spherosomes were further characterized by extraction withhot 5% (w/v) trichloroacetic acid three times. The residuewas dried and analyzed. The trichloroacetic acid supernatantwas neutralized with NaOH and a heavy white precipitateappeared. This was sedimented by centrifugation at 2300g for10 min, dissolved in fresh 5% (w/v) trichloroacetic acid,precipitated by neutralization, and the process was repeatedonce more. The final product, a white powder insoluble inwater, was dried and analyzed. Carbon and hydrogen weredetermined in an elemental analyzer and ash content wasdetermined by pyrolysis in a Pregl micro-muffle.

Preparation of Samples for Electron Microscopy. Proteinbodies in 0.1 M citrate buffer, pH 6.2, were centrifuged at17,000g for 10 min, and the supernatant was discarded andreplaced with 5% glutaraldehyde in 0.05 M phosphate buffer,pH 7.2. The pellet was resuspended in glutaraldehyde and leftfor 1 hr in ice. Spherosomes were sedimented by centrifugationat 2,300g for 3 min, and the 50% (v/v) acetone supernatantwas discarded and replaced with glutaraldehyde. The pelletwas resuspended in glutaraldehyde and left for 1 hr in ice. Bothparticles were then centrifuged at 2,300g for 10 min and theglutaraldehyde was removed. The pellets were then resus-pended in a small amount of agar at 50 C. When the agarsolidified, the block was chopped into small pieces and storedin 0.05 M phosphate buffer, pH 7.0. The agar blocks, afterdehydration in ethanol and propylene oxide, were embeddedin epon-araldite mixture. Samples were sectioned with adiamond knife and double stained with uranyl acetate and leadcitrate.

RESULTSTwo distinctly different subcellular particles. protein bodies

and spherosomes. were isolated from whole grain, separated

Table I. Compositioni of Particles Isolated from Whole Graiji,Embryo, and Aleuronie Tissue

Components are expressed as a percentage of oven dry weight ofacetone-dehydrated particles.

ComponentsParticle Source Particle Type

Nitrogen Phos- 'Soluble Polysac- Poly-phorus sugars charide phenol

Whole grain Protein body 10.95 0.37 0.17 17.10 0.46Embryo Protein body 15.20 1.85 0.10 4.44 NDAleurone Protein body 7.36 0.59 15.90 13.25Whole grain Spherosome 4.98 11.54 1.01 2.17 0.03Embryo Spherosome 4.50 12.26 0.34 1.62 0.01Aleurone !Spherosome 3.30 12.05 2.17 0.20

Table II. Metals Associated wvith Particles Isolated fromi WholeGraili, Emnbryo, antd Aleu-ronie Tissue

Metal content is expressed as a percentage of oven dry weightof acetone-dehydrated particles.

Metal

Particle Source Particle TypeCalcium gs-ium Potassium! Iron

Whole grain Protein body 0.02 0.01 0.02 0.09Embryo Protein body 0.01 0.27 0.04 0.12Aleurone Protein body 0.01 0.21 0.06 0.10Whole grain Spherosome 0.21 6.12 1.28 0.15Embryo Spherosome 0.05 7.15 1.62 0.15Aleurone Spherosome 0.27 6.68 2.03 0.10

aleurone tissue, and separated embryos. The major componentsof these particles are shown in Table I. Using the conventionalconversion factor of 6.25, the protein contents of proteinbodies varies from 46% in the aleurone layer to 95% in theembryo with whole grain preparations intermediate. Sphero-somes were considerably lower in protein, but varying muchless with location, the range being 20.6% to 31.2%. The phos-phorus distribution varies inversely to the protein content.Spherosomes were all extremely high in phosphorus andprotein bodies quite low.

Soluble sugars in whole grain and embryo particles werelow and excessive pigmentation prevented colorimetric assayof soluble sugars in particles from the aleurone layer. Poly-saccharide was a significant component of the protein bodiesfrom whole grain and aleurone layer, but much lower in thosefrom the embryo. All spherosomes were very low in polysac-charide content. Protein bodies from aleurone and whole grainsources were always more pigmented than those from embryotissue and this is reflected in the polyphenol content (Table I).Protein bodies have very low metal contents (Table II),whereas in spherosomes, Mg is a major component, beingalways in excess of 6% followed by K which was always greaterthan 1.25%. There was also more Ca in the spherosomes butthe Fe content was fairly evenly distributed throughout bothparticles from all sources.A more detailed analysis of spherosomes is shown in Table

III. The material soluble in trichloroacetic acid has a C, H, andP content close to that reported by Johnson and Tate (14) fortrihydrated sodium phytate (C, 7.6%; H, 1.8%; P, 19.1%).Furthermore, this compound moves similarly to authenticsodium phytate under high voltage electrophoresis under theconditions described by Johnson and Tate (14). This acid-

2 Plant Physiol. Vol. 55, 1975



SORGHUM PROTEIN BODIES AND SPHEROSOMES

soluble component of spherosomes is most likely a salt ofphytic acid. The acid-insoluble component is high in C and N,and low in ash, which is consistent with a protein. This frac-tion is approximately 83% protein, whereas the original particlewas only about 34% protein.

Although both particles were obtained from whole grain,separated aleurone, and embryo tissue, the relative yields ofthese particles were not similar. Protein bodies were the majorproduct obtained from whole grain (Table IV), whilespherosomes predominated in aleurone and embryo prepara-tions. The embryo was a particularly rich source of particles.Under the electron microscope, the protein bodies from

whole grain, show an obviously granule-like structure (Figs. 1and 2). The particles have well defined edges possibly sur-rounded by a membrane. They range in diameter from about0.5 to 3.5 ,um. Many of these particles have areas of darkermaterial arranged in concentric rings within the body of thegranule. In Figure 2 these darker areas are about 0.04 Mmin diameter. Embryo protein bodies are considerably different(Fig. 3). They do not have such a well defined border andare more irregular than those seen in Figure 1. They have asimilar size range to those in whole grain, but often containelectron transparent vacuoles, which are never seen in otherprotein bodies. The internal structure is also different; in theseparticles it is much less of a monolithic mass and has morethe appearance of a collection of very small granules. Proteinbodies from aleurone cells are somewhat similar to those fromembryo (Fig. 4). These particles also have a diffuse border asseen in the particles from embryo. They are generally smallerthan the other protein bodies.

Spherosomes from all three sources were very similar underthe electron microscope. (Figs. 5-8). They appear as smallvesicles with a distinct electron dense border and electron trans-parent interior. On higher magnification, these particles do notshow a conventional membrane structure, but have a ratherdiffuse border which varies in thickness. (Figs. 5 and 7). The

Table III. Chemical Composition of Spherosomes from WholeGraini and of Trichloroacetic Acid-soluble and -insoluble

Components of ParticlesData are expressed as percentage of oven dry weight of acetone-

dehydrated particles.

Sample

Chemical ComponentWhole spherosome Acid-soluble Acid-insolubleWholespbersome material material

Carbon 26.10 8.58 49.70Hydrogen 3.95 2.07 6.38Nitrogen 5.42 0.30 14.05Phosphorus 10.71 18.59 0.26Ash 40.90 69.30 0.87

Table IV. Yield of Particles from Whole Grain, Embryo, andAleurone Tissue

Yield of Acetone-dehydrated

Particle Source Weight of Starting ParticlesParticlSourceMaterialProtein body Spherosome

g %

diameter of these individual particles is about 0.1 pm, but theyoccur in clumps and chains with a length much greater thanthis.

DISCUSSION

Analytical and electron microscopic data confirm that pro-tein bodies and spherosomes are distinctly different sub-cellular particles. The polysaccharide component of proteinbodies from whole grain is possibly contaminating starchgranules. A few small starch grains were observed under thelight microscopes in samples of these protein bodies. However,the polysaccharide content of aleurone cell protein bodies isnot due to contaminating starch granules, since samples ofthese particles under the light microscope showed very fewstarch granules. Protein bodies from barley aleurone tissue maycontain about 30% carbohydrate (20). Aleurone grains ofbarley have been shown, by cytochemical procedures, to con-

tain a protein-carbohydrate body (11) and isolated aleuronecell contents from rice, also contained about 8% carbo-hydrate (25). In each case, the materials studied were essen-

tially starch-free. It is possible that the protein bodies isolatedfrom sorghum aleurone tissue are composed of a protein-carohydrate mixture. The nature of this carbohydrate is atpresent unknown.The greater concentration of the protein bodies in whole

grain preparations suggests these are located primarily in theendosperm which is known to be the major site of proteinbodies in sorghum (23). Under the electron microscope, proteinbodies isolated from whole grains have an appearance verysimilar in size and structure to those described in sections ofsorghum endosperm by Seckinger and Wolf (23), and do notappear to be altered during isolation. These authors also ob-served concentric rings of darker staining material as shownin Figure 2. Protein bodies from mature grains have not beenisolated in large quantities previously, because as the graindevelops, the protein bodies and starch granules pack togetherand become tightly bound and difficult to separate (4). Theprocedure reported here is able to disperse the starch proteinmixture and yields protein granules essentially free of starchwith similar structure to those observed in situ (23).The granular and vacuolated appearance of the protein

bodies isolated from emryo has also been observed in sectionsof sorghum embryo (7). The vacuoles were described as phytingloboids, but no datum was presented to substantiate this.Embryo protein bodies do contain more P than those fromwhole grain or aleurone (Table I) and this is probably phyticacid. Protein bodies from sorghum aleurone layer have notbeen isolated and studied in detail previously, so little com-

parative data are available. Our data suggest, however, thatthese are a complex mixture of protein, carbohydrate, andpolyphenolic materials.

Spherosomes from all sources have a much more uniformcomposition (Tables I and II). The large phytic acid com-

ponent of these particles is probably in the form of a potas-sium magnesium salt. Similar conclusions have been drawn byTanaka et al. (25) and by Pomeranz (22), who studied rice and

barley aleurone tissue, respectively.It is possible that in aleurone cells the spherosomes sur-

round the protein bodies. This juxtaposition of two particleshas been reported in aleurone cells by several workers (3, 11,16, 20). The smaller particles covering the surface of thealeurone grain have been designated spherosomes (11, 16, 21)and, consequently, particles described here are probablyspherosomes. The aleurone cell particles observed by Stevens

(24) and by Tanaka (25) are, possibly, combinations of proteinbodies coated by densely packed spherosomes. This would

Whole grain 2000 1.9 0.8Embryo 82.5 5.5 14.6Aleurone 300 1.2 2.7

3Plant Physiol. Vol. 55, 1975



ADAMS AND NOVELLIE

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FIGS. 1-4. Protein bodies of sorghum. 1: Protein bodies from whole sorghum. These particles have discrete edges possibly surrounded by amembrane (X 45,000). 2: Higher power image of protein body of whole grain showing areas of darker material arranged in concentric rings(X 84,000). 3: Protein bodies from sorghum embryo. These particles are more irregular than those in whole grain preparations and have a granu-lar mass (X 27,500). 4: Protein bodies from sorghum aleurone tissue showing a diffuse border and granular internal structure (X 132,000).

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Plant Physiol. Vol. 55, 1975 SORGHUM PROTEIN BODIES AND SPHEROSOMES

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ADAMS AND NOVELLIE

account for the high phytic acid content of aleurone cellcontents isolated from rice (25) and for the solubility character-istics of the aleurone cell contents of wheat (24). Thespherosomes prepared from sorghum contain small but sig-nificant amounts of soluble sugars (Table I), soluble protein,and soluble enzymes (1), despite being prepared in an aqueousmedium. This result also suggests that they have a vacuolarstructures as has been observed in spherosomes (11, 16, 21).Under the electron microscope, this vacuolar structure is

quite evident (Figs. 5 and 6). The vacuole wall is possibly aprotein phytic acid complex containing soluble materials inside.Spherosomes in aleurone cells of germinating barley stainedwith lead, appear as vesicles with a diffuse wide border verysimilar to that shown in Figures 5 and 7 (6). Spherosomes havebeen observed in situ by several workers (11, 16, 21) but havenot been previously isolated from ungerminated seeds. Sphero-somes have also been observed in germinated seedlings (8) andhave a somewhat similar appearance, although Frey-Wyssling(8) suggests that spherosomes from corn coleorhiza have a unitmembrane border which is not the case in sorghum grainspherosomes. At this stage, however, little is known of the rela-tionship between spherosomes in ungerminated seeds and thosein actively growing seedlings.

Acknowledgments-Appreciation is expressed to N. v.d. W. Liebenberg of theNational Food Research Institute for preparing the electron micrographs and toT. C. Robberts for skilled technical assistance.

LITERATURE CITED

1. ADAMS, C. A. AND L. NOVELLIE. 1975. Acid hydrolases and autolytic propertiesof protein bodies and spherosomes isolated from uingerminated seeds ofSorghum bicolor (Linn.) Moench. Plant Physiol. 55: 7-11.

2. BUTTROSE, M. S. 1963. Ultrastructure of the developing wheat endosperm.Aust. J. Biol. Sci. 16: 305-317.

3. BUrTTROSE, M. S. 1963. Ultrastructure of the developing aleurone cells of wheatgrain. Aust. J. Biol. Sci. 16: 768-774.

4. CHRISTIANSON, D. D., H. C. NIELSEN, U. KHOO, M. J. WOLF, AND J. S. WALL.1969. Isolation and chemical composition of protein bodies and matrix pro-teins in corn endosperm. Cer. Chem. 46: 372-381.

5. CLEGG, K. M. 1956. The application of the anthrone reagent to the estimationof starch in cereals. J. Sci. Food Agr. 7: 40-44.

6. EB, A. A. VAN DER AND P. J. NIEUwIDoRP. 1967. Electron microscopic structure

of the aleurone cells of barley during germination. Acta Bot. N6er. 15: 690-699.

7. FULCHER, R. G., T. P. O'BRIEN, AND D. H. SIMMONDS. 1972. Localization ofarginine-rich proteins in mature seeds of some members of the gramineae.Aust. J. Biol. Sci. 25: 487-497.

8. FREY-WYSSLING, A., E. GRIESHAaER, AND K. MtHLETHALER. 1963. Origin ofspherosomes in plant cells. J. Ultrastruct. Res. 8: 506-516.

9. GRAHAM, J. S. D., R. K. MORTON, AND J. K. RAIsoN. 1963. Isolation and char-acterization of protein bodies from developing wheat endosperm. Aust. J.Biol. Sci. 16: 375-383.

10. HiN-TON, J. J. C. 1953. The distribution of protein in the maize kernel in com-parison with that in wheat. Cer. Chem. 30: 441-445.

11. JACOBSEN, J. V., R. B. KNOX, AND N. A. PYLIOTIS. 1971. The structure andcomposition of aleurone grains in the barley aleurone layer. Planta 101: 189-209.

12. JENN-IN'GS, A. C., R. K. MORTON, AND B. A. PALE. 1963. Cytological studies ofprotein bodies of developing wheat endosperm. Aust. J. Biol. Sci. 16: 366-374.

13. JERUJMANIS, J. 1972. Uber die Veranderung der Polyphenole im Verlauf desMalzens und Maischens. Brauwissenschaft 25: 313-322.

14. JOHNsoN, L. F. AND M. E. TATE. 1969. Structure of "phytic acids". Can. J.Chem. 47: 63-73.

15. JOHNSTON, F. B. AND H. STERN. 1957. Mvlass isolation of viable wheat embryos.Nature 179: 160-161.

16. JONES, R. L. 1969. The fine structure of barley aleurone cells. Planta 85: 359-375.

17. Ksoo, U. AND M. J. WOLF. 1970. Origin and development of protein granules inmaize endosperm. Amer. J. Bot. 57: 1042-1050.

18. MITSUDA, H., K. YASUMOTO, K. MUROKAMI, T. KusANo, AND H. KISHIDA. 1967.Studies on the proteinaceous subcellular particles in rice endosperm: electronmicroscopy and isolation. Agr. Biol. Chem. 31: 293-300.

19. ORY, R. L. 1972. Enzyme activities associated with protein bodies of seeds. In:G. E. Inglett, ed., Symposium: Seed Proteins. The AVI Publishing Co.,Westport, Conn. pp. 86-98.

20 ORY, R. L. AND K. W. HENNINGSEN. 1969. Enzymes associated with proteinbodies isolated from ungerminated barley seeds. Plant Physiol. 44: 1488-1498.

21. PALEG, L. AND B. HYDE. 1964. Physiological effects of gibberellic acid. VII.Electron microscopy of barley aleurone cells. Plant Physiol. 39: 673-680.

22. POMERANZ, Y. 1973. Structure and mineral composition of cereal aleurone cellsas shown by scanning electron microscopy. Cer. Chem. 50: 504-511.

23. SECKINGER, H. L. AND M. J. WOLF. 1973. Sorghum protein ultrastructure as itrelates to composition. Cer. Chem. 50: 455465.

24. STEVENS, D. J. 1973. The aleurone layer of wheat. IV. Effects of extraction byaqueous media. J. Sci. Food Agr. 24: 847-854.

25. TANAKA, K., T. YOSHIDA, K. ASADA, AND Z. KASAI. 1973. Subcellular particlesisolated from aleurone layer of rice seeds. Arch. Biochem. Biophys. 155: 136-143.

26. THoMAs, R. L., R. W. SHEARD, AND J. R. MOaER. 1967. Comparison of con-ventional and automated procedures for nitrogen, phosphorus and potas-sium analysis of plant material using a single digestion. Agron. J. 59: 240-243.

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