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Fine Structure of Endosperm Protein Bodies


Protoplasma 73, 475--479 (1971) 9 by Springer-Verlag 1971

F i n e Structure of E n d o s p e r m Protein B o d i e s in Setaria lutescens ( G r a m i n e a e ) 1
THOMAS L. ROST Department of Biology, Brookhaven National Laboratory, Upton, Long Island, New York, U.S.A. With 4 Figures Received May 24, 1971 Summary Endosperm protein bodies are membrane bound. Internally, each body shows a pattern of dark and light concentric layers. A median dense core may also be present. These bodies stain for protein with mercuric bromphenol blue, but not for acid phosphatase.

1. Introduction
Protein bodies are storage organelles commonly found in grass caryopses. MITSUDA et aI. (1969) isolated protein bodies from rice endosperm which contained alternating light and dark layers. These layers appeared granular and were apparent only after staining with uranyl acetate and lead citrate. Layering within each body was concentrated near the pheriphery. KHOO and WOLF (1970) observed somewhat similarly layered protein bodies in corn kernels 16 days after fertilization. JENNINGS et al. (1963) and Oily and HENNINGSEN (1969) observed protein bodies from wheat and barley embryos which also showed peripheral layering; they speculated that such bodies might be related to phytin deposition. In this paper, endosperm protein bodies are described from yellow foxtail grass (Setaria lutescens); their fine structure, histochemistry, and possible function are discussed.

2. Materials and Methods
Caryopses were quartered and fixed at 4~ C for 2 hours in 0.1 M phosphate-buffered 4% glutaraldehyde at pH 7.3. Tissues were post-fixed 1 hour in 1% OsO4 at 4 C in the same buffer. A graded series of ethanol to propylene oxide was used for dehydration; embedding 1 Based on a portion of a dissertation submitted to the Graduate College of Iowa State University in partial fulfillment of the requirements for the Ph.D. degree.

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TH. L. Rosw

was in an Araldite/Epon mixture (Araldite 502-10ml, Epon 812-13ml, DDSA 30mI, DMP-30-1.5 ml). Sections were cut on an LKB iII ultramicrotome with a DuPont diamond knife, stained with lead citrate, and observed on a Hitachi HU-11 C electron microscope. Paraffin (61~ C Tissuemat) embedded tissues were similarly fixed in glutaraldehyde and prepared as described in ROST and LERSTEN(1971). The Go~ol~I (1952) method was used on sections reacted for acid phosphatase. Mercuric bromphenol blue coupled with a pepsin extraction control was used for protein histochemistry(MAZlAet al., 1953).
3. O b s e r v a t i o n s and D i s c u s s i o n

Protein bodies in the endosperm are removed by pepsin digestion; they also stain deeply with mercuric bromphenol blue (Fig. 1). They are concentrated in peripheral cells of the endosperm, becoming more scattered and sparce toward the inside. The aleurone layer, also of endosperm origin, does not contain these bodies. Each endosperm protein body is membrane bound, roughly spherical and 1-2/zm in diameter (Figs. 3 and 4). Some bodies are lobed and others appear to aggregate into small clusters (Fig. 3). A subtle internal structure of alternating light and dark concentric layers can be seen in sections stained with lead citrate (Figs. 3 and 4). In some bodies a dense central core was observed. Sections treated for acid phosphatase did not show positive activity in, or around, the endosperm protein bodies, suggesting that these bodies lack storage phosphate (Fig. 2). Acid phosphatase activity has been demonstrated to be associated with protein bodies in the embryos of this species (RosT 1971); it has also been reported in protein bodies of several other plant seeds (MATII.E 1968, POUX 1965, YATSt3 and JAcKs 1968). The internally layered structure of Setaria endosperm protein bodies is similar to those of 16 day old corn kernels as reported by KHoo and WoLf (1970). They differ from the peripherally layered bodies in rice endosperm (M1TstmA et al. 1969) only in the position of the layered pattern. This type of protein body has been observed only in the endosperm of three grasses; rice, corn, and S. lutescens. The significance of endosperm protein body structure relative to its nutritive function must, at this point, remain speculative. The internal concentric layering may indicate differential deposition of protein during body formation. It may also indicate the presence of two or more different types of protein, or possibly some nonprotein material other than phytin. Fig. 1. Paraffin section through endosperm stained with mercuric bromphenol blue. The highest concentration of protein bodies per cell is toward the outside. White grains within the endosperm cells are starch granules. Line scale 100 ibm Fig. 2. Paraffin section reacted for acid phosphatase and counterstained with fast green. The endosperm region shows no dark positive reaction sites. Embryo protein bodies are highly reactive. Line scale 100 ~m Fig. 3. Electron micrograph of portion of three peripheral endosperm cells. Protein bodies are 1-2,urn in diameter with an alternating light and dark internal layer pattern. Some bodies appear lobed, while some may aggregate. Line scale 1 um

Fine Structure of Endosperm in Setaria lutescens

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Figs. 1-3

478

TH. L. RosT

Fig. 4. Each endosperm protein body is membrane bound (arrow). Their granular appearance and layered structure can be seen. Line scale 0.25 ~ m

Fine Structure of Endosperm in Setaria lutescens

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References
GO3aORI, G., 1952: Microscopic histochemistry: Principles and practices. Chicago, Ill.: Univ. of Chicago Press. JENNmGS, A. C., R. K. MORTON, and B. A. PAZK, 1963: Cytological studies of protein bodies of developing wheat endosperm. Aust. J. Biol. Sci. 16 366--374. KHoo, U., and M. J. WOLF, 1970: Origin and development of protein granules in maize endosperm. Amer. J. Bot. 57, 1042--1050. MATIL~, P., 1968: Aleurone vacuoles as lysosomes. Pflanzenphysiologie 58, 365--368. MAzIA, D., P. A. BI~zw~R, and M. ALFEI/T, 1953: The cytochemical staining and measurement of proteins with mercuric bromphenol blue. Biol. Bull. 104, 57--67. MITSODA, H . , K. MURAKAMI, T. KUSANO, and K. YASUMOTO, 1969: Fine structure of protein bodies isolated from rice endosperm. Arch. Biochem. Biophys. 130, 678--680. ORY, R. L., and K. W. I-I~NNINGSEN, 1969: Enzymes associated with protein bodies from ungerminated barley seeds. Plant Physiol. 44, 1488--1498. Poux, N., 1965: Localisation de l'activit6 phosphatasique acide et des phosphates dans les grains d'aleurone. I. Grains d'aleurone renfermant a la lois glohoides et cristalloides. J. Microscopie 4, 771--782. Rosx, T. L., 1971: Structural and histochemical investigations of dormant and non-dormant caryopses of Setaria lutescens (Gramineae). Unpub. P h . D . Thesis. Library, Iowa State Univ., Ames, Iowa. - - a n d N. R. LERSTEN, 1970: Transfer aleurone cell,s in Setaria lutescens (Gramineae). Protoplasma 71, 403--408. YATSU, L. Y., and T. J. JAcks, 1968: Association of lysosomal activity with aleurone grains in plant cells. Arch. Biochem. Biophys. 124, 466--471. Author's address: Dr. THOMAS L. ROST, Department of Biology, Brookhaven National Laboratory, Upton, Long Island, NY 11973, U.S.A.

Herausgeber, Eigentiimer und Verleger: Springer-Verlag, M61kerbastei 5, A-1011 Wien. - - Ffir den Textteil verantwortlieh: Dr. Wilhelm Schwabl, M61kerbastei 5, A-1011 Wien. - - Ffir den Anzeigenteil verantwortlich= Bruno Schweder, Schweizertalstrage 8-10, A-1130 Wien Druck: Adolf Holzhausens Nfg., Kandlgasse 19-21, A-1070 Wien Printed in Austria


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