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ACTA PHYSIOLOGIAE PLANTARUM Vol. 21. No. 4. 1999:383-390
Associations of maize protein bodies with cytoskeleton, membranes, and ribosomes in the endosperm of wild type and opaque-2 mutant Bratislav StankoviO a,*, Shunnosuke Abe b Kishu Azama b Koichi Shibatab Yoko Ro b Stanislaw Weidner c, Eric Davies a aBotany Department, North Carolina State University, Raleigh, NC 27695-7612, USA bLab of Cell and Molecular Biology, Ehime University, Matsuyama 790, Japan CDept Biochem, Fac Biol, Warmia and Masuria University in Olsztyn, Kortowo, P1. L6dzki, PL-10-718 Olsztyn, Poland *To whom correspondence should be addressed current address: Wisconsin Center for Space Automation & Robotics (WCSAR), College of Engineering, 1415 Engineering Dr., 2348 Engineering Hall, Madison, WI 53706, U.S.A., Fax: 608-262-4958; E-mail:
[email protected]
K e y words: cytoskeleton, endosperm, opaque-2, polyribosomes, protein bodies, Zea mays
Abstract Maize endosperm was homogenized in a cytoskeletonstabilizing buffer, filtered and layered on gradients of 20-80 % sucrose and analyzed by monitoring their UV absorbance. A major peak of UV-light absorbing material was detected on the gradient, at about 60-65 % sucrose (density of approximately . . . . . 1.3 g.ml- 1 ). Bxochemlcal, fluorescence microscopic, and lmmunoblot analyses of this peak showed that it consisted of protein bodies associated with actin, membranes, and RNA (ribosomes). Seeds of wild type and opaque-2mutant were then homogenized, the homogenate was modified using detergents and/or cytoskeleton-disrupting agents, and centrifuged on sucrose gradients. In wild type maize endosperm, detergent treatment caused the major peak (protein bodies) to increase in density so that they sediment further down the gradient. However, in opaque-2 the protein bodies formed a broader, but smaller peak which, upon treatment with detergent, generated protein bodies which pelleted to the bottom of the gradient. Analysis of gradient fractions by gel electrophoresis and immuno-blotting showed that both the wild type and the mutant had cytoskeleton proteins in the upper regions (soluble, non-polymerized micro-
filaments and microtnbules) as well as in the peak regions. Comparisons of both the UV-absorbance profiles and the immunoblot data suggest that the protein bodies from the two maize types associate differently with the membranes and the cytoskeleton.
List of abbreviations: CSB, cytoskeleton-stabilizing buffer; DiOC6, 3,3'-dihexyloxacarbocyanine iodide; DOC, deoxycholic acid, sodium salt; EFIc~, elongation factor 1~; EGTA, ethylene glycol-bis (13-aminoethyl ether) N,N,N',N'-tetraacetic acid; ER, endoplasmic reticulum; HEPES, (N- [2-Hydroxyethyl]piperazine-N=-[2-ethanesulfonic acid]); PMSF, phenylmethylsulfonyl fluoride; PTE, polyoxyethylene-10-tridecyl ether; RER, rough endoplasmic reticulum; SBP, streptavidin-binding protein; S D S - P A G E , sodium d o d e c y l sulfate-polyacrylamide gel electrophoresis
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B. STANKOVIC, S. ABE, K. AZAMA, K. SHIBATA, Y. ITO, S. WEIDNER & E. DAVIES
Introduction Maize endosperm is becoming a useful system to study the plant cytoskeleton and is likely to provide novel insights into cytoskeletal structure and function in plants. Various reports have suggested a possible role of the cytoskeleton in RNA localization and/or protein synthesis in this tissue (Abe et al. 1991, Davies et al. 1993, 1998, Stankovi6 et al. 1993, Clore et al. 1996). The cells of the endosperm accumulate large amounts of starch and ~torage proteins (zeins), which are cotranslationally inserted into the lumen of the rough endoplasmic reticulum (RER) where they coalesce into spherical protein bodies (Larkins and Hurkman 1978). These protein bodies were shown to be surrounded by, and enmeshed in, a network of actin microfilaments (Abe et al. 1991). Later, Davies et al. (1993) provided biochemical evidence for the existence of cytoskeleton-membrane-bound polysomes in this tissue, while Stankovi6 et al. (1993) provided fluorescence microscope evidence demonstrating associations between filamentous actin, polysomes and protein bodies following homogenization of developing endosperm in a cytoskeletal stabilizing buffer (CSB). However, treatment with non-ionic detergent, which removed most of the ER membranes, did not dislodge the polysomes from around the protein bodies nor their colocalization with actin (Stankovi6 et al. 1993). More recently, Clore et al. (I 996) used indirect immunofluorescence and confocal microscopy to show that protein bodies are enmeshed in complexes of EF-1 ~ and actin and are found juxtaposed with microtubules. These studies raised the prospect that the cytoskeleton may be involved in attaching zein polysomes to the ER that surrounds the protein bodies. Maize has several mutants which affect the protein content and physical properties of the endosperm, and one of these, the maize opaque-2 mutant, has an altered pattern of endosperm protein biosynthesis resulting in low yield and reduced protein content (Mertz et al. 1964). This involves mutation of a transcription factor that results in a soft, floury endosperm. A major cytological outcome of this mutation is that opaque-2 has smaller protein bodies than does the wild type when endosperm tissue is examined by electron microscopy (Geetha et al.
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1991). Even though the protein bodies are smaller in opaque-2 and the protein content is different, it is not known whether the associations between the cytoskeleton, the membranes, and the polysomes are different from those in the wild type. T h e main aim of this work was to use several different experimental approaches to investigate the nature of the complex cytoskeleton-membrane-ribosome associations in the maize endosperm. We employed biochemical fractionation, density gradient centrifugation, gel electrophoresis and immunoblotting, and fluorescence microscopy to provide further evidence for the existence of cytoskeletonmembrane-bound ribosomes in maize endosperm. We obtained data which suggest that the interactions of protein bodies with the cytoskeleton/membrane complex are different in the endosperm of wild type maize from those in the opaque-2 mutant.
Materials and Methods Plant material and isolation o f cytoskeleton and membrane fractions
Maize (Zea mays L.) W64A and opaque-2 seeds were kindly donated by Prof. Brian Larkins, University of Arizona, Tucson, AZ, USA, and by Prof. Becky Boston, North Carolina State University, Raleigh, NC, USA. These were harvested 21 days after pollination, frozen in liquid nitrogen, and stored at -80 °C until analysis. The seeds were homogenized in a mortar and pestle using 10 volumes of CSB (5 mM HEPES, 3.2 mM KOH, 10 mM Mg(OAc)2, 2 mM EGTA, 1 mM PMSF, pH 7.5) as described by Abe and Davies (1991) supplemented with 7 % (w/v) sucrose (Ito et al. 1994). In some instances, the homogenates were modified by addition of Tris to destabilize the microfilaments, by addition of the non-ionic detergent, polyoxyethylene 10-tridecyl ether (PTE), to dissolve the membranes, or both, as indicated in the text. Other homogenized samples were modified by addition of buffer U (200 mM Tris-HC1, pH 8.5, 50 mM KCI, 25 mM MgC12, 2 mM EGTA, 100 ~ml-1 heparin, 2 % PTE, 1 % DOC), to destabilize both the cytoskeleton and the membranes, thereby releasing attached polysomes. Extracts were filtered through Miracloth and aliquots layered on gradients con-
A S S O C I A T I O N S O F M A I Z E P R O T E I N B O D I E S ...
sisting of 20-80 % (w/v) sucrose in buffer C (CSB lacking EGTA and PMSF), centrifuged for various times at 250,000 x g and scanned at 254 nm using an Isco (Isco Inc., Lincoln, NE, USA) gradient fractionator (model 640) with a UA-5 absorbance monitor. Samples were collected from the gradients and used for fluorescence microscopy, gel electrophoresis and Western blotting.
Results
Extraction of maize endosperm in a buffer (CSB), designed to preserve cytoskelcton integrity, followed by gradient centrifugation, furnished a small peak of monosomes (M) towards the top of the gradient, a small shoulder corresponding to polysomes (P) near the middle, and a large peak (LP) towards the bottom, at about 60-65 % sucrose concentration, i.e., a density of approximately 1.3 g.m1-1 (Fig. 1).
Gel electrophoresis and immunoblotting Protein samples were subjected to SDS-PAGE and Western blotting as previously described (Abe and Davies 1991). Detection of three cytoskeletal proteins (actin, c~- and g-tubulin) was performed simultaneously with monoclonal antibodies (Amersham, UK) using the triple-blotting technique (Ito et al. 1994).
The components in the gradient were investigated
E tt"q
8e-
Fluorescence microscopy
O
.8 < 20%
20%
80%
b
LP
C
80%
Fig. 1. Absorbance profile of maize endospermextract. Wild type maize endosperm was homogenized, filtered through Miracloth, layered onto 20-80 % (w/v) sucrose gradients in buffer C, centrifngedfor 80 min at 250,000 x g, and tube contents monitoredat 254 nm using an Isco gradientfractionator (model 640) with a UA-5 monitor. M, monosomes;P, polysomes; LP, large peak. by employing "variable-term" sucrose density centrifugation, as previously done with pea epicotyls (Davies and Abe 1995), using samples isolated in CSB alone (Fig. 2). In the sample centrifuged for 15 min, the only material visible was the large peak (LP), which had already reached its isopicnic point
Double-staining was performed whenever possible. Rhodamine-phalloidin fluorescence was observed under a green excitation filter (546 nm), whereas DiOC6 and thiazole orange werc observed under a blue excitation filter (480 nm) and thus could not be examined in the same specimen.
LP
M
0~
-e
Staining of the homogenate with fluorescent dyes was performed as described in Stankovid et al. (I993). Rhodamine-phalloidin was used to visualize actin, 3,3'-dihexyloxacarbocyanine iodide (DiOC6) was used to visualize membranes, and thiazole orange was use to visualize RNA (ribosomes). All fluorescently-labelled probes were obtained from Molecular Probes (Eugene, OR, USA). Observation of the samples under the appropriate filters of a Nikon epifluorescence microscope and photographic documentation were done as described in Stankovid et al. (1993).
a
LP
A
Fig. 2. A b s o r b a n c e profiles of
LP
d
maize endospermextracts subject to variable-term density gradient centrifugation.Identicalaliquotsof wild type maize endosperm homogenates were layeredonto 2080 % (w/v) sucrose gradients in btffferC, sptm at 250,000 x g for 025 to 6 h and monitoredat 254 nm. a, 0.25 h; b, 0.75 h; c, 2 h; d, 6 h. The darkenedregionsonthegradients correspond to free polysomes, which sediment slowly throughthe gradient.
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B. STANKOVIC, S. ABE, K. AZAMA, K. SHIBATA, Y. 1TO, S. WEIDNER & E. DAVIES
of 1.3 g.ml -t (Fig. 2a). By 45 min (Fig. 2b) a small monosome peak (M) and a region of polysomes (P) had appeared (shaded regions) and these slowly migrated through the gradient until by 6 h (Fig. 2d) the monosome was a small shoulder on the large peak, and the polysomes began to pellet. Analysis of gradients centrifuged for extended periods of time suggests that these free polysomes pellet to the bottom of the gradient within about 50-70 h (data not shown). To investigate the character of materials in the large peak, fractions collected from the peak region in Fig. 1A were stained for actin, membranes, and RNA (ribosomes) as described in Stankovi6 et al. (1993) observed under the fluorescence microscope (Fig. 3). It should be pointed out that the fluorescent images were far less clear in these gradient samples (which contain high levels of sucrose) than those from total extract (Stankovi6 et al. 1993). The phase contrast image (Fig. 3a) shows two clusters of protein bodies in the center of the field, and a smaller cluster towards the right. All of these protein bodies were surrounded by actin (Fig. 3b). When similar samples were double-stained with different fluorochromes, co-localization of both RNA (Fig. 3c) with actin (Fig. 3d) and ER membranes (Fig. 3e) with actin (Fig. 3f) was observed. These data suggest that the large peak consists of protein bodies surrounded by membranes, cyto-
skeleton and polysomes (presumably zein synthesizing polysomes). To our knowledge, no gradient centrifugation comparisons have been made between wild type and the opaque-2 mutant, and so we examined the absorbance profiles of both types. We also looked at the effect of various chemicals on the behavior of the absorbance profile, both in wild type plants (W64A) and in the opaque-2 mutant (Fig. 4). When endosperm tissue was homogenized in CSB alone, wild type maize exhibited the typical monosome peak and the "large peak" (Fig. 4a). Addition of a non-ionic detergent (0.5 % PTE) to the homogenate resulted in a decrease in the amount of monosomes, polysomes, and the large peak and by a shift in the location of the peak towards denser regions in the gradient (Fig. 4b). When the homogenate was isolated in CSB and subsequently modified to contain 0.2 M Tris, there was an increase in monosomes and polysomes, and a decrease in the large peak (Fig. 4c). When both detergent (0.5 % PTE) and 0.2 M Tris were added to the homogenate prior to layering onto gradients, there was an increase in polysomes on the gradient, but a decrease in the material in the peak region, which appeared as 3 overlapping peaks (Fig. 4d). Finally, addition of buffer U (Abe and Davies 1995) to the homogenate caused an increase in monosomes and polysomes compared
Fig. 3. Fluorescencemicroscopyof componentsin the "peak" fraction. Wild type maize endosperm was ground in CSB, layered on gradients, centrifuged, and aliquots from the peak region (LP in Fig. 1) were stained and viewed trader the fluorescencemicroscope. Samplescorrespondto: a, phase contrast; b, identical samplestained for aetin; c, RNA; d, identical sample stainedfor actin; e, ER membranes; f, identical samplestained for actin. Bar = 10larn. Fluorescencemicroscopywas hampered becausethe material was collected from gradients and contains approximately63 % sucrose. 386
ASSOCIATIONS OF MAIZE PROTEIN BODIES ...
~-
,p~
e
20%
80%
Fig. 4. Absorbance profiles of wild-type and opaque-2 extracted in different buffers. Maize seeds were homogenized in CSB, filtered through Miracloth, modified as indicated, layered on a 20-80 % (w/v) sucrose gradient in buffer C, gradients centrifuged for 80 rain at 250,000 xg and monitored at 254 nm. Samples correspond to: a-e, wild type, homogenized in: a, CSB only; b, CSB plus 0.5 % PTE; c, CSB plus 0.2 M Tris; d, CSB plus 0.5 % PTE and 0.2 M Tris; e, buffer U; f-j, opaque-2; f, CSB only; g, CSB plus 0.5 % PTE; h, CSB plus 0.2 M Tris; i CSB plus 0.5 % ~ and 0.2 M Tris; j, buffer U.
with the control (Fig. 4a), but a decrease in the large peak (Fig. 4e) which again appeared to be 3 overlapping peaks as seen with P T E + Tris (Fig. 4d). I n the opaque-2 mutant ground in CSB alone (Fig. 4f), the m o n o s o m e p e a k was greater, implying less active protein synthesis (Davies and Larkins1980), while the "large p e a k " was smaller and broader, sedimented less distance into the gradient and lacked the sharpness o f that seen with the wild type (Fig. 4a). W h e n detergent (0.5 % PTE) was added to the h o m o g e n a t e o f the opaque-2 mutant, the large p e a k disappeared entirely f r o m the profile (Fig. 4g) rather than exhibiting just a small shift in
13-tul c~-tul
Fig. 5. Immunoblots of electrophoresed proteins obtained from sucrose gradients. Samples from wild type and opaque-2 endosperm tissue, isolated in CSB alone, were subjected to gradient centrifugation and fractionated. Each fraction was precipitated with 4 volumes of acetone, dissolved in SDS buffer, and denatured by heating at 100 *Cfor 5 min. Electrophoresis in 10 % polyacrylamide gels and blotting onto PVDF membranes (Millipore) were conducted as described by Abe and Davies (1995). The Western blots were triple-probed with antibodies to actin, ct- and ~-tubulin. Samples correspond to: a, b, absorbance profile and the corresponding immunoblot of wild type endosperm; c, d, absorbance profile and the corresponding immtmoblot of opaque-2 endosperm. The individual fractions are numbered from top to bottom of the gradient. SBP, Streptavidin-binding protein. sedimentation in the wild type (Fig. 4b). Addition o f 0.2 M Tris to the h o m o g e n a t e o f opaque-2 en-
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B. STANKOVIC, S. ABE, K. AZAMA, K. SHIBATA, Y. ITO, S. WEIDNER & E. DA VIES
dosperm caused an increase in monosomes and a slight lessening of the large peak (Fig. 4h), similar to that seen with wild type (Fig. 4c), while addition of both detergent and Tris (Fig. 4i) and buffer U (Fig. 4j) caused the disappearance of the large peak and a substantial increase in polysomes (presumably released from the large peak). To investigate the localization of cytoskeletal proteins on the gradients, proteins in each fraction of the gradients from tissue homogenized in CSB alone were precipitated, dissolved in sample buffer, electrophoresed and triple-blotted with antibodies to the three cytoskeleton proteins, ~x- and 13-tubulin and actin (Ito et al. 1994). Absorbance profiles and corresponding immunoblots were obtained for both wild type (Fig. 5a, b) and opaque-2 (Fig. 5c, d) endosperm. In wild type samples, all three cytoskeletal proteins were detected at the top of the gradient. Actin and 13-tubulin were detected in fractions 9-1 I, corresponding to the large peak region (Fig. 5b). In accordance with previous observations (Ito et al. 1994), streptavidin-binding proteins (SBP) were also detected in the major peak (fractions 9-11, Fig. 5b). Zein proteins were detected by their nonspecific antibody binding in samples from fractions 10-12 (Fig. 5b). In opaque-2 samples, the large peak reached its isopicnic point in lighter regions of the gradient, corresponding to fractions 7-t 1 (Fig. 5c). All three cytoskeletal proteins were detected at the top of the gradient, while B-tubulin was also found in fractions 7-9, actin in fractions 9-10, SBP in fractions 9-11, and zeins in fractions 9-12 (Fig. 5d).
Discussion The specially-designed cytoskeleton-stabilizing buffer (CSB) enabled isolation of the plant cytoskeleton as large fragments of F-actin (microfilaments) rather than small fragments or monomeric G-actin (Abe and Davies 1991). This buffer was successfully used to show that protein bodies in wild type maize endosperm were surrounded by, and enmeshed in, a network of microfilaments and that the polysomes were attached to the cytoskeleton rather than to the membranes (Abe et al. 1991, Stankovi6 et al. 1993, Davies et al. 1993). In this report, we use three different approaches (biochemical fractionation, fluorescence microscopy, and
388
immuno-blotting) to provide further evidence for the existence of interactions among cytoskeleton, protein bodies, membranes, and ribosomes in maize endosperm and to show differences between wild type and the opaque-2 mutant. Wild type maize endosperm tissue ground in CSB and centrifuged on heavy (20-80 %) sucrose gradients reveals 3 distinct regions: a small peak of monosomes towards the top, a region of polysomes near the middle, and a massive "peak" of UV-light absorbing material near the bottom (Fig. 1). Variable-term sucrose density centrifugation shows that the monosomes and free polysomes slowly migrate through the gradient, while the "large peak" reaches its isopicnic point within 15 min (Fig. 2a-d) as found earlier with pea epicotyls (Ito et al. 1994). Earlier experiments using total homogenates of maize endosperm indicated that protein bodies were surrounded by membranes, cytoskeleton, and ER, with the polysomes being attached via the cytoskeleton to the protein bodies (Stankovi6 et al. 1993). Here we show that these protein body-cytoskeletonpolysome complexes are the main constituents of the "large peak", since these fractions show numerous protein bodies encircled with actin, and colocalizing with RNA and membranes (Fig. 3). The data presented in Figure 4 suggest that protein bodies in wild type are different from, and more homogenous than, those in opaque-2. In wild type samples isolated in CSB, the protein body peak is sharp and high, while in opaque-2 it is broad and fiat. After detergent treatment, the wild-type peak shifts a little towards denser regions, while the opaque-2 mutant shifts completely off the gradient. These findings are consistent with the idea that the protein bodies in wild type are large and uniform and that their membrane/protein ratio is low. This causes them to shift only slightly to denser regions of the gradient when the membranes are removed by detergent. In contrast, protein bodies in opaque2 are much smaller (Geetha et al. 1991) and would thus have a higher membrane/protein ratio making them less dense when the membrane is still present. Since polysomes have been shown to be attached to the surface of protein bodies and remain associated even after detergent treatment (Stankovi6 et al. 1993), and since RNA is more dense than protein,
ASSOCIATIONS OF MAIZE PROTEIN BODIES ...
the greater increase in density of opaque-2 protein bodies after detergent treatment could be due to their greater polysome/protein body ratio. Both types of protein bodies are shifted slightly towards lighter regions, concomitant with an increase in "free" polysomes seen on the gradient after treatment with Tris, a cytoskeleton-disrupting agent. This implies that both kinds of protein bodies normally bear polysomes, and that they have similar protein body-cytoskeleton associations. M a n y of these interpretations are supported by data in Figure 5. The sharp peak (Fig. 5a) and the almost total coincidence of SBP, actin, ct-tubulin, and most of the zein in wild type (Fig. 5b) suggests that they have but one major type of protein body. However, the broad peak (Fig. 5c) and the presence of most of the cytoskeleton proteins in fractions lighter than those containing zeins, suggest that opaque-2 has two types of protein bodies: smaller (lighter) ones almost devoid of zein, with cytoskeleton proteins (and presumably polysomes) attached; and larger (denser) ones with abundant amounts of zein, but lacking cytoskeleton, perhaps lacking polysomes, and certainly lacking cytoskeleton-associated polysomes that are most active in protein synthesis (Davies et al. 1998).
In summary, the results reported here support the evidence presented earlier for the existence of complex associations among protein bodies, ribosomes, cytoskeleton, and membranes in maize endosperm. Since the function of polysomes is to translate mRNA into proteins, their attachment to the cytoskeleton surrounding the protein bodies in maize might be to regulate the process of protein body formarion, including the synthesis of specific types of zein. Part of the effect of the opaque-2 mutation appears to be in modifying protein bodies so+ that some of those synthesizing zein are not associated with the cytoskeleton, thereby rendering them less efficient in zein synthesis.
Acknowledgments This work was supported in part by the Sasagawa Scientific Research Grant from Japan Science Society to S.A., and by the North CaroIina Agricultural Research Service (project # 06446) to E.D.
References Abe S., and Davies E. 1991. Isolation of F-actin from pea stems: Evidence from fluorescence microscopy. Protoplasma 163, 51-61, Abe, S., You, W., and Davies, E, 1991. Protein bodies in maize endosperm are enclosed by and enmeshed in Factin. Protoplasma 165, 139-149. Clore, A.M., Dannenhoffer, J.M, and Larkins, B.A. 1996. EF-I~ is associated with a cytoskeletal network surrounding protein bodies in maize endosperm cells. Plant Cell 8, 2003-2014. Davies, E., and Larkins, B.A. 1980. Ribosomes. In Plant Biochemistry: A comprehensive treatise, Vol. I, P.K. Stumpf and E.E. Conn, eds, N.E. Tolbert, vol. ed (New York: Academic Press), pp. 413-435. Davies, E., and Abe, S. 1995. Methods for isolation and analysis of polyribosomes. In Methods in Cell Biology, Vol 50, part B: Methods in Plant Cell Biology, D.W. Galbraith, D.P. Bourque, and H.J. Bohnert, eds (New York, Academic Press), pp. 209-222. Davies, E., Comer, E.C., Lionberger, J.M., Stankovi~, B., and Abe, S. 1993. Cytoskeleton-bound polysomes in plants. III. Polysome-cytoskeleton-membrane interactions in maize endosperm. Cell Biol. Int. 17, 331340. Davies, E., Abe, S., Larkins, B.A., Clore, A.M., Quatrano, R.S., and Weidner, S. 1998. The role of the cytoskeleton in plant protein synthesis. In: A look beyond transcription: Mechanisms determining mRNA stability and translation in plants, J. Bailley-Serres and D.R. Gallic, eds (Amer. Soc. Plant Physiol.), pp 115-124. Geetha, K.B., Lending, C.R, Lopes, M.A., Wallace. J.C., and Larkins, B.A. 1991. Opaque-2 modifiers increase y-zein synthesis and alter its spatial distribution in maize endosperm. Plant Cell 3, 1207-1219. Ito, Y., Abe, S., and Davies, E. 1994. Co-localization of cytoskeleton proteins and polysomes with a membrane fraction from peas. J. Expt. Bot. 45,253-259. Larkins, B.A., and Hurkman, W.J. 1978. Synthesis and deposition of zein in protein bodies of maize endosperm. Plant Physiol. 62, 256-263. Mertz, E.T., Bates, L.S., and Nelson, O.E. 1964. Mutant gene that changes protein composition and increases lysine content of maize endosperm. Science 145, 279280. Stankovi6, B., Abe, S., and Davies, E. 1993. Co-localization of polysomes, cytoskeleton, and membranes with protein bodies from maize endosperm. Protoplasma 177, 66-72.
ReceivedMarch 30, 1999; acceptedMay 24, 1999
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