Structure and composition of protein bodies from wild‐type and high‐lysine barley endosperm

Protein bodies were isolated from 13 and 28 day old endosperms of barley mutant 1508 and its wild type, Bomi barley. The fine structure of the isolated protein bodies was determined by electron microscopy, and the proteins present in the preparations characterized by amino-acid analysis and SDS-polyacrylamidegel electrophoresis. Sections through pellets of isolated protein bodies from both the mutant and the wild type revealed protein body structures corresponding with those observed in sections through the intact starchy endosperms. The majority of the wild-type protein bodies was homogeneous spheres accompanied with a granular component. Particles with the same structure were present in the protein body preparation from the mutant, where, however, the granular component was the most prominent. Amino-acid composition and SDS-polyacrylamide gel electrophoresis of the proteins from the protein body preparation revealed that the wild-type protein bodies contained large amounts of prolamines (the storage protein group which is soluble in 55 % isopropanol) and some glutelins (the storage proteins soluble in dilute alkali), whereas the mutant protein bodies have glutelin as the major component and little prolamines. It is suggested that the homogeneous protein body component represents a storage organelle with a high concentration of prolamines, and the granular component a storage organelle with a high concentration of glutelins.

Genes leading to an increased lysine content of cereal endosperm proteins have been found in maize (MERTZ et al. 1964;NELSON et al. 1965), barley (MUNCK et al. 1970DOLL^^ al. 1974) and sorghum (SINGH and AXTELL 1974). Such single gene mutants provide a potentially useful tool in studies on storage protein synthesis and its regulation. The increased lysine content of the endosperm proteins from highlysine cereals is a result of alterations in storage protein composition and amount of free amino acids (MERTZ et al. 1964;MUNCK et a]. 1970;MUNCK 1972;MERTZ et al. 1974;BRANDT 1975). In the case of opaque-2, floury-2 maize and barley mutant 1508 the deposition of lysine-poor prolamines is reduced and compensated for by an increased synthesis of lysine-rich proteins. This change in protein composition is reflected by an altered development and structure of the protein bodies (WOLF et al. 1967;MUNCK and VON WETTSTEIN 1975), which are the principal protein stores of the cereal endosperm.
In an attempt to further characterize the effects of the 1508 mutation on storage protein formation, protein bodies of Bomi barley and its mutant 1508 were isolated at two different stages of endosperm development and their protein composition studies.
Material and methods genized for 5 min. in a mortar by a gradual addition of 1 + 2 + 2 ml of an extraction buffer consisting of 150 mM TRIS pH 7.5, 10 mM KCI, 1 mM MgCI,, 1 mM EDTA and 1396 sucrose. Starch and cell debris were removed by centrifugation at 900 g for 15 min. The resulting supernatant was layered on the top of a sucrose gradient made up in the following way from the top towards the bottom of the tube: 5 ml 2096 sucrose, 20 ml linear sucrose gradient 20-60:4, and a cushion of 5 ml 600; sucrose. After centrifugation at 40,000 g for 16 hr the bands had reached equilibrium, and the gradient was eluted by penetrating the bottom of the tube. The OD,,, of the eluate was measured continuously, and 2.5 ml fractions were collected. To each fraction 2.5 ml extraction buffer were added and the contents pelleted by centrifugation at 45,000 g for 15 min.
The sediment was washed twice in 2 ml extraction buffer for electron microscopy or electrophoresis.
Electron microscopy. -The pellet was fixed for two hr in 6:; glutaraldehyde, dissolved in 0,05 M phosphate buffer, pH 7.5, and postfixed with 2;: osmium tetroxide for two hr, dehydrated through an alcohol series and embedded in Spurr's lowviscosity epoxy resin. Sections were cut on a Reichert UM-2 microtome, contrasted with uranylacetate and lead citrate and examined in a Siemens Elmiskop I A.
Amino-acid cinalysis. -Automatic amino-acid analysis took place after hydrolysis in vacuum. Nitrogen determinations were done by micro Kjeldahl on the lyophilized pellets.

Results and discussion
Protein bodies (aleuron grains, cf. FREY WYSSLING and MUHLETHALER 1965, p. 159) from sunflower cotyledons have been prepared from cell homogenates by isopycnic density gradient centrifugation (SCHNARRENBERGER et al. 1972). When homogenates of wild-type barley endosperm harvested 28 days after fertilization were subjected to gradient centrifugation a broad opalescent band was present between the center and the bottom of the gradient. This band appeared as the major peak in the OD,,, profile of the gradient (Fig. lB, solid line). The high 280 nm absorption of the top of the gradient is due to the sample layer with its high content of dissolved proteins. The isopycnic density of the sedimented particles ranges from 1.21 -1.29 gX ccl, slightly lower than the isopycnic density of 1.26-1.36 gXcc-' found for protein bodies from mature sunflower cotyledons (SCHNARRENBERGER et al. 1972). The sedimented particles of this band were collected and further purified by pelleting and resuspension in extraction buffer. Fig. 2A shows a section through a portion of cells from a 28-day old Bomi barley endosperm with an aggregate of protein bodies inside a vacuole. The developing protein bodies in the starchy endosperm of barley consist of two components, the homogeneously structured spheres (a) and a granular component (b) in which the homogeneous spheres are embedded (MUNCK and comm.). The homogeneous spheres correspond to what has been called protein bodies in maize (KHOO and WOLF 1970) and wheat (BUTTROSE 1963). Fig. 2B, which is a section through the purified pellets reveals that the preparation of the isolated protein bodies contains both components, the homogeneously structured spheres (a) and the granular component (b). The components of this preparation have an appearance that is similar to the protein bodies isolated by ORY and HENNING-SEN (1 969) in barley and by ADAMS and NOVELLIE (1975) in sorghum. The protein bodies of the starchy endosperm cells are morphologically very different from the aleuron grains of the aleuron cells in the cereals (JACOBSEN et al. 1971) or the aleuron grains of dicotyledonous seeds. Aleuron grains containing the phytate-rich globoids and the carbohydrate-rich crystalloids have been isolated from aleurone layers of rice (TANAKA et al. 1973) and distinguished from the protein bodies of the starchy endosperm. Since the latter comprise the vast majority when the whole endosperm is ground up it is not surprising that aleuron The gradients were eluted after centrifugation at 40,000 g for 16 hr and the optical density of the fractions determined at = 280 nm.
grains were not detected in the present preparations.
The isopycnic density of the isolated protein bodies from the 28-day old mutant 1508 endosperm was 1.13-1.24gXcc-l, i.e. lower than that of Bomi protein bodies (Fig. lB, broken line). A section through a portion of a cell from a mutant 1508 endosperm (Fig. 2C) shows the protein bodies to consist mainly of a granular component (b) in which few homogeneous spheres (a) are embedded (MUNCK and VON WETTSTEIN 1975; VON WETTSTEIN, pers.comm.). The protein bodies have thus a very different morphology than those of the wild type. The section through the pellet of the isolated protein bodies from the mutant (Fig.  2D) reveals that the morphology has been preserved during isolation. Homogeneous spheres are embedded in the granular component. The strong osmiophily of the homogeneous component in Figure 2C is observed in some fixations and not in others and may be caused by different degrees of compaction during protein body formation (VON WETTSTEIN, pers.comm.).
Protein bodies were also isolated from 13-day old endosperms (Fig. LA). At this early stage of development both wild-type and mutant endosperm homogenates contained protein bodies with isopycnic densities ranging from 1.13-1.24 The nitrogen content of the protein body preparations from the 28-day old endosperms was 7.4% for the mutant as compared to 11 % for the wild type. The low value for mutant 1508 signifies a high content of non-protein material in the preparation.
The amino-acid composition of the proteins in g x cc-l.
Hereditas 81. 1975 the protein body preparations from 28-day old endosperms is given in Table 1. The amino-acid composition of the wild-type proteins resembles that of the barley prolamines as they are extracted from mature seeds ( ) except for glutamic acid and proline which amount to only of the content determined for wild-type prolamines at maturity. Likewise, the proteins in the preparations with mutant protein bodies contained 309; less glutamic acid and proline than the mutant prolamines at maturity. Since the amino-acid composition of both the mutant and the wild-type prolamines isolated from 28-day old endosperms is similar to the amino-acid composition of prolamines isolated from mature kernels (A. BRANDT, pers.comm.), the low content of glutamic acid and proline is not due to the young age of the endosperms. The amino-acid composition indicates that the isolated fractions contain a mixture of prolamines and other proteins.

ZL
SDS-polyacrylamide gel electrophoresis of the fractions from the gradients revealed that the composition of the mutant protein bodies was very different from that of the wild type, and the wild-type band pattern of the protein body peak resembled that of barley prolamines (B K B I E , pers. comm.). The individual proteins were deposited with almost identical intensity during the period studied, i.e. from 13 to 28 days after fertilization. Fig. 3 depicts representative gels of the proteins extracted from the fractions of Fig. 1B. Coelectrophoresis of protein body proteins and prolamines extracted from mature kernels with 55 94 isopropanol revealed that both the wild type and the mutants contained polypeptides in the protein bodies with the same electrophoretic mobility as the major bands of the prolamines. These polypeptides were, however, much less prominent in the extracts of the mutant protein bodies. It has earlier been demonstrated by immunoelectrophoresis of extracts from isolated barley protein bodies that those contain prolamines (TRONIER et al. 1971). The present investigation confirms this conclusion. Furthermore, the altered morphology of the protein bodies in mutant 1508, characterized by the paucity of the homogeneous component is likely to be related t o the reduction in the major prolamine bands and the recognition of bands not visible in the gel patterns of the extracts from wild-type protein bodies.
No new bands appear when total extracts of reduced endosperm proteins of the mutant are subjected to SDS-polyacrylamidegel electrophoresis, but the relative amounts of the different endosperm proteins are altered. Since the albumins and globulins were already extracted when the tissue was homogenized a major portion of the proteins associated with the mutant protein bodies must belong t o the glutelins (the storage protein group soluble in dilute alkali).
The above-mentioned results on amino-acid composition indicated the protein bodies of both the wild type and the mutant to contain prolamines and other proteins. It is, therefore, apparent that the wild-type protein bodies contain large amounts of prolamines and some glutelins, whereas the mutant protein bodies have glutelin as a major component and little prolamines. The morphological difference between wild type and mutant intimates that the homogeneous component represents a storage organelle with a high concentration of prolamines and the granular component a storage organelle with high concentration of glutelins. CHRISTIANSON et al. (1974) have isolated protein bodies by zonal centrifugation from wild-type maize and its high-lysine mutant opaque-2 and characterized them by scanning electron micros-  Fig. 1 B. A typical prolamine band pattern is found in fractions 6 to 10 for the wild type proteins (B). The mutant proteins of fractions 4 to 8 gave an altered pattern with a pronounced relative deficiency of some of the most prominent bands of the wild type. The arrows indicate the prolamine bands, i.e. the polypeptides present in a alcoholic extract of a mature wild type endosperm.
copy. The opaque-2 starchy endosperm appears to contain fewer and smaller protein bodies than the wild-type endosperm (WOLF et al. 1967;CHRISTIANSON et al. 1974). It will be of interest to compare the fine structure and protein composition of the small opaque-2 protein bodies with those of the wild-type maize protein bodies. This should give additional information as to whether or not the opaque-2 gene is homologous to the 1508 gene. So far I have been unable to demonstrate the presence of an insoluble protein in my gradient either by OD,,, elution profile or by electrophoresis. More studies are, however, necessary to insure that a glutelin-2 like protein is absent in mature barley endosperm. Some proteins associated with the opaque-2 protein bodies are soluble in 70% ethanol, which establishes their prolamine character (CHRISTIAN- SON et al. 1974). Using a similar extraction procedure it was impossible to extract measureable amounts of prolamines from mutant 1508 protein bodies.
Acknowfedgmenfs. -I thank Professor D. von Wettstein for very helpful advice and the permission to use the electron micrographs shown in Fig. 2A