Planta
Planta 137, 185-193 (1977)
9 by Springer-Verlag 1977
Development of Aleurone and Sub-aleurone Layers in Maize D.J. Kyle* and E.D. Styles Department of Biology, University of Victoria, Victoria, B.C. V8W 2Y2, Canada
Abstract. Electron-microscope studies indicate that the aleurone tissue of maize (Zea mays L.) starts developing approximately 10-15 days after pollination in stocks that take ca. 40 days for the aleurone to mature completely. Development commences when specialized endosperm cells adjacent to the maternal nucellar layer start to differentiate. Differentiation is characterized by the formation of aleurone protein bodies and spherosomes. The protein bodies of the aleurone layer have a vacuolar origin whereas the protein bodies of the immediate underlying endosperm cells appear to develop from protrusions of the rough endoplasmic reticulum. Thus, two morphologically and developmentally distinct types of protein bodies are present in these adjacent tissues. The spherosomes of the aleurone layer form early in the development of this tissue and increase in number as the tissue matures. During the final stages of maturation, these spherosomes become closely apposed to the aleurone grains and the plasma membrane. No further changes are apparent in the structure of the aleurone cells after 40 days from pollination when the caryopsis begins to desiccate. Key words: Aleurone - Protein bodies - Spherosomes - Endosperm - Zea.
Introduction
The aleurone tissue of the Gramineae is a special layer of cells surrounding the starchy endosperm of the seed, except in the area adjacent to the embryo. It is characterized by a large number of protein granDepartment of Plant Science, University of Alberta, Edmonton, Alta T6G 2E3, Canada *
Present address:
ules or aleurone grains, and spherosomes. The aleurone layer of maize is particularly useful for investigations on the structural and biochemical changes that occur during development of this tissue since each ear may contain up to 1000 genetically equivalent caryopses (kernels) from a single controlled ,cross. In addition, the maize caryopses are large and the aleurone is only one cell layer thick, ensuring rapid penetration of fixatives. The development of the aleurone tissue was first studied with the electron microscope by Buttrose (1963) and more recently by Morrison et al. (1975) in wheat. Other workers have described the fine structure of the aleurone tissue in dry kernels of barley (Jones, 1969; Jacobsen et al., 1971; Taiz and Jones, 1973), wheat (Fulcher etal., 1972) and sorghum (Adams and Novellie, 1975), but only in wheat has it been studied in terms of embryogenetic development. The subcellular components of the developing aleurone layer of maize were first described by Mottier in 1921 with the aid of the light microscope. Electron-microscopical studies on the intracellular structures of maize caryopses have been restricted to the endosperm (Duvick, 1961; Khoo and Wolf, 1970) and the scutellum (Longo and Longo, 1969). In this study we describe the development of the fine structure of the aleurone and adjoining tissues of maize, with particular reference to the origin and structure of protein bodies and spherosomes.
Material and Methods Seeds of a coloured-aleurone strain of the W22 inbred line of L. from our own stocks were grown to maturity under field conditions. A number of plants were self-pollinated and indiZea mays
186
D.J. Kyle and E.D. Styles: Aleurone Development in Maize
vidual ears were harvested at 5-day intervals from 5 to 40 days post-pollination, Developing caryopses were removed at random from a 1-cm-wide band, 2 cm from the base of the ear. The outer pericarp layer was stripped from the caryopses of 15 days and older since it represents a penetration barrier for the fixative. In caryopses younger than 15 days, the pericarp could not be removed without considerable structural damage to the underlying tissues. A small, 1-mm section of tissue was cut from the top of the caryopsis. The majority of the underlying endosperm was peeled off and the aleurone layer was immediately fixed in cold 3% glutaraldehyde buffered with 50 mM sodium cacodylate buffer (pH 7.2) for 24 h at 4 ~ After three washes of 1 h each in the same cold buffer, the samples were post-fixed in 2% OsO4 in 50 mM cacodylate buffer for 6 h at 4 ~C. The samples were again washed 3 times for 1 h in cold buffer, and dehydrated in a graded acetone series of 25%, 50%, 75% and 2 x 100% for 30 min each, followed by propylene oxide. The tissue pieces were then transferred to a 1 : 1 mixture of propylene oxide and Epon-812 for 6 h, followed by pure Epon-812 for 12 h. They were then transferred to fresh resin and hardened at 60~ for 48 h. Thin sections were cut with a glass knife on an ultramicrotome and mounted on 120-mesh formvar-coated grids. The grids were
Results Kernels sampled at 5 and l0 days post-pollination (representing tissues 4 and 9 days old, respectively, since fertilization occurs about 24 h after pollination) were very small, and identification of a distinctive aleurone layer in this material was difficult. The maternal tissues of nucellus and pericarp, normally dead and desiccated in the mature caryopsis contained intact mitochondria, plastids, and a diffuse nucleus ( F i g . 2). T h e e n d o s p e r m o f t h e s e y o u n g s e e d s h a d milky texture and appearance. F i g u r e 1 is a d i a g r a m a t i c i n t e r p r e t a t i o n o f t h e sequence of changes occurring during the development
15 D A Y S
5 DAYS
;..;o
stained with uranyl acetate for 2 h, followed by Reynold's lead citrate for 3 min. All electron-microscopic observations were made with a Phillips EM 300 electron microscope.
o
/o o 2+ PD
/.|
0o"
0| DAYS
PD
0 f
25
--
J 40
DAYS
Fig. 1. A schematic diagram of the developmental sequence of a typical aleurone layer cell of maize. 5 days post-pollination the cells are columnar, contain few organelles and large vacuoles. 15 days post-pollination the cells are more cuboidal, there is visual evidence of protein synthesis, and the aleurone protein bodies have begun to form. 25 days post-pollination the cell size has changed very little, protein bodies have developed further and spherosomes are more apparent. 40 days post-pollination the cell wall has thickened and a slight reduction of total cell volume has occurred. The spherosomes completely surround the fully developed aleurone protein bodies and line the periphery of the plasmalemma. Golgi bodies are more obvious at this stage. N nucleus; P plastid; V vacuole; PD plasmodesmata; VP vacuolar precipitate; S spherosome; M mitochondria; P B protein body; P R polyribosomes; G Golgi body
Fig. 2. 10 days posl-pollination. The cells of the aleurone layer are small and contain little substructure other than the nucleus and vacuoles. The adjacent nucellar layer has not yet collapsed, x 5500
Fig. 3. 15 dayspost-pollination. The initial stages of aleurone protein body development has commenced as denoted by vacuolar precipitates. Note that the vacuoles are about one fifth the size of those for the 10-day cells in Figure 2. Ribosomes in the cytoplasm are predominantly aggregated into polysomes rather than on endopIasmic reticulum, x 30,000 Fig. 4. 15 days post-pollination. Spherosomes are generally located around the periphery of the aleurone cell whereas the mitochondria, plastids, and forming aleurone protein bodies are nearer the centrally located nucleus, x 19,000
188
of a maize aleurone cell 1. The aleurone cells of the 5-to 10-day stage are characterized by large vacuoles and a diffuse cytoplasm (Fig. 2). Developmental changes in the aleurone cell are most rapid between 10 and 15 days post-pollination. The most abvious change is the reduction in size of the large vacuoles characteristic of the younger tissue by about 80% (Fig. 3). In addition, the numbers of mitochondria and ribosome-like particles have increased. Two nucleoli may be present in the large, diffuse nucleus. Nucleolar organizers at this stage are shown in Figure 4. Spherosomes, which are bound by an atypical half-unit membrane (Fig. 11), are found throughout the cytoplasm. Electron-transparent, unit-membranebound vacuoles representing the remnants of the initial large vacuoles are present, and in many cases an electron-dense precipitate can be seen within these vacuoles (Figs. 3, 4). The vacuoles, mitochondria and plastids are typically adjacent to the central nucleus whereas the spherosomes are located nearer the periphery of the cell (Fig. 4). Demarcation between the aleurone and the adjacent outer endosperm cells is still difficult at the 15-day stage. The entire nucellar layer, however, has collapsed and forms a distinct layer between the aleurone and pericarp tissues. The walls of the Neurone cells have thickened only slightly since the earlier stages, and plasmodesmata can be seen not only between neighbouring aleurone cells (Fig. 4), but between aleurone and endosperm cells also (Fig. 9). At 20 days, the vacuolar precipitates in the aleurone cells have increased in size to fill the vacuoles entirely (Fig. 5), and they have taken on the appear1AL=aleurone layer; A P B ~ a l e u r o n e protein body; C W = c e l l wall; D B = d e n s e body of the aleurone protein body; E L = e n d o sperm layer; EPB = endosperm protein body ; G = Golgi body ; M = mitochondrion; MT = microtubules; NL = nucellar layer ; NP = nuclear pore; N O = n u c l e o l a r organizer; P = p l a s t i d ; P D = p l a s m o desmata; PM = peripheral matrix of aleurone protein body; PR = polysomes; R = r i b o s o m e s ; R E R = r o u g h endoplasmic reticulum; S = spherosome; SG ~ starch grain; TC = electron-transparent core of aIeurone protein body; V = v a c n o l e ; V P = v a c u o l a r precipitate
D.J. Kyle and E.D. Styles: Aleurone Development in Maize
ance of an aleurone protein body. A positive reaction with toluidine blue indicates that these bodies are at least partly proteinaceous in content. Protein bodies are forming in the sub-Neurone endosperm cells during this period of protein body formation in the Neurone. The endosperm protein bodies are, on the average, slightly smaller, more uniform in size, and have a different substructural appearance than their counterparts in the Neurone layer (Fig. 6). They appear to be formed directly from rough ER (Figs. 7, 8), and ribosomes can be seen attached to the outer surface of the protein-body membrane (Fig. 6). The numbers of protein bodies in the endosperm cells increase from day 20 to maturity of the tissue. By 25 days post-pollination, the Neurone cells are almost completely filled with small spherosomes (average diameter about 1 i~m). The Neurone grains now are more electron dense, and electron-transparent pits are seen within many of the grains (Fig. 9). The rate of cell-wall thickening increases between 25 and 30 days, and maximum wall thickness of 1-2 gm is achieved by 35-40 days post-pollination. By 35 days the aleurone protein bodies have fully matured. Their structure is shown in Figures 10 and 11. The dense body of the grain has shrunken away from the vacuole wall and is now showing a less dense fibrous ground substance in the peripheral matrix. A large, electron-transparent core can be seen within the dense body of the grain; the grain may also contain some fibrous substructure (Fig. 11). Maize aleurone protein-bodies range in size from 1.0 to 1.5 gm, making them not only different in appearance, but much smaller in size than Neurone proteinbodies described for wheat (Morrison et al., 1975) or barley (Jacobsen et al., 1971). Spherosomes aggregate around the aleurone protein bodies only after they have fully developed (35-40 days post-pollination). The spherosomes are surrounded by a membrane, but where it is adjacent to a unit membrane of the aleurone protein body (Fig. 11, insets), it is clear that the spherosome mere-
Fig. 5 . 2 0 days post-pollination. The amount of precipitate within the vacuole has increased but the internal structure of the developing aleurone protein body is still diffuse, x 45,000 Fig. 6. 25 days pos~-pollination. The developing endosperm protein bodies can be seen, with clearly defined ribosomes on the surface. • 46,000 Fig. 7. 30 days post-pollination. Formation of endosperm protein bodies may commence by intracisternal dilations of the rough ER. Note the unusually large endosperm protein body in upper right. • 35,000 Fig. 8. 30 days post-pollination. Very small endosperm protein bodies can also be seen associated with rough ER. Formation of these protein bodies may also be from swellings at the terminus of the rough ER (arrow). x 35,000 Fig. 9. 25 days post-pollination. The aleurone protein bodies now appear much more densely packed than in earlier stages. Note that intercellular communication (plasmodesmata) is occurring between cells of the aleurone layer and cells of the endosperm layer, x 40,000
D.J. Kyle and E,D. Styles: Aleurone Development in Maize
189
Fig. 10. 35 days post-pollination. A mature aleurone protein body with its substructure defined in terms of a peripheral matrix, the dense body, a n d a central transparent core. • 43,000 Fig. 11.35 days post-pollination. The final stage of aleurone protein body development is demonstrated by the apposition of spherosomes to the protein-body surface. Higher magnification of these interfaces clearly shows that the spherosome m e m b r a n e does not have the tripartite structure of a normal unit m e m b r a n e (insets). x 52,000; insets x 70,000
Fig. 12. 35 days post-pollination. The mature aleurone tissue changes very little from now to desiccation. Note the preponderance of Golgi bodies, x 20,000 Fig. 13. 35 days post-pollination. At this stage further development of the aleurone layer has stopped but protein bodies continue to be formed in the underlying endosperm (arrow). x 22,000
192
D.J. Kyle and E.D. Styles: Aleurone Development in Maize
brane does not have the normal tripartite structure. As the maize kernel reaches full maturity and begins to dehydrate, the spherosomes completely surround the aleurone protein bodies and line the inside periphery of the plasma membrane. Fewer mitochondria and plastids are seen in 35day-old aleurone cells, but Golgi bodies are more prevalent than in previous stages. Almost the entire volume of the cytoplasm now consists of spherosomes and aleurone protein bodies (Fig. 12). The small sub-aleurone endosperm cells retain their 25-day-old appearance even after 40 days (Fig. 13). They are conspicuous by their lack of the large starch grains typical of the deeper endosperm cells. The continued production of protein bodies and the presence of numerous mitochondria indicates that these cells are still active after the aleurone cells have matured.
Discussion Protein Bodies
The aleurone layer of maize represents a highly specialized, structurally complex layer of endosperm cells. The size and substructure of mature maize aleurone protein-bodies is markedly different from that of similar bodies in other species (Morrison et al., 1975; Rest and Vaughan, 1972; Jacobsen et al., 1971 ; Briarty et aI., I969) as weli as from that of the underlying endosperm tissue. Figure 14 represents a scheme for the development of the protein bodies of the maize aleurone layer as deduced from our electron-microscopic observations. Visible developmental changes began by 15 days post-pollination. At this time, regions of dense (protein) precipitate appeared in the center of the
cell vacuoles. Morrison et al. (1975) have described a similar occurrence during the initial stages of the development of protein bodies in wheat aleurone. The vacuolar precipitates in the wheat aleurone, however, were much more densely staining than those of maize; they showed compact concentric layers and were therefore described as the Type I phytin inclusions such as those originally described by Fulcher (1972). The amount of rough endoplasmic reticulum (ER) present at this stage is extremely small compared to the numbers of free ribosomes seen. This observation indicates that the vacuolar precipitates or the protein component of the vacuolar precipitates originates from the free cytoplasmic ribosomes rather than from rough ER. The protein accumulates in the vacuole over the next 15 days and condenses into an electrondense spherical structure that has an electron-transparent core and is surrounded by a fibrous ground substance (peripheral matrix). In some protein bodies, the electron-transparent core can also be seen to contain a small amount of ground substance similar to the peripheral matrix as in Figure 11. The protein bodies of the endosperm differ from those of the aleurone tissue in structure and origin. Khoo and Wolf (1970) have described the formation of maize endosperm bodies from rough ER either by cisternal dilations or enlargements of the ends of the hR. The presence of polysomes on the surface of these bodies has been shown by Burr and Burr (1976). Our observations lead us also to believe that the endosperm protein bodies are formed from rough ER (Figs. 7, 8). [n addition, ribosomes can be cleariy discerned on the surface of these protein bodies (Fig. 6) whereas they were never seen on the surface of the aleurone protein bodies. The substructures of the aleurone and endosperm protein bodies also differs significantly.Thus, the aleurone layer which is derived from the endosperm tissue, has differentiated
I 15 D A Y S
20
DAYS
25 DAYS
35 D A Y S
Fig. 14. The development of aleurone protein bodies in maize. Protein precipitates are first detected in vacuoles about 15 days from pollination. Protein continues to accumulate for an additional 10 days. 25 days post-pollination the protein condenses forming a large electron transparent core. The fully developed protein body is denoted by the apposition of spherosomes to the protein body surface
D.J. Kyle and E_D. Styles: Ateurone Development in Maize
in maize to the extent that a different mechanism of protein-body development is operating and as a result two distinct classes of protein bodies can be identified in these adjacent tissues.
193 The authors thank Mr. Jack Dietrich for his invaluable technical assistance and helpful consultations throughout this study. We also wish to thank Mr. B. Zytaruk for his help in preparing the photomicrographs.
References
Spherosomes High-magnification views of spherosomes from the aleurone tissue demonstrate the peculiarity of the limiting membrane. Rather than the typical tripartite structure of a unit membrane only a single limiting line can be seen around these bodies. This is particularly clear when seen adjacent to a typical unit membrane such as that surrounding protein bodies (Fig. 11, insets). Yastu and Jacks were first to describe the "half-unit membrane" in spherosomes from peanut seeds Arachis hypogaea (1972). They interpret these membranes as representing a phospholipid monolayer with the apolar regions directed toward the center of the lipid-containing spherosomes. This hypothesis would also explain the observation of limiting membranes without the typical tripartite structure around the spherosomes of the maize aleurone tissue. Frey-Wyssling et al. (1963) and Schwartzenbach (1971) have proposed hypotheses involving an origin for spherosomes from the ER. Smith (1974) differentiates between spherosomes and oil bodies, suggesting that spherosomes are fi-glycerophosphatase positive and are produced by the ER while oil bodies are deposits o f " reserve oil" which are produced adjacent to areas of electron-dense particulate matter. We have observed no small spherosome inclusions as required by the hypothesis of Schwartzenbach. On the other hand, although many areas of electron-dense particulate matter similar to those described by Smith were seen, in no case were developing prospherosomes seen associated with them. Spherosomes of the maize aleurone develop rapidly between 10 and 15 days postpollination, and thus further information on the origin of these bodies might be obtained by studying Neurone development during this crucial time period. This research was funded by NRC of Canada and the University of Victoria.
Adams, C.A., Novellie, L. : Composition and structure of protein bodies and spherosomes isolated from ungerminated seeds of Sorghum biocolor (Linn.) Moench. Plant Physiol. 55, 1-6 (1975) Briaty, L.G., Coult, D.A., Boulter, D.: Protein bodies of developing seeds of Viciafaba. J. Exp. Bot. 20, 358-372 (1969) Burr, B., Burr, F.A. : Zein synthesis in maize endosperm by polyribosomes attached to protein bodies. Proc. Nat. Acad. Sci. USA 73, 515-519 (1976) Buttrose, M.S. : Ultrastructure of developing aleurone cells of the wheat grain. Austr. J. Biol. Sci. 16, 768-774 (1963) Duvick, D.N.: Protein granules of maize endosperm ceils. Cereal Chem. 38, 374-385 (1961) Frey-Wyssling, A., Grieshaber, E., Miihlethaler, K.: Origin of spherosomes in plants. J. Ultrastruct. Res. 8, 506-516 (i963) Fulcher, R.G., O'Brien, T.P., Lee, J.W.: Studies on the aleurone layer. I. Conventional and fluorescence microscopy of the cell wall with emphasis on phenol-carbohydrate complexes in wheat. Austr. J. Biol. Sci. 25, 23-34 (1972) Jacobsen, J.V., Knox, R.B., Pyliotis, N.A. : The structure and composition of aleurone grain in the barley aleurone layer. Planta (Berl.) 101, 189-209 (1971) Jones, R.L.: The fine structure of barley aleurone cells. Planta (Berl.) 85, 359 375 (1969) Khoo, V., Wolf, M.J. : Origin and development of protein grannies in maize endosperm. Amer. J. Bot. 57, 1042 1050 (1970) Longo, C.P., Longo, G.P.: The development of glyoxysomes in peanut cotyledons and maize scutella. Plant Physiol. 45, 249-254 (1969) Morrison, I.N., Kuo, J., O'Brien, T.P.: Histochemistry and fine structure of developing wheat aleurone cells. Planta (Bed.) 123, 105 116 (1975) Mottier, D.M.: On certain plastids, with special reference to the protein bodies of Zea, Ricinus, and ConophoIisl Ann. Bot. 35, 349-369 (I921) Rest, J.A., Vaughan, J.G.: The development of protein and oil bodies in the seed of Sinapis alba L. Planta (Berl.) 105, 245-262 (1972) Schwartzenbacb, A.M. : Observations on spherosome membranes. Cytobiol. 4, 145-147 (1971) Smith, C.O. : The ultrastructural development of spherosomes and oil bodies in the developing embryo of Crambe abyssinica. Planta (Berl.) 119, 125-142 (i974) Taiz, L., Jones, R.L.: Plasmodesmata and an associated cell wall component in barley aleurone tissue. Amer. J. Bot. 60, 67-75 (1973) Yatsu, L.Y., Jacks, T,J. : Spherosome membranes. Half unit-membranes. Plant Physiol. 49, 737-943 (1972)
Received 2 February ; accepted 22 August 1977
Planta 137, 185-193 (1977)
9 by Springer-Verlag 1977
Development of Aleurone and Sub-aleurone Layers in Maize D.J. Kyle* and E.D. Styles Department of Biology, University of Victoria, Victoria, B.C. V8W 2Y2, Canada
Abstract. Electron-microscope studies indicate that the aleurone tissue of maize (Zea mays L.) starts developing approximately 10-15 days after pollination in stocks that take ca. 40 days for the aleurone to mature completely. Development commences when specialized endosperm cells adjacent to the maternal nucellar layer start to differentiate. Differentiation is characterized by the formation of aleurone protein bodies and spherosomes. The protein bodies of the aleurone layer have a vacuolar origin whereas the protein bodies of the immediate underlying endosperm cells appear to develop from protrusions of the rough endoplasmic reticulum. Thus, two morphologically and developmentally distinct types of protein bodies are present in these adjacent tissues. The spherosomes of the aleurone layer form early in the development of this tissue and increase in number as the tissue matures. During the final stages of maturation, these spherosomes become closely apposed to the aleurone grains and the plasma membrane. No further changes are apparent in the structure of the aleurone cells after 40 days from pollination when the caryopsis begins to desiccate. Key words: Aleurone - Protein bodies - Spherosomes - Endosperm - Zea.
Introduction
The aleurone tissue of the Gramineae is a special layer of cells surrounding the starchy endosperm of the seed, except in the area adjacent to the embryo. It is characterized by a large number of protein granDepartment of Plant Science, University of Alberta, Edmonton, Alta T6G 2E3, Canada *
Present address:
ules or aleurone grains, and spherosomes. The aleurone layer of maize is particularly useful for investigations on the structural and biochemical changes that occur during development of this tissue since each ear may contain up to 1000 genetically equivalent caryopses (kernels) from a single controlled ,cross. In addition, the maize caryopses are large and the aleurone is only one cell layer thick, ensuring rapid penetration of fixatives. The development of the aleurone tissue was first studied with the electron microscope by Buttrose (1963) and more recently by Morrison et al. (1975) in wheat. Other workers have described the fine structure of the aleurone tissue in dry kernels of barley (Jones, 1969; Jacobsen et al., 1971; Taiz and Jones, 1973), wheat (Fulcher etal., 1972) and sorghum (Adams and Novellie, 1975), but only in wheat has it been studied in terms of embryogenetic development. The subcellular components of the developing aleurone layer of maize were first described by Mottier in 1921 with the aid of the light microscope. Electron-microscopical studies on the intracellular structures of maize caryopses have been restricted to the endosperm (Duvick, 1961; Khoo and Wolf, 1970) and the scutellum (Longo and Longo, 1969). In this study we describe the development of the fine structure of the aleurone and adjoining tissues of maize, with particular reference to the origin and structure of protein bodies and spherosomes.
Material and Methods Seeds of a coloured-aleurone strain of the W22 inbred line of L. from our own stocks were grown to maturity under field conditions. A number of plants were self-pollinated and indiZea mays
186
D.J. Kyle and E.D. Styles: Aleurone Development in Maize
vidual ears were harvested at 5-day intervals from 5 to 40 days post-pollination, Developing caryopses were removed at random from a 1-cm-wide band, 2 cm from the base of the ear. The outer pericarp layer was stripped from the caryopses of 15 days and older since it represents a penetration barrier for the fixative. In caryopses younger than 15 days, the pericarp could not be removed without considerable structural damage to the underlying tissues. A small, 1-mm section of tissue was cut from the top of the caryopsis. The majority of the underlying endosperm was peeled off and the aleurone layer was immediately fixed in cold 3% glutaraldehyde buffered with 50 mM sodium cacodylate buffer (pH 7.2) for 24 h at 4 ~ After three washes of 1 h each in the same cold buffer, the samples were post-fixed in 2% OsO4 in 50 mM cacodylate buffer for 6 h at 4 ~C. The samples were again washed 3 times for 1 h in cold buffer, and dehydrated in a graded acetone series of 25%, 50%, 75% and 2 x 100% for 30 min each, followed by propylene oxide. The tissue pieces were then transferred to a 1 : 1 mixture of propylene oxide and Epon-812 for 6 h, followed by pure Epon-812 for 12 h. They were then transferred to fresh resin and hardened at 60~ for 48 h. Thin sections were cut with a glass knife on an ultramicrotome and mounted on 120-mesh formvar-coated grids. The grids were
Results Kernels sampled at 5 and l0 days post-pollination (representing tissues 4 and 9 days old, respectively, since fertilization occurs about 24 h after pollination) were very small, and identification of a distinctive aleurone layer in this material was difficult. The maternal tissues of nucellus and pericarp, normally dead and desiccated in the mature caryopsis contained intact mitochondria, plastids, and a diffuse nucleus ( F i g . 2). T h e e n d o s p e r m o f t h e s e y o u n g s e e d s h a d milky texture and appearance. F i g u r e 1 is a d i a g r a m a t i c i n t e r p r e t a t i o n o f t h e sequence of changes occurring during the development
15 D A Y S
5 DAYS
;..;o
stained with uranyl acetate for 2 h, followed by Reynold's lead citrate for 3 min. All electron-microscopic observations were made with a Phillips EM 300 electron microscope.
o
/o o 2+ PD
/.|
0o"
0| DAYS
PD
0 f
25
--
J 40
DAYS
Fig. 1. A schematic diagram of the developmental sequence of a typical aleurone layer cell of maize. 5 days post-pollination the cells are columnar, contain few organelles and large vacuoles. 15 days post-pollination the cells are more cuboidal, there is visual evidence of protein synthesis, and the aleurone protein bodies have begun to form. 25 days post-pollination the cell size has changed very little, protein bodies have developed further and spherosomes are more apparent. 40 days post-pollination the cell wall has thickened and a slight reduction of total cell volume has occurred. The spherosomes completely surround the fully developed aleurone protein bodies and line the periphery of the plasmalemma. Golgi bodies are more obvious at this stage. N nucleus; P plastid; V vacuole; PD plasmodesmata; VP vacuolar precipitate; S spherosome; M mitochondria; P B protein body; P R polyribosomes; G Golgi body
Fig. 2. 10 days posl-pollination. The cells of the aleurone layer are small and contain little substructure other than the nucleus and vacuoles. The adjacent nucellar layer has not yet collapsed, x 5500
Fig. 3. 15 dayspost-pollination. The initial stages of aleurone protein body development has commenced as denoted by vacuolar precipitates. Note that the vacuoles are about one fifth the size of those for the 10-day cells in Figure 2. Ribosomes in the cytoplasm are predominantly aggregated into polysomes rather than on endopIasmic reticulum, x 30,000 Fig. 4. 15 days post-pollination. Spherosomes are generally located around the periphery of the aleurone cell whereas the mitochondria, plastids, and forming aleurone protein bodies are nearer the centrally located nucleus, x 19,000
188
of a maize aleurone cell 1. The aleurone cells of the 5-to 10-day stage are characterized by large vacuoles and a diffuse cytoplasm (Fig. 2). Developmental changes in the aleurone cell are most rapid between 10 and 15 days post-pollination. The most abvious change is the reduction in size of the large vacuoles characteristic of the younger tissue by about 80% (Fig. 3). In addition, the numbers of mitochondria and ribosome-like particles have increased. Two nucleoli may be present in the large, diffuse nucleus. Nucleolar organizers at this stage are shown in Figure 4. Spherosomes, which are bound by an atypical half-unit membrane (Fig. 11), are found throughout the cytoplasm. Electron-transparent, unit-membranebound vacuoles representing the remnants of the initial large vacuoles are present, and in many cases an electron-dense precipitate can be seen within these vacuoles (Figs. 3, 4). The vacuoles, mitochondria and plastids are typically adjacent to the central nucleus whereas the spherosomes are located nearer the periphery of the cell (Fig. 4). Demarcation between the aleurone and the adjacent outer endosperm cells is still difficult at the 15-day stage. The entire nucellar layer, however, has collapsed and forms a distinct layer between the aleurone and pericarp tissues. The walls of the Neurone cells have thickened only slightly since the earlier stages, and plasmodesmata can be seen not only between neighbouring aleurone cells (Fig. 4), but between aleurone and endosperm cells also (Fig. 9). At 20 days, the vacuolar precipitates in the aleurone cells have increased in size to fill the vacuoles entirely (Fig. 5), and they have taken on the appear1AL=aleurone layer; A P B ~ a l e u r o n e protein body; C W = c e l l wall; D B = d e n s e body of the aleurone protein body; E L = e n d o sperm layer; EPB = endosperm protein body ; G = Golgi body ; M = mitochondrion; MT = microtubules; NL = nucellar layer ; NP = nuclear pore; N O = n u c l e o l a r organizer; P = p l a s t i d ; P D = p l a s m o desmata; PM = peripheral matrix of aleurone protein body; PR = polysomes; R = r i b o s o m e s ; R E R = r o u g h endoplasmic reticulum; S = spherosome; SG ~ starch grain; TC = electron-transparent core of aIeurone protein body; V = v a c n o l e ; V P = v a c u o l a r precipitate
D.J. Kyle and E.D. Styles: Aleurone Development in Maize
ance of an aleurone protein body. A positive reaction with toluidine blue indicates that these bodies are at least partly proteinaceous in content. Protein bodies are forming in the sub-Neurone endosperm cells during this period of protein body formation in the Neurone. The endosperm protein bodies are, on the average, slightly smaller, more uniform in size, and have a different substructural appearance than their counterparts in the Neurone layer (Fig. 6). They appear to be formed directly from rough ER (Figs. 7, 8), and ribosomes can be seen attached to the outer surface of the protein-body membrane (Fig. 6). The numbers of protein bodies in the endosperm cells increase from day 20 to maturity of the tissue. By 25 days post-pollination, the Neurone cells are almost completely filled with small spherosomes (average diameter about 1 i~m). The Neurone grains now are more electron dense, and electron-transparent pits are seen within many of the grains (Fig. 9). The rate of cell-wall thickening increases between 25 and 30 days, and maximum wall thickness of 1-2 gm is achieved by 35-40 days post-pollination. By 35 days the aleurone protein bodies have fully matured. Their structure is shown in Figures 10 and 11. The dense body of the grain has shrunken away from the vacuole wall and is now showing a less dense fibrous ground substance in the peripheral matrix. A large, electron-transparent core can be seen within the dense body of the grain; the grain may also contain some fibrous substructure (Fig. 11). Maize aleurone protein-bodies range in size from 1.0 to 1.5 gm, making them not only different in appearance, but much smaller in size than Neurone proteinbodies described for wheat (Morrison et al., 1975) or barley (Jacobsen et al., 1971). Spherosomes aggregate around the aleurone protein bodies only after they have fully developed (35-40 days post-pollination). The spherosomes are surrounded by a membrane, but where it is adjacent to a unit membrane of the aleurone protein body (Fig. 11, insets), it is clear that the spherosome mere-
Fig. 5 . 2 0 days post-pollination. The amount of precipitate within the vacuole has increased but the internal structure of the developing aleurone protein body is still diffuse, x 45,000 Fig. 6. 25 days pos~-pollination. The developing endosperm protein bodies can be seen, with clearly defined ribosomes on the surface. • 46,000 Fig. 7. 30 days post-pollination. Formation of endosperm protein bodies may commence by intracisternal dilations of the rough ER. Note the unusually large endosperm protein body in upper right. • 35,000 Fig. 8. 30 days post-pollination. Very small endosperm protein bodies can also be seen associated with rough ER. Formation of these protein bodies may also be from swellings at the terminus of the rough ER (arrow). x 35,000 Fig. 9. 25 days post-pollination. The aleurone protein bodies now appear much more densely packed than in earlier stages. Note that intercellular communication (plasmodesmata) is occurring between cells of the aleurone layer and cells of the endosperm layer, x 40,000
D.J. Kyle and E,D. Styles: Aleurone Development in Maize
189
Fig. 10. 35 days post-pollination. A mature aleurone protein body with its substructure defined in terms of a peripheral matrix, the dense body, a n d a central transparent core. • 43,000 Fig. 11.35 days post-pollination. The final stage of aleurone protein body development is demonstrated by the apposition of spherosomes to the protein-body surface. Higher magnification of these interfaces clearly shows that the spherosome m e m b r a n e does not have the tripartite structure of a normal unit m e m b r a n e (insets). x 52,000; insets x 70,000
Fig. 12. 35 days post-pollination. The mature aleurone tissue changes very little from now to desiccation. Note the preponderance of Golgi bodies, x 20,000 Fig. 13. 35 days post-pollination. At this stage further development of the aleurone layer has stopped but protein bodies continue to be formed in the underlying endosperm (arrow). x 22,000
192
D.J. Kyle and E.D. Styles: Aleurone Development in Maize
brane does not have the normal tripartite structure. As the maize kernel reaches full maturity and begins to dehydrate, the spherosomes completely surround the aleurone protein bodies and line the inside periphery of the plasma membrane. Fewer mitochondria and plastids are seen in 35day-old aleurone cells, but Golgi bodies are more prevalent than in previous stages. Almost the entire volume of the cytoplasm now consists of spherosomes and aleurone protein bodies (Fig. 12). The small sub-aleurone endosperm cells retain their 25-day-old appearance even after 40 days (Fig. 13). They are conspicuous by their lack of the large starch grains typical of the deeper endosperm cells. The continued production of protein bodies and the presence of numerous mitochondria indicates that these cells are still active after the aleurone cells have matured.
Discussion Protein Bodies
The aleurone layer of maize represents a highly specialized, structurally complex layer of endosperm cells. The size and substructure of mature maize aleurone protein-bodies is markedly different from that of similar bodies in other species (Morrison et al., 1975; Rest and Vaughan, 1972; Jacobsen et al., 1971 ; Briarty et aI., I969) as weli as from that of the underlying endosperm tissue. Figure 14 represents a scheme for the development of the protein bodies of the maize aleurone layer as deduced from our electron-microscopic observations. Visible developmental changes began by 15 days post-pollination. At this time, regions of dense (protein) precipitate appeared in the center of the
cell vacuoles. Morrison et al. (1975) have described a similar occurrence during the initial stages of the development of protein bodies in wheat aleurone. The vacuolar precipitates in the wheat aleurone, however, were much more densely staining than those of maize; they showed compact concentric layers and were therefore described as the Type I phytin inclusions such as those originally described by Fulcher (1972). The amount of rough endoplasmic reticulum (ER) present at this stage is extremely small compared to the numbers of free ribosomes seen. This observation indicates that the vacuolar precipitates or the protein component of the vacuolar precipitates originates from the free cytoplasmic ribosomes rather than from rough ER. The protein accumulates in the vacuole over the next 15 days and condenses into an electrondense spherical structure that has an electron-transparent core and is surrounded by a fibrous ground substance (peripheral matrix). In some protein bodies, the electron-transparent core can also be seen to contain a small amount of ground substance similar to the peripheral matrix as in Figure 11. The protein bodies of the endosperm differ from those of the aleurone tissue in structure and origin. Khoo and Wolf (1970) have described the formation of maize endosperm bodies from rough ER either by cisternal dilations or enlargements of the ends of the hR. The presence of polysomes on the surface of these bodies has been shown by Burr and Burr (1976). Our observations lead us also to believe that the endosperm protein bodies are formed from rough ER (Figs. 7, 8). [n addition, ribosomes can be cleariy discerned on the surface of these protein bodies (Fig. 6) whereas they were never seen on the surface of the aleurone protein bodies. The substructures of the aleurone and endosperm protein bodies also differs significantly.Thus, the aleurone layer which is derived from the endosperm tissue, has differentiated
I 15 D A Y S
20
DAYS
25 DAYS
35 D A Y S
Fig. 14. The development of aleurone protein bodies in maize. Protein precipitates are first detected in vacuoles about 15 days from pollination. Protein continues to accumulate for an additional 10 days. 25 days post-pollination the protein condenses forming a large electron transparent core. The fully developed protein body is denoted by the apposition of spherosomes to the protein body surface
D.J. Kyle and E_D. Styles: Ateurone Development in Maize
in maize to the extent that a different mechanism of protein-body development is operating and as a result two distinct classes of protein bodies can be identified in these adjacent tissues.
193 The authors thank Mr. Jack Dietrich for his invaluable technical assistance and helpful consultations throughout this study. We also wish to thank Mr. B. Zytaruk for his help in preparing the photomicrographs.
References
Spherosomes High-magnification views of spherosomes from the aleurone tissue demonstrate the peculiarity of the limiting membrane. Rather than the typical tripartite structure of a unit membrane only a single limiting line can be seen around these bodies. This is particularly clear when seen adjacent to a typical unit membrane such as that surrounding protein bodies (Fig. 11, insets). Yastu and Jacks were first to describe the "half-unit membrane" in spherosomes from peanut seeds Arachis hypogaea (1972). They interpret these membranes as representing a phospholipid monolayer with the apolar regions directed toward the center of the lipid-containing spherosomes. This hypothesis would also explain the observation of limiting membranes without the typical tripartite structure around the spherosomes of the maize aleurone tissue. Frey-Wyssling et al. (1963) and Schwartzenbach (1971) have proposed hypotheses involving an origin for spherosomes from the ER. Smith (1974) differentiates between spherosomes and oil bodies, suggesting that spherosomes are fi-glycerophosphatase positive and are produced by the ER while oil bodies are deposits o f " reserve oil" which are produced adjacent to areas of electron-dense particulate matter. We have observed no small spherosome inclusions as required by the hypothesis of Schwartzenbach. On the other hand, although many areas of electron-dense particulate matter similar to those described by Smith were seen, in no case were developing prospherosomes seen associated with them. Spherosomes of the maize aleurone develop rapidly between 10 and 15 days postpollination, and thus further information on the origin of these bodies might be obtained by studying Neurone development during this crucial time period. This research was funded by NRC of Canada and the University of Victoria.
Adams, C.A., Novellie, L. : Composition and structure of protein bodies and spherosomes isolated from ungerminated seeds of Sorghum biocolor (Linn.) Moench. Plant Physiol. 55, 1-6 (1975) Briaty, L.G., Coult, D.A., Boulter, D.: Protein bodies of developing seeds of Viciafaba. J. Exp. Bot. 20, 358-372 (1969) Burr, B., Burr, F.A. : Zein synthesis in maize endosperm by polyribosomes attached to protein bodies. Proc. Nat. Acad. Sci. USA 73, 515-519 (1976) Buttrose, M.S. : Ultrastructure of developing aleurone cells of the wheat grain. Austr. J. Biol. Sci. 16, 768-774 (1963) Duvick, D.N.: Protein granules of maize endosperm ceils. Cereal Chem. 38, 374-385 (1961) Frey-Wyssling, A., Grieshaber, E., Miihlethaler, K.: Origin of spherosomes in plants. J. Ultrastruct. Res. 8, 506-516 (i963) Fulcher, R.G., O'Brien, T.P., Lee, J.W.: Studies on the aleurone layer. I. Conventional and fluorescence microscopy of the cell wall with emphasis on phenol-carbohydrate complexes in wheat. Austr. J. Biol. Sci. 25, 23-34 (1972) Jacobsen, J.V., Knox, R.B., Pyliotis, N.A. : The structure and composition of aleurone grain in the barley aleurone layer. Planta (Berl.) 101, 189-209 (1971) Jones, R.L.: The fine structure of barley aleurone cells. Planta (Berl.) 85, 359 375 (1969) Khoo, V., Wolf, M.J. : Origin and development of protein grannies in maize endosperm. Amer. J. Bot. 57, 1042 1050 (1970) Longo, C.P., Longo, G.P.: The development of glyoxysomes in peanut cotyledons and maize scutella. Plant Physiol. 45, 249-254 (1969) Morrison, I.N., Kuo, J., O'Brien, T.P.: Histochemistry and fine structure of developing wheat aleurone cells. Planta (Bed.) 123, 105 116 (1975) Mottier, D.M.: On certain plastids, with special reference to the protein bodies of Zea, Ricinus, and ConophoIisl Ann. Bot. 35, 349-369 (I921) Rest, J.A., Vaughan, J.G.: The development of protein and oil bodies in the seed of Sinapis alba L. Planta (Berl.) 105, 245-262 (1972) Schwartzenbacb, A.M. : Observations on spherosome membranes. Cytobiol. 4, 145-147 (1971) Smith, C.O. : The ultrastructural development of spherosomes and oil bodies in the developing embryo of Crambe abyssinica. Planta (Berl.) 119, 125-142 (i974) Taiz, L., Jones, R.L.: Plasmodesmata and an associated cell wall component in barley aleurone tissue. Amer. J. Bot. 60, 67-75 (1973) Yatsu, L.Y., Jacks, T,J. : Spherosome membranes. Half unit-membranes. Plant Physiol. 49, 737-943 (1972)
Received 2 February ; accepted 22 August 1977
