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Tubular structures in endoplasmic reticulum of cultured broccoli
Printed in Sweden Copyright ~ 1974 by Academic Press, Inc. All rights of reproduction in any form reserved
92
J. ULTRASTRUCTURERESEARCH48, 92-101 (1974)
Tubular Structures in Endoplosmic Reticulum of Cultured Broccoli S. G. QUAN,1 E. Y. C m * and S. M. CAPLIN
Department of Biology, California State University, Los Angeles, Los Angeles, California 90032, and *Department of Pathology, School of Medicine, University of Washington, Seattle, Washington 98195 Received October 29, 1973, and in revised form January 24, 1974 Tubular structures were found within the cisternae of smooth-endoplasmic reticulum (ER) of root tip cells of broccoli pith tissues in culture. The straight, unbranched tubules were 26-34 nm in diameter and more than 1 #m in length. They were found only within parenchyma and differentiating sieve tube cells, but not in fully differentiated cells. No tubules were found in root-ER of germinated seeds. A scheme for the development of these tubules is presented. Within the endoplasmic reticulum (ER) of a variety of animal cells tubular structures 18-26 n m in diameter have recently been reported (1). I n plants similar structures 29-30 n m in diameter have also been observed in E R of egg, zygote, and y o u n g embryo of cotton (15), as well as 12-15 n m diameter tubules in E R of Coleus p a r e n c h y m a cells redifferentiating into tracheary elements (13). Broccoli pith grown in tissue culture (23) were observed to contain tubular structures within developing roots. Tubular structures were f o u n d in developing sieve tube, p a r e n c h y m a and meristematic cells but not in callus tissue. The tubules and their development are described.
METHODS A N D MATERIALS Broccoli (Brassica oleracea var. italiea) stem inflorescences were obtained from the local supermarket. These were sterilized by immersion in 10% Clorox with a trace amount of Tween 80 for 5 minutes, then rinsed in several changes of sterile double-distilled water. Pith explants weighing 6-8 mg were cut from 2-mm transverse slices of the inflorescence axis using a 1.8-mm cannula. Five explants were inoculated in 30 ml of liquid White's basal (30) medium containing, in ppm, 0.1 naphthalene acetic acid, 0.3 kinetin, 25 casein hydrolyzate, 100 myo-inositol, 2.5 calcium pantotbenate, and 2% sucrose. Cultures were grown in 1 Present address: Pasadena Foundation for Medical Research, 99 North E1 Molino Avenue, Pasadena, California 91101.
ENDOPLASMIC RETICULUM TUBULES
93
FIG. 1. Overall distribution of endoplasmic reticulum (ER) tubules. Cross section of the outer cells in the root meristem region. Three cells contain ER tubules (single arrow). Cell on the left shows ER with swollen cisternae ends (double arrow) connected by a narrow strand of smooth ER. × 12 000.
94
QUAN, CHI AND CAPLIN
125-ml flasks in a rotary shaker (Psychrotherm, New Brunswick Scientific) with a 1-inch radius set at 120 rpm. Fluorescent lights were adjusted to 200 ft-c at the.!evel of the cultures and a 12-hour photoperiod. The growth period was 14 days at 28°G.) Small pieces of callus tissue were removed from undifferentiated cultures, from cultures with differentiated roots, and from roots germinated from seeds. These were fixed in a mixture of 3% paraformaldehyde and 5% glutaraldehyde in 0.1 M phosphate buffer at pH 6.8 under vacuum for 2 hours at room temperature, then washedin buffer and postfixed in buffered 2% osmium tetroxide for 15-18 hours at 4°C. After dehydration in an acetone graded series into propylene oxide, the tissues were embedded in E p o n 812. Sections were cut with a diamond knife on a Porter-Blum MT-2 ultramicrotome, stained successively with uranyl acetate and Sato's Lead Stain (25), and then examined with'an RCA EMU-3G.
RESULTS T u b u l a r structures were f o u n d within swollen E R f r o m the root meristematic region of the cultured pith explants (Fig. 1). N o similar structures w e r e f o u n d in the root cap cells. However, similar tubules were observed frequently :in differentiating sieve elements a n d were morphologically different f r o m tubules observed in the P-protein bodies (Fig. 2) of y o u n g sieve cells a n d cytoplasmic m i c r o t u b u l e s (Fig. 3). The fine structure of the tubules in s m o o t h - E R show a hollow center a n d measure 26-34 n m in diameter. I n section, the t u b u l e s appear to have s u b u n i t s (Fig. 4, arrow) b u t n o distinct or regular p a t t e r n could always be detected, i n l o n g i t u d i n a l a n d obliquely l o n g i t u d i n a l sections tubules were more t h a n 1 # m 10ng (Fig. 5). These were mostly straight, or occasionally curved, with walls u n i f o r m in thickness a n d diameter. F r e q u e n t l y b o t h l o n g i t u d i n a l a n d cross sections of tubules appear within
FIG. 2. Relationship of the ER tubules to rough-ER (rER), nucleus (N), and P-protein body (PP). Cross section of the tubules are in the center of a differentiating sieve element. Nuclear pores are evident. Subunits (arrow) may be seen in cross section, x 71 000. FIG. 3. Cytoplasmic microtubules (arrow) near the cell wall (W). × 29 000. FIG. 4. Cross and longitudinal sections of mature tubules within a cell. Some of the tubules appear to be piled on top of one another and at various angles to each other. Subunits in the tubule wall (arrow) may be observed. × 100 000. FIG. 5. Section of an individual ER cisterna containing tubules. Longitudinal, cross, and oblique sections can be observed, x 54 000. FIGS. 6-9. ER relationships and configuration to tubules. FIG. 6. Tubules oriented in different directions within ER. Cross and longitudinal sections of the tubules are interspersed. Longitudinal tubules appear to be either piled or on different planes. x 18 000. Fla. 7. Dilated rough-ER (dER) filled with a proteinaceous material. Relationship between the ER and a dilated cisterna neck (double arrow) showing continuity. Below the dilated ER is a swollen srnooth-ER cisterna filled with tubules (single arrow), x 15000. FIG. 8. Cell near center of root showing different configurations of swollen tubule containing ER cisternae. × 15 000. FI~. 9. Tubules within four separate ER cisternae, all about the same stage of development. x 34 000.
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a single swollen ER cisterna (Fig. 6). Aggregated and piled tubules were also observed within the cell (Fig. 7). Serial sections of the meristematic region showed that the distribution of the tubules was approximately the same throughout. One striking difference between cells on the periphery and those more central was the size of the tubule containing cisternae. Cells near the root surface usually had more swollen cisternae (Fig. 9) than the more central cells (Fig. 8). However, no discernible correlation could be made between the size of the cisternae and the number of tubules. The sequence of events during the development of tubular structures in swollen E R cisternae is shown in Figs. 10-14. Initially, ER containing amorphous material, probably proteinaceous, shows a concurrent gradual buildup of tubule walls (Fig. 11, arrow) and a decrease in amorphous matrix. The core of the tubules is at first very dense and the walls are irregular in diameter. DISCUSSION The existence of tube-like structures within ER have been reported (1, 7, 13-I6, 21, 26, 29). With these observations questions have been raised (16, 21, 27) concerning the origin, function, and possible relationships to cytoplasmic microtubules. ER tubules located in rough-ER were either crooked or curved (15, 16), whereas tubules in broccoli observed within smooth-ER were mostly straight. Previously reported ER tubules were aligned in one direction (1, 13, 14, 26, 27), whereas our tubules were frequently oriented in many directions (Fig. 6). Location in the cell was another difference among broccoli tubules, ER containing tubules in other plants and animals, and cytoplasmic microtubules in general. Hepler and Newcomb (13) found "fibrils" measuring 12-16 nm in diameter in ER long the cell wall of Coleus. Steer and Newcomb (27) found a high concentration of tubules (29 and 55-66 nm) throughout the perinuclear cytoplasm of bean, whereas tubules in broccoli (26-34 nm in diameter) were randomly distributed in the cytoplasm. Cytoplasmic microtubules in broccoli were found along the cell wall and rarely in any other part of the cell. They were not observed as frequently as tubules contained in ER. Cytoplasmic microtubules (Fig. 3) and the ER tubules (Fig. 5) may be distinguished as follows. ER-tubule diameter is 26-34 nm with a dense wall 2-7 Fins. 10-14. Developmental sequence of ER tubules. FIG. 10. Clusters of many swollen smooth-ER (arrows) filled with electron dense material. × 26 000. FIG. 11. Tubules beginning to form (arrows) and emerge from the mass of material present in the cisternae, x 17 000. Fins. 12 and 13. Further differentiation (arrows) of tubules from electron dense material. Note less dense and more even background of cisterna interior. Fig. 12. × 28 000. Fig. 13. x 23 000. F~e. 14. Mature tubules in smooth-ER. ER cisterna membrane appears to be continuous (arrow) with the nuclear envelope (NE). Greatly extended mitochondria (M) appear. × 37 000.
100
QUAN, CHI AND CAPLIN
nm thick and a lumen l 5-23 nm in diameter, while cytoplasmic microtubule diameter is 13-20 nm with a 3-6 nm wall and a light interior 7-11 nm in diameter. Unlike the tubules in bean (27), cotton (15), osteosarcoma (16), and cerebral tissue (1), which are found within and sometimes contiguous with the ER membrane, the tubules seen in this investigation were free of connections during their ontogeny. Other ER tubules have been suggested to develop from outgrowths of the ER membrane. In endothelial cells and macrophages anastomosing tubules, 24-27 nm in diameter, appear to form by invagination or budding of the rough-ER membrane (1). Cotton tubules 29-31 nm in diameter (15) were observed to be formed by nuclear or ER membrane invagination and frequently to branch and fuse with each other. In bean, two tubule sizes were present (27). Tubules 29 nm in diameter were formed in clusters from swollen elements of the ER. Larger tubules, 56-66 nm, were suggested to have come from disaggregation and reassembly of small tubules, or intercalation of subunits into small tubules. The intracisternal tubules in Vacuolaria (14) are thought to be formed from proteins synthesized by ribosomes, transported into cisternae (24) and assembled at nucleating sites on the ER wall or by subunits added to existing tubules. The scheme of tubular development in broccoli shows a decrease in the amorphous matrix (Figs. 11-13), which is probably proteinaceous. The matrix decrease suggests that the tubules form by self-assembly from subunits in the matrix. Hepler and Newcomb (13) suggest a similar developmental sequence for their Coleus "fibrils." Several functions for ER containing tubules have been suggested. Tubules in cotton function to reduce cell volume (15). O'Brien suggested that tubules in the bean may play a role in maintaining cell shape and also perhaps cytoplasmic streaming (21). Hepler and Newcomb's small tubules or "fibrils" in Coleus represent developmental stages of secondary wall microfibrils (13). Conversely the function of the tubule structures may be species specific as in mastigoneme production within the Golgi body in brown algae and the golden alga Ochromonas (8-10), and in flagellar hair formation within ER of the yellow-green alga Vacuolaria
virescens (14). ER containing tubules morphologically often resemble plant vacuoles. Recent studies of vacuolation in plants (2, 11) provide strong evidence for vacuoles as derivatives of ER (2-4, 19, 20, 22) but not of dictyosomes (18, 28). Concurrently it has also been suggested that dictyosomes and vacuoles represent two different specializations of the ER (2, 20). With this idea in mind, it is conceivable that the developmental pathway of ER tubules in broccoli could be similar to theca (17) and mastigoneme development (8-10) and to cell wall formation in Pleurochrysis and
Cricosphaera (5, 6). The discrepancies between frequency and orientation of tubules in different
ENDOPLASMICRETICULUMTUBULES
101
organisms may be related to the metabolism of the cell in relation to the organism. Specific materials may accumulate in the cisternae in response to the physiological state of the cell. Broccoli tissue examined here was grown in vitro. Growth in culture provides different conditions for growth from in situ conditions. Perhaps tubule structures in the ER cisternae are formed in response to culture conditions, or to eliminate or transport metabolic by-products. The absence of the ER tubules in roots germinated from seeds may support this idea. F r o m these observations it must be concluded that in tissue cultures the metabolism of the cells under consideration is changed in such a way that material capable of condensing into tubules is accumulated in the ER cisternae. REFERENCES 1. 2. 3. 4. 5. 6.
7. 8. 9.
10. 11. ]2. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30.
BARINGER,J. R. and SWOVELAND, P., J. Ultrastruct. Res. 41, 270 (1972). BERJAK, P., Ann. Bot. 36, 73 (1972). BERJAK, P. and VILL~ERS,T. A., New PhytoL 69, 929 (1970). BRACKER,C. E., GROVE,S. N., HEINTZ,C. E. and MORRO,D. J., Cytobiologie 4, 1 (1971). BROWN, R. M., J. Cell Biol. 41, 109 (1969). -ibid. 59, 35a (1973). BONNETT, H. T. and NEWCOMB, E. H., or. Cell Biol. 27, 423 (1965). BoucK, G. B., J. Cell Biol. 40, 446 (1969). -ibid. 50, 362 (1971). - - - - Advan. Cell Mol. Biol. 2, 237 (1972). F[NERAN, B. A., d. Ultrastruct. Res. 43, 75 (1973). GIFrORD, E. M. and STEWART, K. D., J. Cell Biol. 33, 131 (1967). HzWER, P. K. and Nsweo~B, E. H., J. Cell Biol. 20, 529 (1964). H~YWOOD, P., or. Ultrastruct. Res. 39, 608 (1972). JENSEN, W. A., J. Ultrastruct. Res. 22, 296 (1968). JENSON, A. B., SVJUT, H. J., SM~Trt, M. N. and RAPe, F., Cancer 27, 1440 (1971). MANTON, I. and PARKE, M., J. Mar. BioL Ass. U.K. 45, 743 (1965). MARINOS, N, G., J. Ultrastruct. Res. 9, 177 (1963). MAT1LE, P. and MOOR, H., Planta (Berl.) 80, 159 (1968). MESQUITA,J. F., J. Ultrastruet. Res. 26, 242 (1969). O'BR~EN, T. P., Z Cell Sci. 2, 557 (1967). Poux, N., J. Microse. (Paris) 1, 55 (1962). QUAN, S. and CAPLIN, S. M., In Vitro 7, 259 (1972). RSD~AN, C. M. and SABATINI,D. D., Proc. Nat. Acad. Sci. U.S. 56, 608 (1966). SATO, T., J. Electronmicrosc. 16, 133 (1967). SCHNEW, E. and DEICr~GRXSER, G., J. Microsc. (Paris) ~4, 361 (1972). STEER, M. W. and NEWCOMt3, E. H., Protoplasma 67, 33 (1969). U~DA, K., Cytologia 31, 461 (1966). VALERI, V., GONCALVZS,R. P., CRUZ, A. R. and LAICINE, E. M., or. Ultrastruet. Res. 35, 197 (1971). WroTE, P. R., Handbook of Plant Tissue Culture. Jacques Cattell Press, Lancaster, Pennsylvania, 1943.
92
J. ULTRASTRUCTURERESEARCH48, 92-101 (1974)
Tubular Structures in Endoplosmic Reticulum of Cultured Broccoli S. G. QUAN,1 E. Y. C m * and S. M. CAPLIN
Department of Biology, California State University, Los Angeles, Los Angeles, California 90032, and *Department of Pathology, School of Medicine, University of Washington, Seattle, Washington 98195 Received October 29, 1973, and in revised form January 24, 1974 Tubular structures were found within the cisternae of smooth-endoplasmic reticulum (ER) of root tip cells of broccoli pith tissues in culture. The straight, unbranched tubules were 26-34 nm in diameter and more than 1 #m in length. They were found only within parenchyma and differentiating sieve tube cells, but not in fully differentiated cells. No tubules were found in root-ER of germinated seeds. A scheme for the development of these tubules is presented. Within the endoplasmic reticulum (ER) of a variety of animal cells tubular structures 18-26 n m in diameter have recently been reported (1). I n plants similar structures 29-30 n m in diameter have also been observed in E R of egg, zygote, and y o u n g embryo of cotton (15), as well as 12-15 n m diameter tubules in E R of Coleus p a r e n c h y m a cells redifferentiating into tracheary elements (13). Broccoli pith grown in tissue culture (23) were observed to contain tubular structures within developing roots. Tubular structures were f o u n d in developing sieve tube, p a r e n c h y m a and meristematic cells but not in callus tissue. The tubules and their development are described.
METHODS A N D MATERIALS Broccoli (Brassica oleracea var. italiea) stem inflorescences were obtained from the local supermarket. These were sterilized by immersion in 10% Clorox with a trace amount of Tween 80 for 5 minutes, then rinsed in several changes of sterile double-distilled water. Pith explants weighing 6-8 mg were cut from 2-mm transverse slices of the inflorescence axis using a 1.8-mm cannula. Five explants were inoculated in 30 ml of liquid White's basal (30) medium containing, in ppm, 0.1 naphthalene acetic acid, 0.3 kinetin, 25 casein hydrolyzate, 100 myo-inositol, 2.5 calcium pantotbenate, and 2% sucrose. Cultures were grown in 1 Present address: Pasadena Foundation for Medical Research, 99 North E1 Molino Avenue, Pasadena, California 91101.
ENDOPLASMIC RETICULUM TUBULES
93
FIG. 1. Overall distribution of endoplasmic reticulum (ER) tubules. Cross section of the outer cells in the root meristem region. Three cells contain ER tubules (single arrow). Cell on the left shows ER with swollen cisternae ends (double arrow) connected by a narrow strand of smooth ER. × 12 000.
94
QUAN, CHI AND CAPLIN
125-ml flasks in a rotary shaker (Psychrotherm, New Brunswick Scientific) with a 1-inch radius set at 120 rpm. Fluorescent lights were adjusted to 200 ft-c at the.!evel of the cultures and a 12-hour photoperiod. The growth period was 14 days at 28°G.) Small pieces of callus tissue were removed from undifferentiated cultures, from cultures with differentiated roots, and from roots germinated from seeds. These were fixed in a mixture of 3% paraformaldehyde and 5% glutaraldehyde in 0.1 M phosphate buffer at pH 6.8 under vacuum for 2 hours at room temperature, then washedin buffer and postfixed in buffered 2% osmium tetroxide for 15-18 hours at 4°C. After dehydration in an acetone graded series into propylene oxide, the tissues were embedded in E p o n 812. Sections were cut with a diamond knife on a Porter-Blum MT-2 ultramicrotome, stained successively with uranyl acetate and Sato's Lead Stain (25), and then examined with'an RCA EMU-3G.
RESULTS T u b u l a r structures were f o u n d within swollen E R f r o m the root meristematic region of the cultured pith explants (Fig. 1). N o similar structures w e r e f o u n d in the root cap cells. However, similar tubules were observed frequently :in differentiating sieve elements a n d were morphologically different f r o m tubules observed in the P-protein bodies (Fig. 2) of y o u n g sieve cells a n d cytoplasmic m i c r o t u b u l e s (Fig. 3). The fine structure of the tubules in s m o o t h - E R show a hollow center a n d measure 26-34 n m in diameter. I n section, the t u b u l e s appear to have s u b u n i t s (Fig. 4, arrow) b u t n o distinct or regular p a t t e r n could always be detected, i n l o n g i t u d i n a l a n d obliquely l o n g i t u d i n a l sections tubules were more t h a n 1 # m 10ng (Fig. 5). These were mostly straight, or occasionally curved, with walls u n i f o r m in thickness a n d diameter. F r e q u e n t l y b o t h l o n g i t u d i n a l a n d cross sections of tubules appear within
FIG. 2. Relationship of the ER tubules to rough-ER (rER), nucleus (N), and P-protein body (PP). Cross section of the tubules are in the center of a differentiating sieve element. Nuclear pores are evident. Subunits (arrow) may be seen in cross section, x 71 000. FIG. 3. Cytoplasmic microtubules (arrow) near the cell wall (W). × 29 000. FIG. 4. Cross and longitudinal sections of mature tubules within a cell. Some of the tubules appear to be piled on top of one another and at various angles to each other. Subunits in the tubule wall (arrow) may be observed. × 100 000. FIG. 5. Section of an individual ER cisterna containing tubules. Longitudinal, cross, and oblique sections can be observed, x 54 000. FIGS. 6-9. ER relationships and configuration to tubules. FIG. 6. Tubules oriented in different directions within ER. Cross and longitudinal sections of the tubules are interspersed. Longitudinal tubules appear to be either piled or on different planes. x 18 000. Fla. 7. Dilated rough-ER (dER) filled with a proteinaceous material. Relationship between the ER and a dilated cisterna neck (double arrow) showing continuity. Below the dilated ER is a swollen srnooth-ER cisterna filled with tubules (single arrow), x 15000. FIG. 8. Cell near center of root showing different configurations of swollen tubule containing ER cisternae. × 15 000. FI~. 9. Tubules within four separate ER cisternae, all about the same stage of development. x 34 000.
7 - 741821 J. U#rastructttre Research
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a single swollen ER cisterna (Fig. 6). Aggregated and piled tubules were also observed within the cell (Fig. 7). Serial sections of the meristematic region showed that the distribution of the tubules was approximately the same throughout. One striking difference between cells on the periphery and those more central was the size of the tubule containing cisternae. Cells near the root surface usually had more swollen cisternae (Fig. 9) than the more central cells (Fig. 8). However, no discernible correlation could be made between the size of the cisternae and the number of tubules. The sequence of events during the development of tubular structures in swollen E R cisternae is shown in Figs. 10-14. Initially, ER containing amorphous material, probably proteinaceous, shows a concurrent gradual buildup of tubule walls (Fig. 11, arrow) and a decrease in amorphous matrix. The core of the tubules is at first very dense and the walls are irregular in diameter. DISCUSSION The existence of tube-like structures within ER have been reported (1, 7, 13-I6, 21, 26, 29). With these observations questions have been raised (16, 21, 27) concerning the origin, function, and possible relationships to cytoplasmic microtubules. ER tubules located in rough-ER were either crooked or curved (15, 16), whereas tubules in broccoli observed within smooth-ER were mostly straight. Previously reported ER tubules were aligned in one direction (1, 13, 14, 26, 27), whereas our tubules were frequently oriented in many directions (Fig. 6). Location in the cell was another difference among broccoli tubules, ER containing tubules in other plants and animals, and cytoplasmic microtubules in general. Hepler and Newcomb (13) found "fibrils" measuring 12-16 nm in diameter in ER long the cell wall of Coleus. Steer and Newcomb (27) found a high concentration of tubules (29 and 55-66 nm) throughout the perinuclear cytoplasm of bean, whereas tubules in broccoli (26-34 nm in diameter) were randomly distributed in the cytoplasm. Cytoplasmic microtubules in broccoli were found along the cell wall and rarely in any other part of the cell. They were not observed as frequently as tubules contained in ER. Cytoplasmic microtubules (Fig. 3) and the ER tubules (Fig. 5) may be distinguished as follows. ER-tubule diameter is 26-34 nm with a dense wall 2-7 Fins. 10-14. Developmental sequence of ER tubules. FIG. 10. Clusters of many swollen smooth-ER (arrows) filled with electron dense material. × 26 000. FIG. 11. Tubules beginning to form (arrows) and emerge from the mass of material present in the cisternae, x 17 000. Fins. 12 and 13. Further differentiation (arrows) of tubules from electron dense material. Note less dense and more even background of cisterna interior. Fig. 12. × 28 000. Fig. 13. x 23 000. F~e. 14. Mature tubules in smooth-ER. ER cisterna membrane appears to be continuous (arrow) with the nuclear envelope (NE). Greatly extended mitochondria (M) appear. × 37 000.
100
QUAN, CHI AND CAPLIN
nm thick and a lumen l 5-23 nm in diameter, while cytoplasmic microtubule diameter is 13-20 nm with a 3-6 nm wall and a light interior 7-11 nm in diameter. Unlike the tubules in bean (27), cotton (15), osteosarcoma (16), and cerebral tissue (1), which are found within and sometimes contiguous with the ER membrane, the tubules seen in this investigation were free of connections during their ontogeny. Other ER tubules have been suggested to develop from outgrowths of the ER membrane. In endothelial cells and macrophages anastomosing tubules, 24-27 nm in diameter, appear to form by invagination or budding of the rough-ER membrane (1). Cotton tubules 29-31 nm in diameter (15) were observed to be formed by nuclear or ER membrane invagination and frequently to branch and fuse with each other. In bean, two tubule sizes were present (27). Tubules 29 nm in diameter were formed in clusters from swollen elements of the ER. Larger tubules, 56-66 nm, were suggested to have come from disaggregation and reassembly of small tubules, or intercalation of subunits into small tubules. The intracisternal tubules in Vacuolaria (14) are thought to be formed from proteins synthesized by ribosomes, transported into cisternae (24) and assembled at nucleating sites on the ER wall or by subunits added to existing tubules. The scheme of tubular development in broccoli shows a decrease in the amorphous matrix (Figs. 11-13), which is probably proteinaceous. The matrix decrease suggests that the tubules form by self-assembly from subunits in the matrix. Hepler and Newcomb (13) suggest a similar developmental sequence for their Coleus "fibrils." Several functions for ER containing tubules have been suggested. Tubules in cotton function to reduce cell volume (15). O'Brien suggested that tubules in the bean may play a role in maintaining cell shape and also perhaps cytoplasmic streaming (21). Hepler and Newcomb's small tubules or "fibrils" in Coleus represent developmental stages of secondary wall microfibrils (13). Conversely the function of the tubule structures may be species specific as in mastigoneme production within the Golgi body in brown algae and the golden alga Ochromonas (8-10), and in flagellar hair formation within ER of the yellow-green alga Vacuolaria
virescens (14). ER containing tubules morphologically often resemble plant vacuoles. Recent studies of vacuolation in plants (2, 11) provide strong evidence for vacuoles as derivatives of ER (2-4, 19, 20, 22) but not of dictyosomes (18, 28). Concurrently it has also been suggested that dictyosomes and vacuoles represent two different specializations of the ER (2, 20). With this idea in mind, it is conceivable that the developmental pathway of ER tubules in broccoli could be similar to theca (17) and mastigoneme development (8-10) and to cell wall formation in Pleurochrysis and
Cricosphaera (5, 6). The discrepancies between frequency and orientation of tubules in different
ENDOPLASMICRETICULUMTUBULES
101
organisms may be related to the metabolism of the cell in relation to the organism. Specific materials may accumulate in the cisternae in response to the physiological state of the cell. Broccoli tissue examined here was grown in vitro. Growth in culture provides different conditions for growth from in situ conditions. Perhaps tubule structures in the ER cisternae are formed in response to culture conditions, or to eliminate or transport metabolic by-products. The absence of the ER tubules in roots germinated from seeds may support this idea. F r o m these observations it must be concluded that in tissue cultures the metabolism of the cells under consideration is changed in such a way that material capable of condensing into tubules is accumulated in the ER cisternae. REFERENCES 1. 2. 3. 4. 5. 6.
7. 8. 9.
10. 11. ]2. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30.
BARINGER,J. R. and SWOVELAND, P., J. Ultrastruct. Res. 41, 270 (1972). BERJAK, P., Ann. Bot. 36, 73 (1972). BERJAK, P. and VILL~ERS,T. A., New PhytoL 69, 929 (1970). BRACKER,C. E., GROVE,S. N., HEINTZ,C. E. and MORRO,D. J., Cytobiologie 4, 1 (1971). BROWN, R. M., J. Cell Biol. 41, 109 (1969). -ibid. 59, 35a (1973). BONNETT, H. T. and NEWCOMB, E. H., or. Cell Biol. 27, 423 (1965). BoucK, G. B., J. Cell Biol. 40, 446 (1969). -ibid. 50, 362 (1971). - - - - Advan. Cell Mol. Biol. 2, 237 (1972). F[NERAN, B. A., d. Ultrastruct. Res. 43, 75 (1973). GIFrORD, E. M. and STEWART, K. D., J. Cell Biol. 33, 131 (1967). HzWER, P. K. and Nsweo~B, E. H., J. Cell Biol. 20, 529 (1964). H~YWOOD, P., or. Ultrastruct. Res. 39, 608 (1972). JENSEN, W. A., J. Ultrastruct. Res. 22, 296 (1968). JENSON, A. B., SVJUT, H. J., SM~Trt, M. N. and RAPe, F., Cancer 27, 1440 (1971). MANTON, I. and PARKE, M., J. Mar. BioL Ass. U.K. 45, 743 (1965). MARINOS, N, G., J. Ultrastruct. Res. 9, 177 (1963). MAT1LE, P. and MOOR, H., Planta (Berl.) 80, 159 (1968). MESQUITA,J. F., J. Ultrastruet. Res. 26, 242 (1969). O'BR~EN, T. P., Z Cell Sci. 2, 557 (1967). Poux, N., J. Microse. (Paris) 1, 55 (1962). QUAN, S. and CAPLIN, S. M., In Vitro 7, 259 (1972). RSD~AN, C. M. and SABATINI,D. D., Proc. Nat. Acad. Sci. U.S. 56, 608 (1966). SATO, T., J. Electronmicrosc. 16, 133 (1967). SCHNEW, E. and DEICr~GRXSER, G., J. Microsc. (Paris) ~4, 361 (1972). STEER, M. W. and NEWCOMt3, E. H., Protoplasma 67, 33 (1969). U~DA, K., Cytologia 31, 461 (1966). VALERI, V., GONCALVZS,R. P., CRUZ, A. R. and LAICINE, E. M., or. Ultrastruet. Res. 35, 197 (1971). WroTE, P. R., Handbook of Plant Tissue Culture. Jacques Cattell Press, Lancaster, Pennsylvania, 1943.