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Observations on the ultrastructure of the plasmalemma in oranges
© 1966 by Academic Press Inc.
640
J. OLTRASTRUCTURERESEARCH16, 640--650 (1966)
Observations on the Ultrastructure of the Plasmalemma in Oranges WILLIAM W. THOMSON
Assistant Professor of Biology, Department of Life Sciences, University of California, Riverside, California Received December 23, 1965 The plasmalemma of parenchyma cells in the orange rind was resolved as a tripartite, asymmetrical membrane. The outer opaque layer was thicker than the inner opaque layer, and this asymmetry was seen in unstained as well as stained material. After lead citrate, uranyl acetate, and a combination of these stains, the dimensions and the staining qualities of the membrane were nearly the same. It was concluded that these stains reacted with the same components of the membrane. After permanganate staining, the dimensions of the membrane and the staining qualities of the membrane differed from the above stains and the conclusion was that permanganate was also staining other components of the membrane. Several variations in the structure of the membrane were observed. These included variations in the thickness of electron dense layers and the findings that the membrane was often symmetrical and frequently composed of five to seven layers. Small electron dense bridges extending across the light zone interposed between the opaque layers were often seen. These bridges were rather regularly spaced which suggested that the membrane was composed of subunits. The unit-membrane concept as proposed by Robertson (I3, 14) is a modification and application of the Danielli-Davson (5) paucimolecular model to the structure of all cell membranes as seen with the electron microscope. The basis of ultrastructure consideration in the unit-membrane theory is that all cellular membranes are composed of two, 20 A, electron dense layers separated by an electron transparent zone 35 A wide. Robertson suggests the two electron dense layers represent protein sheets which are separated from each other by the nonpolar, electron transparent ends of two layers of lipids. Because both dense layers are the same width, the membrane is termed symmetrical. Although several high resolution, electron microscope studies of the plasma membrane of animal cells have been published, only a few such studies on the plasmalemma of plants have appeared. Buvat (2, 3) has observed that in successful preparation of Elodea leaf and bud tissue the plasmalemma was composed of two electron-
PLASMALEMMAIN ORANGES
641
dense layers about 25 ~ thick, separated by an electron transparent space approximately 30 A in width. Grun (8) has also reported that the plasmalemma in young roots of Solanum was a triple-layered structure with a total width of about 75 ~. Although no measurements were presented, Grun concluded that the membrane was symmetrical and similar to the plasmalemma of Elodea. Based on total width and the observations that both inner and outer dense layers of the membrane were of the same approximate width (i.e., a symmetrical membrane), the plasmalemma in these plant tissues could be classified as a unit membrane according to Robertson's definition. The plasma membranes of several different types of animal cells are asymmetrical (7, 15-20), and SjSstrand (15-19) has suggested that the geometrical asymmetry and the probable differences in the chemical composition suggested by the asymmetry, argues against the all-inclusiveness of the unit-membrane concept. Moreover, recent studies have shown that cristae of mitochondria (17, 18), chloroplast membranes (9, 21), and membrane disks of photoreceptors (10, 11), are composed of subunits which are probably globular in nature. These observations tend to weigh against the simple arrangement in membranes of two continuous bimolecular lipid leaflets associated with two layers of protein. The present study is concerned with the asymmetry and structural variations of the plasmalemma of orange fruits.
MATERIALS AND METHODS Valencia and Navel oranges (Citrus sinensis L.) of various stages of ripeness from deep green to orange were used in this study. All fruits were taken from trees in Riverside, California. The rind was peeled from the fruit and most of the mesocarp (albedo) the colorless part of the rind was trimmed away. The remaining epicarp (flavedo) consisting mainly of the outer, colored portion of the rind, was cut into small sections about 2 mm 2 and placed in a fixative. Two different fixatives were used: 2% KMnO~ at room temperature for 1-2 hours; 1% phosphate-buffered osmium for 12-18 hours (21). The section was dehydrated in an acetone series and embedded in Maraglas (1). The blocks were mounted on wooden dowels and thin sections were cut with a PorterBlum MT-2 ultramicrotome. The sections were either picked up on uncoated or Formvarcoated grids; and stained on the grid with either (a) 1% Ba(KMnO4)~ for 10 minutes; (b)lead citrate for 10 minutes (12); (c) saturated uranyl acetate for 1 hour; or (d) uranyl acetate for 1 hour followed by lead citrate for 10 minutes. All staining was done at room temperature. The material was studied with a Hitachi HU 11 electron microscope equipped with a specimen chamber cooling device. All measurements were taken on micrographs that had been enlarged 3-10 times. The measurements were made with a Bausch and Lomb magnifier with a scale where each division equaled 0.1 mm.
642
w . w . THOMSON OBSERVATIONS
Fig. 1 is a low magnification survey micrograph showing the general appearance of an epicarp parencyma cell. The cell organelles are easily recognized; mitochondria, rn; developing chromoplasts, c, with large starch grains, and numerous osmiophilic globules, and the vacuole, v, with bordering tonoplast, t. The plasmalemma is barely distinguishable as a thin, dark, wavy line around the perimeter of the cell. The walls are relatively thick in these cells and pit-pairs, p, with several plasmodesmata are frequently observed. Material fixed in osmium and unstained was very low in contrast, but at high magnification the ectoplast was resolved as tripartite, having two electron dense layers separated by an electron transparent region (Fig. 2). The membrane was asymmetric in that the outer opaque layer averaged 40 A in width and was thicker than the inner opaque layer, which was about 30 A in width (Fig. 2, arrows). The electron translucent region between the two dense layers was about 30 A in width. The total width of the membrane was approximately 105 A. A considerable increase in contrast was obtained with the different stains, and the asymmetrical nature of the membrane was clearly evident and enhanced with all stains employed (Figs. 3-6). The width of the membrane was larger after staining but varied depending on the type of stain used (Table I). The greatest contribution to this variation occurred in the outer opaque layer with much smaller differences in the inner opaque layer. The width of the light interposed layer, however, was practically the same in the stained as in the unstained material. The largest values were found after permanganate staining, the membrane was about 135 A in width, while the outer, opaque layer averaged 65 A in width and the inner opaque layer about 45 A. However, the dimensions of the membrane were nearly the same after any of the other stains were used. The width of the membrane averaged about 120 ]~. The inner
TABLE I DIMENSIONS OF THE PLASMALEMMA AFTER VARIOUS S T A I N I N G PROCEDURES a Uranyl Acetate Stained
(A)
Outer opaque layer 54 _+6 Light interposed layer 28 _+4 Inner opaque layer 38 _+5 Total thickness of membrane 120_+8
Lead Citrate Stained
(h)
54 _+4 27 _+5 37 ± 5 118_+9
Uranyl Acetate and Lead Citrate Ba(KMnO4)2 Stained Stained
(A)
(A)
57 _+4 27 ± 3 37 _+4 121 _+8
64 +_7 26 ± 5 44 + 6 134_+10
a Figures shown represent an average of 20 or more individual measurements.
Unstained
(A)
40 -+6 32 -+5 31 ± 5 103__3
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FIG. 1. Epicarp p a r e n c h y m a cell of the rind of an orange. T h e mitochondria, m a n d developing c h r o m o p l a s t s , c, are easily identified. T h e vacuole, v, is bordered by the tonoplast, t, a n d the plasmal e m m a appears as a thin line along the wall, w. O s m i u m fixed, lead stained, x 8000.
644
w.w.
THOMSON
FIG. 2. The triple-layered plasmalemma as seen with osmium fixation without staining. The arrows indicate areas where the asymmetry of the membrane is evident. Note that the thickest, opaque layer borders the wail, w. x 162,000. F~G. 3. The plasmalemma as seen after osmium fixation and permanganate staining. Both inner and outer opaque layers show the same intensity of staining, but for the most part, the outer layer along the wall, w, is thicker, x 154,000. Fie. 4. The plasmalemma after osmium fixation and uranyl acetate staining. Note that the thicker, opaque layer along the wall, w, is much more intensely stained than the thin, cytoplasmic layer. x 162,000.
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FIG. 5. The triple-layered ptasmalemma after osmium fixation and lead citrate staining. The opaque layer bordering the wall, w, is thicker and more intensely stained than the opaque layer bordering the cytoplasm. × 154,000. Fra. 6. The asymmetrical, triple-layered plasmalemma after osmium fixation and section-staining with uranyl acetate followed by lead citrate. The opaque layer along the wall, w, is wider and much more intensely stained than the inner opaque layer. The arrows indicate regions where the membrane appears more complex than the usual triple-layered structure. × 150,000. FIG. 7. The plasmalemma after permanganate fixation. In general, it appears as an aggregation of electron dense granules along the cell wall, w. The arrows indicate regions that might represent a triple-layered structure. The double membrane, m, just adjacent to the plasmalemma is a limiting membrane of a chloroplast. × 146,000.
646
w . w . THOMSON
opaque layer was about 35 A in width and the outer opaque layer averaged between 55 and 60 A. With permanganate, both inner and outer layers showed a similar increase in density. With all the other stains used there was an asymmetry in staining in that the outer layer showed a marked increase in density while the inner layer was only slightly increased in opacity (compare Fig. 3 to Figs. 4-6). The plasmalemma was not uniformly tripartite (Figs. 8-10), nor was it uniformly asymmetric. In places the membrane consisted of five layers: three electron dense components and two interposed electron transparent regions (Figs. 8 and 10a). Frequently, areas were observed which suggested that some parts of the membrane may even have as many as seven layers (Fig. 9 b). In the regions where the membrane consisted of more than three layers, the general asymmetry of the membrane was not apparent. In many instances, two parts of the outer, opaque layer of the membrane overlapped one another (Fig. 10a). Fine electron dense bridges extending across the electron transparent region connecting the opaque components of the membrane were frequently observed (Figs. 8 and 10, arrows). These bridges were rather regularly spaced, and the membrane appeared to be composed of globular subunits. When potassium permanganate was used as a fixative, the plasmalemma appeared generally as a single dense line in low magnification micrographs. At higher magnification it was resolved not as a discrete membrane but as an irregular aggregate of electron dense granules (Fig. 7). In only a few places was there any suggestion of a tripartite structure (Fig. 7, arrow). DISCUSSION When osmium was used as a fixative and no section-staining was employed, the plasmalemma of orange fruits was revealed as an asymmetric structure. The outer electron dense layer was 40 A thick and the inner electron dense layer was 30 A. Interposed between these layers was an electron transparent zone 30 A in width. After section-staining there was an increase in the density of the electron dense layers and measurements of the opaque layers gave higher values than in the unstained material. However, there was no apparent change in the width of the electron-transparent zone interposed between the two electron dense layers. Since the width of this electron transparent zone remained constant, the stains probably reacted with the same layers that osmium does, as well as with other surface components of the membrane. Uranyl acetate, lead citrate, or uranyl acetate followed by lead citrate produced the same general results as stains. The inner electron dense layer was about 35 A and the outer dense layer about 55 A after any of these stains were employed. Further, these stains had a similar asymmetrical staining pattern. That is, the outer layer was con-
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647
Fins. 8-10. High magnification micrograpbs of the plasmalemma after osmium fixation and permanganate staining. In Figs. 8 and 10 the membrane at a is composed of three electron dense layers and two interposed light zones. At b, in Fig. 9 the membrane appears to have seven layers. The arrows in Figs. 8 and 9 indicate regions where fine electron dense bridges extend from across the light zone and in these regions the membrane appears to be composed of small, globular subunits, x 400,000. s i d e r a b l y increased in density while the inner layer was only slightly e n h a n c e d (Figs. 4-6). These similarities in staining p r o p e r t i e s suggested that these stains react with the same c o m p o n e n t s of the m e m b r a n e . I n c o m p a r i s o n , the widths of the m e m b r a n e a n d each o p a q u e layer was significantly wider after p e r m a n g a n a t e staining. M o r e o v e r , the inner layer also showed a decided increase in density ( c o m p a r e Fig. 3
648
w . w . THOMSON
to Figs. 4-6). These differences in width, and particularly the difference in the density of the inner layer, suggested that permanganate was staining constituents of the membrane in addition to those stained by uranyl and lead salts. Measurements of membranes and other structure revealed by the electron microscope were subject to several sources of error. These sources of error included the possible changes in the membrane induced by the preparatory techniques as well as the somewhat arbitrary problem of defining the limits of a membrane on an electron micrograph. Therefore, in order to establish a basis for comparison and evaluation, two criteria for measurements were used in this study. First, although the membrane was not uniformly tripartite or asymmetrical, it was predominantly so. Thus, measurements were made only where the membrane was clearly tripartite and asymmetric. However, variations from this pattern were not considered any less real. Secondly, accurate measurements were only possible where the membrane was oriented perpendicular to the surface of the section. It was recognized that often, and probably generally, the membrane would be canted in the section and measurements in these regions would be inaccurate since they would reflect the width of the membrane plus the slant of the membrane in the plastic. All measurements were made at points along the membrane where the edge of the membrane was rather clearly delineated. These regions tended to be where the membrane showed the smallest overall dimensions. Many areas where the membrane is clearly delineated are only a few angstroms from regions where the membrane is considerably wider and the boundaries of the membrane are quite diffuse. However, the electron transparent region between the two opaque layers measures about 30 A where the membrane boundaries are sharp as well as in many areas where the boundaries are quite diffuse. This electron transparent region should be smaller or nonexistent if the membrane is on a tilt, and thus the diffuseness of at least some of the surface is attributed to membrane properties per se. This may be due to one or all of the following reasons: a localized accretion or absorption of materials to the membrane, an unequal accumulation in different places of basic membrane components, or to a difference along the membrane in the state of compactness of membrane components. In osmium-fixed preparations Buvat (2, 3) has observed that the plasmalemma of Elodea leaf and bud cells is composed of two opaque layers of the same approximate width separated by a light interspace. Recently, Grun (8) found that the plasmalemma of Solanurn root cells is not clearly resolved with osmium alone but with lead or uranyl stains the membrane is tripartite and symmetrical. However, the plasmalemma of the cells in the orange fruit is tripartite and asymmetrical. Cronshaw and Bouck (4) have also observed that the plasmalemma of developing xylem element in Arena coleoptiles is triple-layered and that the outer opaque layer is usually thicker than the
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649
inner opaque layer. It may be that the asymmetry of the plasmalemma in the cells of the orange and developing xylem reflects a general difference in physiology and/or differentiation between these cells and those more nearly meristematic in which the plasmalemma is symmetrical. However, this variation may also be due to differences in fixations and staining. This question is now being investigated. A basic structural and therefore probably chemical difference exists between the plasmalemma of the cells in orange fruits and the plasma membrane of animal cells. Although the plasma membranes of many animal cells, after permanganate or osmium fixation and section-staining, are asymmetric structures (6, 7, 15, 16, 18-20), this asymmetry is characterized by the inner cytoplasmic layer being thicker than the outer layers. This is exactly the reverse of the pattern found in orange fruits where the outer layer is thicker than the inner. However, Farquhar and Palade (7) found that the usual asymmetrical pattern of the plasma membranes of the epithelial cells of frog skin is reversed if glutaraldehyde followed by osmium is used as a fixative. The plasmalemma of the cells studied in this investigation is not a uniform, asymmetric, triple-layered structure. In many places, the membrane is symmetrical and often composed of five or more layers. Most models tend to convey the idea that the membrane is a uniform structure. However, considering the physiological functions that occur at this boundary, it is not surprising that the plasmalemma has a rather complex structure. Frequently, the electron transparent zone interposed between the two dense layers of the membrane appears discontinuous. Small, opaque bridges extend across the light zone at regular intervals connecting the two dense layers. In these regions the membrane appears to be composed of small subunits. Globular subunits in mitochondrial (17, 18), cytoplasmic (18), chloroplasts (9, 22), and other types of membranes have been described and the prospect that the plasmalemma is likewise composed of subunits is an exciting one. However, until other corroborating investigations with other fixatives are done, we must consider the subunit structure of the plasmalemma only as a strong possibility. The unit membrane hypothesis as proposed by Robertson (I3, 14) is based on the concept that membranes are composed of two continuous layers of lipids coated on each side by a layer of protein. The general asymmetry of the membrane, the variations in the width of the opaque layers, and the probable chemical differences as indicated by the various staining qualities, and the possible occurrence of a subunit structure suggest that the plasmalemma in oranges is more complex than can be explained by the unit membrane hypothesis. Similarly, Sj6strand and his associates (14-20) have shown that the membranes of animal cells are quite variable in structure and have concluded that the unit membrane hypothesis is too limiting to explain all the variations seen.
650
W. W. THOMSON REFERENCES
1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22.
BISALPUTRA,T. and WEIER,T. E., Am. J. Botany 50, 1011 (1963). B~JVAT,R., Ann. Sci. Nat. Botan. Biol. Veget. 11, 122 (1958). -Intern. Rev. Cytol. 14, 41 (1963). CRONSHAW,J. and BoucK, G. B., J. Cell Biol. 24, 415 (1965). DANIZLI~, J. F. and DAVSON, H. A., J. Cellular Comp. Physiol. 5, 495 (1935). EI.FVIN, L. G., J. Ultrastruct. Res. 5, 388 (1961). FARQUHAR,M. G, and PALADE,G. E., J. CellBiol. 26, 263 (1965). GRUN, P., J. Ultrastruct. Res. 9, 198 (1963). HonE, H. R. and HEVTON, A., J. Ultrastruct. Res. 12, 542 (1965). NILSSON, S. E. G., J. Ultrastruct. Res. 11, 581 (1964). - - ibid. 12, 207 (1965). REYNOLDS,E. S., J. Cell Biol. 17, 208 (1963). ROBERTSON~J. D., Biochem. Soc. Symp. (Cambridge, Engl.) 16, 3 (1959). -Symp. Soc. Study Develop. Growth 22, 1 (1964). SJ6STRANO, F. S., in HARRIS, R. J. C., (Ed.), The Interpretation of Ultrastructure, p. 47. Academic Press, New York, 1962. - - - - J. Ultrastruct. Res. 8, 517 (1963). -ibid. 9, 340 (1963). -ibid. 9, 561 (1963). -in BOURNZ, G. H. (Ed.), Cytology and Cell Physiology, p. 311. Academic Press, New York, 1964. SJOSTRAND,F. S. and ELFVIN,L. G., J. Ultrastruct. Res. 7, 504 (1962). THOMSON,W. W., J. Exptl. Botany 16, 169 (1965). WEr~R, T. E., ENGELBRECHT,A. H. P., HARRISON, A. and RISLEY, E. B., J. Ultrastruet. Res. 13, 92 (1965).
640
J. OLTRASTRUCTURERESEARCH16, 640--650 (1966)
Observations on the Ultrastructure of the Plasmalemma in Oranges WILLIAM W. THOMSON
Assistant Professor of Biology, Department of Life Sciences, University of California, Riverside, California Received December 23, 1965 The plasmalemma of parenchyma cells in the orange rind was resolved as a tripartite, asymmetrical membrane. The outer opaque layer was thicker than the inner opaque layer, and this asymmetry was seen in unstained as well as stained material. After lead citrate, uranyl acetate, and a combination of these stains, the dimensions and the staining qualities of the membrane were nearly the same. It was concluded that these stains reacted with the same components of the membrane. After permanganate staining, the dimensions of the membrane and the staining qualities of the membrane differed from the above stains and the conclusion was that permanganate was also staining other components of the membrane. Several variations in the structure of the membrane were observed. These included variations in the thickness of electron dense layers and the findings that the membrane was often symmetrical and frequently composed of five to seven layers. Small electron dense bridges extending across the light zone interposed between the opaque layers were often seen. These bridges were rather regularly spaced which suggested that the membrane was composed of subunits. The unit-membrane concept as proposed by Robertson (I3, 14) is a modification and application of the Danielli-Davson (5) paucimolecular model to the structure of all cell membranes as seen with the electron microscope. The basis of ultrastructure consideration in the unit-membrane theory is that all cellular membranes are composed of two, 20 A, electron dense layers separated by an electron transparent zone 35 A wide. Robertson suggests the two electron dense layers represent protein sheets which are separated from each other by the nonpolar, electron transparent ends of two layers of lipids. Because both dense layers are the same width, the membrane is termed symmetrical. Although several high resolution, electron microscope studies of the plasma membrane of animal cells have been published, only a few such studies on the plasmalemma of plants have appeared. Buvat (2, 3) has observed that in successful preparation of Elodea leaf and bud tissue the plasmalemma was composed of two electron-
PLASMALEMMAIN ORANGES
641
dense layers about 25 ~ thick, separated by an electron transparent space approximately 30 A in width. Grun (8) has also reported that the plasmalemma in young roots of Solanum was a triple-layered structure with a total width of about 75 ~. Although no measurements were presented, Grun concluded that the membrane was symmetrical and similar to the plasmalemma of Elodea. Based on total width and the observations that both inner and outer dense layers of the membrane were of the same approximate width (i.e., a symmetrical membrane), the plasmalemma in these plant tissues could be classified as a unit membrane according to Robertson's definition. The plasma membranes of several different types of animal cells are asymmetrical (7, 15-20), and SjSstrand (15-19) has suggested that the geometrical asymmetry and the probable differences in the chemical composition suggested by the asymmetry, argues against the all-inclusiveness of the unit-membrane concept. Moreover, recent studies have shown that cristae of mitochondria (17, 18), chloroplast membranes (9, 21), and membrane disks of photoreceptors (10, 11), are composed of subunits which are probably globular in nature. These observations tend to weigh against the simple arrangement in membranes of two continuous bimolecular lipid leaflets associated with two layers of protein. The present study is concerned with the asymmetry and structural variations of the plasmalemma of orange fruits.
MATERIALS AND METHODS Valencia and Navel oranges (Citrus sinensis L.) of various stages of ripeness from deep green to orange were used in this study. All fruits were taken from trees in Riverside, California. The rind was peeled from the fruit and most of the mesocarp (albedo) the colorless part of the rind was trimmed away. The remaining epicarp (flavedo) consisting mainly of the outer, colored portion of the rind, was cut into small sections about 2 mm 2 and placed in a fixative. Two different fixatives were used: 2% KMnO~ at room temperature for 1-2 hours; 1% phosphate-buffered osmium for 12-18 hours (21). The section was dehydrated in an acetone series and embedded in Maraglas (1). The blocks were mounted on wooden dowels and thin sections were cut with a PorterBlum MT-2 ultramicrotome. The sections were either picked up on uncoated or Formvarcoated grids; and stained on the grid with either (a) 1% Ba(KMnO4)~ for 10 minutes; (b)lead citrate for 10 minutes (12); (c) saturated uranyl acetate for 1 hour; or (d) uranyl acetate for 1 hour followed by lead citrate for 10 minutes. All staining was done at room temperature. The material was studied with a Hitachi HU 11 electron microscope equipped with a specimen chamber cooling device. All measurements were taken on micrographs that had been enlarged 3-10 times. The measurements were made with a Bausch and Lomb magnifier with a scale where each division equaled 0.1 mm.
642
w . w . THOMSON OBSERVATIONS
Fig. 1 is a low magnification survey micrograph showing the general appearance of an epicarp parencyma cell. The cell organelles are easily recognized; mitochondria, rn; developing chromoplasts, c, with large starch grains, and numerous osmiophilic globules, and the vacuole, v, with bordering tonoplast, t. The plasmalemma is barely distinguishable as a thin, dark, wavy line around the perimeter of the cell. The walls are relatively thick in these cells and pit-pairs, p, with several plasmodesmata are frequently observed. Material fixed in osmium and unstained was very low in contrast, but at high magnification the ectoplast was resolved as tripartite, having two electron dense layers separated by an electron transparent region (Fig. 2). The membrane was asymmetric in that the outer opaque layer averaged 40 A in width and was thicker than the inner opaque layer, which was about 30 A in width (Fig. 2, arrows). The electron translucent region between the two dense layers was about 30 A in width. The total width of the membrane was approximately 105 A. A considerable increase in contrast was obtained with the different stains, and the asymmetrical nature of the membrane was clearly evident and enhanced with all stains employed (Figs. 3-6). The width of the membrane was larger after staining but varied depending on the type of stain used (Table I). The greatest contribution to this variation occurred in the outer opaque layer with much smaller differences in the inner opaque layer. The width of the light interposed layer, however, was practically the same in the stained as in the unstained material. The largest values were found after permanganate staining, the membrane was about 135 A in width, while the outer, opaque layer averaged 65 A in width and the inner opaque layer about 45 A. However, the dimensions of the membrane were nearly the same after any of the other stains were used. The width of the membrane averaged about 120 ]~. The inner
TABLE I DIMENSIONS OF THE PLASMALEMMA AFTER VARIOUS S T A I N I N G PROCEDURES a Uranyl Acetate Stained
(A)
Outer opaque layer 54 _+6 Light interposed layer 28 _+4 Inner opaque layer 38 _+5 Total thickness of membrane 120_+8
Lead Citrate Stained
(h)
54 _+4 27 _+5 37 ± 5 118_+9
Uranyl Acetate and Lead Citrate Ba(KMnO4)2 Stained Stained
(A)
(A)
57 _+4 27 ± 3 37 _+4 121 _+8
64 +_7 26 ± 5 44 + 6 134_+10
a Figures shown represent an average of 20 or more individual measurements.
Unstained
(A)
40 -+6 32 -+5 31 ± 5 103__3
PLASMALEMMA IN ORANGES
643
FIG. 1. Epicarp p a r e n c h y m a cell of the rind of an orange. T h e mitochondria, m a n d developing c h r o m o p l a s t s , c, are easily identified. T h e vacuole, v, is bordered by the tonoplast, t, a n d the plasmal e m m a appears as a thin line along the wall, w. O s m i u m fixed, lead stained, x 8000.
644
w.w.
THOMSON
FIG. 2. The triple-layered plasmalemma as seen with osmium fixation without staining. The arrows indicate areas where the asymmetry of the membrane is evident. Note that the thickest, opaque layer borders the wail, w. x 162,000. F~G. 3. The plasmalemma as seen after osmium fixation and permanganate staining. Both inner and outer opaque layers show the same intensity of staining, but for the most part, the outer layer along the wall, w, is thicker, x 154,000. Fie. 4. The plasmalemma after osmium fixation and uranyl acetate staining. Note that the thicker, opaque layer along the wall, w, is much more intensely stained than the thin, cytoplasmic layer. x 162,000.
PLASMALEMMA IN ORANGES
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FIG. 5. The triple-layered ptasmalemma after osmium fixation and lead citrate staining. The opaque layer bordering the wall, w, is thicker and more intensely stained than the opaque layer bordering the cytoplasm. × 154,000. Fra. 6. The asymmetrical, triple-layered plasmalemma after osmium fixation and section-staining with uranyl acetate followed by lead citrate. The opaque layer along the wall, w, is wider and much more intensely stained than the inner opaque layer. The arrows indicate regions where the membrane appears more complex than the usual triple-layered structure. × 150,000. FIG. 7. The plasmalemma after permanganate fixation. In general, it appears as an aggregation of electron dense granules along the cell wall, w. The arrows indicate regions that might represent a triple-layered structure. The double membrane, m, just adjacent to the plasmalemma is a limiting membrane of a chloroplast. × 146,000.
646
w . w . THOMSON
opaque layer was about 35 A in width and the outer opaque layer averaged between 55 and 60 A. With permanganate, both inner and outer layers showed a similar increase in density. With all the other stains used there was an asymmetry in staining in that the outer layer showed a marked increase in density while the inner layer was only slightly increased in opacity (compare Fig. 3 to Figs. 4-6). The plasmalemma was not uniformly tripartite (Figs. 8-10), nor was it uniformly asymmetric. In places the membrane consisted of five layers: three electron dense components and two interposed electron transparent regions (Figs. 8 and 10a). Frequently, areas were observed which suggested that some parts of the membrane may even have as many as seven layers (Fig. 9 b). In the regions where the membrane consisted of more than three layers, the general asymmetry of the membrane was not apparent. In many instances, two parts of the outer, opaque layer of the membrane overlapped one another (Fig. 10a). Fine electron dense bridges extending across the electron transparent region connecting the opaque components of the membrane were frequently observed (Figs. 8 and 10, arrows). These bridges were rather regularly spaced, and the membrane appeared to be composed of globular subunits. When potassium permanganate was used as a fixative, the plasmalemma appeared generally as a single dense line in low magnification micrographs. At higher magnification it was resolved not as a discrete membrane but as an irregular aggregate of electron dense granules (Fig. 7). In only a few places was there any suggestion of a tripartite structure (Fig. 7, arrow). DISCUSSION When osmium was used as a fixative and no section-staining was employed, the plasmalemma of orange fruits was revealed as an asymmetric structure. The outer electron dense layer was 40 A thick and the inner electron dense layer was 30 A. Interposed between these layers was an electron transparent zone 30 A in width. After section-staining there was an increase in the density of the electron dense layers and measurements of the opaque layers gave higher values than in the unstained material. However, there was no apparent change in the width of the electron-transparent zone interposed between the two electron dense layers. Since the width of this electron transparent zone remained constant, the stains probably reacted with the same layers that osmium does, as well as with other surface components of the membrane. Uranyl acetate, lead citrate, or uranyl acetate followed by lead citrate produced the same general results as stains. The inner electron dense layer was about 35 A and the outer dense layer about 55 A after any of these stains were employed. Further, these stains had a similar asymmetrical staining pattern. That is, the outer layer was con-
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Fins. 8-10. High magnification micrograpbs of the plasmalemma after osmium fixation and permanganate staining. In Figs. 8 and 10 the membrane at a is composed of three electron dense layers and two interposed light zones. At b, in Fig. 9 the membrane appears to have seven layers. The arrows in Figs. 8 and 9 indicate regions where fine electron dense bridges extend from across the light zone and in these regions the membrane appears to be composed of small, globular subunits, x 400,000. s i d e r a b l y increased in density while the inner layer was only slightly e n h a n c e d (Figs. 4-6). These similarities in staining p r o p e r t i e s suggested that these stains react with the same c o m p o n e n t s of the m e m b r a n e . I n c o m p a r i s o n , the widths of the m e m b r a n e a n d each o p a q u e layer was significantly wider after p e r m a n g a n a t e staining. M o r e o v e r , the inner layer also showed a decided increase in density ( c o m p a r e Fig. 3
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to Figs. 4-6). These differences in width, and particularly the difference in the density of the inner layer, suggested that permanganate was staining constituents of the membrane in addition to those stained by uranyl and lead salts. Measurements of membranes and other structure revealed by the electron microscope were subject to several sources of error. These sources of error included the possible changes in the membrane induced by the preparatory techniques as well as the somewhat arbitrary problem of defining the limits of a membrane on an electron micrograph. Therefore, in order to establish a basis for comparison and evaluation, two criteria for measurements were used in this study. First, although the membrane was not uniformly tripartite or asymmetrical, it was predominantly so. Thus, measurements were made only where the membrane was clearly tripartite and asymmetric. However, variations from this pattern were not considered any less real. Secondly, accurate measurements were only possible where the membrane was oriented perpendicular to the surface of the section. It was recognized that often, and probably generally, the membrane would be canted in the section and measurements in these regions would be inaccurate since they would reflect the width of the membrane plus the slant of the membrane in the plastic. All measurements were made at points along the membrane where the edge of the membrane was rather clearly delineated. These regions tended to be where the membrane showed the smallest overall dimensions. Many areas where the membrane is clearly delineated are only a few angstroms from regions where the membrane is considerably wider and the boundaries of the membrane are quite diffuse. However, the electron transparent region between the two opaque layers measures about 30 A where the membrane boundaries are sharp as well as in many areas where the boundaries are quite diffuse. This electron transparent region should be smaller or nonexistent if the membrane is on a tilt, and thus the diffuseness of at least some of the surface is attributed to membrane properties per se. This may be due to one or all of the following reasons: a localized accretion or absorption of materials to the membrane, an unequal accumulation in different places of basic membrane components, or to a difference along the membrane in the state of compactness of membrane components. In osmium-fixed preparations Buvat (2, 3) has observed that the plasmalemma of Elodea leaf and bud cells is composed of two opaque layers of the same approximate width separated by a light interspace. Recently, Grun (8) found that the plasmalemma of Solanurn root cells is not clearly resolved with osmium alone but with lead or uranyl stains the membrane is tripartite and symmetrical. However, the plasmalemma of the cells in the orange fruit is tripartite and asymmetrical. Cronshaw and Bouck (4) have also observed that the plasmalemma of developing xylem element in Arena coleoptiles is triple-layered and that the outer opaque layer is usually thicker than the
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inner opaque layer. It may be that the asymmetry of the plasmalemma in the cells of the orange and developing xylem reflects a general difference in physiology and/or differentiation between these cells and those more nearly meristematic in which the plasmalemma is symmetrical. However, this variation may also be due to differences in fixations and staining. This question is now being investigated. A basic structural and therefore probably chemical difference exists between the plasmalemma of the cells in orange fruits and the plasma membrane of animal cells. Although the plasma membranes of many animal cells, after permanganate or osmium fixation and section-staining, are asymmetric structures (6, 7, 15, 16, 18-20), this asymmetry is characterized by the inner cytoplasmic layer being thicker than the outer layers. This is exactly the reverse of the pattern found in orange fruits where the outer layer is thicker than the inner. However, Farquhar and Palade (7) found that the usual asymmetrical pattern of the plasma membranes of the epithelial cells of frog skin is reversed if glutaraldehyde followed by osmium is used as a fixative. The plasmalemma of the cells studied in this investigation is not a uniform, asymmetric, triple-layered structure. In many places, the membrane is symmetrical and often composed of five or more layers. Most models tend to convey the idea that the membrane is a uniform structure. However, considering the physiological functions that occur at this boundary, it is not surprising that the plasmalemma has a rather complex structure. Frequently, the electron transparent zone interposed between the two dense layers of the membrane appears discontinuous. Small, opaque bridges extend across the light zone at regular intervals connecting the two dense layers. In these regions the membrane appears to be composed of small subunits. Globular subunits in mitochondrial (17, 18), cytoplasmic (18), chloroplasts (9, 22), and other types of membranes have been described and the prospect that the plasmalemma is likewise composed of subunits is an exciting one. However, until other corroborating investigations with other fixatives are done, we must consider the subunit structure of the plasmalemma only as a strong possibility. The unit membrane hypothesis as proposed by Robertson (I3, 14) is based on the concept that membranes are composed of two continuous layers of lipids coated on each side by a layer of protein. The general asymmetry of the membrane, the variations in the width of the opaque layers, and the probable chemical differences as indicated by the various staining qualities, and the possible occurrence of a subunit structure suggest that the plasmalemma in oranges is more complex than can be explained by the unit membrane hypothesis. Similarly, Sj6strand and his associates (14-20) have shown that the membranes of animal cells are quite variable in structure and have concluded that the unit membrane hypothesis is too limiting to explain all the variations seen.
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W. W. THOMSON REFERENCES
1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22.
BISALPUTRA,T. and WEIER,T. E., Am. J. Botany 50, 1011 (1963). B~JVAT,R., Ann. Sci. Nat. Botan. Biol. Veget. 11, 122 (1958). -Intern. Rev. Cytol. 14, 41 (1963). CRONSHAW,J. and BoucK, G. B., J. Cell Biol. 24, 415 (1965). DANIZLI~, J. F. and DAVSON, H. A., J. Cellular Comp. Physiol. 5, 495 (1935). EI.FVIN, L. G., J. Ultrastruct. Res. 5, 388 (1961). FARQUHAR,M. G, and PALADE,G. E., J. CellBiol. 26, 263 (1965). GRUN, P., J. Ultrastruct. Res. 9, 198 (1963). HonE, H. R. and HEVTON, A., J. Ultrastruct. Res. 12, 542 (1965). NILSSON, S. E. G., J. Ultrastruct. Res. 11, 581 (1964). - - ibid. 12, 207 (1965). REYNOLDS,E. S., J. Cell Biol. 17, 208 (1963). ROBERTSON~J. D., Biochem. Soc. Symp. (Cambridge, Engl.) 16, 3 (1959). -Symp. Soc. Study Develop. Growth 22, 1 (1964). SJ6STRANO, F. S., in HARRIS, R. J. C., (Ed.), The Interpretation of Ultrastructure, p. 47. Academic Press, New York, 1962. - - - - J. Ultrastruct. Res. 8, 517 (1963). -ibid. 9, 340 (1963). -ibid. 9, 561 (1963). -in BOURNZ, G. H. (Ed.), Cytology and Cell Physiology, p. 311. Academic Press, New York, 1964. SJOSTRAND,F. S. and ELFVIN,L. G., J. Ultrastruct. Res. 7, 504 (1962). THOMSON,W. W., J. Exptl. Botany 16, 169 (1965). WEr~R, T. E., ENGELBRECHT,A. H. P., HARRISON, A. and RISLEY, E. B., J. Ultrastruet. Res. 13, 92 (1965).