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Further observations on the Golgi apparatus and its functions in cells of the wheat seedling
© 1967 by Academic Press Inc.
j. ULTRASTRUCTURERESEARCH18, 287--303 (1967)
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Further Observations on the Golgi Apparatus and Its Functions in Cells of the Wheat Seedling J. D. PICKETT-HEAPS
The John Curtin School of Medical Research, Canberra., A.C.T., Australia Received April 1, ]966 Radioautographic experiments confirm previous work indicating that Golgi bodies in root epidermal cells contribute material to the wall, and, in telophase cells, preferentially to the cell plate. Observations on other cells suggest that the endoplasmic reticulum and the Golgi apparatus are involved in the synthesis of wall precursors. In stomatal cells, vesicles probably derived from the Golgi apparatus are apparently discharged into the vacuole. Further study of the Golgi apparatus in several types of cells, and some preliminary histochemical work, indicates that progressive changes can occur across the cisternal stack, and that the cisternae of some Golgi bodies do not necessarily all have identical functions. The functions of the Golgi apparatus in the cell have been the subject of much speculation by cytologists. Their role in the production of material that is subsequently incorporated into the wall in plant cells, however, has been well documented. This was very clearly shown by Mollenhauer et al. (21) in the root cap cells of maize, and the hypertrophied nature of the Golgi bodies in this type of cell, first described by Whaley et al. (33), was later found in the root cap cells of some other plants (8, 24). Mollenhauer (19) clearly demonstrated that the Golgi apparatus was modified during maturation of the cell to give this characteristic form. Whaley and Mollenhauer (34) showed that during mitosis in root epidermal cells the cell plate forms by the fusion of vesicles that are derived from the Golgi apparatus; this was confirmed by Frey-Wyssling et al. (9), who also noted that the vesicles could be absorbed into older walls of the cells. Sievers (30) observed the phenomenon in the root hair cells of wheat. During cell plate initiation in root meristematic cells, Porter and Machado (27) noted that the young wall was formed from a similar fusion of vesicular components, but it was not possible to identify positively the source of these vesicles. In other types of plant tissue, incorporation of vesicular components into the wall is also apparent. It has been most clearly demonstrated during the course of wall synthesis in the rapidly growing pollen tube (15, 29). It appears to be a
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possible means of wall synthesis in xylem (35), phloem (36), and several other types of plant cells (11, 14, 24). Using recently introduced radioautographic techniques, Northcote and PickettHeaps (22) demonstrated unambiguously the passage of labeled material from the Golgi bodies into the wall in root cap cells. This material accumulated in the slime layer of the tissue, and chemical analysis of the labeled polysaccharides indicated that the radioactive components detected radioautographically contained mainly glucose and galactose and were therefore probably pectic in nature. In wheat xylem cells the Golgi body again can be labeled near a massive accumulation of radioactivity in the wall, although it would also appear that the endoplasmic reticulum plays an important part in the synthesis and/or deposition of the wall thickenings (23). It should not be assumed, however, that the sole function of the Golgi apparatus in plant cells is concerned with the synthesis and transport of wall material. An association of the organelle with vacuolation is possible, and Marinos suggested that small prevacuolar bodies could arise by enlargement of the entire intramembranous space of a Golgi cisterna. In the single-celled Euglena, which cannot be regarded as being a typical plant cell, Brandes (2) and Sommer and Blum (31) showed that the Golgi apparatus is associated with the formation of lysosomes, exhibiting an "acid-phosphatase" type of reaction when the cells are incubated in a Gomori medium. The observations reported in this article are further extensions of those described previously (22, 24, 25, 26); they were made primarily to investigate two possibilities: (a) that the cisternae and associated vesicles in some Golgi bodies were not all identical in appearance and function; (b) that the Golgi body is involved in vacuolation as well as cell wall deposition. MATERIALS AND METHODS Wheat seeds (Triticttm vulgare) were washed and germinated on damp filter paper in daylight at room temperature. Segments of coleoptile and root tips were fixed for electron microscopy either in 4 % potassium permanganate containing sodium and calcium chlorides (24), or in a 6 To buffered glutaraldehyde/osmium system, containing calcium chloride (25). For radioautographic experiments, intact or excised root tips and coleoptile segments were incubated either in a solution of D-glucose-6-~H (1.3 C/mmole, from the Radiochemical Centre, Amersham, Bucks.) or D-glucose 1-3H (350-500 mC/mmole, from the New England Nuclear Corporation, Boston, Massachusetts) in glass distilled water, at a concentration of 0.5-2.0 mC/ml. After an appropriate period of incubation (5 minutes to 4 hours), the tissues were washed and immediately fixed in either the permanganate or the glutaraldehyde fixatives mentioned above. For attempts at the localization of enzyme activity, the tissues were fixed in 6 % glutaraldehyde buffered to pH 7 with Veronal-acetate buffer, for ½ hour at room temperature. It
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was later found that such a buffering system is suspect (12), but excellent morphological preservation was nevertheless obtained; phosphate buffers, of course, cannot be used in such histochemical experiments. After a thorough wash, the specimens were incubated in the medium of Wachstein and Meisel (32) at pH 5, using ~-glycerophosphate as the substrate. The incubation lasted 1-4 hours at 37°C. Controls were run concurrently using: (a) no substrate, (b) adenosine triphosphate (ATP) as a substrate, and (c) c~-glycerophosphate as a substrate at pH 7. All these samples were well washed after the incubation, briefly exposed to a weak solution of ammonium sulfide, and washed again. They were then postfixed in osmium tetroxide as normal. All specimens were dehydrated in ethanol, embedded in Araldite, and sectioned on a mechanical advance or Porter-Blum microtome. Radioautographic specimens were coated with emulsion by a slightly modified method of Caro and van Tubergen (5); for details, see Northcote and Pickett-Heaps (22). All sections were stained with lead (18) for 5-15 minutes, except those examined for histochemical localizations which were not stained at all. Sections taken from specimens fixed in the glutaraldehyde/osmium system were further stained for ~3--1hour in a saturated solution of uranium acetate in 50 % ethanol. Sections were examined at 80 kV in a Philips EM 100 or EM 200 electron microscope.
OBSERVATIONS
Root epidermal cells' During the radioautographic study of the function of the Golgi apparatus in root cap cells (22), the Golgi bodies in the nearby root epidermal cells were frequently observed to be labeled, and further investigations indicated that these organelles were involved in wall synthesis and cell-plate formation. In radioautographic experiments, the walls of epidermal cells were always found to contain a large concentration of radioactive derivatives of the labeled glucose, most of the radioactivity being found at the outer facing wall surface, as in the interphase cells in Fig. 3. The Golgi bodies were also labeled to some extent in these circumstances. When the time of exposure of the tissue to labeled glucose was of short duration (ca. 10 minutes), the Golgi bodies were quite highly labeled as compared to the labeling of the wall and the rest of the cell (Fig. 1). As the time of incubation increased, there was an increasing accumulation of the labeled material at the external wall surface. During the longer periods of incubation, the starch grains in the plastids of these cells also became appreciably labeled. In previous radioautographic investigations (22), the radioactivity could be localized over the Golgi apparatus or the associated vesicles in root cap cells, since these organelles were quite large in comparison to the radioautographic resolution achieved. A similar localization could not be so clearly shown in the case of the epidermal cells, since both the organelle itself and the vesicles apparently derived from it were much smaller with respect to the radioautographic resolution obtainable. However, high magnification pictures did strongly suggest that radioactive derivatives were
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localized within the organelle itself a n d its vesicles (Fig. 4). The G o l g i bodies were also labeled in e p i d e r m a l cells at telophase d u r i n g a n d after cell plate initiation. W h e n the tissue had been p r e v i o u s l y exposed to solutions of tritiated glucoses for m o r e t h a n a b o u t 30 minutes, y o u n g walls between recently divided cells were always massively labeled when c o m p a r e d to nearby, older transverse walls [see plate L, N o r t h c o t e a n d P i c k e t t - H e a p s (22)], a n d this a c c u m u l a t i o n of labeled m a t e r i a l pres u m a b l y started d u r i n g plate f o r m a t i o n . If the r o o t was cut transversely, it was possible to find some sections which passed t h r o u g h the plate region of an early telophase cell a p p r o x i m a t e l y in the plane of the plate. I n this event, the m a j o r a c c u m u l a t i o n of labeled m a t e r i a l in the cell coincided in p o s i t i o n to the region occupied b y the collection of cell plate vesicles; m a n y n e a r b y G o l g i bodies were also l a b e l e d (Fig. 2). Subsequent fusion of r a d i o a c t i v e vesicles w o u l d o b v i o u s l y l e a d to such a highly labeled y o u n g cell wall. Before a n d d u r i n g cell plate f o r m a t i o n , some directive force m o v e s the vesicles to the plate region a n d then aligns t h e m across the a p p r o p r i a t e regions of the cell, a n d it has been suggested t h a t the i n t e r z o n a l spindle m i c r o t u b u l e s are involved in this process (7, 25, 26). R a d i o a u t o g r a p h y can p r o v i d e a clear illustration of the result of such a directive force. W h e n transverse sections of late telophase e p i d e r m a l cells were t a k e n f r o m tissue that h a d been previously e x p o s e d to tritiated glucose for fairly l o n g p e r i o d s (ca. 2 hours), the r a d i o a u t o g r a p h s showed t h a t the cell plate was highly labeled as expected. However, the n e a r b y region of the o u t e r cell wall in the same cell c o n t a i n e d a c o n s p i c u o u s l y low a m o u n t of r a d i o a c t i v e derivatives, when c o m p a r e d
ep g gc m n
cell plate Golgi apparatus guard cell mitochondrion nucleus
Key to abbreviations se subsidiary cell of stomatal complex th xylem wall thickening v vacuole w cell wall
FIGS. 1-4. Radioautographs of root epidermal cells fixed in permanganate. FIG. 1. Longitudinal section through cells exposed to G-6-SH for 10 minutes. Most of the incorporated radioactivity is associated with the Golgi bodies (g) of the cells; the outermost wall surface has accumulated some labeled material, x 5300. FIG. 2. Transverse section through cell plate region of a telophase cell, exposed to G-6-~H for 30 minutes. Many vesicles collect between the daughter nuclei prior to the formation of the cell plate. The concentration of vesicles coincides in position on the autographs with the concentration of radioactivity. Nearby Golgi bodies (g) are also labeled, x 8600. FIG. 3. Transverse section (as in Fig. 2) of a root tip exposed to G-1-3H for 2 hours. The section cut the central, late telophase cell approximately in the plane of the undulating cell plate, which therefore shows up as a twisting profile. In the interphase cells on either side, most of the radioactivity has been incorporated into the outer-facing wall surface; this contrasts with the telophase cell where the activity is located over the cell plate, not over the outer wall. x 3400. FIG. 4. Interphase cell, incubated in a solution of G-6-SH for 10 minutes. Radioactivity is associated with a Golgi body (g) and a vesicle that is being incorporated into the wall (w). x 50,000.
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with the usual pattern of incorporation observable in neighboring cells (Fig. 3). T h e central cell in Fig. 3 has been sectioned in the plane of recently f o r m e d cell plate, which, undulating across the cell, presented a twisted profile in this section. Close inspection of the cell plate clearly showed its " b e a d e d " nature, due to the uneven distribution of vesicular contents sandwiched between the new plasmalemma membranes [as in Fig. 2b of Whaley and Mollenhauer (34)]. Attempts to show phosphatase activity at the ultrastructural level in r o o t tip cells has initially met with little success, this apparently being mainly due to the problem of penetration into the cell of the components of the incubation mixture. However, the preliminary experiments have indicated that the Golgi bodies in epidermal cells can show a positive reaction when incubated in a G o m o r i medium (Fig. 5); this reaction was not f o u n d at either p H 7 or in the absence of the substrate at p H 5, or in either case using A T P as a substrate, and it was not observed in all epidermal cells examined. Most, however, showed clear indications of the reaction, which manifested itself as an electron-dense deposit in the cisternae (and frequently in an associated vesicle) of the organelle; the degree of reaction was progressively greater across the stack (Fig. 6). It was not obvious what this histochemical reaction was related to morphologically; the wall, nucleus, and often the vacuoles of the cell showed varying degrees of positive reaction, but this was not specific for any of the incubation media used.
Mer&tematic root tip cells It was h o p e d that radioautographic methods would elucidate the possible function of the Golgi b o d y in the formation of the cell plate in meristematic root tip cells. Porter and M a c h a d o (27) first described cell plate formation in such cells but were not able to identify the source of the cell plate vesicles. Their contents were electron
FIGS. 5 and 6. Glutaraldehyde/osmium-fixed cells that have been incubated in a Gomori medium at acid pH (see text). FIG. 5. Longitudinal section of root epidermal cell. Reaction products are visible in the nucleus, wall, vacuoles, and Golgi bodies (g); the reaction of the nucleus, wall, and vacuoles was not specific for this particular incubation system, x 5800. FIG. 6. Reaction product associated with the Golgi apparatus and its vesicles. The reaction is progressively more pronounced across the stack, x 26,000. Fins. 7-10. Radioautograms of root meristematic cells fixed in permanganate and sectioned longitudinally. F~G. 7. Telophase cell, exposed to G-6-3H for 4 hours. Massive incorporation of labeled material into the cell plate is evident; nearby older walls contain little radioactivity. × 8000. FIG. 8. Telophase cell after brief (10 minutes) exposure to G-6-SH at high activity (5 mC/ml). The cell plate contains some radioactivity, and under these conditions, the Golgi bodies (g) are sometimes labeled (small arrows). × 13,000. FIG. 9. Telophase cell exposed to G-6-SH for 30 minutes. The cell plate (cp) is seen between the daughter nuclei (n); some radioactivity is associated with it. Nearby elements of endoplasmic reticulum (arrowed) are often labeled in such experiments, x 17,000.
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transparent, as were both the vesicles associated with the nearby Golgi bodies, and also the tubular elements of endoplasmic reticulum in the region (which were probably also giving rise to vesicles). A large number of radioautographic experiments cannot be said to have completely clarified the situation. Glucose-l-SH (G-I-~H) never gave any significant labeling patterns in meristematic root tip cells. When glucose-6-SH (G-6-SH) was used, the amount of radioactivity incorporated into the cell plate varied, increasing as the time of incubation was prolonged. During plate formation, the vesicles contained or were associated with radioactive material, though never to the extent detectable in nearby telophase epidermal cells. The Golgi bodies in the interzone region were occasionally labeled, particularly when a very short, intense pulse of radioactive glucose was applied to the tissue (Fig. 8). Over longer periods of incubation however, the Golgi bodies in the interzone region were scarcely labeled at all when compared with the labeling of the cell plate, and in these circumstances radioactivity was more frequently associated with the nearby elements of endoplasmic reticulum (Fig. 9). During further growth of the young cell wall, there was often a marked accumulation of radioactivity in the wall when the times of incubation were long (1-4 hours, Fig. 7). As before, the Golgi bodies were almost invariably free of activity, and the endoplasmic reticulum was the only organelle consistently labeled near such walls (Fig. 10).
CoIeoptile parenchyma cells Incorporation of material derived from G-6-SH was often observed into the walls of the highly vacuolated parenchyma cells. Again, the nearby Golgi bodies were almost invariably unlabeled, but the endoplasmic reticulum was often seen to be associated with the radioactivity present in the cytoplasm near the walls (Fig. 13).
Phloem and Xylem Tissues In radioautographic experiments on root tips, young sieve-tube cells almost always showed a fairly massive incorporation into the wails, of tritiated derivatives formed FIG. 10. Longitudinal section through meristematic cells, exposed to G-6-SH for 1 hour. The transverse wall is fairly heavily labeled, and nearby elements of the endoplasmic reticulum are also associated with radioactivity. The Golgi bodies near such older walls are generally unlabeled. × 16,000. FIG. 11. Longitudinal section through a young sieve-tube cell, from a root tip exposed to G-6-~H for 2 hours, and fixed in glutaraldehyde/osmium. The wall (w) and a nearby Golgi apparatus (g) is labeled, x 33,000. FIG. 12. Young sieve-tube cell in a root tip, fixed in permanganate. The Golgi bodies are associated with two distinct types of vesicles; most appear small and gray in the photograph, and a few are larger and electron transparent, x 18,000. FIG. 13. Transverse section through three highly vacuolated coleoptile cortical cells, after exposure to G-6-SH for 2 hours and fixation in permanganate. Vacuoles (v), wall (w), and mitochondria (rn) are marked; a triangular air space is present between the cells. The walls are labeled, and nearby elements of endoplasmic reticulum are also often associated with radioactivity (arrows). In such older cells, the Golgi bodies are not normally labeled under these conditions. × 13,500.
;ii
:4
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from both G-6-BH and G-1-SH; nearby Golgi bodies were often also labeled (Fig. 11). For some reason that is not quite clear, the sieve tubes of the coleoptile showed little incorporation of tritiated glucose into either the cytoplasm or wall. This may be associated with the fact that, whereas the root tips were generally used intact, coleoptile tissue was always excised for the incubation procedure (the sieve tubes being susceptible to damage by turgor pressure changes). In the permanganate-fixed image of young sieve tube cells, vesicles associated with the Golgi bodies appeared to be of two types, since generally some larger, electrontransparent vesicles were present adjacent to the cisternae (Fig. 12). A possible fate of these vesicles cannot yet be suggested. In phloem parenchyma cells in the coleoptile the Golgi bodies were occasionally closely associated with small vacuolar components (Fig. 15), in a very similar fashion to that described in barley by Marinos (17). This was uncommon, however, and was not generally observed in other types of cells. Some radioautographic evidence suggests that the Golgi bodies can supply wall material during the formation of secondary thickening of the xylem wall in wheat coleoptile. This has been described elsewhere (23). During the las t stages of xylem differentiation, wall deposition (as measured by the incorporation of radioactive derivatives into the thickenings) had virtually ceased. At this stage, the cytoplasm showed a progressive deterioration, and cell organelles (particularly the mitochondria and plastids) were disintegrating. However, Golgi bodies were still present, and micrographs indicated that some vesicles were appreciably different from the remainder around the stack of cisternae (Fig. 14). In much older coleoptile tissues, the parenchyma cells around xylem vessels become highly vacuolated; m a n y large, electrontransparent vesicles were usually present in the cytoplasm, and similar components were apparently being derived from the Golgi bodies (Fig. 16). Generally only one of the cisternae in a given stack was associated with the enormously distended vesicle.
Guard cells in the stomata The formation of the stomatal complex in wheat leaf epidermis has already been described in detail at the ultrastructural level (26). By taking tissue at later stages of FIG. 14. Longitudinal section through a maturing xylem vessel in the coleoptile, fixed in glutaraldehyde/osmium; wall thickenings (th) are marked. The Golgi bodies at this stage of maturation are associated with two types of vesicular components; most appear small and gray, and some (arrowed) are larger and more electron transparent (cf. Fig. 12). × 26,000. FIG. 15. Phloem parenchyma cell, fixed in glutaraldehyde/osmium. The Golgi apparatus in the micrograph is very closely associated with a small vacuolar body, near the main cell vacuole (v). × 58,000. FIG. 16. Xylem parenchyma cell fixed in glutaraldehyde/osmium. One cisterna of the Golgi body is apparently producing a very large, clear vesicle (cf. Figs. 20 and 22). Other vesicular components are more usual in size and content. × 38,000. FIG. 17. Longitudinal section through young stomatal complex in leaf epidermis, fixed in glutaraldehyde/osmium. The guard cells (gc) are flanked by subsidiary cells (sc). Vacuoles (v) have appeared in the cells. The wall between the cells has thickened in the central region before splitting to form the stomatal pore; this wall does not entirely separate the guard cells (arrow). x 5500.
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growth, it has been possible to describe the postmitotic development in these cells, which gives rise to the characteristic pore structure in the epidermis of the leaf. After the symmetrical third division [see Pickett-Heaps and Northcote (26)], the complex consists of two guard cells (oblong or biconcave in overall view when the two are seen in longitudinal section), and each of these has a subsidiary cell on the outside, making the whole complex biconvex in surface view (Fig. 17). It was an invariable feature of the guard cells, confirming earlier observations (3), that they were not completely separated from one another by the wall between them; gaps were always found at each end of this wall, allowing the cytoplasm of the two cells to be continuous at all stages of growth so far observed (e.g., Figs. 17 and 18). Soon after the basic complex had been formed, all the cells elongated, and the guard cells, changing their shape became fatter. A vacuole appeared in each end of each guard cell, and these vacuoles rapidly increased in size as the cells elongated (Figs. 17 and 18). The central region of the wall between the guard cells thickened considerably (Fig. 17) prior to this region of the wall splitting apart to form the stomatal pore (Fig. 18). A large number of microtubules were always associated with this region of the wall. The cytoplasm of the guard cell was very characteristic in its appearance at this stage, since it contained numbers of large, electron-transparent, membrane-bounded bodies (Figs. 18 and 19). From the sections, it would appear that these bodies were being discharged into the vacuoles at each end of the cell (Figs. 18 and 19). Furthermore, some of the vesicles were associated with the Golgi bodies in the cells, and closer examination revealed instances when the membranes of one or two cisternae in the stack of the Golgi body, were clearly continuous with the membranes of the large vesicles. Other cisternae in the same stack were associated with much smaller vesicles, these being the more usual, darker-staining components (Fig. 20). During later stages of maturation, the cisternae of the endoplasmic reticulum occasionally became enormously distended, apparently producing a similar body or vesicle (Fig. 21). The Golgi bodies as before were generally also associated with the larger as well as some smaller type of vesicles (Fig. 21). Epidermal hair cells are also present in the epidermis of wheat leaves, and in sections of these cells too, the Golgi bodies were associated in a similar fashion with large vesicles that were apparently being discharged into the central vacuole of the rapidly elongating cells. DISCUSSION As mentioned in the introduction, the incorporation of vesicles, derived from the Golgi body, into the cell-plate and wall of root epidermal cells has been described by several authors. The results presented here confirm these observations. It has not been possible to demonstrate unambiguously the time course of the passage
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of the vesicles from the Golgi apparatus to the wall, as has been done in the root cap cells (22), but the steady accumulation of radioactivity in the walls of the cells over longer periods of incubation make it reasonable to conclude that at least part of the radioactive wall material passes to the wall via the Golgi apparatus. The Golgi bodies and outer wall of the epidermal cells were always far more heavily labeled than those of internal, more undifferentiated root tip cells; this is almost certainly because the epidermal cells must synthesize large amounts of wall materials (since the outer wall of the root tip is very thick) and probably some mucilaginous compounds, and this stimulated rate of wall synthesis would be reflected by the heavier labeling of the outer-facing wall and the organelles concerned in its deposition. When Ledbetter and Porter (16) first reported the presence of microtubules in root tip cells, they noted that these organelles were found during telophase between the daughter nuclei following mitosis. Later, Esau and Gill (7) and Pickett-Heaps and Northcote (25, 26) suggested that the microtubules probably directed the vesicles to the plate region and aligned the young wall in the correct position in the cytoplasm. Radioautography demonstrates such a highly directional movement of the cell plate vesicles; in Fig. 3, labeled wall material produced in the interzone region of a telophase cell during the incubation in tritiated glucose had clearly been directed almost exclusively into the cell plate. Very little radioactivity was detected in the adjacent part of the outer-facing wall of this cell, while the two neighboring cells show the typical concentration of labeled derivatives in the thick outer wall. Since this particular photograph was taken from tissue fixed in permanganate, the microtubules themselves are, of course, not visible. The role played by the Golgi body in cell plate and wall formation in meristematic root tip cells is still somewhat open to speculation. From the appearance of many micrographs [as in Porter and Machado (27)], it seems most likely that vesicles derived from the Golgi apparatus do contribute to the new wall material and plasmalemma. The Golgi bodies were seldom labeled even when near a heavily labeled wall; this may be due to the fact that the radioactivity of the cell plate or wall represents an accumulation of the label over the incubation period, whereas the labeling of the Golgi bodies might merely represent the transitory concentration of labeled precursors present in the organelle. A similar dilemma exists in accounting for the labeling pattern of the Golgi apparatus associated with xylem wall growth (23). The most striking labeling of the Golgi bodies observed so far, occurs in cells which are producing large amounts of wall or similar type of material, namely the outer root cap and the root epidermal cells. The labeling pattern of cytoplasmic organelles suggests that the endoplasmic reticulum also plays a part in wall synthesis. This has been noted in the case of xylem wall thickening (23): many observations made on the distribution of incorporated
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labeled derivatives into the cell plate, and older walls (in both root and coleoptile) suggest that the endoplasmic reticulum m a y well be at least as important as the Golgi bodies in the deposition of such walls, since this is generally the only adjacent cell organelle consistently labeled (e.g., Figs. 10 and 13). However, the results can be interpreted in other ways, and a real clarification of the situation probably awaits the use of more complex and sophisticated labeled precursors. It may be profitable to compare the results with those obtained by other workers who have examined protein synthesis in vertebrate cells with radioautographic methods. For example, Caro and Palade (4) have demonstrated the appearence of labeled material sequentially in the endoplasmic reticulum, Golgi complex, and finally zymogen granules, in pancreatic exocrine cells. A function of the Golgi apparatus in the process of cell vacuolation has been sought in wheat cells. The organelle is occasionally seen to be closely associated with prevacuolar bodies [Fig. 15; cf. Marinos (17)]. However, in some of the vacuolating cells examined, vesicles derived from the Golgi apparatus, were apparently being incorporated directly into the vacuole (Figs. 16, 18, and 19). In a few cases, even the endoplasmic reticulum could be involved in this process (Fig. 21). This is most clearly seen during the postmitotic development of the stomatal complex and in epidermal hair cells. The increase in size and change in shape of the guard cells coincided with the appearance and enlargement of the vacuoles in these cells [compare Figs. 17 and 18 with Fig. 17 from Pickett-Heaps and Northcote (26)], and the two phenomena are almost certainly interrelated. In several types of cells, the vesicles found at the periphery of the Golgi cisternae did not appear to be all the same, either in size or in content. It is therefore possible that the individual cisternae o f such organelles were not all performing the same functions in the cell. For example, in stomatal cells, it is possible that some cisternae were giving rise to vacuolar fluid, whereas others were perhaps involved in production of material for purposes such as wall deposition (see Figs. 20 and 22). In some cells (e.g., Figs. 12 and 14), it is not possible yet to suggest reasons for this disparity or FrG. 18. As for Fig. 17, but the stomatal complex is more mature. Vacuolation is well under way. The guard cells have elongated and swollen; the wall between them has split to form the stomatal pore. Many large vesicular components are visible in the cytoplasm of the guard cells, some of these (arrowed) being apparently discharged into the vacuoles (v) x 4250. FIG. 19. Serial section of Fig. 18, at high magnification. Some large vesicular components (arrowed) are apparently being discharged into the vacuole, and others are associated with Golgi bodies (g). x 10,000. FIG. 20. Golgi body from the same cell shown in Fig. 19. One of the large vesicles is clearly continuous with the membranes of a Golgi cisterna, x 33,000. Fro. 21. As for Fig. 17, but the stomatal complex is much older. A portion of the endoplasmic reticulum (arrowed) is very swollen, possibly giving rise to a large vesicle, x 29,000. FiG. 22. A Golgi body from the cell shown in Fig. 21. It is very similar to those seen in younger stomatal cells (cf. Fig. 20). x 25,500.
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guess the fate of the vesicles. Some of the smaller vesicles may even have been about to fuse with the Golgi cisternae; no way has yet been devised to clarify this possibility. It may be an important concept if the cisternae were being formed at one face (see below). Very recently Cunningham et al. (6) have clearly shown m a n y differences in the nature of two types of vesicles associated with isolated plant Golgi bodies. Such a difference in the function of the individual cisternae can be contrasted with the progressive changes that are sometimes seen to occur across the stack of membranes. Mollenhauer and Whaley (20) have suggested that the cisternae might mature as they move across the stack, large vesicles being shed on one side and new cisternae being added on the other. The process is easy to visualize in the case of the Golgi bodies in root cap cells. Such a hypothesis could easily explain the gradation of some enzymatic activity that is evident in the Golgi bodies of the root epidermal cells (Fig. 6). The nature of this reaction is obscure, and it is perhaps unwise to describe it in terms of acid phosphatase activity although the preliminary results indicate that it is a fairly specific reaction, occurring at a low pH. It seems likely that the phenomenon indicates some gradation of biochemical and physiological activity across the stack of membranes. This is different from the previous case in that the vesicles produced by the Golgi bodies in these cells appear uniform in size and electron opacity, and all seem destined to be incorporated into the wall. Poux (28) shows a similar association of phosphatase activity with the Golgi bodies in other wheat cells. These and the previous results described might be compared with the numerous observations of phosphatase activity in animal cells [e.g., by Novikoff and his associates (13)], and also with more unspecific asymmetric staining reactions of the cisternal stack (10) and associated vesicles (1) in other cells. Much of the experimental work described here was carried out while the author was working in Cambridge with Dr. D. H. Northcote; the author is greatly indebted to Dr. Northcote for his interest in the work and critical appraisal of the ideas that led to this paper. The author also gratefully acknowledges the receipt of an Agricultural Research Council Studentship, during the tenure of which much of the experimental work was carried out.
REFERENCES 1. BAINTON,D. F. and FARQUHAR,M. G., J. Cell BioL 28, 277 (1966). 2. BRANDES,D., J. Ultrastruct. Res. 12, 63 (1965). 3. BROWN, W. V. and JOHNSON, S. C., Am. J. Botany 49, 110 (1962). 4. CARO, L. G. and PALADE, G. E.. J. CellBiol. 20, 473 (1964). 5. CARO, L. G. and VAN TUBERGEN, R. P., J. CellBioL 15, 173 (1962). 6. CUNNINGHAM, W. P., MORRt~, D. J. and MOLLENHAUER, H. H., J. Cell Biol. 28, 169 (1966). 7. ESAU, K. and GILL, R. H., Planta 67, 168 (1965).
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8. FABERGI~,A. C. and LEWIS, C. W., J. Cell Biol. 15, 579 (1962). 9. FREY-WYSSLING,A., L6~Ez-S~Ez, J. F. and M/3HLETHALER,K., J. Ultrastract. Res. 10, 422 (1964). 10. FRIEND, D. S. and MURRAY, M. J., Am. J. Anat. 117, 135 (1965). 11. GANTT, E. and ARNOrT, H. J., Am. J. Botany 52, 82 (1965). 12. HOLT, S. J. and HICKS, R. M., Nature 191, 832 (1961). 13. HOLTZMAN,E., and NOVIKOFr, A. B., J. Cell Biol. 27, 651 (1965). 14. JENSEN, W. A., Am. J. Botany 52, 238 (1965). 15. LARSON,D. A., Am. J. Botany 52, 139 (1965). 16. LEDBETrER, M. C. and PORTER, K. R., J. Cell Biol. 19, 239 (1963). 17. MARINOS,N. G., J. Ultrastruct. Res. 9, 177 (1963). 18. MmLONm, G., J. Biophys. Biochem. CytoI. 11, 736 (1961). 19. MOLLENHAUER,H. H , J. Ultrastruct. Res. 12, 439 (1965). 20. MOLLENHAUER,H. H. and WHALEY, W. G., J. Cell Biol. 17, 222 (1963). 21. MOLLENHAUER,H. H., WHALEY, W. G. and LEECH, J. H., J. Ultrastruct. Res. 5, 193 (1961). 22. NORTHCOTE,D. H. and PICKETT-HEArS,J. D., Biochem. J. 98, 159 (1966). 23. PICKETT-HEAPS,J. D., Planta 71, 1 (1966). 24. PICKETT-HEArS,J. D. and NORTHCOTE, D. H., J. ExptL Botany 17, 20 (1966). 25. - - - - J. Cell. Sci. 1, 109 (1966). 26. - ibM. 1, 121 (1966). 27. PORTER, K. R. and MACHADO,R. D., J. Biophys. Biochem. Cytol. 7, 167 (1960). 28. Poux, N., J. Microscopie 2, 485 (1963). 29. ROSEN, W. G., GAWLIK, S. R., DASHEK,W. V. and SEIGESMUND,K. A., Am. J. Botany 51, 61 (1964). 30. SIEVERS,A., Protoplasma 56, 188 (1963). 31. SOMMER,J. R. and BLUM, J. J., J. CellBiol. 24, 235 (1965). 32. WACHSTEIN,M. and MEISEL, E., Am. J. Clin. PathoI. 27, 13 (1957). 33. WHALEY,W. G., KEPHART,J. E. and MOLLENHAU~R,H. H., Am. J. Botany 46, 743 (1959). 34. WHALEY,W. G. and MOLLENHAUER,H. H., J. Cell Biol. 17, 216 (1963). 35. WOODINO,F. B. P. and NORTHCOTE,D. H., J. Cell Biol. 23, 327 (1964). 36. - ibid. 24, 117 (1965).
j. ULTRASTRUCTURERESEARCH18, 287--303 (1967)
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Further Observations on the Golgi Apparatus and Its Functions in Cells of the Wheat Seedling J. D. PICKETT-HEAPS
The John Curtin School of Medical Research, Canberra., A.C.T., Australia Received April 1, ]966 Radioautographic experiments confirm previous work indicating that Golgi bodies in root epidermal cells contribute material to the wall, and, in telophase cells, preferentially to the cell plate. Observations on other cells suggest that the endoplasmic reticulum and the Golgi apparatus are involved in the synthesis of wall precursors. In stomatal cells, vesicles probably derived from the Golgi apparatus are apparently discharged into the vacuole. Further study of the Golgi apparatus in several types of cells, and some preliminary histochemical work, indicates that progressive changes can occur across the cisternal stack, and that the cisternae of some Golgi bodies do not necessarily all have identical functions. The functions of the Golgi apparatus in the cell have been the subject of much speculation by cytologists. Their role in the production of material that is subsequently incorporated into the wall in plant cells, however, has been well documented. This was very clearly shown by Mollenhauer et al. (21) in the root cap cells of maize, and the hypertrophied nature of the Golgi bodies in this type of cell, first described by Whaley et al. (33), was later found in the root cap cells of some other plants (8, 24). Mollenhauer (19) clearly demonstrated that the Golgi apparatus was modified during maturation of the cell to give this characteristic form. Whaley and Mollenhauer (34) showed that during mitosis in root epidermal cells the cell plate forms by the fusion of vesicles that are derived from the Golgi apparatus; this was confirmed by Frey-Wyssling et al. (9), who also noted that the vesicles could be absorbed into older walls of the cells. Sievers (30) observed the phenomenon in the root hair cells of wheat. During cell plate initiation in root meristematic cells, Porter and Machado (27) noted that the young wall was formed from a similar fusion of vesicular components, but it was not possible to identify positively the source of these vesicles. In other types of plant tissue, incorporation of vesicular components into the wall is also apparent. It has been most clearly demonstrated during the course of wall synthesis in the rapidly growing pollen tube (15, 29). It appears to be a
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possible means of wall synthesis in xylem (35), phloem (36), and several other types of plant cells (11, 14, 24). Using recently introduced radioautographic techniques, Northcote and PickettHeaps (22) demonstrated unambiguously the passage of labeled material from the Golgi bodies into the wall in root cap cells. This material accumulated in the slime layer of the tissue, and chemical analysis of the labeled polysaccharides indicated that the radioactive components detected radioautographically contained mainly glucose and galactose and were therefore probably pectic in nature. In wheat xylem cells the Golgi body again can be labeled near a massive accumulation of radioactivity in the wall, although it would also appear that the endoplasmic reticulum plays an important part in the synthesis and/or deposition of the wall thickenings (23). It should not be assumed, however, that the sole function of the Golgi apparatus in plant cells is concerned with the synthesis and transport of wall material. An association of the organelle with vacuolation is possible, and Marinos suggested that small prevacuolar bodies could arise by enlargement of the entire intramembranous space of a Golgi cisterna. In the single-celled Euglena, which cannot be regarded as being a typical plant cell, Brandes (2) and Sommer and Blum (31) showed that the Golgi apparatus is associated with the formation of lysosomes, exhibiting an "acid-phosphatase" type of reaction when the cells are incubated in a Gomori medium. The observations reported in this article are further extensions of those described previously (22, 24, 25, 26); they were made primarily to investigate two possibilities: (a) that the cisternae and associated vesicles in some Golgi bodies were not all identical in appearance and function; (b) that the Golgi body is involved in vacuolation as well as cell wall deposition. MATERIALS AND METHODS Wheat seeds (Triticttm vulgare) were washed and germinated on damp filter paper in daylight at room temperature. Segments of coleoptile and root tips were fixed for electron microscopy either in 4 % potassium permanganate containing sodium and calcium chlorides (24), or in a 6 To buffered glutaraldehyde/osmium system, containing calcium chloride (25). For radioautographic experiments, intact or excised root tips and coleoptile segments were incubated either in a solution of D-glucose-6-~H (1.3 C/mmole, from the Radiochemical Centre, Amersham, Bucks.) or D-glucose 1-3H (350-500 mC/mmole, from the New England Nuclear Corporation, Boston, Massachusetts) in glass distilled water, at a concentration of 0.5-2.0 mC/ml. After an appropriate period of incubation (5 minutes to 4 hours), the tissues were washed and immediately fixed in either the permanganate or the glutaraldehyde fixatives mentioned above. For attempts at the localization of enzyme activity, the tissues were fixed in 6 % glutaraldehyde buffered to pH 7 with Veronal-acetate buffer, for ½ hour at room temperature. It
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was later found that such a buffering system is suspect (12), but excellent morphological preservation was nevertheless obtained; phosphate buffers, of course, cannot be used in such histochemical experiments. After a thorough wash, the specimens were incubated in the medium of Wachstein and Meisel (32) at pH 5, using ~-glycerophosphate as the substrate. The incubation lasted 1-4 hours at 37°C. Controls were run concurrently using: (a) no substrate, (b) adenosine triphosphate (ATP) as a substrate, and (c) c~-glycerophosphate as a substrate at pH 7. All these samples were well washed after the incubation, briefly exposed to a weak solution of ammonium sulfide, and washed again. They were then postfixed in osmium tetroxide as normal. All specimens were dehydrated in ethanol, embedded in Araldite, and sectioned on a mechanical advance or Porter-Blum microtome. Radioautographic specimens were coated with emulsion by a slightly modified method of Caro and van Tubergen (5); for details, see Northcote and Pickett-Heaps (22). All sections were stained with lead (18) for 5-15 minutes, except those examined for histochemical localizations which were not stained at all. Sections taken from specimens fixed in the glutaraldehyde/osmium system were further stained for ~3--1hour in a saturated solution of uranium acetate in 50 % ethanol. Sections were examined at 80 kV in a Philips EM 100 or EM 200 electron microscope.
OBSERVATIONS
Root epidermal cells' During the radioautographic study of the function of the Golgi apparatus in root cap cells (22), the Golgi bodies in the nearby root epidermal cells were frequently observed to be labeled, and further investigations indicated that these organelles were involved in wall synthesis and cell-plate formation. In radioautographic experiments, the walls of epidermal cells were always found to contain a large concentration of radioactive derivatives of the labeled glucose, most of the radioactivity being found at the outer facing wall surface, as in the interphase cells in Fig. 3. The Golgi bodies were also labeled to some extent in these circumstances. When the time of exposure of the tissue to labeled glucose was of short duration (ca. 10 minutes), the Golgi bodies were quite highly labeled as compared to the labeling of the wall and the rest of the cell (Fig. 1). As the time of incubation increased, there was an increasing accumulation of the labeled material at the external wall surface. During the longer periods of incubation, the starch grains in the plastids of these cells also became appreciably labeled. In previous radioautographic investigations (22), the radioactivity could be localized over the Golgi apparatus or the associated vesicles in root cap cells, since these organelles were quite large in comparison to the radioautographic resolution achieved. A similar localization could not be so clearly shown in the case of the epidermal cells, since both the organelle itself and the vesicles apparently derived from it were much smaller with respect to the radioautographic resolution obtainable. However, high magnification pictures did strongly suggest that radioactive derivatives were
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localized within the organelle itself a n d its vesicles (Fig. 4). The G o l g i bodies were also labeled in e p i d e r m a l cells at telophase d u r i n g a n d after cell plate initiation. W h e n the tissue had been p r e v i o u s l y exposed to solutions of tritiated glucoses for m o r e t h a n a b o u t 30 minutes, y o u n g walls between recently divided cells were always massively labeled when c o m p a r e d to nearby, older transverse walls [see plate L, N o r t h c o t e a n d P i c k e t t - H e a p s (22)], a n d this a c c u m u l a t i o n of labeled m a t e r i a l pres u m a b l y started d u r i n g plate f o r m a t i o n . If the r o o t was cut transversely, it was possible to find some sections which passed t h r o u g h the plate region of an early telophase cell a p p r o x i m a t e l y in the plane of the plate. I n this event, the m a j o r a c c u m u l a t i o n of labeled m a t e r i a l in the cell coincided in p o s i t i o n to the region occupied b y the collection of cell plate vesicles; m a n y n e a r b y G o l g i bodies were also l a b e l e d (Fig. 2). Subsequent fusion of r a d i o a c t i v e vesicles w o u l d o b v i o u s l y l e a d to such a highly labeled y o u n g cell wall. Before a n d d u r i n g cell plate f o r m a t i o n , some directive force m o v e s the vesicles to the plate region a n d then aligns t h e m across the a p p r o p r i a t e regions of the cell, a n d it has been suggested t h a t the i n t e r z o n a l spindle m i c r o t u b u l e s are involved in this process (7, 25, 26). R a d i o a u t o g r a p h y can p r o v i d e a clear illustration of the result of such a directive force. W h e n transverse sections of late telophase e p i d e r m a l cells were t a k e n f r o m tissue that h a d been previously e x p o s e d to tritiated glucose for fairly l o n g p e r i o d s (ca. 2 hours), the r a d i o a u t o g r a p h s showed t h a t the cell plate was highly labeled as expected. However, the n e a r b y region of the o u t e r cell wall in the same cell c o n t a i n e d a c o n s p i c u o u s l y low a m o u n t of r a d i o a c t i v e derivatives, when c o m p a r e d
ep g gc m n
cell plate Golgi apparatus guard cell mitochondrion nucleus
Key to abbreviations se subsidiary cell of stomatal complex th xylem wall thickening v vacuole w cell wall
FIGS. 1-4. Radioautographs of root epidermal cells fixed in permanganate. FIG. 1. Longitudinal section through cells exposed to G-6-SH for 10 minutes. Most of the incorporated radioactivity is associated with the Golgi bodies (g) of the cells; the outermost wall surface has accumulated some labeled material, x 5300. FIG. 2. Transverse section through cell plate region of a telophase cell, exposed to G-6-~H for 30 minutes. Many vesicles collect between the daughter nuclei prior to the formation of the cell plate. The concentration of vesicles coincides in position on the autographs with the concentration of radioactivity. Nearby Golgi bodies (g) are also labeled, x 8600. FIG. 3. Transverse section (as in Fig. 2) of a root tip exposed to G-1-3H for 2 hours. The section cut the central, late telophase cell approximately in the plane of the undulating cell plate, which therefore shows up as a twisting profile. In the interphase cells on either side, most of the radioactivity has been incorporated into the outer-facing wall surface; this contrasts with the telophase cell where the activity is located over the cell plate, not over the outer wall. x 3400. FIG. 4. Interphase cell, incubated in a solution of G-6-SH for 10 minutes. Radioactivity is associated with a Golgi body (g) and a vesicle that is being incorporated into the wall (w). x 50,000.
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with the usual pattern of incorporation observable in neighboring cells (Fig. 3). T h e central cell in Fig. 3 has been sectioned in the plane of recently f o r m e d cell plate, which, undulating across the cell, presented a twisted profile in this section. Close inspection of the cell plate clearly showed its " b e a d e d " nature, due to the uneven distribution of vesicular contents sandwiched between the new plasmalemma membranes [as in Fig. 2b of Whaley and Mollenhauer (34)]. Attempts to show phosphatase activity at the ultrastructural level in r o o t tip cells has initially met with little success, this apparently being mainly due to the problem of penetration into the cell of the components of the incubation mixture. However, the preliminary experiments have indicated that the Golgi bodies in epidermal cells can show a positive reaction when incubated in a G o m o r i medium (Fig. 5); this reaction was not f o u n d at either p H 7 or in the absence of the substrate at p H 5, or in either case using A T P as a substrate, and it was not observed in all epidermal cells examined. Most, however, showed clear indications of the reaction, which manifested itself as an electron-dense deposit in the cisternae (and frequently in an associated vesicle) of the organelle; the degree of reaction was progressively greater across the stack (Fig. 6). It was not obvious what this histochemical reaction was related to morphologically; the wall, nucleus, and often the vacuoles of the cell showed varying degrees of positive reaction, but this was not specific for any of the incubation media used.
Mer&tematic root tip cells It was h o p e d that radioautographic methods would elucidate the possible function of the Golgi b o d y in the formation of the cell plate in meristematic root tip cells. Porter and M a c h a d o (27) first described cell plate formation in such cells but were not able to identify the source of the cell plate vesicles. Their contents were electron
FIGS. 5 and 6. Glutaraldehyde/osmium-fixed cells that have been incubated in a Gomori medium at acid pH (see text). FIG. 5. Longitudinal section of root epidermal cell. Reaction products are visible in the nucleus, wall, vacuoles, and Golgi bodies (g); the reaction of the nucleus, wall, and vacuoles was not specific for this particular incubation system, x 5800. FIG. 6. Reaction product associated with the Golgi apparatus and its vesicles. The reaction is progressively more pronounced across the stack, x 26,000. Fins. 7-10. Radioautograms of root meristematic cells fixed in permanganate and sectioned longitudinally. F~G. 7. Telophase cell, exposed to G-6-3H for 4 hours. Massive incorporation of labeled material into the cell plate is evident; nearby older walls contain little radioactivity. × 8000. FIG. 8. Telophase cell after brief (10 minutes) exposure to G-6-SH at high activity (5 mC/ml). The cell plate contains some radioactivity, and under these conditions, the Golgi bodies (g) are sometimes labeled (small arrows). × 13,000. FIG. 9. Telophase cell exposed to G-6-SH for 30 minutes. The cell plate (cp) is seen between the daughter nuclei (n); some radioactivity is associated with it. Nearby elements of endoplasmic reticulum (arrowed) are often labeled in such experiments, x 17,000.
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transparent, as were both the vesicles associated with the nearby Golgi bodies, and also the tubular elements of endoplasmic reticulum in the region (which were probably also giving rise to vesicles). A large number of radioautographic experiments cannot be said to have completely clarified the situation. Glucose-l-SH (G-I-~H) never gave any significant labeling patterns in meristematic root tip cells. When glucose-6-SH (G-6-SH) was used, the amount of radioactivity incorporated into the cell plate varied, increasing as the time of incubation was prolonged. During plate formation, the vesicles contained or were associated with radioactive material, though never to the extent detectable in nearby telophase epidermal cells. The Golgi bodies in the interzone region were occasionally labeled, particularly when a very short, intense pulse of radioactive glucose was applied to the tissue (Fig. 8). Over longer periods of incubation however, the Golgi bodies in the interzone region were scarcely labeled at all when compared with the labeling of the cell plate, and in these circumstances radioactivity was more frequently associated with the nearby elements of endoplasmic reticulum (Fig. 9). During further growth of the young cell wall, there was often a marked accumulation of radioactivity in the wall when the times of incubation were long (1-4 hours, Fig. 7). As before, the Golgi bodies were almost invariably free of activity, and the endoplasmic reticulum was the only organelle consistently labeled near such walls (Fig. 10).
CoIeoptile parenchyma cells Incorporation of material derived from G-6-SH was often observed into the walls of the highly vacuolated parenchyma cells. Again, the nearby Golgi bodies were almost invariably unlabeled, but the endoplasmic reticulum was often seen to be associated with the radioactivity present in the cytoplasm near the walls (Fig. 13).
Phloem and Xylem Tissues In radioautographic experiments on root tips, young sieve-tube cells almost always showed a fairly massive incorporation into the wails, of tritiated derivatives formed FIG. 10. Longitudinal section through meristematic cells, exposed to G-6-SH for 1 hour. The transverse wall is fairly heavily labeled, and nearby elements of the endoplasmic reticulum are also associated with radioactivity. The Golgi bodies near such older walls are generally unlabeled. × 16,000. FIG. 11. Longitudinal section through a young sieve-tube cell, from a root tip exposed to G-6-~H for 2 hours, and fixed in glutaraldehyde/osmium. The wall (w) and a nearby Golgi apparatus (g) is labeled, x 33,000. FIG. 12. Young sieve-tube cell in a root tip, fixed in permanganate. The Golgi bodies are associated with two distinct types of vesicles; most appear small and gray in the photograph, and a few are larger and electron transparent, x 18,000. FIG. 13. Transverse section through three highly vacuolated coleoptile cortical cells, after exposure to G-6-SH for 2 hours and fixation in permanganate. Vacuoles (v), wall (w), and mitochondria (rn) are marked; a triangular air space is present between the cells. The walls are labeled, and nearby elements of endoplasmic reticulum are also often associated with radioactivity (arrows). In such older cells, the Golgi bodies are not normally labeled under these conditions. × 13,500.
;ii
:4
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from both G-6-BH and G-1-SH; nearby Golgi bodies were often also labeled (Fig. 11). For some reason that is not quite clear, the sieve tubes of the coleoptile showed little incorporation of tritiated glucose into either the cytoplasm or wall. This may be associated with the fact that, whereas the root tips were generally used intact, coleoptile tissue was always excised for the incubation procedure (the sieve tubes being susceptible to damage by turgor pressure changes). In the permanganate-fixed image of young sieve tube cells, vesicles associated with the Golgi bodies appeared to be of two types, since generally some larger, electrontransparent vesicles were present adjacent to the cisternae (Fig. 12). A possible fate of these vesicles cannot yet be suggested. In phloem parenchyma cells in the coleoptile the Golgi bodies were occasionally closely associated with small vacuolar components (Fig. 15), in a very similar fashion to that described in barley by Marinos (17). This was uncommon, however, and was not generally observed in other types of cells. Some radioautographic evidence suggests that the Golgi bodies can supply wall material during the formation of secondary thickening of the xylem wall in wheat coleoptile. This has been described elsewhere (23). During the las t stages of xylem differentiation, wall deposition (as measured by the incorporation of radioactive derivatives into the thickenings) had virtually ceased. At this stage, the cytoplasm showed a progressive deterioration, and cell organelles (particularly the mitochondria and plastids) were disintegrating. However, Golgi bodies were still present, and micrographs indicated that some vesicles were appreciably different from the remainder around the stack of cisternae (Fig. 14). In much older coleoptile tissues, the parenchyma cells around xylem vessels become highly vacuolated; m a n y large, electrontransparent vesicles were usually present in the cytoplasm, and similar components were apparently being derived from the Golgi bodies (Fig. 16). Generally only one of the cisternae in a given stack was associated with the enormously distended vesicle.
Guard cells in the stomata The formation of the stomatal complex in wheat leaf epidermis has already been described in detail at the ultrastructural level (26). By taking tissue at later stages of FIG. 14. Longitudinal section through a maturing xylem vessel in the coleoptile, fixed in glutaraldehyde/osmium; wall thickenings (th) are marked. The Golgi bodies at this stage of maturation are associated with two types of vesicular components; most appear small and gray, and some (arrowed) are larger and more electron transparent (cf. Fig. 12). × 26,000. FIG. 15. Phloem parenchyma cell, fixed in glutaraldehyde/osmium. The Golgi apparatus in the micrograph is very closely associated with a small vacuolar body, near the main cell vacuole (v). × 58,000. FIG. 16. Xylem parenchyma cell fixed in glutaraldehyde/osmium. One cisterna of the Golgi body is apparently producing a very large, clear vesicle (cf. Figs. 20 and 22). Other vesicular components are more usual in size and content. × 38,000. FIG. 17. Longitudinal section through young stomatal complex in leaf epidermis, fixed in glutaraldehyde/osmium. The guard cells (gc) are flanked by subsidiary cells (sc). Vacuoles (v) have appeared in the cells. The wall between the cells has thickened in the central region before splitting to form the stomatal pore; this wall does not entirely separate the guard cells (arrow). x 5500.
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growth, it has been possible to describe the postmitotic development in these cells, which gives rise to the characteristic pore structure in the epidermis of the leaf. After the symmetrical third division [see Pickett-Heaps and Northcote (26)], the complex consists of two guard cells (oblong or biconcave in overall view when the two are seen in longitudinal section), and each of these has a subsidiary cell on the outside, making the whole complex biconvex in surface view (Fig. 17). It was an invariable feature of the guard cells, confirming earlier observations (3), that they were not completely separated from one another by the wall between them; gaps were always found at each end of this wall, allowing the cytoplasm of the two cells to be continuous at all stages of growth so far observed (e.g., Figs. 17 and 18). Soon after the basic complex had been formed, all the cells elongated, and the guard cells, changing their shape became fatter. A vacuole appeared in each end of each guard cell, and these vacuoles rapidly increased in size as the cells elongated (Figs. 17 and 18). The central region of the wall between the guard cells thickened considerably (Fig. 17) prior to this region of the wall splitting apart to form the stomatal pore (Fig. 18). A large number of microtubules were always associated with this region of the wall. The cytoplasm of the guard cell was very characteristic in its appearance at this stage, since it contained numbers of large, electron-transparent, membrane-bounded bodies (Figs. 18 and 19). From the sections, it would appear that these bodies were being discharged into the vacuoles at each end of the cell (Figs. 18 and 19). Furthermore, some of the vesicles were associated with the Golgi bodies in the cells, and closer examination revealed instances when the membranes of one or two cisternae in the stack of the Golgi body, were clearly continuous with the membranes of the large vesicles. Other cisternae in the same stack were associated with much smaller vesicles, these being the more usual, darker-staining components (Fig. 20). During later stages of maturation, the cisternae of the endoplasmic reticulum occasionally became enormously distended, apparently producing a similar body or vesicle (Fig. 21). The Golgi bodies as before were generally also associated with the larger as well as some smaller type of vesicles (Fig. 21). Epidermal hair cells are also present in the epidermis of wheat leaves, and in sections of these cells too, the Golgi bodies were associated in a similar fashion with large vesicles that were apparently being discharged into the central vacuole of the rapidly elongating cells. DISCUSSION As mentioned in the introduction, the incorporation of vesicles, derived from the Golgi body, into the cell-plate and wall of root epidermal cells has been described by several authors. The results presented here confirm these observations. It has not been possible to demonstrate unambiguously the time course of the passage
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of the vesicles from the Golgi apparatus to the wall, as has been done in the root cap cells (22), but the steady accumulation of radioactivity in the walls of the cells over longer periods of incubation make it reasonable to conclude that at least part of the radioactive wall material passes to the wall via the Golgi apparatus. The Golgi bodies and outer wall of the epidermal cells were always far more heavily labeled than those of internal, more undifferentiated root tip cells; this is almost certainly because the epidermal cells must synthesize large amounts of wall materials (since the outer wall of the root tip is very thick) and probably some mucilaginous compounds, and this stimulated rate of wall synthesis would be reflected by the heavier labeling of the outer-facing wall and the organelles concerned in its deposition. When Ledbetter and Porter (16) first reported the presence of microtubules in root tip cells, they noted that these organelles were found during telophase between the daughter nuclei following mitosis. Later, Esau and Gill (7) and Pickett-Heaps and Northcote (25, 26) suggested that the microtubules probably directed the vesicles to the plate region and aligned the young wall in the correct position in the cytoplasm. Radioautography demonstrates such a highly directional movement of the cell plate vesicles; in Fig. 3, labeled wall material produced in the interzone region of a telophase cell during the incubation in tritiated glucose had clearly been directed almost exclusively into the cell plate. Very little radioactivity was detected in the adjacent part of the outer-facing wall of this cell, while the two neighboring cells show the typical concentration of labeled derivatives in the thick outer wall. Since this particular photograph was taken from tissue fixed in permanganate, the microtubules themselves are, of course, not visible. The role played by the Golgi body in cell plate and wall formation in meristematic root tip cells is still somewhat open to speculation. From the appearance of many micrographs [as in Porter and Machado (27)], it seems most likely that vesicles derived from the Golgi apparatus do contribute to the new wall material and plasmalemma. The Golgi bodies were seldom labeled even when near a heavily labeled wall; this may be due to the fact that the radioactivity of the cell plate or wall represents an accumulation of the label over the incubation period, whereas the labeling of the Golgi bodies might merely represent the transitory concentration of labeled precursors present in the organelle. A similar dilemma exists in accounting for the labeling pattern of the Golgi apparatus associated with xylem wall growth (23). The most striking labeling of the Golgi bodies observed so far, occurs in cells which are producing large amounts of wall or similar type of material, namely the outer root cap and the root epidermal cells. The labeling pattern of cytoplasmic organelles suggests that the endoplasmic reticulum also plays a part in wall synthesis. This has been noted in the case of xylem wall thickening (23): many observations made on the distribution of incorporated
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labeled derivatives into the cell plate, and older walls (in both root and coleoptile) suggest that the endoplasmic reticulum m a y well be at least as important as the Golgi bodies in the deposition of such walls, since this is generally the only adjacent cell organelle consistently labeled (e.g., Figs. 10 and 13). However, the results can be interpreted in other ways, and a real clarification of the situation probably awaits the use of more complex and sophisticated labeled precursors. It may be profitable to compare the results with those obtained by other workers who have examined protein synthesis in vertebrate cells with radioautographic methods. For example, Caro and Palade (4) have demonstrated the appearence of labeled material sequentially in the endoplasmic reticulum, Golgi complex, and finally zymogen granules, in pancreatic exocrine cells. A function of the Golgi apparatus in the process of cell vacuolation has been sought in wheat cells. The organelle is occasionally seen to be closely associated with prevacuolar bodies [Fig. 15; cf. Marinos (17)]. However, in some of the vacuolating cells examined, vesicles derived from the Golgi apparatus, were apparently being incorporated directly into the vacuole (Figs. 16, 18, and 19). In a few cases, even the endoplasmic reticulum could be involved in this process (Fig. 21). This is most clearly seen during the postmitotic development of the stomatal complex and in epidermal hair cells. The increase in size and change in shape of the guard cells coincided with the appearance and enlargement of the vacuoles in these cells [compare Figs. 17 and 18 with Fig. 17 from Pickett-Heaps and Northcote (26)], and the two phenomena are almost certainly interrelated. In several types of cells, the vesicles found at the periphery of the Golgi cisternae did not appear to be all the same, either in size or in content. It is therefore possible that the individual cisternae o f such organelles were not all performing the same functions in the cell. For example, in stomatal cells, it is possible that some cisternae were giving rise to vacuolar fluid, whereas others were perhaps involved in production of material for purposes such as wall deposition (see Figs. 20 and 22). In some cells (e.g., Figs. 12 and 14), it is not possible yet to suggest reasons for this disparity or FrG. 18. As for Fig. 17, but the stomatal complex is more mature. Vacuolation is well under way. The guard cells have elongated and swollen; the wall between them has split to form the stomatal pore. Many large vesicular components are visible in the cytoplasm of the guard cells, some of these (arrowed) being apparently discharged into the vacuoles (v) x 4250. FIG. 19. Serial section of Fig. 18, at high magnification. Some large vesicular components (arrowed) are apparently being discharged into the vacuole, and others are associated with Golgi bodies (g). x 10,000. FIG. 20. Golgi body from the same cell shown in Fig. 19. One of the large vesicles is clearly continuous with the membranes of a Golgi cisterna, x 33,000. Fro. 21. As for Fig. 17, but the stomatal complex is much older. A portion of the endoplasmic reticulum (arrowed) is very swollen, possibly giving rise to a large vesicle, x 29,000. FiG. 22. A Golgi body from the cell shown in Fig. 21. It is very similar to those seen in younger stomatal cells (cf. Fig. 20). x 25,500.
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guess the fate of the vesicles. Some of the smaller vesicles may even have been about to fuse with the Golgi cisternae; no way has yet been devised to clarify this possibility. It may be an important concept if the cisternae were being formed at one face (see below). Very recently Cunningham et al. (6) have clearly shown m a n y differences in the nature of two types of vesicles associated with isolated plant Golgi bodies. Such a difference in the function of the individual cisternae can be contrasted with the progressive changes that are sometimes seen to occur across the stack of membranes. Mollenhauer and Whaley (20) have suggested that the cisternae might mature as they move across the stack, large vesicles being shed on one side and new cisternae being added on the other. The process is easy to visualize in the case of the Golgi bodies in root cap cells. Such a hypothesis could easily explain the gradation of some enzymatic activity that is evident in the Golgi bodies of the root epidermal cells (Fig. 6). The nature of this reaction is obscure, and it is perhaps unwise to describe it in terms of acid phosphatase activity although the preliminary results indicate that it is a fairly specific reaction, occurring at a low pH. It seems likely that the phenomenon indicates some gradation of biochemical and physiological activity across the stack of membranes. This is different from the previous case in that the vesicles produced by the Golgi bodies in these cells appear uniform in size and electron opacity, and all seem destined to be incorporated into the wall. Poux (28) shows a similar association of phosphatase activity with the Golgi bodies in other wheat cells. These and the previous results described might be compared with the numerous observations of phosphatase activity in animal cells [e.g., by Novikoff and his associates (13)], and also with more unspecific asymmetric staining reactions of the cisternal stack (10) and associated vesicles (1) in other cells. Much of the experimental work described here was carried out while the author was working in Cambridge with Dr. D. H. Northcote; the author is greatly indebted to Dr. Northcote for his interest in the work and critical appraisal of the ideas that led to this paper. The author also gratefully acknowledges the receipt of an Agricultural Research Council Studentship, during the tenure of which much of the experimental work was carried out.
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