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Sieve element pores in Nicotiana pith culture
© 1970 by Academic Press, Inc.
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s. tJLTRASTRUCTURERESEARCH32, 458-471 (1970)
Sieve E l e m e n t Pores in Nicotiana Pith C u l t u r e 1 RICHARD ANDERSON AND JAMES CRONSHAW
Department of Biological Sciences, University of California, Santa Barbara, California 93106 Received January 9, 1970 Sieve elements were induced to form in cultured pith cylinders of Nieotiana tabacum L. (var. Wisconsin 38). Both xylem and phloem differentiate in isolated nodules at the periphery of the cylinders. The mature sieve elements closely resemble sieve elements in intact plants but are more irregular in shape and usually not highly elongated. The formation of pores in the phloem sieve plates is similar to that observed in intact plants. Callose platelets are deposited around plasmodesmata, then the plasmodesmata enlarge to form the mature perforation. Dense slime plugs are only rarely observed. The majority of sieve plate pores are plugged by a combination of P-proteins, ER, and callose, but there are also numerous unplugged pores. We interpret the observation of numerous unplugged pores as further evidence that in vivo pores may not be plugged in tobacco, There are an increasing number of observations which indicate that in vivo sieve plate pores of Nicotiana are not plugged but contain a loose array of P-protein. Plugged pores, like slime plugs and heavy callose deposits, appear to be wound reactions. We observed unplugged pores in plants which were frozen in liquid nitrogen and then fixed at 4°C (5). Later we found that if pores were blocked by starch grains near cut surfaces they were devoid of P-protein (1). Pores with loosely arranged P-protein were also found in some wilted plants and plants thinly sliced before fixation (2). Wooding (24) investigated the ultrastructure of sieve elements induced in tobacco callus. He observed a few pores which were not plugged with P-protein. Electron microscopic observations of the development of sieve plate pores were first made on Cucurbita by Frey-Wyssling and Muller (15), who studied the perforation process observing the wall microfibrils in shadowed preparations. Esau, Cheadle, and Risley (12) later investigated Cucurbita using fixed and sectioned material. They observed that endoplasmic reticulum became applied over the plasmodesmata of the future sieve plates. Callose platelets formed beneath the endoplasmic reticulum and then expanded in thickness and diameter. The middle lamella was removed, the callose z The study was supported by National Science Foundation Grant GB 12371 to Dr. J. Cronshaw.
SIEVE ELEMENT PORES IN NICOTIANA PITH
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platelets fused, then the platelets were eroded to produce the mature pore. Northcote and Wooding (20) and Evert et al. (13) observed a similar sequence of events in Aeer and Cucurbita, but reported that perforation began with enlargement of the plasmodesmata in the middle lamella. Bouck and Cronshaw (3) observed similar development in pea except that the association of the ER with the pore sites was no more frequent than with other wall regions. Wooding (24) reported that pore formation in Nieotiana callus sieve elements proceeded by essentially the same process as in Acer. In the present investigation we have examined the fine structure of differentiating and mature sieve elements induced in tobacco pith culture. Numerous unplugged pores were observed in these in vitro sieve elements. The development of the sieve plate pores in the pith culture sieve element has been found to be similar to that of sieve elements in intact plants.
MATERIALS AND METHODS Tobacco plants (Nicotiana tabacum L. var. Wisconsin 38) were grown in a greenhouse until they were about 1 m tall. After defoliation the stems were cut into 3 cm segments which were sterilized in 50 % Clorox for 1 minute. The segments were then rinsed in several changes of sterile water. Sections about 2 mm thick were shaved off each end of the segments to remove cells contaminated with Clorox. Pith cylinders were then cut out with a 1/4-inch cork borer. The cylinders were inserted 4 mm into an agar growth medium in 3-inch culture tubes. The growth medium was the basal medium of Murashige and Skoog (18) to which were added 4 % sucrose and 0.5 or 1 nag/liter indole-3-acetic acid or naphthalene acetic acid. Cylinders were incubated at 26°C for 3-5 weeks, and then were prepared for electron microscopy. The pith cylinders were fixed in formaldehyde-glutaraldehyde in 0.1 M pH 6.8 phosphate buffer (16). They were either cut in half or placed in the fixative whole. The vials of fixative and specimens were placed in a vacuum chamber, and the pressure was alternately lowered and raised during the fixation period of 2 hours. This procedure removed air from the specimens and facilitated penetration of the fixative. The cylinders were then sliced into disks less than 1 mm thick which were washed in buffer for 3 hours (3 changes). The sections were postfixed in 2 % osmium tetroxide in 0.1 M pH 6.8 phosphate buffer overnight at 4°C. They were dehydrated with a graded acetone series and then infiltrated with Epon 812 epoxy resin. The specimens were recut just before polymerization. The blocks were sectioned with a diamond knife on a Porter-Blum MTI ultramicrotome. Sections were stained with uranyl acetate and lead (17), and examined and photographed with a Siemens Elmiskop I.
RESULTS Under the growth conditions of this investigation, cell division occurred in the periphery of the pith cylinders from the base of the cylinders to about 1 cm above the level of the medium. Nodules containing xylem and phloem differentiated only in
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the zone above the level of the medium. Xylem cells usually differentiated first, at the center of the nodules. A r o u n d this core some of the cells differentiated into phloem. The shape of the sieve elements was variable; they were often elongated but were irregular in section. Sieve elements at early stages of differentiation resemble meristematic cells in intact plants. Callose platelets f o r m in the wall a r o u n d the plasmodesmata of the future sieve plates (Figs. 1 and 2). The platelets enlarge laterally and in thickness (Figs. 3-6). Endoplasmic reticulum is usually associated with the platelets (Figs. 4-6). The plasmodesmata are lined by the plasma m e m b r a n e f r o m their inception and have a central tubule. The plasmodesmata enlarge in the region of the middle lamella to produce a median nodule (Fig. 6). As the plasmodesmata enlarge further, callose and middle lamella are eroded to produce the pores of the mature sieve elements. The sequence of pore differentiation does not always correspond closely to the changes occurring in the cytoplasm. We have not observed stages of the actual perforation process. As j u d g e d by the difficulty of finding pores at stages of perforation, it is likely that actual perforation is quite rapid, as suggested by Esau, Cheadle, and Risley (12). The mature sieve elements in tobacco tissue culture closely resemble the sieve elements of intact plants (5, 7). The mitochondria, endoplasmic reticulum, plastids, and P-protein are essentially the same as those components in intact plants. The development and distribution of P-protein is described in another c o m m u n i c a t i o n (6). The P-protein in cultured sieve elements is generally distributed t h r o u g h o u t the cell lumina; dense slime plugs are not generally formed, but occasional ones have been Key to abbreviations
C
callose ER endoplasmic reticulum M mitochondrion M L middle lamella P pore
Pl Pp i'd SE SP
plastid p-protein proplastid sieve element sieve plate
FIGS. 1 and 2. Nicotiana tabacum. Fig. 1. Electron micrograph of cells in early stages of sieve element differentiation. Small amounts of callose are around the plasmodesmata, which are the future pore sites (arrows). Normal cytoplasmic components are present, including proplastids (Pd) and mitochondria (M). Fig. 2. A more highly magnified view of a developing pore showing one of the callose platelets and the plasmodesma (arrow). Fig. 1, x 9000; Fig. 2, x 36,000. FIGS. 3-6. Nicotiana tabacum. Series of stages in pore development. Fig. 3. Conical callose platelets (C) have formed around the plasmodesmata on either side of the middle lamella. The plasmodesmata are lined by the plasma membrane (arrow). Figs. 4 and 5. The plasmodesma is slightly enlarged in the region of the middle lamella. Lengths of endoplasmic reticulum lie close to the plasma membrane over the callose platelets. Fig. 6. The median nodule of a pore at this stage is considerably enlarged. Indications of structures, possibly membranes, are visible within the nodule. Fig. 3, x 55,000; Fig. 4, x 43,000; Fig. 5, x 44,000; Fig. 6, x 52,000. FIG, 7. Nicotiana tabacum. Electron micrograph of a sieve plate associated with an unusually dense slime plug. P-protein is compacted in the pores, x 28,000. FIG. 8. Nicotiana tabacum. Electron micrograph of pore blocked by ER and constricted by callose. There is no dense slime plug. P-protein of the tubular (Pp) type is in the lumina of the cells. × 45.600.
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FIGS. 9 and 10. Nicotiana tabacum. Electron micrographs of portions of sieve plates. The P-protein is fairly evenly distributed in the region of the sieve plates. The pores in Fig. 9 are loosely packed with P-protein while those of Fig. 10 contain widely spaced P-protein. One of the pores of Fig. 10 is blocked by some ER. Deposition of callose has constricted the diameter of the pores. Fig. 9, × 16,500; Fig. 10, × 20,000. observed (Fig. 7). The majority of sieve element pores are plugged with P-protein a n d / o r endoplasmic reticulum (Figs. 8 and 9, one of the pores in Fig. 10). However, m a n y sieve plate pores are not plugged with P-protein (Figs. 10-12). Because of favorable section orientation, the sieve element of Fig. 11 appears very similar to the sieve elements of intact plants. The cell is elongated, with a sieve plate at either end. P-protein is dispersed t h r o u g h o u t the lumen and is rather evenly distributed. The modified plastids are concentrated near the sieve plates. The sieve plate pores are large, with little callose, and are not plugged by P-protein. This sieve element is part of a sieve tube of undetermined length. Fig. 12 depicts portions of two sieve elements whose slime bodies have only partially dispersed. The sieve plate pores in such sieve elements contain very little P-protein. FIG. l 1. Nicotiana tabacum. Electron micrograph of a portion of a linear row of sieve elements and associated cells. The pores in the two sieve plates are large and are not plugged with P-protein. Little callose is deposited in the pores. The P-protein is scattered throughout most of the cell lumina. × 4900
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The pore visible in Fig. 12 is devoid of P-protein. A portion of another sieve element with partially dispersed P-protein is included in Fig. 1 I. Callose was present in all the pores observed. The majority were almost completely filled by callose (Fig. 8). The contents of these pores are tightly compressed. In numerous other pores, however, there is less callose and less compression of the pore contents (Figs. 10, 11). When a sieve element in a plant becomes nonfunctional, a mass of definitive callose is deposited on the sieve plates (11). The cytoplasmic contents disorganize, and the sieve element may be crushed by surrounding cells. Sieve elements in culture appear to undergo the same process. Especially in older cultures, masses of definitive callose separate intact sieve elements from those which have become disorganized (Fig. 13). The disorganized sieve elements may also be crushed by the growth of surrounding cells. Starch granules occasionally persist in the crushed cells. A connection between a sieve element and an associated parenchyma cell is shown in Fig. 14. There is a single opening surrounded by callose on the sieve element side; there is no callose on the parenchyma cell side. Many channels from the parenchyma cell connect with a single channel Of the sieve element. The connections are lined by the plasma membrane. A tubule is visible at the open arrow in Fig. 14. A section of endoplasmic reticulum appears to extend into one of the branches. The connections between companion cells and sieve elements in many plants resemble this type of branched connection. The lower cell in Fig. 14 is probably comparable to the companion cells of intact plants. DISCUSSION Sieve elements in Nicotiana pith culture differentiate in nodules at the periphery of the pith cylinders. The sieve plate pores develop from plasmodesmata in the region of the future sieve plates. Callose platelets form in the wall around each plasmodesma. The plasmodesma develops a median nodule in the region of the middle lamella then enlarges to form a wide perforation in the mature sieve element. This process parallels that described for Aeer by Northcote and Wooding (20) and for Cucurbita by Esau, Cheadle, and Risley (12). The protoplasmic contents of mature sieve elements in tissue culture closely resemble those in intact plants. They form short sieve tubes in the isolated nodules. Slime plugs, if present, are usually only slightly developed. The sieve plate pores are generally closed by P-protein and callose, but in many cases the pores are not plugged
Fio. 12. Nicotiana tabacum. Electron micrograph of two sieve elements whose P-protein bodies have not completely dispersed. The sieve plate pores are nearly devoid of P-protein, and there is little callose in the pores, x 9100.
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and there is little callose. In some sieve elements there are dense slime plugs and plugged pores. Thus, the pore-closing mechanisms of in vivo sieve elements may also function in cultured sieve elements. Sieve elements are very susceptible to damage during fixation procedures. The main changes induced by manipulation are formation of slime plugs (4, 10, 19), plugging of the sieve plate pores (1, 2, 5, 8, 20), and deposition of callose (5, 9, 10, 21). These responses are most likely mechanisms to seal off the sieve tubes when they are damaged in nature. The mechanism for initiation of callose deposition is unknown. The mechanism of slime plug formation seems to be based on the very function of the sieve tube. The high sucrose concentration in the translocate creates a high hydrostatic pressure in the sieve tube. When the sieve tube is severed, or when its semipermeability is locally destroyed by fixative, the pressure is released and an accelerated flow is initiated. Slime plugs form as the sieve plates filter out P-protein and starch grains dislodged by the flow. The length of the sieve tube probably affects the extent of disruption which occurs when a sieve tube is fixed or cut. Sieve elements at quite a distance from a cut appear to contribute to the flow toward the cut. This is apparent from the observation that slime plugs may form up to 1.5 cm from a cut although they decrease in intensity (4, 10). Ford and Peel (14) have also shown that, if the sieve tubes of a willow stem are cut, sieve elements more than 16 cm away f r o m the cut may contribute to the exudation. This discussion of slime plug formation is pertinent to an understanding of the relative insensitivity to manipulation demonstrated by in vitro sieve elements. The length of cultured sieve tubes is unknown but must not exceed a few millimeters. If such short sieve tubes are severed or disrupted by fixative there should be less potential for flow than in long sieve tubes. Another factor contributing to the insensitivity of in vitro sieve tubes may be the nature of the cell contents. Since in vitro sieve tubes are not connected to sugar sources such as a leaf, their contents may not have the high hydrostatic pressure of in vivo sieve tubes. These two factors, short length and probable low pressure, may combine to account for the general lack of slime plugs and the presence of some unplugged pores. The sieve plate pores in the majority of sieve elements were plugged even though a dense slime plug did not form (Figs. 8 and 9). Probably m a n y of these were plugged before immersion into fixative, especially those plugged with E R (Figs. 8 and 10). FIG. 13. Nicotiana tabacum. Electron micrograph of a sieve plate with definitive callose, and associated cells. The cytoplasm of one of the cells has become disorganized while the other sieve element appears normal. × 19,600. FIG. 14. Nicotiana tabacum. Electron micrograph of the branched connections between a sieve element and an adjacent cell which is probably a companion cell. The open arrow indicates a tubule in one of the branches. A length of endoplasmic reticulum at the arrow appears to be associated with a structure in the connection. × 105,000.
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However, others were probably closed by the deposition of callose as described by Northcote and Wooding (20) and Cronshaw and Anderson (5). According to these authors, when callose is deposited in pores as a wound response, the contents of the pores may be compressed. It is likely that this same mechanism operates to close the pores in cultured sieve elements. Wooding (23, 24) reported less callose in cultured sieve elements than in those of intact plants. We have observed a wide range in the amount of callose deposited in the sieve plate pores. The sieve elements with unplugged pores generally have less callose than those with plugged pores. Similar observations were made on cotton by Shih and Currier (21) and on wilted tobacco plants by Anderson and Cronshaw (2), who found that sieve elements with open pores had little callose in the pores. The in vitro tobacco sieve element protoplasts are structurally similar to intact plant sieve dements. They undergo similar differentiation processes, and can form definitive callose deposits on their sieve plates. The same mechanisms of slime plug formation, callose deposition, and pore plugging can operate as in intact plants. But because of shorter length, and possibly lower sucrose concentration these mechanisms may fail to operate in vitro. We have also observed the failure of pore closing mechanisms in tobacco plants prepared by special techniques. Pore plugging mechanisms may be disrupted in plants frozen in liquid nitrogen (5). When plants are wilted to a certain extent the pore plugging mechanisms fail to operate and the pores remain unplugged (2). If sieve tubes are cut into short lengths many of the pores are unplugged (2). In sieve elements near cut edges the pore blocking mechanisms operate effectively, but in many cases pores are blocked by starch grains rather than P-protein (1). None of these results by themselves are definitive, but taken together they indicate that in tobacco, plugged pores are artifacts resulting from disturbance of the sieve tubes. REFERENCES 1. ANDERSON, R. and CRONSHAW, J., J. Ultrastruct. Res. 29, 50 (1969). ANDERSON,R. and CRONSHAW,J., Planta, in press. BOUCK,G. B. and CRONSHAW,J., J. Cell Biol. 25, 79 (1965). CRAFTS,A. S., Am. J. Botany 26, 172 (1939). CRONSHAW,J. and ANDERSON,R., J. Ultrastruct. Res. 27, 134 (1969).
2. 3. 4. 5. 6. 7. 8. 9. 10. 11. ]2. 13.
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In press.
CRONSHAW, J. and ESAU, K., d. Cell Biol. 34, 801 (1967). CRONSHAW,J. and ESAU, K., ar. Cell Biol. 38, 292 (1968). CURRIER,H. B., Am. J. Botany 44, 478 (1957). ENGLEMAN,E. M., Ann. Botany 29, 83 (1965).
ESAU, K., Plant Anatomy. Wiley, New York, 1965. ESAU, K., CHEADLE, V. I. and RISLEY, E. B., Botan. Gaz. 123, 233 (1962). EVERT, R. F., MURMANIS,L. and SACHS, I. B., Ann. Botany 30, 563 (1966).
SIEVE ELEMENTPORES IN NICOTIANAPITH 14. 15. 16. 17. 18. 19: 20. 21. 22. 23. 24.
FORD, J. and PEEL, A. J., J. Exptl. Botany 17, 522 (1966). FREY-WYSSLING,A. and MULLER, H. R., J. UItrastruct. Res. 1, 38 (1957). KARNOVS~Y,M. J., J. Cell Biol. 27, 137A (1965). MILLONIG, G., J. Biophys. Biochem. Cytol. 11, 736 (1961). MURASmGE, T. and SKOOG, F., Physiol. Plantarum 15, 473 (1962). NAGELI, C., S.B. Bayer. Akad. Wiss. 1, 212 (1861). NORTHCOTE,D. H. and WOODING, F. B., Proc. Roy. Soc. B163, 524 (1965). SHIH, C. Y. and CURRIER, H. B., Am. J. Botany 56, 464 (1969). WEATHERLEY,P. E., PEEL, A. J. and HILL, G. P., J. Exptl. Botany 10, 1 (1959). WOODING, F. B. P., Planta 83, 99 (1968). -ibid. 85, 284 (1969).
3t -- 701829 J. Ultrastructure Research
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Sieve E l e m e n t Pores in Nicotiana Pith C u l t u r e 1 RICHARD ANDERSON AND JAMES CRONSHAW
Department of Biological Sciences, University of California, Santa Barbara, California 93106 Received January 9, 1970 Sieve elements were induced to form in cultured pith cylinders of Nieotiana tabacum L. (var. Wisconsin 38). Both xylem and phloem differentiate in isolated nodules at the periphery of the cylinders. The mature sieve elements closely resemble sieve elements in intact plants but are more irregular in shape and usually not highly elongated. The formation of pores in the phloem sieve plates is similar to that observed in intact plants. Callose platelets are deposited around plasmodesmata, then the plasmodesmata enlarge to form the mature perforation. Dense slime plugs are only rarely observed. The majority of sieve plate pores are plugged by a combination of P-proteins, ER, and callose, but there are also numerous unplugged pores. We interpret the observation of numerous unplugged pores as further evidence that in vivo pores may not be plugged in tobacco, There are an increasing number of observations which indicate that in vivo sieve plate pores of Nicotiana are not plugged but contain a loose array of P-protein. Plugged pores, like slime plugs and heavy callose deposits, appear to be wound reactions. We observed unplugged pores in plants which were frozen in liquid nitrogen and then fixed at 4°C (5). Later we found that if pores were blocked by starch grains near cut surfaces they were devoid of P-protein (1). Pores with loosely arranged P-protein were also found in some wilted plants and plants thinly sliced before fixation (2). Wooding (24) investigated the ultrastructure of sieve elements induced in tobacco callus. He observed a few pores which were not plugged with P-protein. Electron microscopic observations of the development of sieve plate pores were first made on Cucurbita by Frey-Wyssling and Muller (15), who studied the perforation process observing the wall microfibrils in shadowed preparations. Esau, Cheadle, and Risley (12) later investigated Cucurbita using fixed and sectioned material. They observed that endoplasmic reticulum became applied over the plasmodesmata of the future sieve plates. Callose platelets formed beneath the endoplasmic reticulum and then expanded in thickness and diameter. The middle lamella was removed, the callose z The study was supported by National Science Foundation Grant GB 12371 to Dr. J. Cronshaw.
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platelets fused, then the platelets were eroded to produce the mature pore. Northcote and Wooding (20) and Evert et al. (13) observed a similar sequence of events in Aeer and Cucurbita, but reported that perforation began with enlargement of the plasmodesmata in the middle lamella. Bouck and Cronshaw (3) observed similar development in pea except that the association of the ER with the pore sites was no more frequent than with other wall regions. Wooding (24) reported that pore formation in Nieotiana callus sieve elements proceeded by essentially the same process as in Acer. In the present investigation we have examined the fine structure of differentiating and mature sieve elements induced in tobacco pith culture. Numerous unplugged pores were observed in these in vitro sieve elements. The development of the sieve plate pores in the pith culture sieve element has been found to be similar to that of sieve elements in intact plants.
MATERIALS AND METHODS Tobacco plants (Nicotiana tabacum L. var. Wisconsin 38) were grown in a greenhouse until they were about 1 m tall. After defoliation the stems were cut into 3 cm segments which were sterilized in 50 % Clorox for 1 minute. The segments were then rinsed in several changes of sterile water. Sections about 2 mm thick were shaved off each end of the segments to remove cells contaminated with Clorox. Pith cylinders were then cut out with a 1/4-inch cork borer. The cylinders were inserted 4 mm into an agar growth medium in 3-inch culture tubes. The growth medium was the basal medium of Murashige and Skoog (18) to which were added 4 % sucrose and 0.5 or 1 nag/liter indole-3-acetic acid or naphthalene acetic acid. Cylinders were incubated at 26°C for 3-5 weeks, and then were prepared for electron microscopy. The pith cylinders were fixed in formaldehyde-glutaraldehyde in 0.1 M pH 6.8 phosphate buffer (16). They were either cut in half or placed in the fixative whole. The vials of fixative and specimens were placed in a vacuum chamber, and the pressure was alternately lowered and raised during the fixation period of 2 hours. This procedure removed air from the specimens and facilitated penetration of the fixative. The cylinders were then sliced into disks less than 1 mm thick which were washed in buffer for 3 hours (3 changes). The sections were postfixed in 2 % osmium tetroxide in 0.1 M pH 6.8 phosphate buffer overnight at 4°C. They were dehydrated with a graded acetone series and then infiltrated with Epon 812 epoxy resin. The specimens were recut just before polymerization. The blocks were sectioned with a diamond knife on a Porter-Blum MTI ultramicrotome. Sections were stained with uranyl acetate and lead (17), and examined and photographed with a Siemens Elmiskop I.
RESULTS Under the growth conditions of this investigation, cell division occurred in the periphery of the pith cylinders from the base of the cylinders to about 1 cm above the level of the medium. Nodules containing xylem and phloem differentiated only in
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the zone above the level of the medium. Xylem cells usually differentiated first, at the center of the nodules. A r o u n d this core some of the cells differentiated into phloem. The shape of the sieve elements was variable; they were often elongated but were irregular in section. Sieve elements at early stages of differentiation resemble meristematic cells in intact plants. Callose platelets f o r m in the wall a r o u n d the plasmodesmata of the future sieve plates (Figs. 1 and 2). The platelets enlarge laterally and in thickness (Figs. 3-6). Endoplasmic reticulum is usually associated with the platelets (Figs. 4-6). The plasmodesmata are lined by the plasma m e m b r a n e f r o m their inception and have a central tubule. The plasmodesmata enlarge in the region of the middle lamella to produce a median nodule (Fig. 6). As the plasmodesmata enlarge further, callose and middle lamella are eroded to produce the pores of the mature sieve elements. The sequence of pore differentiation does not always correspond closely to the changes occurring in the cytoplasm. We have not observed stages of the actual perforation process. As j u d g e d by the difficulty of finding pores at stages of perforation, it is likely that actual perforation is quite rapid, as suggested by Esau, Cheadle, and Risley (12). The mature sieve elements in tobacco tissue culture closely resemble the sieve elements of intact plants (5, 7). The mitochondria, endoplasmic reticulum, plastids, and P-protein are essentially the same as those components in intact plants. The development and distribution of P-protein is described in another c o m m u n i c a t i o n (6). The P-protein in cultured sieve elements is generally distributed t h r o u g h o u t the cell lumina; dense slime plugs are not generally formed, but occasional ones have been Key to abbreviations
C
callose ER endoplasmic reticulum M mitochondrion M L middle lamella P pore
Pl Pp i'd SE SP
plastid p-protein proplastid sieve element sieve plate
FIGS. 1 and 2. Nicotiana tabacum. Fig. 1. Electron micrograph of cells in early stages of sieve element differentiation. Small amounts of callose are around the plasmodesmata, which are the future pore sites (arrows). Normal cytoplasmic components are present, including proplastids (Pd) and mitochondria (M). Fig. 2. A more highly magnified view of a developing pore showing one of the callose platelets and the plasmodesma (arrow). Fig. 1, x 9000; Fig. 2, x 36,000. FIGS. 3-6. Nicotiana tabacum. Series of stages in pore development. Fig. 3. Conical callose platelets (C) have formed around the plasmodesmata on either side of the middle lamella. The plasmodesmata are lined by the plasma membrane (arrow). Figs. 4 and 5. The plasmodesma is slightly enlarged in the region of the middle lamella. Lengths of endoplasmic reticulum lie close to the plasma membrane over the callose platelets. Fig. 6. The median nodule of a pore at this stage is considerably enlarged. Indications of structures, possibly membranes, are visible within the nodule. Fig. 3, x 55,000; Fig. 4, x 43,000; Fig. 5, x 44,000; Fig. 6, x 52,000. FIG, 7. Nicotiana tabacum. Electron micrograph of a sieve plate associated with an unusually dense slime plug. P-protein is compacted in the pores, x 28,000. FIG. 8. Nicotiana tabacum. Electron micrograph of pore blocked by ER and constricted by callose. There is no dense slime plug. P-protein of the tubular (Pp) type is in the lumina of the cells. × 45.600.
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FIGS. 9 and 10. Nicotiana tabacum. Electron micrographs of portions of sieve plates. The P-protein is fairly evenly distributed in the region of the sieve plates. The pores in Fig. 9 are loosely packed with P-protein while those of Fig. 10 contain widely spaced P-protein. One of the pores of Fig. 10 is blocked by some ER. Deposition of callose has constricted the diameter of the pores. Fig. 9, × 16,500; Fig. 10, × 20,000. observed (Fig. 7). The majority of sieve element pores are plugged with P-protein a n d / o r endoplasmic reticulum (Figs. 8 and 9, one of the pores in Fig. 10). However, m a n y sieve plate pores are not plugged with P-protein (Figs. 10-12). Because of favorable section orientation, the sieve element of Fig. 11 appears very similar to the sieve elements of intact plants. The cell is elongated, with a sieve plate at either end. P-protein is dispersed t h r o u g h o u t the lumen and is rather evenly distributed. The modified plastids are concentrated near the sieve plates. The sieve plate pores are large, with little callose, and are not plugged by P-protein. This sieve element is part of a sieve tube of undetermined length. Fig. 12 depicts portions of two sieve elements whose slime bodies have only partially dispersed. The sieve plate pores in such sieve elements contain very little P-protein. FIG. l 1. Nicotiana tabacum. Electron micrograph of a portion of a linear row of sieve elements and associated cells. The pores in the two sieve plates are large and are not plugged with P-protein. Little callose is deposited in the pores. The P-protein is scattered throughout most of the cell lumina. × 4900
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The pore visible in Fig. 12 is devoid of P-protein. A portion of another sieve element with partially dispersed P-protein is included in Fig. 1 I. Callose was present in all the pores observed. The majority were almost completely filled by callose (Fig. 8). The contents of these pores are tightly compressed. In numerous other pores, however, there is less callose and less compression of the pore contents (Figs. 10, 11). When a sieve element in a plant becomes nonfunctional, a mass of definitive callose is deposited on the sieve plates (11). The cytoplasmic contents disorganize, and the sieve element may be crushed by surrounding cells. Sieve elements in culture appear to undergo the same process. Especially in older cultures, masses of definitive callose separate intact sieve elements from those which have become disorganized (Fig. 13). The disorganized sieve elements may also be crushed by the growth of surrounding cells. Starch granules occasionally persist in the crushed cells. A connection between a sieve element and an associated parenchyma cell is shown in Fig. 14. There is a single opening surrounded by callose on the sieve element side; there is no callose on the parenchyma cell side. Many channels from the parenchyma cell connect with a single channel Of the sieve element. The connections are lined by the plasma membrane. A tubule is visible at the open arrow in Fig. 14. A section of endoplasmic reticulum appears to extend into one of the branches. The connections between companion cells and sieve elements in many plants resemble this type of branched connection. The lower cell in Fig. 14 is probably comparable to the companion cells of intact plants. DISCUSSION Sieve elements in Nicotiana pith culture differentiate in nodules at the periphery of the pith cylinders. The sieve plate pores develop from plasmodesmata in the region of the future sieve plates. Callose platelets form in the wall around each plasmodesma. The plasmodesma develops a median nodule in the region of the middle lamella then enlarges to form a wide perforation in the mature sieve element. This process parallels that described for Aeer by Northcote and Wooding (20) and for Cucurbita by Esau, Cheadle, and Risley (12). The protoplasmic contents of mature sieve elements in tissue culture closely resemble those in intact plants. They form short sieve tubes in the isolated nodules. Slime plugs, if present, are usually only slightly developed. The sieve plate pores are generally closed by P-protein and callose, but in many cases the pores are not plugged
Fio. 12. Nicotiana tabacum. Electron micrograph of two sieve elements whose P-protein bodies have not completely dispersed. The sieve plate pores are nearly devoid of P-protein, and there is little callose in the pores, x 9100.
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and there is little callose. In some sieve elements there are dense slime plugs and plugged pores. Thus, the pore-closing mechanisms of in vivo sieve elements may also function in cultured sieve elements. Sieve elements are very susceptible to damage during fixation procedures. The main changes induced by manipulation are formation of slime plugs (4, 10, 19), plugging of the sieve plate pores (1, 2, 5, 8, 20), and deposition of callose (5, 9, 10, 21). These responses are most likely mechanisms to seal off the sieve tubes when they are damaged in nature. The mechanism for initiation of callose deposition is unknown. The mechanism of slime plug formation seems to be based on the very function of the sieve tube. The high sucrose concentration in the translocate creates a high hydrostatic pressure in the sieve tube. When the sieve tube is severed, or when its semipermeability is locally destroyed by fixative, the pressure is released and an accelerated flow is initiated. Slime plugs form as the sieve plates filter out P-protein and starch grains dislodged by the flow. The length of the sieve tube probably affects the extent of disruption which occurs when a sieve tube is fixed or cut. Sieve elements at quite a distance from a cut appear to contribute to the flow toward the cut. This is apparent from the observation that slime plugs may form up to 1.5 cm from a cut although they decrease in intensity (4, 10). Ford and Peel (14) have also shown that, if the sieve tubes of a willow stem are cut, sieve elements more than 16 cm away f r o m the cut may contribute to the exudation. This discussion of slime plug formation is pertinent to an understanding of the relative insensitivity to manipulation demonstrated by in vitro sieve elements. The length of cultured sieve tubes is unknown but must not exceed a few millimeters. If such short sieve tubes are severed or disrupted by fixative there should be less potential for flow than in long sieve tubes. Another factor contributing to the insensitivity of in vitro sieve tubes may be the nature of the cell contents. Since in vitro sieve tubes are not connected to sugar sources such as a leaf, their contents may not have the high hydrostatic pressure of in vivo sieve tubes. These two factors, short length and probable low pressure, may combine to account for the general lack of slime plugs and the presence of some unplugged pores. The sieve plate pores in the majority of sieve elements were plugged even though a dense slime plug did not form (Figs. 8 and 9). Probably m a n y of these were plugged before immersion into fixative, especially those plugged with E R (Figs. 8 and 10). FIG. 13. Nicotiana tabacum. Electron micrograph of a sieve plate with definitive callose, and associated cells. The cytoplasm of one of the cells has become disorganized while the other sieve element appears normal. × 19,600. FIG. 14. Nicotiana tabacum. Electron micrograph of the branched connections between a sieve element and an adjacent cell which is probably a companion cell. The open arrow indicates a tubule in one of the branches. A length of endoplasmic reticulum at the arrow appears to be associated with a structure in the connection. × 105,000.
1'1
7J
, f
11
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However, others were probably closed by the deposition of callose as described by Northcote and Wooding (20) and Cronshaw and Anderson (5). According to these authors, when callose is deposited in pores as a wound response, the contents of the pores may be compressed. It is likely that this same mechanism operates to close the pores in cultured sieve elements. Wooding (23, 24) reported less callose in cultured sieve elements than in those of intact plants. We have observed a wide range in the amount of callose deposited in the sieve plate pores. The sieve elements with unplugged pores generally have less callose than those with plugged pores. Similar observations were made on cotton by Shih and Currier (21) and on wilted tobacco plants by Anderson and Cronshaw (2), who found that sieve elements with open pores had little callose in the pores. The in vitro tobacco sieve element protoplasts are structurally similar to intact plant sieve dements. They undergo similar differentiation processes, and can form definitive callose deposits on their sieve plates. The same mechanisms of slime plug formation, callose deposition, and pore plugging can operate as in intact plants. But because of shorter length, and possibly lower sucrose concentration these mechanisms may fail to operate in vitro. We have also observed the failure of pore closing mechanisms in tobacco plants prepared by special techniques. Pore plugging mechanisms may be disrupted in plants frozen in liquid nitrogen (5). When plants are wilted to a certain extent the pore plugging mechanisms fail to operate and the pores remain unplugged (2). If sieve tubes are cut into short lengths many of the pores are unplugged (2). In sieve elements near cut edges the pore blocking mechanisms operate effectively, but in many cases pores are blocked by starch grains rather than P-protein (1). None of these results by themselves are definitive, but taken together they indicate that in tobacco, plugged pores are artifacts resulting from disturbance of the sieve tubes. REFERENCES 1. ANDERSON, R. and CRONSHAW, J., J. Ultrastruct. Res. 29, 50 (1969). ANDERSON,R. and CRONSHAW,J., Planta, in press. BOUCK,G. B. and CRONSHAW,J., J. Cell Biol. 25, 79 (1965). CRAFTS,A. S., Am. J. Botany 26, 172 (1939). CRONSHAW,J. and ANDERSON,R., J. Ultrastruct. Res. 27, 134 (1969).
2. 3. 4. 5. 6. 7. 8. 9. 10. 11. ]2. 13.
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CRONSHAW, J. and ESAU, K., d. Cell Biol. 34, 801 (1967). CRONSHAW,J. and ESAU, K., ar. Cell Biol. 38, 292 (1968). CURRIER,H. B., Am. J. Botany 44, 478 (1957). ENGLEMAN,E. M., Ann. Botany 29, 83 (1965).
ESAU, K., Plant Anatomy. Wiley, New York, 1965. ESAU, K., CHEADLE, V. I. and RISLEY, E. B., Botan. Gaz. 123, 233 (1962). EVERT, R. F., MURMANIS,L. and SACHS, I. B., Ann. Botany 30, 563 (1966).
SIEVE ELEMENTPORES IN NICOTIANAPITH 14. 15. 16. 17. 18. 19: 20. 21. 22. 23. 24.
FORD, J. and PEEL, A. J., J. Exptl. Botany 17, 522 (1966). FREY-WYSSLING,A. and MULLER, H. R., J. UItrastruct. Res. 1, 38 (1957). KARNOVS~Y,M. J., J. Cell Biol. 27, 137A (1965). MILLONIG, G., J. Biophys. Biochem. Cytol. 11, 736 (1961). MURASmGE, T. and SKOOG, F., Physiol. Plantarum 15, 473 (1962). NAGELI, C., S.B. Bayer. Akad. Wiss. 1, 212 (1861). NORTHCOTE,D. H. and WOODING, F. B., Proc. Roy. Soc. B163, 524 (1965). SHIH, C. Y. and CURRIER, H. B., Am. J. Botany 56, 464 (1969). WEATHERLEY,P. E., PEEL, A. J. and HILL, G. P., J. Exptl. Botany 10, 1 (1959). WOODING, F. B. P., Planta 83, 99 (1968). -ibid. 85, 284 (1969).
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