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Sieve plate pores of nicotiana
© 1969 by Academic Press, Inc.
134
J. ULTRASTRUCTURE RESEARCH 27, 134--148 (1969)
Sieve Plate Pores of Nicotiana' JAMES CRONSHAWAND RICHARD ANDERSON
Department of Biological Sciences, University of California, Santa Barbara, California 93106 Received October 9, 1968, and in revised form December 2, 1968 Nicotiana tabacum L. (var. Wisconsin 38) piants were fixed for light and electron microscopy using solutions of glutaraldehyde, acrolein, and formaldehydeglutaraldehyde. The plants were (a) cut before fixation at room temperature or (b) fixed as whole plants at room temperature, or (c) rapidly frozen and transferred to cold fixative. Good fixation was obtained by direct chemical fixation of either cut or whole plants. After rapid freezing, fixation was inferior but still satisfactory. Glutaraldehyde and formaldehyde-glutaraldehyde-fixed material showed sieve plate pores that were filled with P-protein. Acrolein-fixed material showed mainly sieve plate pores filled with P-protein, but some pores were observed containing loosely arranged P-protein. In contrast, after rapid freezingcold chemical fixation, numerous "open" pores and pores with loosely arranged P-protein were seen. It is suggested that these results support the mass flow hypothesis of translocation. After rapid freezing-cold fixation much less callose was observed around the sieve plate pores than after room temperature fixation with the same fixative. Enzymatic reactions and callose deposition must take place during fixation procedures at room temperature. The pores in the sieve plates of mature phloem sieve elements develop during the differentiation of the cells by a dissolution of wall material. The question of the nature of the pore contents is a classical one in the literature, and its answer is essential to an understanding of the mechanism of long-range transport of nutrients in the phloem. Early observations by Preston and co-workers (23, 24) indicated that in Cucurbita, Sorbus, and Vitis the pores contained electron opaque material which was continuous with similar material at one or both surfaces of the sieve plate. The material had been fixed using osmium tetroxide. Schumacher and Kollmann (35) and Kollmann (29) studied Passiflora after osmium fixation and described dense connecting strands running through the sieve plate pores. Ziegler (39) observed dense connecting strands in Heracleum, and Hohl (25) depicted similar material in Datura. These observations raised doubts as to whether longdistance transport of nutrients in the phloem could be by a mass-flow mechanism. z The study was supported by National Science Foundation Grant GB 6271 to Dr. J. Cronshaw.
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In contrast to these results, Esau and Cheadle using potassium permanganate as a fixative (12-14) demonstrated open pores in Cucurbita. The so-called "mictoplasm" was said to be continuous through the pores (14). When glutaraldehyde was introduced as a fixative (34), it appeared to yield excellent fixation of plant cells (31) and especially of the phloem tissue (2). Bouck and Cronshaw (2) described dense plugging material in the pores of the sieve elements of Pisum fixed with either glutaraldehyde or acrolein. More recent studies using glutaraldehyde fixation have shown pores filled with electron opaque material in Acer (33, 38), Nicotiana (6), Dioscorea (1), Phaseolus (30), Tilia (20), and Primula (36). Pores containing loosely organized P-protein have been demonstrated in the monocotyledons EIodea (10) and Nymphoides (27). The validity of the observations of sieve plate pores being filled with P-protein was questioned when our observations showed that in sugar beet plants infected with the beet yellows virus, some sieve plate pores were occupied by virus particles instead of P-protein (15). The results were interpreted as indicating a mass movement of the virus particles through the sieve tubes together with translocated photosynthate. Later observations on Cucurbita (7, 8) showed varying conditions of the sieve plate pores and their association with P-protein. The conditions ranged from filled to unfilled. Most investigators have tended to conclude that P-protein can accumulate in the pores as a result of accelerated flow caused by pressure changes in the conduit induced by cutting. The present investigation has shown that good fixation of whole plants can be obtained either by direct fixation or by fixation after rapid killing in liquid nitrogen. By these methods unfilled and loosely filled pores have been demonstrated in tobacco sieve plates and it has also been shown that enzymatic reactions and callose deposition may take place during normal fixation procedures. MATERIALS AND METHODS Tobacco plants (Nicotiana tabacum L. var. Wisconsin 38) were grown under greenhouse conditions until the stems were 3-5 cm and the largest leaves 10-20 cm. Plants were watered on a regular schedule and harvested for fixation about 10 A.M. Specimens for light and electron microscopy were taken from a standard position at the base of the petiole of a 10-15 cm leaf according to the following procedures. Three fixing solutions were used: (a) a combination of glutaraldehyde and formaldehyde in 0.1 M pH 6.8 phosphate buffer (28); (b) 3 % glutaraldehyde in 0.1 M p H 6.8 phosphate buffer; (c) 10 % acrolein in 0.05 or 0.1 M p H 6.8 phosphate buffer. Three main fixation techniques were employed: (a) One-centimeter segments were cut from the standard position on the petiole into the fixing solution. After fixation for 0.5 hour, the inner third of these segments was cut into disks and the remainder of the material was discarded. Fixation was continued for a further 1.5 hours and then the disks were washed in buffer for 3 hours (3 changes) and postfixed in 2 % osmium tetroxide
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in 0.1 M pH 6.8 phosphate buffer overnight in the refrigerator. Dehydration was with a graded acetone series, and the specimens were embedded in Epon 812 epoxy resin. When necessary the specimens were cut into smaller pieces. (b) Whole plant fixation. Pl~r~ts were rinsed free of vermiculite on their roots then carefully immersed in fixative for 1.5 hours. Sections under 1 mm were cut from the standard position on the petiole, and fixation was continued 1.5 hgurs longer. After fixation the specimens were washed in buffer for 3 hours and postfixed in osmium tetroxide overnight in the refrigerator. Dehydration and embedding was as for (a). (c) Rapid freezing. Plants were bound with string to facilitate their insertion into the freezing chamber, quickly removed from their pots and immersed in an isopentane-methylcyclohexane mixture cooled by liquid nitrogen (26). The fixing solutions were cooled until ice crystals just began to form, and the plants were transferred to the cooled fixative for 3-4 hours. They were then allowed to gradually warm at room temperature for 1 hour. Sections were cut from the standard position on the petiole as before, and fixation was continued for a further 1.5 hours. Postfixation, dehydration, and embedding were as in (a). In later experiments it was found that better fixation was made possible by omitting the isopentane-methylcyclohexane mixture and freezing the plants directly in liquid nitrogen. The blocks were trimmed to section the external phloem, and sectioning was with a diamond knife on a Porter-Blum MT1 ultramicrotome. Sections were cut at 0.5-1.5 tt for light microscopy and showing silver interference colors for electron microscopy. For light microscopy the sections were viewed using either phase contrast or Nomarski interference contrast optics. For electron microscopy the thin sections were stained with uranyl acetate and lead (32) and viewed and photographed with a Siemens Elmiskop I.
RESULTS In an earlier r e p o r t f r o m this l a b o r a t o r y (6), electron m i c r o g r a p h s were published of Nicotiana tabacum sieve plate pores illustrating the relationship of P - p r o t e i n t o the pore. This material h a d been processed for electron m i c r o s c o p y after samples
Key to abbreviations C callose SP sieve plate CC companion cell P pore SE sieve element V vacuole FrG. 1. Nicotiana tabacum. Whole plant glutaraldehyde fixation. Electron micrograph of a portion of a sieve element (SE) and an adjacent parenchyma cell. The sieve element appears mature with a fully developed pore in the sieve plate. P-protein is sparsely distributed in the lumen and plastids (P) are arranged at the periphery of the cell. Cisternae of endoplasmic reticulum can be seen adjacent to the plasma membrane. There is good preservation of the organelles and general cytoplasmic features. The adjacent parenchyma cell has well preserved cytoplasm with plastids, mitochondria, endoplasmic reticulum, ribosomes, and vacuoles (V). × 18,000. FIGS. 2 and 3. Nicotiana tabaeum. Light micrographs of specimens similar to the one depicted in Fig. 1. Fig. 2 was photographed using Nomarski interference contrast optics, and Fig. 3 using normal phase contrast optics. The general arrangement of the cytoplasmic components in the sieve element (SE) and adjacent companion cell can be seen. The nucleus and the vacuoles in the companion cell contrast sharply with the condition of the sieve element, which has no nucleus and no vacuoles. There is no evidence in the light micrographs of P-protein or strands leading from the sieve plate pores. × 680.
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had been cut from plants and fixed in glutaraldehyde or formaldehyde-glutaraldehyde fixatives. In the published micrographs, as in all the material examined, the sieve plate pores were densely filled with P-protein. Preservation of the sieve elements, companion cells and parenchyma cells was judged as excellent with little distortion of organelles or general cytoplasmic arrangement with the possible exception of hydrostatic flow in the sieve elements. In the present study we obtained similar preservation by fixing whole plants in glutaraldehyde and formaldehyde-glutaraldehyde fixatives and have reasoned that if the whole plant is fixed before cutting there should be no changes due to hydrostatic flow caused by pressure changes upon cutting the phloem conduits. The quality of fixation of sieve elements and adjacent companion cells from whole plants fixed in glutaraldehyde is illustrated in Fig. 1. The organelles are well preserved both in the sieve element and companion cell. The sieve element illustrated is at an advanced stage of differentiation when the vacuolar membrane is no longer present. The companion cell is vacuolate, and the vacuoles are well preserved. It is of interest that the plastids of the sieve element are arranged in a parietal position and one apparently blocks a sieve plate pore. The appearance of material fixed in this way and sectioned for the light microscope is shown in Figs. 2 and 3. These photomicrographs are of serial sections, one (Fig. 2) obtained using Carl Zeiss Nomarski interference contrast optics and the other using Carl Zeiss phase contrast optics. The companion cell illustrated has dense cytoplasm with nucleus and vacuoles distinct and obvious. In contrast, the sieve elements appear devoid of cytoplasmic components with the exception of the peripherally arranged plastids. There is no indication of strands in the sieve element. The sieve plate pores of material fixed as whole plants in glutaraldehyde are comparable to those of plants which are cut before fixation. In all the material examined after fixation as whole plants in glutaraldehyde the pores of the sieve plates have large callose cylinders and are filled with P-protein. A median section of a sieve plate pore is shown in Fig. 4. The pore is filled with P-protein which frays out into the lumen of the sieve element. The P-protein is of the tubular type. The condition of the pore is obviously similar to those of plants cut before fixation in glutaraldehyde (cf. Fig. 10, ref. 6). Similar results are obtained by fixing whole plants in formaldehyde-glutaraldehyde. A comparable micrograph of a pore in a sieve plate from a plant fixed as a whole plant in formaldehyde-glutaraldehyde is presented in Fig. 5. Again the pore is similar to those from samples cut before fixation (cf. Figs. 11 and FIGS. 4 and 5. Nicotiana tabacum. Fig. 4. Whole plant glutaraldehyde fixation; Fig. 5 whole plant formaldehyde-glutaraldehyde fixation. Sections of sieve plates showing pores (P) filled with P-protein and surrounded by large callose cylinders (C). The P-protein frays out into the lumen of the cell and is of the tubular type. Fig. 4, x 40,500; Fig. 5, x 46,500.
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FIGS. 6 and 7. Nicotiana tabacum. Whole plant acrolein fixation. The sieve plate pores (P) are surrounded by callose cylinders (C). However, the callose deposition is not as heavy as with glutaraldehyde and formaldehyde glutaraldehyde fixatives. The P-protein in the pores may be densely packed as in Fig. 6 or loosely packed as in Fig. 7. Fig. 6, × 48,000; Fig. 7, × 43,500.
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FIG. 8. Nicotiana tabacum. Electron micrograph of a s ~ t i o n through sieve elements (SE) and adjacent companion cells (CC). The specimen was fixed in cold glutaraldehyde after killing in liquid nitrogen. The general cytoplasmic preservation is not as good as with glutaraldehyde fixation alone. Most of the organelles are fairly well preserved, however, and the general cytoplasmic features are comparable to those of specimens fixed in glutaraldehyde alone, × 6600. FIG. 9. Nicotiana tabacum. Nomarski interference contrast photograph of a specimen similar to that in Fig, 10. The sieve elements (SE) contain some strands of P-protein. The dense cytoplasmic contents and vacuoles of the adjacent parenchyma cells are evident. × 660.
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13, ref. 6). The pore is filled with P-protein, and a large callose cylinder is present. The tubules of P-protein are very well preserved with formaldehyde-glutaraldehyde. Acrolein fixation also gives similar results for plants cut before fixation or fixed as whole plants. With acrolein fixation the preservation of organelles is similar to that by glutaraldehyde or formaldehyde-glutaraldehyde with the exception of microtubules and tubular P-protein. With occasional exceptions P-protein is not preserved in the tubular form but appears as fibrils, and cytoplasmic microtubules are not apparent. The sieve plate pores have less extensive callose cylinders than after glutaraldehyde or formaldehyde-glutaraldehyde fixation (Figs. 6 and 7) and show a variation in their P-protein contents. In some pores the P-protein is loosely arranged (Fig. 7) while others are densely plugged (Fig. 6). In general, as with glutaraldehyde or formaldehyde-glutaraldehyde, fixation of whole plants yields results similar to those obtained from plants cut before fixation. In all combinations of fixative and technique, slime plugs are invariably present. The plugs are not dense; commonly P-protein concentration is only slightly different on the two sides of the plate. When plants are rapidly frozen in liquid nitrogen (or methyl cyclohexane-isopentane mixture surrounded by liquid nitrogen) before cold fixation by chemical methods, general fixation is inferior to that obtained by r o o m temperature methods. There is some vesiculation of the cytoplasm, plasmolysis in s o m e cells, and some distortion of organelles. Sieve element plastids usually rupture and liberate their starch grains. Despite generally inferior fixation, differences are observed which we consider significant. These concern the observable callose and the relation of the P-protein to the sieve plate pores. Fig. 8 illustrates a sieve tube and adjacent parenchyma cells showing the quality of fixation obtainable with r a p i d freezing-glutaraldehyde fixation. There is some distortion of organelles and some mitochondria have been ruptured. The free starch grains in the sieve tube indicate that sieve element plastids have been ruptured. A dense slime plug and a strand of P-protein are also present. A Nomarski interference contrast micrograph of similar material is shown in Fig. 9. In the sieve element are some strands which are probably P-protein. In the adjacent companion and parenchyma cells dense cytoplasmic components and the vacuoles appear to be intact. There is a wide variation of slime plug formation in rapidly frozen material. Some FIGS. 10-12. Nicotiana tabacum. Sections of sieve plates showing arrangements of P-protein in relation to the sieve plate pores after liquid fiitrogen killing, acrolein fixation. The condition of the sieve plate pores varies. In Fig. 10 the sieve plat~ pores are filled with P-protein material, and in Fig. 11 the P-protein is loosely arranged within the pores. In Fig. 12 the pores are nearly empty of P-protein. Fig. 10, x 15,800; Fig. 11, x 17,400; Fig. 12, x 13,000. FIG. 13. Nicotiana tabacum. Section of a portion of a sieve plate taken from a specimen killed in liquid nitrogen and fixed in formaldehyde-glutaraldehyde. The P-protein material is loosely arranged within the sieve plate pores. Starch grains liberated from the plastids are apparent, x 23,500.
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TABLE I SIZE OF Nicotiana
SIEVE PLATE PORES AFTER VARIOUS FIXATION TECHNIQUESa Number of Plants
Glutaraldehyde Acrolein Formaldehyde-glutaraldehyde Rapid freezing-glutaraldehyde Rapid freezing-acrolein Rapid freezing-formaldehydeglutaraldebyde
Pore Size (#)
P Valueb
5 3 4 9 3
0.22±0.02 0.34 ± 0.02 0.16 ± 0.01 0.46 ± 0.01 0.43 ± 0.02
(30) (42) (26) (270) (79)
5
0.61+_0.03 (42)
Wall Pore e (/1)
---< .00l < .02
0.85±0.03 0.76 ± 0.03 0.76 ± 0.02 0.68 ± 0.01 0.71 +_0.01
(30) (42) (26) (270) (79)
<.001
0.74±0.03 (42)
a Numerals in parentheses indicate the number of measurements made to determine the mean value. All mean values ±sm b p value with respect to the corresponding value derived from unfrozen specimens. The number of plants in each experiment determined the degrees of freedom. c The term "wall pore" has been used for the outer diameter of the eallose cylinder measured at the middle lamella and is the size of the pore in the microfibril-containing part of the sieve plate.
plates possess well d e v e l o p e d plugs (Fig. 14) while others do n o t (Fig. 12). Large quantities of P - p r o t e i n m a y be forced t h r o u g h pores as in Fig. 8 to p r o d u c e p r o t r u sions similar to those observed by Crafts (5) a n d Buvat (4). It is significant t h a t after freezing a n d chemical fixation m a n y open pores are observed (Figs. 12, 13, 15, a n d 16). I n a d d i t i o n to pores t h a t are p l u g g e d with P - p r o t e i n a n d those t h a t are empty, there are m a n y i n t e r m e d i a t e c o n d i t i o n s with loosely a r r a n g e d P - p r o t e i n in the sieve plate pores. Figures 10, 11, a n d 12 show sieve plates of r a p i d l y frozen, acrolein-fixed plants. I n Fig. 10 the sieve plate pores are plugged, in Fig. 11 the plugs are less dense, a n d in Fig. 12 the sieve plate pores are open. Fig. 13 shows loosely a r r a n g e d P - p r o t e i n in the sieve plate pores of a sieve element f r o m r a p i d l y frozen f o r m a l d e h y d e - g l u t a r a l d e h y d e fixed material. Figs. 14, 15, a n d 16 are of sieve plates of r a p i d l y frozen glutaraldehyde-fixed m a t e r i a l illustrating v a r i a t i o n in the degree of b l o c k i n g the pores. I n Fig. 15 an o p e n p o r e a n d plugged pores are present in the same sieve plate. It is considered significant t h a t with the exception of acrolein-fixed m a t e r i a l no o p e n p o r e s are o b s e r v e d after chemical fixation alone, b u t t h a t n u m e r o u s open pores are o b s e r v e d after the freezing-chemical fixation procedures. It m a y be significant t h a t after acro-
FIos. 14-16. Nieotiana tabacum. Sections of sieve plates from specimens killed in liquid nitrogen and fixed in glutaraldehyde. Fig. 14 shows an accumulation of P-protein adjacent to a sieve plate, and the sieve plate pores are partially filled with P-protein. Fig. 15 shows a sieve plate with two sieve plate pores filled with P-protein and one pore virtually devoid of P-protein. Fig. 16 shows a sieve plate with pores containing loosely organized P-protein. Fig. 14, × 11,200; Fig. 15, × 12,500; Fig. 16, × 45,000.
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lein fixation alone some loosely packed pores are observed, as acrolein is generally considered to be a more rapidly penetrating fixative. The size of the callose cylinder appears smaller after rapid freezing, and a study has been made of the pore size and the wall pore size (the term "wall pore" refers to the outer diameter of the callose cylinder measured at the middle lamella) for all the fixation procedures. The results are presented in Table I. The pore size differs significantly with the fixation procedure. The size of the pore in the wall without the callose cylinder, however, is fairly constant, indicating that the amount of callose deposition varies with the fixation method used. With room temperature fixation, the pore size is smallest with formaldehyde-glutaraldehyde and largest with acrolein. |n all cases the pore size is significantly larger when chemical fixation is preceded by freezing. Thus, enzymatic reactions resulting in callose deposition appear to be rapidly stopped by freezing but must be functional during room temperature chemical fixation. DISCUSSION Bullivant and Ames (3) have recently summarized numerous attempts at the preservation of biological structures for the electron microscope by rapid freezing. With the exception of structures demonstrated by the freeze etch technique, most investigators have found that rapid freezing methods do not give preservation as good as that of chemical fixation techniques. Rapid freezing techniques have been used for phloem tissue studies by several workers and have been particularly successful at the light microscope level (10, 1I, 18, 19). In addition to rapid freezing methods, many attempts have been made using specialized fixation techniques to minimize the injury response of sieve elements (10, 11, 17, 19, 27). The present study has shown that general protoplasmic preservation of plants frozen by liquid nitrogen and fixed at low temperature is inferior to that of chemical fixation alone at room temperature. Some artifacts are created including vesiculation of the cytoplasm, disruption of some organelles, and in some cells even plasmolysis. However, the general preservation is fairly good, and we consider the observations on the sieve plate pores significant. If chemical fixation with glutaraldehyde or formaldehyde-glutaraldehyde is preceded by rapid freezing, open pores can be demonstrated. Without rapid freezing the sieve plate pores are consistently plugged with P-protein; also with rapid freezing the pore size is larger and the amount of callose associated with the pores is less. Johnson (27) has recently examined the sieve plate pores of Nymphoides peltata using specialized fixation and freeze etch techniques. Filaments (P-protein) appeared in and near the sieve plate pores. Johnson suggests that the filaments are normally present in the pores. In the published micrographs the filaments appear to be loosely
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arranged in the pores (Ref. 27, Figs. 4, 5, 8, 16 and 17), and the arrangement is similar to that for many of the pores we have observed in the present investigation. Callose is deposited by enzyme-mediated reactions (21) which must be either stopped or drastically slowed by the rapid freezing and chemical fixation at low temperatures. It is of general interest that during room temperature fixation these enzymatic reactions can take place to such an extent that a considerable amount of callose is formed. Callose cylinders associated with sieve plate pores have been generally regarded as wound artifacts. Currier and Strugger (9) described callose formation in response to injury, and Eschrich (17) found that deposition of callose may occur in seconds. Callose deposition on sieve plates may be prevented by rapid fixation (19, 40), by avoiding the stimulus of cutting the plant before fixation (t6, cf. present results), or by employing a chelating agent to remove calcium from the sieve elements (17). However, Engleman (11) found a certain minimum amount of callose in material prepared by several rapid-freezing techniques. He concluded that this quantity is a real component of the intact plant. The present results support this conclusion. Although the amount of callose in the pore cylinders is much less after rapidfreezing techniques some is always present. Eschrich (16, 17) and Northcote and Wooding (33) have suggested that since extensive callose deposition may occur during room temperature fixation, P-protein in the pores would be compressed into the amorphous mass typically observed. Acrolein appears to halt this compression faster than does glutaraldehyde. The pores tend to remain large and loosely filled with P-protein. Loosely packed pores in rapidly frozen plants may likewise depend on rapid arrest of callose deposition. Most appear to contain enough P-protein to form a dense plug if more callose had been deposited. With regard to the present observations it is possible that either open pores or plugged pores or both are artifacts due to the preparation procedures. Freezing or thawing may dislocate the P-protein from the pores and create an artifact of open pores. On the other hand, plugged pores such as those depicted in Fig. 10 may be artifacts formed during slime plug formation. We believe the latter possibility is more likely. The observation that some open pores are present in the sieve plate is obviously significant with reference to the problems of interpreting translocation in relation to sieve tube structure. In view of the recent discussion of this problem by one of us (6), it is sufficient here to point out that a demonstration of open pores obviously supports the mass-flow hypothesis and recent physiological results (22, 37). In further support of the view that the pores in sieve elements are open, are our recent results showing that pores blocked by moving starch grains may be devoid of P-protein. These results will be the subject of another communication, but together with the present and previous results (15) they provide an accumulation of evidence in favor of open pores in functional sieve elements.
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REFERENCES 1. BEHNKE, H. D., Z. Pflanzenphysiol. 53, 214 (1965). BoUcK, G. and CRONSHAW, J., J. Cell Biol. 25, 79 (1965). BULLIVANT,S. and AMES, A., J. Cell Biol. 29, 435 (1966). BUVAT, M. R., Compt. Rend. Acad. Sci. 250, 1528 (1960). CRAFTS, A. S., Translocation in Plants. Holt, New York, 1961. 6. CRONSHAW, J. and ESAU, K., J. Cell Biol. 34, 801 (1967). 7. - ibid. 38, 25 (1968). 8. - ibid. 38, 292 (1968). 9. CURRIER,H. B. and STRUGGER,S., Protoplasma 45, 552 (1956). 10. CURRIER, H. B. and SHIrL C. Y., Am. J. Botany 55, 145 (1968). 11. ENGLEMAN,E. M., Ann. Botany 29, 83 (1965). 12. ESAU, K. and CHEADLE,V. I., Proe. Natl. Aead. Sci. U.S. 47, 1716 (1961). 13. ESAU, K., CHEADLE,V. I. and RISLEY, E. B., Botan. Gaz. 123, 233 (1962). 14. ESAU, K. and CHEADLE,V. I., Univ. California Publ. Botany 36, 253 (1965). 15. ESAU, K., CRONSHAW,J. and HOEFERT,L. L., J. Cell Biol. 32, 71 (1967). 16. ESCHRICn, W., Planta 59, 243 (1963). 17. - ibid. 65, 280 (1965). 18. ESCHRICH,W. and CURRIER, H. B., Stain Teehnol. 39, 303 (1964). 19. EVERT, R. F. and DERR, W. F., Am. J. Botany 51, 552 (1964). 20. EVERT, R. F. and MURMANIS,L., Am. J. Botany 52, 95 (1965). 21. FEINGOLD,D. S., NEUFELD, E. F. and HASSlD, W. Z., J. BioL Chem. 233, 783 (1958). 22. FORD, J. and PEEL, A. J., J. Exptl. Botany 17, 522 (1966). 23. HEPTON, C. E. L., PRESTON, R. D. and RIPLEY, G. W., Nature 176, 868 (1955). 24. HEVTON,C. E. L. and PRESTON, R. D., J. Exptl. Botany 11, 381 (1960). 25. HOHL, H. R., Ber. Sehweiz. Botan. Ges. 70, 395 (1960).
2. 3. 4. 5.
26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40.
JENSEN,W. A., Botanical Histochemistry. Freeman, San Francisco, California, 1962. JOHNSON,R. P. C., Planta 81, 314 (1968). KARNOVSKY,M. J., J. Cell Biol. 27, 137A (1965). KOLLMAbrN,R., Planta 55, 67 (1960). LAFLi~CnE,D., J. Microseopie 5, 493 (1966). LEDBETTER,M. C. and PORTER, K. R., J. Cell Biol. 19, 239 (1963). MILLONIG,G., J. Biophys. Bioehem. Cytol. 11, 736 (1961). NORTHCOTE,D. H. and WOODING, F. B. P., Proe. Roy. Soe. B163, 524 (1965). SABATINI,D. C., BENSCH, K. and BARRNETT,R. J., J. Cell Biol. 17, 19 (1963). SC~IUMACHER,W. R. and KOLLMANN,R., Ber. Deut. Botan. Ges. 72, 176 (1959). TAMULEVICH,S. R. and EVERT, R. F., Planta 69, 319 (1966). WEATHERLEY,P. E., Progr. Biophys. 13, 242 (1963). WOODING,F. B. P., Protoplasma 64, 315 (1967). ZIEGLER,H., Planta 55, 1 (1960). ZIMMERMAN,M. H., Ann. Rev. Plant Physiol. 11, 167 (1960).
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J. ULTRASTRUCTURE RESEARCH 27, 134--148 (1969)
Sieve Plate Pores of Nicotiana' JAMES CRONSHAWAND RICHARD ANDERSON
Department of Biological Sciences, University of California, Santa Barbara, California 93106 Received October 9, 1968, and in revised form December 2, 1968 Nicotiana tabacum L. (var. Wisconsin 38) piants were fixed for light and electron microscopy using solutions of glutaraldehyde, acrolein, and formaldehydeglutaraldehyde. The plants were (a) cut before fixation at room temperature or (b) fixed as whole plants at room temperature, or (c) rapidly frozen and transferred to cold fixative. Good fixation was obtained by direct chemical fixation of either cut or whole plants. After rapid freezing, fixation was inferior but still satisfactory. Glutaraldehyde and formaldehyde-glutaraldehyde-fixed material showed sieve plate pores that were filled with P-protein. Acrolein-fixed material showed mainly sieve plate pores filled with P-protein, but some pores were observed containing loosely arranged P-protein. In contrast, after rapid freezingcold chemical fixation, numerous "open" pores and pores with loosely arranged P-protein were seen. It is suggested that these results support the mass flow hypothesis of translocation. After rapid freezing-cold fixation much less callose was observed around the sieve plate pores than after room temperature fixation with the same fixative. Enzymatic reactions and callose deposition must take place during fixation procedures at room temperature. The pores in the sieve plates of mature phloem sieve elements develop during the differentiation of the cells by a dissolution of wall material. The question of the nature of the pore contents is a classical one in the literature, and its answer is essential to an understanding of the mechanism of long-range transport of nutrients in the phloem. Early observations by Preston and co-workers (23, 24) indicated that in Cucurbita, Sorbus, and Vitis the pores contained electron opaque material which was continuous with similar material at one or both surfaces of the sieve plate. The material had been fixed using osmium tetroxide. Schumacher and Kollmann (35) and Kollmann (29) studied Passiflora after osmium fixation and described dense connecting strands running through the sieve plate pores. Ziegler (39) observed dense connecting strands in Heracleum, and Hohl (25) depicted similar material in Datura. These observations raised doubts as to whether longdistance transport of nutrients in the phloem could be by a mass-flow mechanism. z The study was supported by National Science Foundation Grant GB 6271 to Dr. J. Cronshaw.
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In contrast to these results, Esau and Cheadle using potassium permanganate as a fixative (12-14) demonstrated open pores in Cucurbita. The so-called "mictoplasm" was said to be continuous through the pores (14). When glutaraldehyde was introduced as a fixative (34), it appeared to yield excellent fixation of plant cells (31) and especially of the phloem tissue (2). Bouck and Cronshaw (2) described dense plugging material in the pores of the sieve elements of Pisum fixed with either glutaraldehyde or acrolein. More recent studies using glutaraldehyde fixation have shown pores filled with electron opaque material in Acer (33, 38), Nicotiana (6), Dioscorea (1), Phaseolus (30), Tilia (20), and Primula (36). Pores containing loosely organized P-protein have been demonstrated in the monocotyledons EIodea (10) and Nymphoides (27). The validity of the observations of sieve plate pores being filled with P-protein was questioned when our observations showed that in sugar beet plants infected with the beet yellows virus, some sieve plate pores were occupied by virus particles instead of P-protein (15). The results were interpreted as indicating a mass movement of the virus particles through the sieve tubes together with translocated photosynthate. Later observations on Cucurbita (7, 8) showed varying conditions of the sieve plate pores and their association with P-protein. The conditions ranged from filled to unfilled. Most investigators have tended to conclude that P-protein can accumulate in the pores as a result of accelerated flow caused by pressure changes in the conduit induced by cutting. The present investigation has shown that good fixation of whole plants can be obtained either by direct fixation or by fixation after rapid killing in liquid nitrogen. By these methods unfilled and loosely filled pores have been demonstrated in tobacco sieve plates and it has also been shown that enzymatic reactions and callose deposition may take place during normal fixation procedures. MATERIALS AND METHODS Tobacco plants (Nicotiana tabacum L. var. Wisconsin 38) were grown under greenhouse conditions until the stems were 3-5 cm and the largest leaves 10-20 cm. Plants were watered on a regular schedule and harvested for fixation about 10 A.M. Specimens for light and electron microscopy were taken from a standard position at the base of the petiole of a 10-15 cm leaf according to the following procedures. Three fixing solutions were used: (a) a combination of glutaraldehyde and formaldehyde in 0.1 M pH 6.8 phosphate buffer (28); (b) 3 % glutaraldehyde in 0.1 M p H 6.8 phosphate buffer; (c) 10 % acrolein in 0.05 or 0.1 M p H 6.8 phosphate buffer. Three main fixation techniques were employed: (a) One-centimeter segments were cut from the standard position on the petiole into the fixing solution. After fixation for 0.5 hour, the inner third of these segments was cut into disks and the remainder of the material was discarded. Fixation was continued for a further 1.5 hours and then the disks were washed in buffer for 3 hours (3 changes) and postfixed in 2 % osmium tetroxide
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CRONSHAW AND ANDERSON
in 0.1 M pH 6.8 phosphate buffer overnight in the refrigerator. Dehydration was with a graded acetone series, and the specimens were embedded in Epon 812 epoxy resin. When necessary the specimens were cut into smaller pieces. (b) Whole plant fixation. Pl~r~ts were rinsed free of vermiculite on their roots then carefully immersed in fixative for 1.5 hours. Sections under 1 mm were cut from the standard position on the petiole, and fixation was continued 1.5 hgurs longer. After fixation the specimens were washed in buffer for 3 hours and postfixed in osmium tetroxide overnight in the refrigerator. Dehydration and embedding was as for (a). (c) Rapid freezing. Plants were bound with string to facilitate their insertion into the freezing chamber, quickly removed from their pots and immersed in an isopentane-methylcyclohexane mixture cooled by liquid nitrogen (26). The fixing solutions were cooled until ice crystals just began to form, and the plants were transferred to the cooled fixative for 3-4 hours. They were then allowed to gradually warm at room temperature for 1 hour. Sections were cut from the standard position on the petiole as before, and fixation was continued for a further 1.5 hours. Postfixation, dehydration, and embedding were as in (a). In later experiments it was found that better fixation was made possible by omitting the isopentane-methylcyclohexane mixture and freezing the plants directly in liquid nitrogen. The blocks were trimmed to section the external phloem, and sectioning was with a diamond knife on a Porter-Blum MT1 ultramicrotome. Sections were cut at 0.5-1.5 tt for light microscopy and showing silver interference colors for electron microscopy. For light microscopy the sections were viewed using either phase contrast or Nomarski interference contrast optics. For electron microscopy the thin sections were stained with uranyl acetate and lead (32) and viewed and photographed with a Siemens Elmiskop I.
RESULTS In an earlier r e p o r t f r o m this l a b o r a t o r y (6), electron m i c r o g r a p h s were published of Nicotiana tabacum sieve plate pores illustrating the relationship of P - p r o t e i n t o the pore. This material h a d been processed for electron m i c r o s c o p y after samples
Key to abbreviations C callose SP sieve plate CC companion cell P pore SE sieve element V vacuole FrG. 1. Nicotiana tabacum. Whole plant glutaraldehyde fixation. Electron micrograph of a portion of a sieve element (SE) and an adjacent parenchyma cell. The sieve element appears mature with a fully developed pore in the sieve plate. P-protein is sparsely distributed in the lumen and plastids (P) are arranged at the periphery of the cell. Cisternae of endoplasmic reticulum can be seen adjacent to the plasma membrane. There is good preservation of the organelles and general cytoplasmic features. The adjacent parenchyma cell has well preserved cytoplasm with plastids, mitochondria, endoplasmic reticulum, ribosomes, and vacuoles (V). × 18,000. FIGS. 2 and 3. Nicotiana tabaeum. Light micrographs of specimens similar to the one depicted in Fig. 1. Fig. 2 was photographed using Nomarski interference contrast optics, and Fig. 3 using normal phase contrast optics. The general arrangement of the cytoplasmic components in the sieve element (SE) and adjacent companion cell can be seen. The nucleus and the vacuoles in the companion cell contrast sharply with the condition of the sieve element, which has no nucleus and no vacuoles. There is no evidence in the light micrographs of P-protein or strands leading from the sieve plate pores. × 680.
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had been cut from plants and fixed in glutaraldehyde or formaldehyde-glutaraldehyde fixatives. In the published micrographs, as in all the material examined, the sieve plate pores were densely filled with P-protein. Preservation of the sieve elements, companion cells and parenchyma cells was judged as excellent with little distortion of organelles or general cytoplasmic arrangement with the possible exception of hydrostatic flow in the sieve elements. In the present study we obtained similar preservation by fixing whole plants in glutaraldehyde and formaldehyde-glutaraldehyde fixatives and have reasoned that if the whole plant is fixed before cutting there should be no changes due to hydrostatic flow caused by pressure changes upon cutting the phloem conduits. The quality of fixation of sieve elements and adjacent companion cells from whole plants fixed in glutaraldehyde is illustrated in Fig. 1. The organelles are well preserved both in the sieve element and companion cell. The sieve element illustrated is at an advanced stage of differentiation when the vacuolar membrane is no longer present. The companion cell is vacuolate, and the vacuoles are well preserved. It is of interest that the plastids of the sieve element are arranged in a parietal position and one apparently blocks a sieve plate pore. The appearance of material fixed in this way and sectioned for the light microscope is shown in Figs. 2 and 3. These photomicrographs are of serial sections, one (Fig. 2) obtained using Carl Zeiss Nomarski interference contrast optics and the other using Carl Zeiss phase contrast optics. The companion cell illustrated has dense cytoplasm with nucleus and vacuoles distinct and obvious. In contrast, the sieve elements appear devoid of cytoplasmic components with the exception of the peripherally arranged plastids. There is no indication of strands in the sieve element. The sieve plate pores of material fixed as whole plants in glutaraldehyde are comparable to those of plants which are cut before fixation. In all the material examined after fixation as whole plants in glutaraldehyde the pores of the sieve plates have large callose cylinders and are filled with P-protein. A median section of a sieve plate pore is shown in Fig. 4. The pore is filled with P-protein which frays out into the lumen of the sieve element. The P-protein is of the tubular type. The condition of the pore is obviously similar to those of plants cut before fixation in glutaraldehyde (cf. Fig. 10, ref. 6). Similar results are obtained by fixing whole plants in formaldehyde-glutaraldehyde. A comparable micrograph of a pore in a sieve plate from a plant fixed as a whole plant in formaldehyde-glutaraldehyde is presented in Fig. 5. Again the pore is similar to those from samples cut before fixation (cf. Figs. 11 and FIGS. 4 and 5. Nicotiana tabacum. Fig. 4. Whole plant glutaraldehyde fixation; Fig. 5 whole plant formaldehyde-glutaraldehyde fixation. Sections of sieve plates showing pores (P) filled with P-protein and surrounded by large callose cylinders (C). The P-protein frays out into the lumen of the cell and is of the tubular type. Fig. 4, x 40,500; Fig. 5, x 46,500.
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FIGS. 6 and 7. Nicotiana tabacum. Whole plant acrolein fixation. The sieve plate pores (P) are surrounded by callose cylinders (C). However, the callose deposition is not as heavy as with glutaraldehyde and formaldehyde glutaraldehyde fixatives. The P-protein in the pores may be densely packed as in Fig. 6 or loosely packed as in Fig. 7. Fig. 6, × 48,000; Fig. 7, × 43,500.
SIEVE PLATE PORES OF Nicotiana
141
FIG. 8. Nicotiana tabacum. Electron micrograph of a s ~ t i o n through sieve elements (SE) and adjacent companion cells (CC). The specimen was fixed in cold glutaraldehyde after killing in liquid nitrogen. The general cytoplasmic preservation is not as good as with glutaraldehyde fixation alone. Most of the organelles are fairly well preserved, however, and the general cytoplasmic features are comparable to those of specimens fixed in glutaraldehyde alone, × 6600. FIG. 9. Nicotiana tabacum. Nomarski interference contrast photograph of a specimen similar to that in Fig, 10. The sieve elements (SE) contain some strands of P-protein. The dense cytoplasmic contents and vacuoles of the adjacent parenchyma cells are evident. × 660.
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13, ref. 6). The pore is filled with P-protein, and a large callose cylinder is present. The tubules of P-protein are very well preserved with formaldehyde-glutaraldehyde. Acrolein fixation also gives similar results for plants cut before fixation or fixed as whole plants. With acrolein fixation the preservation of organelles is similar to that by glutaraldehyde or formaldehyde-glutaraldehyde with the exception of microtubules and tubular P-protein. With occasional exceptions P-protein is not preserved in the tubular form but appears as fibrils, and cytoplasmic microtubules are not apparent. The sieve plate pores have less extensive callose cylinders than after glutaraldehyde or formaldehyde-glutaraldehyde fixation (Figs. 6 and 7) and show a variation in their P-protein contents. In some pores the P-protein is loosely arranged (Fig. 7) while others are densely plugged (Fig. 6). In general, as with glutaraldehyde or formaldehyde-glutaraldehyde, fixation of whole plants yields results similar to those obtained from plants cut before fixation. In all combinations of fixative and technique, slime plugs are invariably present. The plugs are not dense; commonly P-protein concentration is only slightly different on the two sides of the plate. When plants are rapidly frozen in liquid nitrogen (or methyl cyclohexane-isopentane mixture surrounded by liquid nitrogen) before cold fixation by chemical methods, general fixation is inferior to that obtained by r o o m temperature methods. There is some vesiculation of the cytoplasm, plasmolysis in s o m e cells, and some distortion of organelles. Sieve element plastids usually rupture and liberate their starch grains. Despite generally inferior fixation, differences are observed which we consider significant. These concern the observable callose and the relation of the P-protein to the sieve plate pores. Fig. 8 illustrates a sieve tube and adjacent parenchyma cells showing the quality of fixation obtainable with r a p i d freezing-glutaraldehyde fixation. There is some distortion of organelles and some mitochondria have been ruptured. The free starch grains in the sieve tube indicate that sieve element plastids have been ruptured. A dense slime plug and a strand of P-protein are also present. A Nomarski interference contrast micrograph of similar material is shown in Fig. 9. In the sieve element are some strands which are probably P-protein. In the adjacent companion and parenchyma cells dense cytoplasmic components and the vacuoles appear to be intact. There is a wide variation of slime plug formation in rapidly frozen material. Some FIGS. 10-12. Nicotiana tabacum. Sections of sieve plates showing arrangements of P-protein in relation to the sieve plate pores after liquid fiitrogen killing, acrolein fixation. The condition of the sieve plate pores varies. In Fig. 10 the sieve plat~ pores are filled with P-protein material, and in Fig. 11 the P-protein is loosely arranged within the pores. In Fig. 12 the pores are nearly empty of P-protein. Fig. 10, x 15,800; Fig. 11, x 17,400; Fig. 12, x 13,000. FIG. 13. Nicotiana tabacum. Section of a portion of a sieve plate taken from a specimen killed in liquid nitrogen and fixed in formaldehyde-glutaraldehyde. The P-protein material is loosely arranged within the sieve plate pores. Starch grains liberated from the plastids are apparent, x 23,500.
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TABLE I SIZE OF Nicotiana
SIEVE PLATE PORES AFTER VARIOUS FIXATION TECHNIQUESa Number of Plants
Glutaraldehyde Acrolein Formaldehyde-glutaraldehyde Rapid freezing-glutaraldehyde Rapid freezing-acrolein Rapid freezing-formaldehydeglutaraldebyde
Pore Size (#)
P Valueb
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0.22±0.02 0.34 ± 0.02 0.16 ± 0.01 0.46 ± 0.01 0.43 ± 0.02
(30) (42) (26) (270) (79)
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0.61+_0.03 (42)
Wall Pore e (/1)
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0.85±0.03 0.76 ± 0.03 0.76 ± 0.02 0.68 ± 0.01 0.71 +_0.01
(30) (42) (26) (270) (79)
<.001
0.74±0.03 (42)
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plates possess well d e v e l o p e d plugs (Fig. 14) while others do n o t (Fig. 12). Large quantities of P - p r o t e i n m a y be forced t h r o u g h pores as in Fig. 8 to p r o d u c e p r o t r u sions similar to those observed by Crafts (5) a n d Buvat (4). It is significant t h a t after freezing a n d chemical fixation m a n y open pores are observed (Figs. 12, 13, 15, a n d 16). I n a d d i t i o n to pores t h a t are p l u g g e d with P - p r o t e i n a n d those t h a t are empty, there are m a n y i n t e r m e d i a t e c o n d i t i o n s with loosely a r r a n g e d P - p r o t e i n in the sieve plate pores. Figures 10, 11, a n d 12 show sieve plates of r a p i d l y frozen, acrolein-fixed plants. I n Fig. 10 the sieve plate pores are plugged, in Fig. 11 the plugs are less dense, a n d in Fig. 12 the sieve plate pores are open. Fig. 13 shows loosely a r r a n g e d P - p r o t e i n in the sieve plate pores of a sieve element f r o m r a p i d l y frozen f o r m a l d e h y d e - g l u t a r a l d e h y d e fixed material. Figs. 14, 15, a n d 16 are of sieve plates of r a p i d l y frozen glutaraldehyde-fixed m a t e r i a l illustrating v a r i a t i o n in the degree of b l o c k i n g the pores. I n Fig. 15 an o p e n p o r e a n d plugged pores are present in the same sieve plate. It is considered significant t h a t with the exception of acrolein-fixed m a t e r i a l no o p e n p o r e s are o b s e r v e d after chemical fixation alone, b u t t h a t n u m e r o u s open pores are o b s e r v e d after the freezing-chemical fixation procedures. It m a y be significant t h a t after acro-
FIos. 14-16. Nieotiana tabacum. Sections of sieve plates from specimens killed in liquid nitrogen and fixed in glutaraldehyde. Fig. 14 shows an accumulation of P-protein adjacent to a sieve plate, and the sieve plate pores are partially filled with P-protein. Fig. 15 shows a sieve plate with two sieve plate pores filled with P-protein and one pore virtually devoid of P-protein. Fig. 16 shows a sieve plate with pores containing loosely organized P-protein. Fig. 14, × 11,200; Fig. 15, × 12,500; Fig. 16, × 45,000.
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lein fixation alone some loosely packed pores are observed, as acrolein is generally considered to be a more rapidly penetrating fixative. The size of the callose cylinder appears smaller after rapid freezing, and a study has been made of the pore size and the wall pore size (the term "wall pore" refers to the outer diameter of the callose cylinder measured at the middle lamella) for all the fixation procedures. The results are presented in Table I. The pore size differs significantly with the fixation procedure. The size of the pore in the wall without the callose cylinder, however, is fairly constant, indicating that the amount of callose deposition varies with the fixation method used. With room temperature fixation, the pore size is smallest with formaldehyde-glutaraldehyde and largest with acrolein. |n all cases the pore size is significantly larger when chemical fixation is preceded by freezing. Thus, enzymatic reactions resulting in callose deposition appear to be rapidly stopped by freezing but must be functional during room temperature chemical fixation. DISCUSSION Bullivant and Ames (3) have recently summarized numerous attempts at the preservation of biological structures for the electron microscope by rapid freezing. With the exception of structures demonstrated by the freeze etch technique, most investigators have found that rapid freezing methods do not give preservation as good as that of chemical fixation techniques. Rapid freezing techniques have been used for phloem tissue studies by several workers and have been particularly successful at the light microscope level (10, 1I, 18, 19). In addition to rapid freezing methods, many attempts have been made using specialized fixation techniques to minimize the injury response of sieve elements (10, 11, 17, 19, 27). The present study has shown that general protoplasmic preservation of plants frozen by liquid nitrogen and fixed at low temperature is inferior to that of chemical fixation alone at room temperature. Some artifacts are created including vesiculation of the cytoplasm, disruption of some organelles, and in some cells even plasmolysis. However, the general preservation is fairly good, and we consider the observations on the sieve plate pores significant. If chemical fixation with glutaraldehyde or formaldehyde-glutaraldehyde is preceded by rapid freezing, open pores can be demonstrated. Without rapid freezing the sieve plate pores are consistently plugged with P-protein; also with rapid freezing the pore size is larger and the amount of callose associated with the pores is less. Johnson (27) has recently examined the sieve plate pores of Nymphoides peltata using specialized fixation and freeze etch techniques. Filaments (P-protein) appeared in and near the sieve plate pores. Johnson suggests that the filaments are normally present in the pores. In the published micrographs the filaments appear to be loosely
SIEVE PLATE PORES OF
Nicotiana
I47
arranged in the pores (Ref. 27, Figs. 4, 5, 8, 16 and 17), and the arrangement is similar to that for many of the pores we have observed in the present investigation. Callose is deposited by enzyme-mediated reactions (21) which must be either stopped or drastically slowed by the rapid freezing and chemical fixation at low temperatures. It is of general interest that during room temperature fixation these enzymatic reactions can take place to such an extent that a considerable amount of callose is formed. Callose cylinders associated with sieve plate pores have been generally regarded as wound artifacts. Currier and Strugger (9) described callose formation in response to injury, and Eschrich (17) found that deposition of callose may occur in seconds. Callose deposition on sieve plates may be prevented by rapid fixation (19, 40), by avoiding the stimulus of cutting the plant before fixation (t6, cf. present results), or by employing a chelating agent to remove calcium from the sieve elements (17). However, Engleman (11) found a certain minimum amount of callose in material prepared by several rapid-freezing techniques. He concluded that this quantity is a real component of the intact plant. The present results support this conclusion. Although the amount of callose in the pore cylinders is much less after rapidfreezing techniques some is always present. Eschrich (16, 17) and Northcote and Wooding (33) have suggested that since extensive callose deposition may occur during room temperature fixation, P-protein in the pores would be compressed into the amorphous mass typically observed. Acrolein appears to halt this compression faster than does glutaraldehyde. The pores tend to remain large and loosely filled with P-protein. Loosely packed pores in rapidly frozen plants may likewise depend on rapid arrest of callose deposition. Most appear to contain enough P-protein to form a dense plug if more callose had been deposited. With regard to the present observations it is possible that either open pores or plugged pores or both are artifacts due to the preparation procedures. Freezing or thawing may dislocate the P-protein from the pores and create an artifact of open pores. On the other hand, plugged pores such as those depicted in Fig. 10 may be artifacts formed during slime plug formation. We believe the latter possibility is more likely. The observation that some open pores are present in the sieve plate is obviously significant with reference to the problems of interpreting translocation in relation to sieve tube structure. In view of the recent discussion of this problem by one of us (6), it is sufficient here to point out that a demonstration of open pores obviously supports the mass-flow hypothesis and recent physiological results (22, 37). In further support of the view that the pores in sieve elements are open, are our recent results showing that pores blocked by moving starch grains may be devoid of P-protein. These results will be the subject of another communication, but together with the present and previous results (15) they provide an accumulation of evidence in favor of open pores in functional sieve elements.
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REFERENCES 1. BEHNKE, H. D., Z. Pflanzenphysiol. 53, 214 (1965). BoUcK, G. and CRONSHAW, J., J. Cell Biol. 25, 79 (1965). BULLIVANT,S. and AMES, A., J. Cell Biol. 29, 435 (1966). BUVAT, M. R., Compt. Rend. Acad. Sci. 250, 1528 (1960). CRAFTS, A. S., Translocation in Plants. Holt, New York, 1961. 6. CRONSHAW, J. and ESAU, K., J. Cell Biol. 34, 801 (1967). 7. - ibid. 38, 25 (1968). 8. - ibid. 38, 292 (1968). 9. CURRIER,H. B. and STRUGGER,S., Protoplasma 45, 552 (1956). 10. CURRIER, H. B. and SHIrL C. Y., Am. J. Botany 55, 145 (1968). 11. ENGLEMAN,E. M., Ann. Botany 29, 83 (1965). 12. ESAU, K. and CHEADLE,V. I., Proe. Natl. Aead. Sci. U.S. 47, 1716 (1961). 13. ESAU, K., CHEADLE,V. I. and RISLEY, E. B., Botan. Gaz. 123, 233 (1962). 14. ESAU, K. and CHEADLE,V. I., Univ. California Publ. Botany 36, 253 (1965). 15. ESAU, K., CRONSHAW,J. and HOEFERT,L. L., J. Cell Biol. 32, 71 (1967). 16. ESCHRICn, W., Planta 59, 243 (1963). 17. - ibid. 65, 280 (1965). 18. ESCHRICH,W. and CURRIER, H. B., Stain Teehnol. 39, 303 (1964). 19. EVERT, R. F. and DERR, W. F., Am. J. Botany 51, 552 (1964). 20. EVERT, R. F. and MURMANIS,L., Am. J. Botany 52, 95 (1965). 21. FEINGOLD,D. S., NEUFELD, E. F. and HASSlD, W. Z., J. BioL Chem. 233, 783 (1958). 22. FORD, J. and PEEL, A. J., J. Exptl. Botany 17, 522 (1966). 23. HEPTON, C. E. L., PRESTON, R. D. and RIPLEY, G. W., Nature 176, 868 (1955). 24. HEVTON,C. E. L. and PRESTON, R. D., J. Exptl. Botany 11, 381 (1960). 25. HOHL, H. R., Ber. Sehweiz. Botan. Ges. 70, 395 (1960).
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