PARTICLES AND MICROTUBULES IN VASCULAR CELLS OF PINUS STROBUS L. DURING CELL WALL FORMATION

Neu'Phytol (1971) 70, 1089-1093. PARTICLES AND MICROTUBULES IN VASCULAR CELLS OF PINUS STROBUS L. DURING CELL WALL FORMATION BY LIDIJA MURMANIS Forest Products Laboratory, * Forest Service, U.S. Department of Agriculture (Received 15 April 1971) SUMMARY Particles in the plasmalemma-cell wall area are reported in fixed and sectioned material of differentiating vascular cells oipimis strobus L. The particles, from 13 nm to 16 nm in diameter, arise fromthe superficial layer ofcytoplasmandreach the plasmalemma-cell wall area apparently by a 'membrane flow.' Frequently the particles are in close spatial association with microtubules at the plasmalemma-cell wall area. The particles observed here are thought identical with those observed on the outer surface of plasmalemma by the freeze-etching method. INTRODUCTION Both particles and microtubules have been related to the formation of cell walls. Particles were first observed by the freeze-etching method on the outer surface of plasmalemma in yeast cells (Moor and Muhlethaler, 1963); since then they have been found in all cells examined by this method (Muhlethaler, 1965). It was thought that particles could not be preserved by the conventional fixation procedures for electron microscopy (Muhlethaler, 1967). However, they have occasionally been reported in association with developing cell walls from sectioned plant tissues after glutaraldehyde-osmium tetroxide fixation (Roland, 1967; Murmanis and Sachs, 1969; Robards, 1969). The existence of microtubules became apparent when glutaraldehyde fixation was introduced in electron microscopy; microtubules were observed first in plant cells by Ledbetter and Porter (1963). In subsequent studies, microtubules have been found in various types of plant cells (e.g. Hepler and Newcomb, 1964; Wooding and Northcote, 1964; Cronshaw and Bouck, 1965). The purpose of this investigation was twofold: to demonstrate that particles are present in sectioned and conventionally fixed vascular cells of Pinus strobus L. during synthesis of the cell wall; and to look for the possible spatial association between the particles and the microtubules that has been suggested (Murmanis and Sachs, 1969; Robards, 1969). MATERIALS AND METHODS Samples of cambium with adjacent wood and bark were collected from young, apparently healthy pine trees (Pinus strobus L.) during the intensive growth period (May and June). The method of preparation of the material has been described in detail (Murmanis and Sachs, 1969). * Maintained at Madison, Wis., in co-operation with the University of Wisconsin. 1089

1090 LiDIJA MURMANIS Tissues were fixed in 3% glutaraldehyde in a phosphate buffer and post fixed in 2 / osmium tetroxide in a veronal-acetate builer. Some samples were fixed in aqueous 2 ' potassium permanganate. The material was dehydrated in acetone and embedded in araldite-epon-ddsa mixture by the method of MoUenhauer (1964). OBSERVATIONS In pine, microtubules, 23-27 nm in diameter, have been observed in both the carabial cells and the differentiating vascular cells during both primary and secondary wall formation (Murmanis and Sachs, 1969). In addition, in this work, particles and microtubules were rather frequently found in close spatial association at the plasmalemma-cell wall area; this was also noted by Robards {1969) in differentiating vascular cells of willow and beech. Information on particles is not as abundant as that on microtubules from fixed and sectioned plant material. The reason is that the association of particles with plasmalemma and cell wall in sectioned material becomes exposed only at unusual sectioning planes at planes of extreme obliquity and at those that pass through the cell surface. Particles within the membrane-bound vesicles were observed in an earlier study (Murmanis, 1971}, but their source was not known. It became evident from the oblique sections and superficial sections only that the particles originate from the surface layer of cytoplasm. Apparently the particle-fiiled, membrane-bound vesicles are out-pocketings of the superficial cytoplasm observed at different phases of their progression to the plasmalemma-cell wall area. In an oblique section (Plate i. No. i) superficial cytoplasm, plasmalemma, and an adjacent cell wall in a young xylem ray cell are exposed. Particles, 13-16 nm in diameter, are scattered in the superficial cytoplasm and are embedded within the plasmalemma, a few are outside the plasmalemma. The ends of microtubules, densely covered with particles, project outside the plasmalemma. It appears that an amorphous substance is thrust from the microtubules (uniabelled arrows). Several small peripheral vacuoles are just within the plasmalemma. The tip of a pit border in a maturing tracheid is shown in Plate 1, No. 2. Particles are clustered on the microtubules, and at their tip the clusters are so heavy that the structural outlines of the tubules are almost hidden. Several outpocketings of cytoplasm, densely filled with particles lie in the small peripheral vacuole. The wall of a pit chamber in a maturing tracheid is seen in Plate 2, No. 3. The particles are crowded in the narrow cytoplasmic space between the tonoplast and the plasmalemma. Because of the oblique plane of the section, the membranes, tonoplast and plasmalemma are very indistinct. Microtubules traverse the particles to the cell wall. The outer ends of the microtubules appear to have passed through the plasmalemma. Outpocketings of cytoplasm filled with particles and surrounded by membrane (tonoplast) within the peripheral vacuole can be observed in Plate 2, No. 4. Two of the outpocketings at the right of the micrograph appear to be in the process of pinching off from the superficial cytoplasm. Occasionally, during preparation, the tonoplast is broken; then the particles are also seen loose within the vacuole (Plate 3, No. 5). In No. 5, the surrounding membrane of an outpocketing has fused with the vacuolar tonoplast, and the tonoplast at several places (uniabelled arrows) is in contact with the plasmalemma. On the other side of the cell wall, particles lie in the cytoplasm between the tonoplast and the plasmalemma. The same cell at different sectioning planes is shown in Plate 3, Nos. 6 and 7. A cyto-

Vascular cells of Pinus strobus 1091 plasmic outpocketing, densely filled with particles which extend through the vacuole toward the cell wall can be observed in No. 6. At the cell wall in No. 7, part of the upper outpocketing has reached the plasmalemma; part is still in contact with the tonoplast, suggesting a recent bulging out of cytoplasm into the vacuole. The lower outpocketing, after fusion of membranes, is attached to the plasmalemma. In Plate 4, No. 8, the progression of cytoplasmic outpocketings to the plasmalemmaceil wall area can be observed. A cluster of particles in the superficial layer of cytoplasm and close to the tonoplast is at the right of the micrograph. It is suggested that soon such clusters will probably bulge into the vacuole. A few outpocketings detached from the cytoplasm and filled with particles are in the vacuole, and one of the outpocketings within the vacuole is apparently fusing with the plasmalemma (unlabelled arrow). Particles are occasionally seen in an orderly arrangement (at top and at left of micrograph). The plasmalemma seems to have become folded into a cylinder exposing the outer surface of plasmalemma at the inside of the cylinder in Plate 4, No. 9. At the open end of the cylinder, therefore, in contact with the outer surface of plasmalemma, very fine, scarcely resolvable filaments about 6 nm in diameter are visible. They are shown at higher magnification in Plate 4, No. 10. DISCUSSION Particles observed here ranged from 13 nm to 16 nm in diameter; their size confirmed that of plasmalemma particles recorded by Miihlethaler (1965). Preston (1964) and Robards (1969) found that particles ranged from 20 nm to 30 nm. The author agrees with Robards (1969) that particles often have an indistinct outline that makes it difficult to measure them precisely. In this work the size of particles, an average of about 15 nm, and the site of their origin, the surface layer of cytoplasm, coincides with the size and the location of cytoplasmic ribosomes. Furthermore, as Muhlethaler (1965) also observed, particles were not observed associated with plasmalemma and cell wall after potassium permanganate fixation. These facts point to the obvious similarity between the particles and the c)t:oplasmic ribosomes, but biochemical studies would be necessary to prove this identity. Regardless of their chemical nature, the particles observed here are believed synonymous to the particles observable on the outer surface of plasmalemma by the freeze-etching method. The function of the outpocketing cytoplasm filled with particles into the vacuoles is not quite clear. These outpocketings do not seem necessary to aid particles to reach the plasmalemma because, occasionally, particles in the adjacent cytoplasm can be seen in contact with the plasmalemma lacking outpocketings. On the contrary, the outpocketings further complicate the association between particles and plasmalemma which involves, first, the fusion between the membrane surrounding the particles and the tonoplast {both of the same membrane type) and second, the fusion between the tonoplast and the plasmalemma. Therefore, outpocketings might more probably be considered a type of regulating mechanism to control the number of particles at the plasmalemma-cell wall area where their eventual function could be to provide an enzyme system in the synthesis of the cell wall. Because the fine, scarcely resolvable filaments, visible at the open end of the cylinder and, presumably, in contact with the outer surface of the plasmalemma, were about 6 nm in diameter (Plate 4, No. 10), it was tempting to assume that these filaments might be of the type observed by Moor and Muhlethaler (1963) protruding from the regularly

1092 LiDijA MURMANIS arranged particle arrays on the outer surface of the plasmalemma in yeast cells after the freeze-etching method. These authors beiieve the filaments in yeast cells correspond to die glucan fibrils. Naturally, the view in No. 10 does not expose the particle arrays or even particles on the outer surface of the plasmalemma within this cytoplasmic fold since freeze-etching was not employed. A close spatial association between the particles and microtubules was observed at the plasmalemma-cell wall area. Frequently microtubules appeared to have penetrated the plasmalemma and become located in the space between the plasmalemma and the cell wall. Usually, particles were seen densely clustered along the outer surface of the microtubules. However, this observation was made from oblique sections; so it is not certain whether the plasmalemma--cell wall space is the true location of the microtubules. If this were the exact location of microtubules and the particles were closely associated with them, it would suggest that particles are not stationary at the outer surface of the plasmalemma as has been suggested (Muhlethaler, 1967). In this study, particles appeared to detach from the plasmalemma and to cluster densely around the outer surface of microtubules. This spatial relation between particles and microtubules suggests that microtubules probably provide structural support for particles at the plasmalemma-cell wall interface. As mentioned, an amorphous substance has been occasionally observed apparently exuding from the outer end of microtubules (Plate i. No. i). This might be the synthetic product of the combined activity of particles and microtubules. Further morphological and biochemical evidence is needed to determine accurately the function and the mechanism of the function of particles and microtubules at the plasmalemraa-cell wall area in the synthesis of the cell wall. REFERENCES CBONSHAW, J. & BOUCK, G. B. (1965). The fine structure of differentiating xylem elements. X CellBkl., 24, 115. HEPUER, P. K. & NEWCOMB, E. H. (1964). Microtubules and fihrils in the cytoplasm of Coleus cells undergoing secondary wail deposition. J. Cell Biol.^ zo, 529. LEDBETTER, M. C. & PoHTEs, K. R. (1963). A 'microtubule' in plant cell fine structure. J. Cell Biol, 19, 239- MoLLENHAtJEH, H. H. (1964). Plastic embedding mixtures for use in electron microscopy. Stain Technol., 39. "I- MOOR, H. & MttHLETHALER, K. (1963). Fine structure in frozen-etched yeast cells. J. Cell Biol., 17, 609. MtJHtETHALER, K. (1965). Growth theories and the development of the cell wall. In: Cellular Ultraslructure of Woody Plants (Ed. by W. A. Ccrt6, Jr), pp. 51-60. Syracuse University Press. MtJHLETjiALER, K. (1967). Uitrastructure and formation of plant cell walls. A. Rev. PI. Physiol., 18, r. MuKMANis, L. (1971). Structural changes in the vascular cambium of Pinus strobus L. during an annual cycle. Ann. Bot., N.S. 35, 133. MuHMANis, L. & SACHS, I. B. (1969). Seasonal development of secondary jcylem in Pi7ms strobus h. Wood Sri. and Tech., 3, 177, PKESTON, R. D. (1964). Structural and mechanical aspects of plant cell walls with particular reference to Bynthesis and growth. In: Formation ofwoodin Forest Trees (Ed. by M. H. Zinunerman), pp. 169-188. Academic Press, New York. RoBABDs, A. W. (1969). Particles associated with developing plant cell walls. Planta, 88, 376. ROLAND, J. C. (1967). Aspects infrastnicturaux des relations existant entre le protoplasme et la paroi des cellules de collenchyme, J. Microscopic, 9, 399. WOODING, F. B. P. & NORTHCOTE, D. H. (1964). The development of the secondary wall of the xylem in Acer pseudoplatama. J. Cell Biol., 23, 327.

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Vascular cells of Pinus strobus 1093 EXPLANATION OF PLATES Key to lettering: oo, cytoplasm outpocketing, cw, cell wall; cy, cytoplasm; cyf, cytoplasm fold; f, filament; mt, raicrotubule; p. particle; pb, pit border; pi, plasmalemma; t, tonoplast; v, vacuole. PLATE I No. I. Oblique section through superficial c>toplasm, plasmalemma, and cell wall of young xylem ray cell. Particles cluster microtubules and are distributed diffusely in cytoplasm and densely in plasmalemma. x 30,000. No. 2. Tip of a pit border in maturing tracheid. Cytoplasm outpocketingsfilledwith particles are in small peripheral vacuole. x 34,000. PLATE 2 No. 3. Wall of pit chamber in maturing tracheid densely aligned by particles; microtubules traverse through particles to cell wall, x 61,600. No. 4. Cytoplasm outpocketings in peripheral vacuole; dense clusters of particles are visible in cytoplasm, x 27,600. PLATE 3 No. 5- Cytoplasm outpocketing, densely filled with particles, in process of fusion with plasmalemma. Clusters of particles are also visible in cytoplasm. Piasmalemma and tonoplast are in contact (unlabelled arrows), x 57,000. No. 6. Outpocketing still in contact "with cytoplasm passing through vacuole toward cell wall. X 25,800. No. 7. Two cytoplasm outpocketings: upper in contact with plasmalemma and tonoplast; and lower attached to plasmalemma. x 30,600. PLATE 4 No. 8. Cytoplasm outpocketings: in process of fusion with plasmalemma (unlabelled arrow); in vacuole. Particles are in regular arrangement at left and at top. x 36,500. No. 9. Cylindrical cytoplasmic fold along cell wall and fine filaments at open end of fold. X 24,000. No. 10. Higher magnification of cytoplasmic fold than that in No. 9. X 48,000.