JOURNAL OF ULTRASTRUCTURE RESEARCH 63, 224-235 (1978)
The Protein Inclusions in Sieve Elements of Cotton ( Gossypium hirsutum L ) 1 KATHERINE ESAU
Department of Biological Sciences, University of California, Santa Barbara, California 93106 Received March 14, 1978 The sieve element of cotton (Gossypium hirsutum L.) contains three kinds of inclusions known to consist of protein. One of these inclusions, an aggregate of filaments that resemble some forms of P protein, is found in the nucleus. The other two are cytoplasmic in origin: a common form of P protein, with filaments indistinctly tubular at first and striated fibrillar later; and a spheroidal inclusion body with a dense core and a paracrystalline mantle. The P protein has a precursory stage represented by a dense mass of apparently anastomosing fibrils. The spheroidal body is seen in the sieve element before any other features identifyingthe cell as a differentiating sieve element become evident, except the lack of grana in the plastids. This kind of body was previously interpreted as a nucleolus released from the disintegrating nucleus during sieve-element ontogeny. In the present study the body was identified in the cytoplasm when the nucleus was still intact and contained a nucleolus. T h e c o t t o n p l a n t is o n e of t h e r e p r e s e n t a t i v e s of d i c o t y l e d o n s i n w h i c h t h e sieve element contains a conspicuous spheroidal i n c l u s i o n b o d y t h a t was o n c e i n t e r p r e t e d as a free n u c l e o l u s e x t r u d e d f r o m t h e d i s i n t e grating nucleus during sieve-element ontogeny. T h i s i n t e r p r e t a t i o n was b a s e d o n e a r l y l i g h t m i c r o s c o p e s t u d i e s ( E n g a r d , 1944; E s a u , 1947}. L a t e r i n v e s t i g a t i o n s , i n c l u d i n g electron microscopy and cytochemical tests ( D e s h p a n d e a n d E v e r t , 1970; I l k e r a n d Currier, 1975; Oberh~iuser a n d K o l l m a n n , 1977), h a v e s h o w n t h a t t h e b o d y is n o t a n u c l e o l u s but a proteinaceous cytoplasmic inclusion c h e m i c a l l y s i m i l a r to t h e P p r o t e i n s as def i n e d b y C r o n s h a w (1974). 'Supported by National Science Foundation Grants BMS 72-02207 and PCM 76-82133.
G o s s y p i u m h i r s u t u m L. was u s e d i n o n e of t h e o r i g i n a l s t u d i e s t h a t led to t h e ass u m p t i o n of n u c l e a r origin of t h e s p h e r o i d a l i n c l u s i o n b o d i e s (Esau, 1947), b u t this species was n o t i n c l u d e d i n t h e i n v e s t i g a t i o n s t h a t s e r v e d to r e i n t e r p r e t t h e n a t u r e of t h e body. A d e v e l o p m e n t a l u l t r a s t r u c t u r a l r e e x a m i n a t i o n of t h e s i e v e - e l e m e n t p r o t o p l a s t i n c o t t o n was t h e r e f o r e c a r r i e d out. T h e s p h e r o i d a l b o d y was s t u d i e d i n j u x t a p o s i t i o n to o t h e r p r o t e i n a c e o u s i n c l u s i o n s i n t h e cell a n d to c e r t a i n o t h e r f e a t u r e s t h a t i d e n t i f y t h e sieve e l e m e n t as such. MATERIALS AND METHODS Collections of leaves and young stems were made from glasshouse-grown seedlings of Gossypium hirsuturn L., upland cotton. The material was fixed at room temperature for 3 hr in Karnovsky's (1965) glutaral-
FIG. 1. Longitudinal section of phloem ceils from a leaf vein. Companion cell (cc), inclusion body (ib), middle lamella (ml), nucleus (n), nuclear inclusion (ni), nascent P protein (npp), parenchyma cell (pa), P protein (pp), sieve element (se), sieve plate (sp). x4800. Fro. 2. Filaments of P protein in immature sieve element from vascular bundle of a petiole. The filaments are indistinctlytubular. Some transections of filaments are indicated by arrowheads. Spiny vesicles at the upper right. × 60 000. FIG. 3. Filaments of P protein in mature sieve element close to sieve plate. From a vascular bundle of a young internode. The filaments are striated fibrillar. Some transections of filaments are indicated by arrowheads. x60 000. FIG. 4. Part of nucleus with fibrillar striated filaments. From the immature sieve element shown in Fig. 1. x60 000. 224 0022-5320/78/0632-0224502.00/0 Copyright © 1978 by Academic Press, Inc.
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dehyde-paraformaldehyde solution brought to pH 6.8 by means of 0.05 phosphate buffer. After a 2-hr washing in three changes of the phosphate buffer, the material was postfixed in 2% OsO4 overnight in a refrigerator, dehydrated in acetone, and embedded in Epon 812. Sections were stained in uranyl acetate and lead citrate. RESULTS
The Differentiating Sieve Element The primary sieve elements in stems, petioles, and large veins are elongated cells with transverse or somewhat inclined sieve plates and thickened lateral walls (Figs. 1 and 9). In minor veins, the sieve elements may deviate from the elongated form, especially where the veins are branched. If the middle lamella is detectable, its position indicates that most of the wall thickening is deposited on the side of the sieve element and that the wall of the adjacent cell remains relatively thin (Figs. 1 and 9). This thickening is small in amount in the primary pit fields so that the wall is deeply pitted (Figs. 1, 16, 20, and 21). In the differentiating sieve element, the elongated nucleus is surrounded by dense cytoplasm interspersed with small vacuoles (Fig. 1). These later fuse into larger vacuoles (Fig. 9). One nucleolus, or sometimes two, may be seen in some sections of the nuclei (Figs. 16 and 20). The heterochromatin is small in amount and most of it is located next to the nuclear envelope. The nucleus frequently has an aggregate of fibrils (Figs. 1 and 4) of the kind that was identified as a proteinaceous inclusion in a number of plant species (cf. Wergin et al., 1970). Below the nucleus seen in Fig. 1 is the spheroidal inclusion body (ib) that used to be called extruded nucleolus. Still farther
down, is a large aggregate of P protein (pp) with a site of origin of this protein at the upper end of the aggregate (npp, nascent P protein). The spheroidal body and the Pprotein aggregate are surrounded by cytoplasm. The immature sieve element contains ribosomes (Figs. 5 and 6), dictyosomes (Figs. 16 and 21), sparse rough endoplasmic reticulum cisternae (Fig. 6), and occasional spiny vesicles (Fig. 2). The plastids have no starch at first (Figs. 14 and 16); later, several rounded starch grains are formed in each plastid (Figs. 5, 9, 20, and 21). The thylakoids are sparse.
The P Protein The P protein is of the type commonly found in dicotyledons. It consists of long filaments that have a tubular form in differentiating sieve elements and a thinner fibriUar form in mature cells. The tubular structure is less compact and the central electron-lucent lumen is less distinct than in the P protein of many other dicotyledons (Fig. 2). The tubular filaments faintly show striations that reflect the helical structure. The fibrillar form appears to be derived from the tubular by stretching, with the resultant decrease in diameter and sharper definition of the striations. The conspicuous alternation of thicker and thinner portions in the extended helices gives the striated fibrils a beaded appearance (Fig. 3). The developmental relation between the tubular and the fibrillar forms is confirmed by occurrence of P-protein filaments that are transitional between the two forms in thickness and in degree of distinctness of the helical structure. In its incipient stage of development the
FIG. 5. Transection of two phloem cells from a vascular bundle of a petiole. Companion cell (cc), dictyosome (d), mitochondrion (m), nucleus (n), nascent P protein (npp), plasmodesma (pd), plastid (pl), P protein (pp). x 12 000. F~G. 6. Enlarged view of part of sieve element from Fig. 5. Endoplasmic reticulum (er). X 18 000. FIc. 7. Part of immature sieve element from longitudinal section of a leaf vein. P-Protein filaments radiate from an aggregate of nascent P protein (npp). x 24 000. FIG. 8. Enlarged view of nascent P protein and associated filaments from Fig. 7. Open arrowheads indicate apparent assembly of filaments, x 60 000.
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P protein consists of dense aggregates of probably anastomosing fibrils (Figs. 1, 5, 7, and 8). Large numbers of P-protein filaments radiate from the masses of nascent P protein (Figs. 7 and 8). The arrangement suggests that the P-protein filaments are assembled from subunits of nascent P protein. In Fig. 8, an alignment of filaments is dimly perceptible within the nascent P-protein mass above (open arrowheads). Ribosomes and other cell components are excluded from the nascent P protein and largely also from the newly formed aggregates of filaments (Figs. 1, 5, and 6). Dilated membranous entities, probably endoplasmic reticulum, may occur nearby and among the new filaments (Figs. 7 and 8). Clusters of ribosomes are present around the growing P-protein masses. As the sieve element continues to differentiate, the nascent P protein disappears (Fig. 9). It probably is used up in the assembly of filaments. The aggregates of the newly formed P-protein filaments are compact (Figs. 1, 5, 6, and 9). Later the filaments become more loosely arranged. The aggregates are then less clearly separated from other cell components, especially the ribosomes. With the method of fixation used, involving no protection from the effects of the first cutting of the tissue, the lumina of mature sieve elements contain thinly dispersed stretched P-protein fibrils (Fig. 9, mse). Similar fibrils accumulate near the sieve plates and in their pores.
The Nuclear Inclusion The nuclear protein inclusion consists of thin fibrillar filaments commonly grouped in loose elongated aggregates (Figs. 1 and 4). The striations indicating helical struc-
ture are less clearly demarcated than those in cytoplasmic P-protein fibrils that are in late stages of stretching (compare Figs. 3 and 4). The nuclear fibrils resemble some forms of P protein that are intermediate between the early tubular and the late striated fibrillar forms. No evidence was obtained that the nuclear inclusion persists after the nucleus disintegrates. If it were released as such during nuclear breakdown, its identification as material distinct from the stretched dispersed P protein would be uncertain.
The Spheroidal Inclusion Body The spheroidal body has a compact core without a discernible substructure and a paracrystalline mantle (Figs. 9 and 10). The paracrystalline material extends to different distances from the periphery of the body and thus gives the surface of the latter a jagged aspect. Depending on the plane of section, the structural units of the mantle appear as rods lying parallel to one another or as parts of an irregular mesh (Figs. 10 and 11). The bodies vary in size but generally their cores are within the size ranges of the nucleoli. In view of the former erroneous interpretation of the spheroidal body as an extruded nucleolus, the timing in the appearance of the body in the cytoplasm was considered in relation to the disintegration of the nucleus in the same cell. Figures 1 and 12-21 give evidence that the body is found in the cytoplasm before the nucleus breaks down and even before most other features identifying the sieve element as such become evident, and that ultrastructurally the body differs from the nucleolus throughout sieveelement development.
FIG. 9. Longitudinal section of phloem cells from a vascular bundle in young internode. Companion cell (cc), inclusion body (ib), immature sieve element (ise), mitochondrion (m), middle lamella (ml), mature sieve element (mse), parenchyma cell ( p a l plastid (pl), P protein (pp), sieve plate (sp). x 7000. FIG. 10. Spheroidal inclusion body with dense amorphous core and paracrystalline mantle. From immature sieve element in a leaf vein. x 60 000. Fro. 11. Section through the paracrystalline mantle of a spheroidal inclusion body. From a mature sieve element in a vascular bundle of young internode. × 60 000.
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In Fig. 12, the two recently formed cells are s e p a r a t e d f r o m one a n o t h e r b y a cell plate t h a t has c o m p l e t e d its growth t o w a r d the lateral walls (arrows) b u t still has large discontinuities in its central part. T h e two sister cells s e e m to be alike except t h a t one of t h e m contains a nearly spheroidal elect r o n - o p a q u e inclusion in the cytoplasm. T h e b o d y does not h a v e a paracrystalline mantle, however; it is i m m a t u r e . T h e t h r e e cells labeled A, B, and C in Fig. 13 are p r o b a b l y derivatives of one cell, the limits of which are indicated with asterisks. T h e sequence of divisions a p p e a r s to h a v e b e e n the following. In the first division, cell C and the precursor of A and B were formed. T h e second division separ a t e d cells A and B. T h e two m o s t recently f o r m e d cell walls are thinner t h a n those m a r k e d with the asterisks and are somew h a t wavy, especially the wall b e t w e e n cells A and B. (Waviness is often seen in recently c o m p l e t e d cell walls.) Cell C is a p a r e n c h y m a cell; it contains chloroplasts (ch). Cell A has an i m m a t u r e inclusion body. T w o rods extending f r o m the b o d y (Fig. 15, arrows) indicate the beginning of paracrystalline m a n t l e development. No P protein is discernible and the plastids lack starch (Fig. 14). T h e nucleus is similar to t h a t in the p a r e n c h y m a cell below. It is intact, b u t the given section does not include the nucleolus. T h e end wall at the open a r r o w h e a d in Fig. 13 is not identifiable as the future sieve plate except by its position in the cell. No special thickening occurs on the side walls. T h e inclusion b o d y and the granaless plastids are the only features
identifying the cell as a differentiating sieve element. T h e central cell in Fig. 16 is s o m e w h a t m o r e a d v a n c e d in differentiation as a sieve e l e m e n t t h a n is cell A in Fig. 13. F r a g m e n t s of p l a s m o d e s m a t a associated with callose serve to localize the pore sites in the developing sieve plate (sp), and the lateral walls are thickened except at the deep pit area. Yet the plastids are still without starch, and no P protein is visible in this section and is not seen in other sections of the same cell. T h e section of the intact nucleus includes a nucleolus. An i m m a t u r e inclusion b o d y is p r e s e n t in the cytoplasm. High magnifications of the b o d y shown in two sections in Figs. 17 and 18 reveal early stages of the paracrystalline m a n t l e development. S h o r t rods extend f r o m the surface of the b o d y here and there, and the f r a g m e n t of the m a n t l e m a r k e d with an asterisk in Fig. 18 a p p e a r s as a mesh. T h e h o m o g e n e o u s core of the b o d y obviously differs f r o m the nucleolus, which is identified by a combination of a granular peripheral region, a finely fibrous center, and an association with c h r o m a t i n (Fig. 19). T h e sieve e l e m e n t shown in two p a r t s in Figs. 20 and 21 illustrates further advance in differentiation. T h e plastids contain starch grains, and the inclusion b o d y (ib) has developed the paracrystalline m a n t l e (the section passed t h r o u g h the m a n t l e only). T h e sieve plate has increased in thickness and has developed thick paired callose pads at the pore sites. T h e nucleus, however, is still intact and contains a nucleolus, No P protein is in view, b u t the
FIG. 12. Transection of phloem cells from a vascular bundle of a petiole. Two sister cells are separated by a cell plate (arrows). Endoplasmic reticulum (er), inclusion body (ib), mitochondrion (m), plastid (pl), × 18 000. FIG. 13. Longitudinal section of phloem cells from a minor vein of a leaf. A, B, and C indicate three ceUsthat resulted from two divisions of a single precursor the longitudinal walls of which are indicated by asterisks. Chloroplast (ch), inclusion body (ib), nucleus (n), plastid (pl), sieve element (se); open arrowhead indicates prospective sieve plate. × 18 000. FIG. 14. Somewhat enlarged part of sieve element from Fig. 13. Inclusion body (ib), mitochondrion (m), plastid (pl). × 24 000. FIG. 15. Enlarged view of inclusion body from Figs. 13 and 14. Arrows mark two fine rods that indicate the beginning of paracrystalline mantle formation. × 60 000.
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adjacent member of the same sieve tube (right) contains this material. The sieve element depicted in Fig. I shows that the P protein is developing--it is associated with nascent P protein--in the presence of an intact nucleus, mature inclusion body, and a sieve plate with plasmodesmata encased in pads of callose. DISCUSSION
The origin of the inclusion body, previously interpreted as an extruded nucleolus that remained in the sieve element after the nucleus was disorganized, was the central subject of the present study. Inclusion bodies of this type occur in numerous dicotyledons (review in Deshpande and Evert, 1970). The concept of nucleolar release in differentiating sieve elements was introduced by Engard (1944) with reference to Rubus. Engard further proposed that in Rubus the free nucleolus constituted the slime body (aggregate of P protein) characteristic of sieve elements in dicotyledons. In reexamining Engard's proposition, Esau (1947) thought she recognized nucleolar release in sieve elements of Rubus, Gossypium, and Eucalyptus. Since, however, the sieve elements of these plants contained P-protein bodies not related to nuclei, she concluded that the "extruded nucleoli" were not slime bodies. As discussed below, the bodies have now been properly identified by means of developmental studies, ultrastructural observations, and cytochemical tests. The data show that the bodies do not originate in nuclei and that they are, indeed, related to the P proteins ("slime"), which are known to assume vat-
ious forms of substructure and aggregation in different dicotyledons (Cronshaw, 1974). Developmental studies serve to determine whether the spheroidal body appears in the cytoplasm while the nucleus is still intact and contains a nucleolus. Deshpande and Evert's (1970) paper includes light microscope views of sieve elements of Tilia, Populus, and Quercus that contain both spheroidal bodies and nuclei with nucleoli. Oberh~iuser and Kollmann (1977) report having occasionally seen intact nuclei and inclusion bodies in the same sieve elements in Passiflora. Ilker and Currier (1975), however, failed to find such combinations in young sieve elements of Xylosma; in fact, these authors saw the inclusion bodies only in mature, and presumably enucleate, sieve elements. In their ultrastructural study, Deshpande and Evert (1970) found a Quercus sieve element in which several fragments of an inclusion body were located next to a nucleolus-containing nucleus. It is an understandably difficult task in electron microscopy to find sections that show, in one cell, a juxtaposition of cell components the developmental relation of which is under examination. Among several differentiating sieve elements with both intact nuclei and inclusion bodies, seen in Gossypium (e.g., Fig. 1), only two showed also a nucleolus in the nucleus. One of these sieve elements was located in a minor vein of a leaf blade (Fig. 16), the other was in a vascular bundle of a young internode (Figs. 20 and 21). The spheroidal body in Gossypium at first has no paracrystalline mantle and superficially resembles a nucleolus. When the nucleus
FIG. 16. Longitudinal section of phloem cells from a minor vein of a leaf. Immature sieve element in center has thick walls. Dictyosome (d), endoplasmic reticulum (er), inclusion body (ib), nucleolus (nu), mitochondrion (m), plastid (pl), sieve plate (sp). x 12 000. FIGS. 17 and 18. Two sections of the inclusion body from Fig. 16. Arrows indicate developing paracrystalline mantle. Asterisks indicate two views of the same fragment of paracrystalline material, x 60 000. FIG. 19. Nucleolus from nucleus in Fig. 16. Chromatin (chr). x 60 000. FIGS. 20 and 21. Two parts of same sieve element from longitudinal section of vascular bundle in young internode. Dictyosome (d), inclusion body (ib), nucleolus (nu), plastid (pl), P protein (pp), sieve plate (sp). In another section of same sieve element, the inclusion body was cut through the core and the sieve plate had more pore sites, x 7000.
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reaches the stage of degeneration the body has a fully developed mantle. The nucleus becomes devoid of chromatic contents (chromatin and nucleolus) before the nuclear envelope disintegrates. Thus, in Gossypium, the spheroidal body is present in the cytoplasm considerably in advance of disruption of nuclear envelope. The formation of this body also precedes the development of P protein, the synthesis of starch in the plastids, and the definition of pore sites in the differentiating sieve plate. The initial developmental stages of the body were not recognized, however. In fact, in one differentiating group of phloem cells, a body was found in one of two sister cells that were in final stages of cytokinesis. It seems appropriate to ask whether that body might not have been present in the precursor of the two new phloem cells, that is, in the cell preparing for mitosis. In cytochemical studies, Ilker and Currier {1975) found that the spheroidal inclusion in Xylosma congestum had chemical properties resembling those of the P protein in the same sieve elements and distinct from those of nucleolar proteins. With toluidine blue at pH 4 the spheroidal bodies stained metachromatically, the nucleoli stained orthochromatically, and, in contrast to the nucleoli, the bodies gave negative results in tests for nucleic acids. The proteins of the body and the P protein were reported to be acidic. Oberhiiuser and Kollmann (1977) also obtained no positive results for nucleic acids in the spheroidal body of Passiflora coerulea sieve elements. Protein tests indicated a similarity between the body and the P protein, but the authors found both proteins to be basic, rather than acidic. The occurrence of conformational changes in the P protein during sieve-element differentiation, involving a loosening and stretching of the helix, is a well-documented phenomenon (e.g., Parthasarathy and Miihlethaler, 1969) and is adequately reviewed by Cronshaw (1974). It seems that in Gossypium the initial tubular form is less
condensed and therefore less evident than in the P proteins of some other dicotyledons. In describing the origin of P protein in a sieve element some authors have stated that tubules were present in the first observable stages in the development of this protein (e.g., Cronshaw and Esau, 1967, Nicotiana; Evert and Deshpande, 1969, Ulmus; Steer and Newcomb, 1969, Coleus). There may be, however, a visible precursory material clearly associated with the emerging P-protein tubules. In Mimosa (Esau, 1971), fine fibrils first form homogeneous aggregates, but later are organized into a three-dimensional system of five- or six-sided compartments. The corners of the compartments seem to be solid at first; later, they assume tubular form. In Phaseolus, a fibrillar percursory material is present in the sieve element at the stage when its protoplast is barely distinguishable from that of adjacent parenchyma cells (Esau, 1978). Eventually, tubules are organized in the fibrillar aggregates. Gossypium sieve elements also reveal a fibrillar precursor spatially related to the initial tubular form of P protein. It seems likely that the precursory aggregates of protein seen in young sieve elements comprise the subunits later assembled into tubules. The fibrillar inclusion in the nuclei of young sieve elements of Gossypium is similar to some stretched forms of P-protein filaments. The sieve elements of Tilia illustrate an even closer resemblance between nuclear inclusions and P protein; the inclusions consist of tubules identical in size and form of aggregation with the tubular cytoplasmic P protein (Evert and Deshpande, 1970). The authors refer to the nuclear inclusion in Tilia as P protein. Nuclear proteinaceous inclusions are widespread in plants in cells other than sieve elements (Wergin et al., 1970, and literature cited). The inclusions are diverse in size and form; may be as large or larger than the nucleoli and have amorphous, fibrillar, paracrystalline, or crystalline fine
PROTEIN INCLUSIONS IN SIEVE ELEMENTS
structure; and occur in healthy and diseased tissues. In view of such common occurrence of nuclear inclusions the question may be raised as to whether the protein inclusions in nuclei of sieve elements should be called P protein. The morphologic identity of the cytoplasmic P protein and the nuclear protein inclusion in Tilia is obvious. It would seem to be plausible to identify as P protein also the nuclear inclusions in Gossypium sieve elements. But, conceivably, a nuclear protein inclusion may greatly deviate from the P protein in the same sieve element. The solution of this terminological problem would be helped if it were known whether during nuclear breakdown the inclusion is released into the cytoplasm and assumes the same relation to the remaining part of the protoplast as does the cytoplasmic P protein. REFERENCES CRONSHAW, J. (1974) P-proteins. in ARONOFF, S.
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(Ed.), Phloem Transport (NATO Advanced Study Institutes: Part a: Life Sciences, Vol. 4), pp. 79-115. Plenum Press, New York. CRONSHAW, J., AND ESAU, K. (1.967) J. Cell Biol. 34, 801-816. DESHPANDE, B. P., AND EVENT, R. F. (1970) J. Ultrastruct. Res. 33, 483-494. ENGARD, C. J. (1944) Univ. Hawaii Res. Publ. 21. ESAU, K. (1947) Amer. J. Bot. 34, 224-233. Eshu, K. (1971) Protoplasma 73, 225-238. ESAU, K. (1978) Ann. Bot. 42, 1-13. EVENT, R. F., AND DESHPANDE, B. P. (1969) Protoplasma 68, 403-432. EVERT, R. F., AND DESHPANDE, B. e. (1970) J. Cell Biol. 44:462-466. ILKER, R., AND CURRIER, H. B. (1975) Protoplasma 85, 127-132. KARNOVSKY,i . J. (1965) J. CellBiol. 27, 137A-138A. OBERHAUSER, R., AND KOLLMANN, R. (1977). Z. Pflanzenphysiol. 84, 61-75. PARTHASARATHY, M. V., AND MOHLETHALER, S. (1969) Cytobiologie 1, 17-36. STEER, M. W., AND NEWCOMB, E. H. (1969) J. Cell Sci. 4, 155-169. WERGIN, W. P., GRUBER, P. J., AND NEWCOMB, E. H. (1970) J. Ultrastruct. Res. 30, 533-557.
The Protein Inclusions in Sieve Elements of Cotton ( Gossypium hirsutum L ) 1 KATHERINE ESAU
Department of Biological Sciences, University of California, Santa Barbara, California 93106 Received March 14, 1978 The sieve element of cotton (Gossypium hirsutum L.) contains three kinds of inclusions known to consist of protein. One of these inclusions, an aggregate of filaments that resemble some forms of P protein, is found in the nucleus. The other two are cytoplasmic in origin: a common form of P protein, with filaments indistinctly tubular at first and striated fibrillar later; and a spheroidal inclusion body with a dense core and a paracrystalline mantle. The P protein has a precursory stage represented by a dense mass of apparently anastomosing fibrils. The spheroidal body is seen in the sieve element before any other features identifyingthe cell as a differentiating sieve element become evident, except the lack of grana in the plastids. This kind of body was previously interpreted as a nucleolus released from the disintegrating nucleus during sieve-element ontogeny. In the present study the body was identified in the cytoplasm when the nucleus was still intact and contained a nucleolus. T h e c o t t o n p l a n t is o n e of t h e r e p r e s e n t a t i v e s of d i c o t y l e d o n s i n w h i c h t h e sieve element contains a conspicuous spheroidal i n c l u s i o n b o d y t h a t was o n c e i n t e r p r e t e d as a free n u c l e o l u s e x t r u d e d f r o m t h e d i s i n t e grating nucleus during sieve-element ontogeny. T h i s i n t e r p r e t a t i o n was b a s e d o n e a r l y l i g h t m i c r o s c o p e s t u d i e s ( E n g a r d , 1944; E s a u , 1947}. L a t e r i n v e s t i g a t i o n s , i n c l u d i n g electron microscopy and cytochemical tests ( D e s h p a n d e a n d E v e r t , 1970; I l k e r a n d Currier, 1975; Oberh~iuser a n d K o l l m a n n , 1977), h a v e s h o w n t h a t t h e b o d y is n o t a n u c l e o l u s but a proteinaceous cytoplasmic inclusion c h e m i c a l l y s i m i l a r to t h e P p r o t e i n s as def i n e d b y C r o n s h a w (1974). 'Supported by National Science Foundation Grants BMS 72-02207 and PCM 76-82133.
G o s s y p i u m h i r s u t u m L. was u s e d i n o n e of t h e o r i g i n a l s t u d i e s t h a t led to t h e ass u m p t i o n of n u c l e a r origin of t h e s p h e r o i d a l i n c l u s i o n b o d i e s (Esau, 1947), b u t this species was n o t i n c l u d e d i n t h e i n v e s t i g a t i o n s t h a t s e r v e d to r e i n t e r p r e t t h e n a t u r e of t h e body. A d e v e l o p m e n t a l u l t r a s t r u c t u r a l r e e x a m i n a t i o n of t h e s i e v e - e l e m e n t p r o t o p l a s t i n c o t t o n was t h e r e f o r e c a r r i e d out. T h e s p h e r o i d a l b o d y was s t u d i e d i n j u x t a p o s i t i o n to o t h e r p r o t e i n a c e o u s i n c l u s i o n s i n t h e cell a n d to c e r t a i n o t h e r f e a t u r e s t h a t i d e n t i f y t h e sieve e l e m e n t as such. MATERIALS AND METHODS Collections of leaves and young stems were made from glasshouse-grown seedlings of Gossypium hirsuturn L., upland cotton. The material was fixed at room temperature for 3 hr in Karnovsky's (1965) glutaral-
FIG. 1. Longitudinal section of phloem ceils from a leaf vein. Companion cell (cc), inclusion body (ib), middle lamella (ml), nucleus (n), nuclear inclusion (ni), nascent P protein (npp), parenchyma cell (pa), P protein (pp), sieve element (se), sieve plate (sp). x4800. Fro. 2. Filaments of P protein in immature sieve element from vascular bundle of a petiole. The filaments are indistinctlytubular. Some transections of filaments are indicated by arrowheads. Spiny vesicles at the upper right. × 60 000. FIG. 3. Filaments of P protein in mature sieve element close to sieve plate. From a vascular bundle of a young internode. The filaments are striated fibrillar. Some transections of filaments are indicated by arrowheads. x60 000. FIG. 4. Part of nucleus with fibrillar striated filaments. From the immature sieve element shown in Fig. 1. x60 000. 224 0022-5320/78/0632-0224502.00/0 Copyright © 1978 by Academic Press, Inc.
All rights of reproductionin any formreserved.
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dehyde-paraformaldehyde solution brought to pH 6.8 by means of 0.05 phosphate buffer. After a 2-hr washing in three changes of the phosphate buffer, the material was postfixed in 2% OsO4 overnight in a refrigerator, dehydrated in acetone, and embedded in Epon 812. Sections were stained in uranyl acetate and lead citrate. RESULTS
The Differentiating Sieve Element The primary sieve elements in stems, petioles, and large veins are elongated cells with transverse or somewhat inclined sieve plates and thickened lateral walls (Figs. 1 and 9). In minor veins, the sieve elements may deviate from the elongated form, especially where the veins are branched. If the middle lamella is detectable, its position indicates that most of the wall thickening is deposited on the side of the sieve element and that the wall of the adjacent cell remains relatively thin (Figs. 1 and 9). This thickening is small in amount in the primary pit fields so that the wall is deeply pitted (Figs. 1, 16, 20, and 21). In the differentiating sieve element, the elongated nucleus is surrounded by dense cytoplasm interspersed with small vacuoles (Fig. 1). These later fuse into larger vacuoles (Fig. 9). One nucleolus, or sometimes two, may be seen in some sections of the nuclei (Figs. 16 and 20). The heterochromatin is small in amount and most of it is located next to the nuclear envelope. The nucleus frequently has an aggregate of fibrils (Figs. 1 and 4) of the kind that was identified as a proteinaceous inclusion in a number of plant species (cf. Wergin et al., 1970). Below the nucleus seen in Fig. 1 is the spheroidal inclusion body (ib) that used to be called extruded nucleolus. Still farther
down, is a large aggregate of P protein (pp) with a site of origin of this protein at the upper end of the aggregate (npp, nascent P protein). The spheroidal body and the Pprotein aggregate are surrounded by cytoplasm. The immature sieve element contains ribosomes (Figs. 5 and 6), dictyosomes (Figs. 16 and 21), sparse rough endoplasmic reticulum cisternae (Fig. 6), and occasional spiny vesicles (Fig. 2). The plastids have no starch at first (Figs. 14 and 16); later, several rounded starch grains are formed in each plastid (Figs. 5, 9, 20, and 21). The thylakoids are sparse.
The P Protein The P protein is of the type commonly found in dicotyledons. It consists of long filaments that have a tubular form in differentiating sieve elements and a thinner fibriUar form in mature cells. The tubular structure is less compact and the central electron-lucent lumen is less distinct than in the P protein of many other dicotyledons (Fig. 2). The tubular filaments faintly show striations that reflect the helical structure. The fibrillar form appears to be derived from the tubular by stretching, with the resultant decrease in diameter and sharper definition of the striations. The conspicuous alternation of thicker and thinner portions in the extended helices gives the striated fibrils a beaded appearance (Fig. 3). The developmental relation between the tubular and the fibrillar forms is confirmed by occurrence of P-protein filaments that are transitional between the two forms in thickness and in degree of distinctness of the helical structure. In its incipient stage of development the
FIG. 5. Transection of two phloem cells from a vascular bundle of a petiole. Companion cell (cc), dictyosome (d), mitochondrion (m), nucleus (n), nascent P protein (npp), plasmodesma (pd), plastid (pl), P protein (pp). x 12 000. F~G. 6. Enlarged view of part of sieve element from Fig. 5. Endoplasmic reticulum (er). X 18 000. FIc. 7. Part of immature sieve element from longitudinal section of a leaf vein. P-Protein filaments radiate from an aggregate of nascent P protein (npp). x 24 000. FIG. 8. Enlarged view of nascent P protein and associated filaments from Fig. 7. Open arrowheads indicate apparent assembly of filaments, x 60 000.
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P protein consists of dense aggregates of probably anastomosing fibrils (Figs. 1, 5, 7, and 8). Large numbers of P-protein filaments radiate from the masses of nascent P protein (Figs. 7 and 8). The arrangement suggests that the P-protein filaments are assembled from subunits of nascent P protein. In Fig. 8, an alignment of filaments is dimly perceptible within the nascent P-protein mass above (open arrowheads). Ribosomes and other cell components are excluded from the nascent P protein and largely also from the newly formed aggregates of filaments (Figs. 1, 5, and 6). Dilated membranous entities, probably endoplasmic reticulum, may occur nearby and among the new filaments (Figs. 7 and 8). Clusters of ribosomes are present around the growing P-protein masses. As the sieve element continues to differentiate, the nascent P protein disappears (Fig. 9). It probably is used up in the assembly of filaments. The aggregates of the newly formed P-protein filaments are compact (Figs. 1, 5, 6, and 9). Later the filaments become more loosely arranged. The aggregates are then less clearly separated from other cell components, especially the ribosomes. With the method of fixation used, involving no protection from the effects of the first cutting of the tissue, the lumina of mature sieve elements contain thinly dispersed stretched P-protein fibrils (Fig. 9, mse). Similar fibrils accumulate near the sieve plates and in their pores.
The Nuclear Inclusion The nuclear protein inclusion consists of thin fibrillar filaments commonly grouped in loose elongated aggregates (Figs. 1 and 4). The striations indicating helical struc-
ture are less clearly demarcated than those in cytoplasmic P-protein fibrils that are in late stages of stretching (compare Figs. 3 and 4). The nuclear fibrils resemble some forms of P protein that are intermediate between the early tubular and the late striated fibrillar forms. No evidence was obtained that the nuclear inclusion persists after the nucleus disintegrates. If it were released as such during nuclear breakdown, its identification as material distinct from the stretched dispersed P protein would be uncertain.
The Spheroidal Inclusion Body The spheroidal body has a compact core without a discernible substructure and a paracrystalline mantle (Figs. 9 and 10). The paracrystalline material extends to different distances from the periphery of the body and thus gives the surface of the latter a jagged aspect. Depending on the plane of section, the structural units of the mantle appear as rods lying parallel to one another or as parts of an irregular mesh (Figs. 10 and 11). The bodies vary in size but generally their cores are within the size ranges of the nucleoli. In view of the former erroneous interpretation of the spheroidal body as an extruded nucleolus, the timing in the appearance of the body in the cytoplasm was considered in relation to the disintegration of the nucleus in the same cell. Figures 1 and 12-21 give evidence that the body is found in the cytoplasm before the nucleus breaks down and even before most other features identifying the sieve element as such become evident, and that ultrastructurally the body differs from the nucleolus throughout sieveelement development.
FIG. 9. Longitudinal section of phloem cells from a vascular bundle in young internode. Companion cell (cc), inclusion body (ib), immature sieve element (ise), mitochondrion (m), middle lamella (ml), mature sieve element (mse), parenchyma cell ( p a l plastid (pl), P protein (pp), sieve plate (sp). x 7000. FIG. 10. Spheroidal inclusion body with dense amorphous core and paracrystalline mantle. From immature sieve element in a leaf vein. x 60 000. Fro. 11. Section through the paracrystalline mantle of a spheroidal inclusion body. From a mature sieve element in a vascular bundle of young internode. × 60 000.
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In Fig. 12, the two recently formed cells are s e p a r a t e d f r o m one a n o t h e r b y a cell plate t h a t has c o m p l e t e d its growth t o w a r d the lateral walls (arrows) b u t still has large discontinuities in its central part. T h e two sister cells s e e m to be alike except t h a t one of t h e m contains a nearly spheroidal elect r o n - o p a q u e inclusion in the cytoplasm. T h e b o d y does not h a v e a paracrystalline mantle, however; it is i m m a t u r e . T h e t h r e e cells labeled A, B, and C in Fig. 13 are p r o b a b l y derivatives of one cell, the limits of which are indicated with asterisks. T h e sequence of divisions a p p e a r s to h a v e b e e n the following. In the first division, cell C and the precursor of A and B were formed. T h e second division separ a t e d cells A and B. T h e two m o s t recently f o r m e d cell walls are thinner t h a n those m a r k e d with the asterisks and are somew h a t wavy, especially the wall b e t w e e n cells A and B. (Waviness is often seen in recently c o m p l e t e d cell walls.) Cell C is a p a r e n c h y m a cell; it contains chloroplasts (ch). Cell A has an i m m a t u r e inclusion body. T w o rods extending f r o m the b o d y (Fig. 15, arrows) indicate the beginning of paracrystalline m a n t l e development. No P protein is discernible and the plastids lack starch (Fig. 14). T h e nucleus is similar to t h a t in the p a r e n c h y m a cell below. It is intact, b u t the given section does not include the nucleolus. T h e end wall at the open a r r o w h e a d in Fig. 13 is not identifiable as the future sieve plate except by its position in the cell. No special thickening occurs on the side walls. T h e inclusion b o d y and the granaless plastids are the only features
identifying the cell as a differentiating sieve element. T h e central cell in Fig. 16 is s o m e w h a t m o r e a d v a n c e d in differentiation as a sieve e l e m e n t t h a n is cell A in Fig. 13. F r a g m e n t s of p l a s m o d e s m a t a associated with callose serve to localize the pore sites in the developing sieve plate (sp), and the lateral walls are thickened except at the deep pit area. Yet the plastids are still without starch, and no P protein is visible in this section and is not seen in other sections of the same cell. T h e section of the intact nucleus includes a nucleolus. An i m m a t u r e inclusion b o d y is p r e s e n t in the cytoplasm. High magnifications of the b o d y shown in two sections in Figs. 17 and 18 reveal early stages of the paracrystalline m a n t l e development. S h o r t rods extend f r o m the surface of the b o d y here and there, and the f r a g m e n t of the m a n t l e m a r k e d with an asterisk in Fig. 18 a p p e a r s as a mesh. T h e h o m o g e n e o u s core of the b o d y obviously differs f r o m the nucleolus, which is identified by a combination of a granular peripheral region, a finely fibrous center, and an association with c h r o m a t i n (Fig. 19). T h e sieve e l e m e n t shown in two p a r t s in Figs. 20 and 21 illustrates further advance in differentiation. T h e plastids contain starch grains, and the inclusion b o d y (ib) has developed the paracrystalline m a n t l e (the section passed t h r o u g h the m a n t l e only). T h e sieve plate has increased in thickness and has developed thick paired callose pads at the pore sites. T h e nucleus, however, is still intact and contains a nucleolus, No P protein is in view, b u t the
FIG. 12. Transection of phloem cells from a vascular bundle of a petiole. Two sister cells are separated by a cell plate (arrows). Endoplasmic reticulum (er), inclusion body (ib), mitochondrion (m), plastid (pl), × 18 000. FIG. 13. Longitudinal section of phloem cells from a minor vein of a leaf. A, B, and C indicate three ceUsthat resulted from two divisions of a single precursor the longitudinal walls of which are indicated by asterisks. Chloroplast (ch), inclusion body (ib), nucleus (n), plastid (pl), sieve element (se); open arrowhead indicates prospective sieve plate. × 18 000. FIG. 14. Somewhat enlarged part of sieve element from Fig. 13. Inclusion body (ib), mitochondrion (m), plastid (pl). × 24 000. FIG. 15. Enlarged view of inclusion body from Figs. 13 and 14. Arrows mark two fine rods that indicate the beginning of paracrystalline mantle formation. × 60 000.
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adjacent member of the same sieve tube (right) contains this material. The sieve element depicted in Fig. I shows that the P protein is developing--it is associated with nascent P protein--in the presence of an intact nucleus, mature inclusion body, and a sieve plate with plasmodesmata encased in pads of callose. DISCUSSION
The origin of the inclusion body, previously interpreted as an extruded nucleolus that remained in the sieve element after the nucleus was disorganized, was the central subject of the present study. Inclusion bodies of this type occur in numerous dicotyledons (review in Deshpande and Evert, 1970). The concept of nucleolar release in differentiating sieve elements was introduced by Engard (1944) with reference to Rubus. Engard further proposed that in Rubus the free nucleolus constituted the slime body (aggregate of P protein) characteristic of sieve elements in dicotyledons. In reexamining Engard's proposition, Esau (1947) thought she recognized nucleolar release in sieve elements of Rubus, Gossypium, and Eucalyptus. Since, however, the sieve elements of these plants contained P-protein bodies not related to nuclei, she concluded that the "extruded nucleoli" were not slime bodies. As discussed below, the bodies have now been properly identified by means of developmental studies, ultrastructural observations, and cytochemical tests. The data show that the bodies do not originate in nuclei and that they are, indeed, related to the P proteins ("slime"), which are known to assume vat-
ious forms of substructure and aggregation in different dicotyledons (Cronshaw, 1974). Developmental studies serve to determine whether the spheroidal body appears in the cytoplasm while the nucleus is still intact and contains a nucleolus. Deshpande and Evert's (1970) paper includes light microscope views of sieve elements of Tilia, Populus, and Quercus that contain both spheroidal bodies and nuclei with nucleoli. Oberh~iuser and Kollmann (1977) report having occasionally seen intact nuclei and inclusion bodies in the same sieve elements in Passiflora. Ilker and Currier (1975), however, failed to find such combinations in young sieve elements of Xylosma; in fact, these authors saw the inclusion bodies only in mature, and presumably enucleate, sieve elements. In their ultrastructural study, Deshpande and Evert (1970) found a Quercus sieve element in which several fragments of an inclusion body were located next to a nucleolus-containing nucleus. It is an understandably difficult task in electron microscopy to find sections that show, in one cell, a juxtaposition of cell components the developmental relation of which is under examination. Among several differentiating sieve elements with both intact nuclei and inclusion bodies, seen in Gossypium (e.g., Fig. 1), only two showed also a nucleolus in the nucleus. One of these sieve elements was located in a minor vein of a leaf blade (Fig. 16), the other was in a vascular bundle of a young internode (Figs. 20 and 21). The spheroidal body in Gossypium at first has no paracrystalline mantle and superficially resembles a nucleolus. When the nucleus
FIG. 16. Longitudinal section of phloem cells from a minor vein of a leaf. Immature sieve element in center has thick walls. Dictyosome (d), endoplasmic reticulum (er), inclusion body (ib), nucleolus (nu), mitochondrion (m), plastid (pl), sieve plate (sp). x 12 000. FIGS. 17 and 18. Two sections of the inclusion body from Fig. 16. Arrows indicate developing paracrystalline mantle. Asterisks indicate two views of the same fragment of paracrystalline material, x 60 000. FIG. 19. Nucleolus from nucleus in Fig. 16. Chromatin (chr). x 60 000. FIGS. 20 and 21. Two parts of same sieve element from longitudinal section of vascular bundle in young internode. Dictyosome (d), inclusion body (ib), nucleolus (nu), plastid (pl), P protein (pp), sieve plate (sp). In another section of same sieve element, the inclusion body was cut through the core and the sieve plate had more pore sites, x 7000.
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reaches the stage of degeneration the body has a fully developed mantle. The nucleus becomes devoid of chromatic contents (chromatin and nucleolus) before the nuclear envelope disintegrates. Thus, in Gossypium, the spheroidal body is present in the cytoplasm considerably in advance of disruption of nuclear envelope. The formation of this body also precedes the development of P protein, the synthesis of starch in the plastids, and the definition of pore sites in the differentiating sieve plate. The initial developmental stages of the body were not recognized, however. In fact, in one differentiating group of phloem cells, a body was found in one of two sister cells that were in final stages of cytokinesis. It seems appropriate to ask whether that body might not have been present in the precursor of the two new phloem cells, that is, in the cell preparing for mitosis. In cytochemical studies, Ilker and Currier {1975) found that the spheroidal inclusion in Xylosma congestum had chemical properties resembling those of the P protein in the same sieve elements and distinct from those of nucleolar proteins. With toluidine blue at pH 4 the spheroidal bodies stained metachromatically, the nucleoli stained orthochromatically, and, in contrast to the nucleoli, the bodies gave negative results in tests for nucleic acids. The proteins of the body and the P protein were reported to be acidic. Oberhiiuser and Kollmann (1977) also obtained no positive results for nucleic acids in the spheroidal body of Passiflora coerulea sieve elements. Protein tests indicated a similarity between the body and the P protein, but the authors found both proteins to be basic, rather than acidic. The occurrence of conformational changes in the P protein during sieve-element differentiation, involving a loosening and stretching of the helix, is a well-documented phenomenon (e.g., Parthasarathy and Miihlethaler, 1969) and is adequately reviewed by Cronshaw (1974). It seems that in Gossypium the initial tubular form is less
condensed and therefore less evident than in the P proteins of some other dicotyledons. In describing the origin of P protein in a sieve element some authors have stated that tubules were present in the first observable stages in the development of this protein (e.g., Cronshaw and Esau, 1967, Nicotiana; Evert and Deshpande, 1969, Ulmus; Steer and Newcomb, 1969, Coleus). There may be, however, a visible precursory material clearly associated with the emerging P-protein tubules. In Mimosa (Esau, 1971), fine fibrils first form homogeneous aggregates, but later are organized into a three-dimensional system of five- or six-sided compartments. The corners of the compartments seem to be solid at first; later, they assume tubular form. In Phaseolus, a fibrillar percursory material is present in the sieve element at the stage when its protoplast is barely distinguishable from that of adjacent parenchyma cells (Esau, 1978). Eventually, tubules are organized in the fibrillar aggregates. Gossypium sieve elements also reveal a fibrillar precursor spatially related to the initial tubular form of P protein. It seems likely that the precursory aggregates of protein seen in young sieve elements comprise the subunits later assembled into tubules. The fibrillar inclusion in the nuclei of young sieve elements of Gossypium is similar to some stretched forms of P-protein filaments. The sieve elements of Tilia illustrate an even closer resemblance between nuclear inclusions and P protein; the inclusions consist of tubules identical in size and form of aggregation with the tubular cytoplasmic P protein (Evert and Deshpande, 1970). The authors refer to the nuclear inclusion in Tilia as P protein. Nuclear proteinaceous inclusions are widespread in plants in cells other than sieve elements (Wergin et al., 1970, and literature cited). The inclusions are diverse in size and form; may be as large or larger than the nucleoli and have amorphous, fibrillar, paracrystalline, or crystalline fine
PROTEIN INCLUSIONS IN SIEVE ELEMENTS
structure; and occur in healthy and diseased tissues. In view of such common occurrence of nuclear inclusions the question may be raised as to whether the protein inclusions in nuclei of sieve elements should be called P protein. The morphologic identity of the cytoplasmic P protein and the nuclear protein inclusion in Tilia is obvious. It would seem to be plausible to identify as P protein also the nuclear inclusions in Gossypium sieve elements. But, conceivably, a nuclear protein inclusion may greatly deviate from the P protein in the same sieve element. The solution of this terminological problem would be helped if it were known whether during nuclear breakdown the inclusion is released into the cytoplasm and assumes the same relation to the remaining part of the protoplast as does the cytoplasmic P protein. REFERENCES CRONSHAW, J. (1974) P-proteins. in ARONOFF, S.
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(Ed.), Phloem Transport (NATO Advanced Study Institutes: Part a: Life Sciences, Vol. 4), pp. 79-115. Plenum Press, New York. CRONSHAW, J., AND ESAU, K. (1.967) J. Cell Biol. 34, 801-816. DESHPANDE, B. P., AND EVENT, R. F. (1970) J. Ultrastruct. Res. 33, 483-494. ENGARD, C. J. (1944) Univ. Hawaii Res. Publ. 21. ESAU, K. (1947) Amer. J. Bot. 34, 224-233. Eshu, K. (1971) Protoplasma 73, 225-238. ESAU, K. (1978) Ann. Bot. 42, 1-13. EVENT, R. F., AND DESHPANDE, B. P. (1969) Protoplasma 68, 403-432. EVERT, R. F., AND DESHPANDE, B. e. (1970) J. Cell Biol. 44:462-466. ILKER, R., AND CURRIER, H. B. (1975) Protoplasma 85, 127-132. KARNOVSKY,i . J. (1965) J. CellBiol. 27, 137A-138A. OBERHAUSER, R., AND KOLLMANN, R. (1977). Z. Pflanzenphysiol. 84, 61-75. PARTHASARATHY, M. V., AND MOHLETHALER, S. (1969) Cytobiologie 1, 17-36. STEER, M. W., AND NEWCOMB, E. H. (1969) J. Cell Sci. 4, 155-169. WERGIN, W. P., GRUBER, P. J., AND NEWCOMB, E. H. (1970) J. Ultrastruct. Res. 30, 533-557.
