Planta 9 by Springer-Verlag 1979
Planta 144, 349-358 (1979)
The Neck Constriction in Plasmodesmata Evidence for a Peripheral Sphincter-Like Structure Revealed by Fixation with Tannic Acid
Peter Olesen Institute of Plant Anatomyand Cytology,Universityof Copenhagen,83 Solvgade,DK-1307 CopenhagenK, Denmark
Abstract. Simple plasmodesmata between mesophyll
and bundle sheath cells in actively expanding leaves of S a l s o l a kaIi L. and roots of E p i l o b i u m h i r s u t u m L. are shown to possess specialized structures, called sphincters, around their neck regions. The sphincters are made visible by the combined effects of tannic acid and heavy metal staining; they are localized just outside that area of the plasmalemma, which forms the collar around the entrance to each plasmodesmos. This localization corresponds to a very active area of the plasmodesmos/plasmalemma complex (i.e. enzyme activity and/or presence of strongly reducing substances). Evidence is presented that these ring structures are structural equivalents to hypothetical sphincters performing some valve function; i.e. participating in the control of rates and directions of symplastic transport of solutes through plasmodesmata. The middle layer of the plasmalemma in the neck region is composed of closely-packed, globular subunits appearing in negative contrast. Apparently, these subunits correspond to particle clusters observed at the plasmodesmatal entrance in freeze-fracture preparations. They appear similar to particle clusters in animal tight junctions, and their possible function in providing electrical coupling via low resistance junctions between plant cells is discussed. Key words: E p i l o b i u m - Plasmodesmata - S a l s o l a Sphincter - Symplast Tannic acid.
Introduction
In recent years discussions on possible functional implications for plasmodesmata in symplastic transport in plants have been frequently centred around the questions: do all plasmodesmata show a tight seal
(neck constriction) between the desmotubule and plasmalemma in their neck region; and what are the ultrastructural details of such tight junctions (Gunning and Robards, 1976). The answers to these questions are of fundamental significance to the understanding of the mechanisms governing the cell-to-cell transport of a great variety of solutes. Until recently it was a widely held view that the desmotubule is the only open pathway (Robards, 1968; Olesen, 1975). But speculations on the feasibility of bidirectional transport through plasmodesmata between mesophyll and bundle sheath cells in Ca-plants (Olesen, 1975; Osmond and Smith, 1976) have strengthened the idea that the cytoplasmic annulus (central cavity) is an operational pathway. Accordingly, Gunning (1976) has suggested the existence of a kind of ultrastructural sphincter which could regulate symplastic transport by dimensional modulations of plasmodesmata. Willison (1976) and Evert et al. (1977) have presented some evidence for the existence of sphincters or plasmodesmatal valves. None of these studies, however, have provided detailed ultrastructural features of such complicated, molecular assemblages. Although freeze-fracturing should be considered the most powerful technique in obtaining morphological information about the desmotubule and plasmalemma in the plasmodesmatal neck, the results have been rather meager (Willison, 1976). Presumably, this is because the fracturing process preferentially produces split membrane faces, and, accordingly, the desmotubule and one half of the membrane break off at the plasmodesmatal entrance revealing no internal details of the neck constriction. Tannic acid used in combination with glutaraldehyde fixation results in an increased contrast of cytomembranes and clarifies the subunit structure of microtubules in thin sections (Tilney et al., 1973). Furthermore, it has been shown (Olesen, 1978) that fixation with tannic acid yields a good qualitative and
0032-0935/79/0144/0349/$02.00
350 quantitative correlation between membrane-associated particles of thylakoid membranes, which become visible in thin sections or by freeze fracturing. The present study investigates the effect of tannic acid on the ultrastructure o f plasmodesmatal neck regions, and this paper presents evidence for a complex ring structure or sphincter in the neck region o f simple, basic-type plasmodesmata.
Materials and Methods Young, actively expanding leaves of Salsola kali L. and apices of thick, fast-growing water roots of Epilobium hirsutum L. were fixed either conventionally, in formaldehyde-glntaraldehyde and osmium tetroxide, or, after addition of 2% tannic acid, to the primary fixative. Processing for electron microscopy and measuring technique was as described previously (Olesen, 1978). The image reinforcement technique of Markham et al. (1963) was employed, using high quality negatives showing cross sections of plasmodesmata at 25,000 or 50,000x primary magnification. Reinforcement diagrams were prepared directly from the original negatives by 16 x photographic enlargement. During preparation and evaluation of the diagrams, the directions and precautions described by Robards (1968) were strictly followed; the number of rotations was extended to cover the whole spectrum between 7 and 18 arcs in each circle.
Results and S o m e Conclusions
1. General Appearance of the Plasmodesmata The main object of this study is the p l a s m o d e s m a t a in the wall separating mesophyll and bundle-sheath cells in the C4-plant Salsola kali (Olesen, 1975). The plasmodesmata between cells within the elongation zone in water roots of Epilobium hirsutum were also included because o f the high, native content o f tanniferous substances in Epilobium roots (cf. Olesen, 1978). In both tissues the p l a s m o d e s m a t a belong to the basic type described by R o b a r d s (1976); i.e. a 15-20 n m desmotubule connected to strands of endoplasmic reticulum is surrounded by the plasmalemma t h r o u g h the wall between neighbouring cells. The distance between the desmotubule and plasmalemma varies in a fixed pattern along the plasmodesmatal canal. Those sectioned t h r o u g h the mid-line show a gap or central cavity (also called the cytoplasmic annulus) between the desmotubule and plasmalemma, whereas, those cut t h r o u g h the neck region show a tight seal between the desmotubule and plasmalemma (" neck constriction", Figs. 1, 3). In almost every case an electron-dense central rod is present and the desm o t u b u l e shows a conspicuous subunit structure. At the entrance of the plasmodesmatal pore the plasmalemma, in m o s t instances, appears slightly raised rela-
P. Olesen: Neck Constriction in Plasmodesmata rive to n o n - p l a s m o d e s m a t a l areas, i.e. the plasmalemma forms collar-like extensions (Figs. 1, 3). In the following, the special structural features of plasmodesmatal neck regions, observed after fixation with tannic acid, are described; the subunit structure of the desmotubule and its relation to the plasmalemma along the plasmodesmatal canal will be the subject o f a later c o m m u n i c a t i o n (Olesen, in preparation). Because o f the relation between average section thickness and plasmodesmatal dimension (cf. Robards, 1976; Figs. 2, 3), more information was obtained f r o m cross sections than f r o m longitudinal sections; this is chiefly because in longitudinal sections the whole o f a plasmodesmos is included in one section and, accordingly, the image superimposes too m u c h (partly irrelevant) information for all details to be resolved. 2. Longitudinal Sections The outer leaflet o f the plasmalemma (i.e. facing the wall) always shows a distinct, electron-opaque appearance and in most instances appears more dense than the inner leaflet (Fig. 3). Thus, tannic acid seemingly introduces a reverse asymmetry o f the plasmalemma c o m p a r e d to the situation after conventional fixation, where the inner layer is m o r e o p a q u e than the outer (e.g. Robards, 1968). Some 20-30 n m particles are frequently f o u n d in the walls close to the plasmalemma (Fig. 3). These particles show a c o m p o site nature with a mosaic of electron-dense and translucent areas ; in this way they bear a superficial resemblance to cytoplasmic ribosomes.
Fig. 11. Longitudinal section of a pit field showing a conspicuous collar (C) and associated sphincter (S) around plasmodesmatal neck regions, x 100,000 Fig. 2. Oblique section showing three different zones of plasmodesmatal structure representing plasmodesmatal transections in midwall (I), inner neck region (II), and outer neck region (III). The sphincters of neighbouring plasmodesmata in region III sometimes coalesce (arrow). • 50,000 Fig. 3. Longitudinal section of single plasmodesmos which in the lower part is sectioned slightly off-center; the collar (C) and sphincter (S) both appear distinct in the upper part. Short arrows denote small electron-transparent particles embedded in the opaque outer leaflet of the plasmalemma in the collar region. Long arrows indicate larger, composite, transparent particles between plasmalemma and the sphincter in the neck region. • 150,000 Figures 1M, 6 9 and 11 represent plasmodesmata between mesophyll and bundle sheath cells in leaves of Salsola kali L. ; Figures 5 and 10 depict plasmodesmata in roots of Epilobium hirsutum L. Unless otherwise stated all micrographs originate from preparations fixed with tannic acid and magnification bar=200 nm
P. Olesen: Neck Constriction in Plasmodesmata
351
352
The area of the plasmalemma showing the highest electron-opacity in the outer leaflet is invariably found in the collar around the plasmodesmatal entrance (Figs. 1, 3). As a matter of fact, the collar, observed as a slight extension of the plasmalemma into the cytoplasm, is mirrored at the plasmodesmatal outer surface as an accumulation of electron-opaque material. In some micrographs (Fig. 3), irregular electron-transparent particles appear half embedded in this opaque material. A consistant feature of the neck region is the presence of a rather diffuse structure extending 40-50 nm into the wall from the opaque outer surface of the plasmodesmatal canal (Figs. 1, 3); its outline is rather fuzzy, and it appears connected to the plasmalemma at a point corresponding to the outer margin or base of the collar. The electron-density of this fuzzy structure is slightly less than that of the opaque material in the outer leaflet of the plasmalemma, but similar to the density of the 20-30 nm particles found close to the plasmalemma in non-plasmodesmatal regions of the wall. The appearance of this structure in differently orientated, longitudinal sections shows that it must be interpreted as a wall-located ring around the plasmodesmatal neck; for the most part, it looks like an extracellular continuation of the collar. 3. Cross Sections of the Neck Region Inspection of a slightly oblique section through the wall (Fig. 2) demonstrates that three different zones of plasmodesmatal structure are evident: (I) a midwall zone where a central cavity (cytoplasmic annulus) is present between the desmotubule and plasmalemma; (II) a zone of intimate contact between the desmotubule and plasmalemma (innermost part of the neck region); and (III) the outermost neck region where a further ring structure is present around each plasmodesma.
A. Neck Region Excluding the Collar. The desmotubule appears tightly pressed against the plasmalemma canal which shows a remarkably constant diameter. The material in the plasmodesmatal canal is highly electron-dense, and, in favourable sections, the subimits of the desmotubule stand out in a clear, negative contrast (Figs. 4, 5, 8). The trilamellar, or unit membrane image of the plasmalemma, is partly obscured by the fact that the dense, inner leaflet is indistinguishable from the very dense material in the central canal. This circumstance makes it impossible to measure the actual inner diameter of the plasmalemma canal. The middle layer of the plasmalemma appears as an electron-translucent ring
P. Olesen: Neck Constriction in Plasmodesmata
with regularly undulate inner and outer surfaces; this appearance indicates a composition of closely-packed, more or less globular subunits. The outer leaflet always shows an extremely high electron-density; its outer surface is characterized by regularly spaced concave notchings which result in a gear wheel-like appearance. High resolution micrographs (Fig. 4) reveal that this appearance is caused by small, bright particles extending outwards into the surrounding wall material. Because of the rather low contrast of this wall material, however, the outer limitation of these particles is poorly resolved.
B. Neck Region Including the Collar. The major departure from sections excluding the collar is the presence of a ring structure around each plasmodesmos (Figs. 2, 9, 10). The outer surface of this structure displays medium electron-density and shows a rather constant diameter of 100-110 nm. Depending on how much cytoplasm is included in the actual section, the ring structure is rather sharply delineated (much low-contrast wall material and little cytoplasm included), or appears to be fuzzy (the section includes more relatively dense cytoplasm and less wall material). At high magnification, varieties of electron-transparent particulate elements (ca. 5-10 nm) are visible in the ring structure and, especially, in the very dense outer leaflet of the plasmalemma (Figs. 4, 5). No doubt this ring structure represents the fuzzy area observed around the neck region in longitudinal sections (Figs. 1, 3) where it appears connected to the outer leaflet of the plasmalemma at the periphery of the collar. In some instances two neighbouring plasmodesmata are so closely packed in the pit field that the ring structure belonging to one of them disturbs that of the other (Fig. 2). Control fixation of the same tissue without tannic acid only gave a hint of such ring structures (Figs. 6, 7), indicating that they are made visible primarily through the binding of tannic acid to some
Figs. 4 and 5. Cross sections of plasmodesmatal neck regions at high magnification. Small bright particles partly embedded in the opaque outer leaflet of the plasmalemma cause a gearwheel-like appearance (Fig. 4, arrow). The bright middle layer of the plasmalemma appears composed of closely spaced subunits, and the subunit structure of the desmotubule is clearly resolved, x 200,000 Figs. 6 and 7. Controls fixed without tannic acid: only a faint indication of a ring structure (sphincter) around the plasmodesmata (arrow in Fig. 6). Figure 6 • 60,000; Figure 7 • 150,000 Fig. 8. Control fixed with tannic acid but only poststained with uranyl acetate (i.e. without lead staining) : no evidence for a sphincter. x 75,000
P. Olesen: Neck Constriction in Plasmodesmata
353
354
specific substance located around the neck. The absence of ring structures in preparations fixed with tannic acid but not stained with lead citrate (Fig. 8) demonstrates, however, that the structures in question are visible through the combined effects of tannic acid and lead. In root cells of Epilobium, with a high native content of tanniferous substances (increased density and contrast of cytoplasmic structures), conspicuous ring structures around the plasmodesmatal entrances were consistently observed whether tannic acid was added during fixation (Figs. 5, 10) or not. 4. Image Reinforcement At high magnification, both cross sections (Fig. 9) and longitudinal sections (Fig. 3) fixed with tannic acid contain some evidence for the existence of larger, composite, electron-transparent particles in the area between the ring structure and the plasmalemma (i.e. around the neck). Favourable micrographs (e.g. Fig. 9) indicate some radial symmetry in this region, and, accordingly, it seems worth-while to try to obtain further details of the ring structure by using the technique of image reinforcement described by Markham et al. (1963). The salient feature of this technique is a reinforcement of structures exhibiting radial symmetry (such structures superimposes during rotation) due to the removal of random noise. Provided that rigo9rous precautions are taken while using it (Robards, 1968), this technique yields positive results, especially when reinforcement images (or diagrams) are compared to the original micrographs. Figures 9-11 show some reinforcement diagrams and the original micrographs from which they were made. In Fig. 9 (SaIsola), where a radial symmetry is indicated in the ring structure, maximum reinforcement is obtained when the photographic paper is rotated through 9 arcs in a circle (n = 9). This result indicates the presence of 9 composite, electron-transparent subunits in the ring structure. In Figl 10 (EpiIobium), where no symmetry is readily observed in the ring structure, maximum reinforcement again occurs at n = 9. The combined evidence from Figures 9 and 10 therefore indicates a basic nine-fold symmetry of the ring structure in plasmodesmata in Salsola and Epilobium. Surprisingly, when the photographic paper was centred with respect to the desmotubule (instead of the ring structure), maximum reinforcement was again obtained at n = 9 (Fig. 11). This result raises further doubt as to the actual number of subunits in the desmotubule, which has been claimed to be 11 (Robards, 1968) or 14 (Zee, 1969) whereas, Robards, in his latest review (Robards, 1976; Fig. 2.2), has depicted 9 subunits. This will be discussed later (Olesen, in preparation).
P. Olesen: Neck Constriction in Plasmodesmata
5. Dimension and Spatial Organization of Structures in the Neck Region Figure 12 is an illustrative diagram of a cross section of the neck region based on the typical appearance of plasmodesmata after fixation with tannic acid. The dimensions of the principal components are given in the diagram; because of the presence of dense material between the desmotubule and plasmalemma, and the thick, opaque covering of the outer leaflet of the latter, it is not possible to compare the dimensions with those in conventionally fixed preparations (Figs. 6, 7) (Robards, 1968). The bright middle layer of the plasmalemma, however, is interpreted as representing intramembranous particles, i.e. particles normally exposed in freeze-fracture studies. The only fairly detailed freeze-fracture study of plasmodesmata available (Willison, 1976) demonstrates accumulations of membrane-associated particles on the fracture face of the plasmalemma, at the junctions between the plasmodesmata and plasmalemma (i.e. where the wall-covered plasmalemma canal in the neck region breaks off during the fracturing process). Furthermore, and of great interest to the present study, the micrographs published by Willison (1976) consistently demonstrate the presence of a collar (slightly extended plasmalemma) around th e plasmodesmatal entrance. Also, a few previously published freeze-fracture micrographs, including plasmodesmata, present some evidence for a collar (Northcote and Lewis, 1968, Fig. 1; Branton and Deamer, 1972, Fig. 17). The observed structural correlation between the present study and freeze-fracture literature suggests that it is worth-while to compare the dimensions of the principal components measured on preparations from conventionally fixed, tannic acid, and freeze-fractured tissues, respectively. Table 1 demonstrates that tee diameter of the collar corresponds roughly to the diameter of the ring structure, which further substantiates the interpretation of longitudinal sections: the ring structure mirrors the plasmalemmal collar and is connected to its outer, opaque surface at the base of the collar. The diameter of the particle clusters around the plasmodesmatal entrance on freeze-fracture micrographs somewhat exceeds the diameter of the bright, middle layer of the plasmalemma in thin Figs. 9-11. Image reinforcement diagrams (A, B, C) and the original micrographs from which they were made. In Figure 9 m a x i m u m reinforcement at n = 9 is obtained in the large, composite, electron-transparent particles between plasmalemma and the sphincter (B). In Figure 10, where no symmetry is readily observed in the original micrograph, reinforcement again occurs at n = 9 (B). Figure 11 shows a similar m a x i m u m reinforcement at n = 9 of the desmotubule subunits (B). Figures 9 10 x 225,000 ; Figure 11 x 450,000
P. Olesen: Neck Constriction in Plasmodesmata
355
356
P. Olesen: Neck Constriction in Plasmodesmata
appear smaller in thin sections as compared to the freeze-fracture view; also, some possible effects of shrinkage during fixation, dehydration, and embedding cannot be excluded.
iiiiiit
cA:, DE 03 477 00: 23 0 8 nm
Discussion
1. The Effect of Tannic Acid Fig. 12. Diagrammatic representation of transverse sections of the neck region in plasmodesmata after fixation with tannic acid. The dimensions referred to are the average of 20 sets of measurements made on image reinforcement diagrams like Figures 9-10. A outer diameter of the ring structure; B outer diameter of the opaque outer leaflet of the plasmalemma; C and D outer and inner diameter of the bright middle layer of the plasmalemma; E outer diameter of the desmotubule
Table 1. Dimensions of the plasmodesmatal neck region after different preparation methods for electron microscopy. The figures in brackets indicate the number of measurements made on respective micrographs Diameter of the ring structure (nm)
Diameter of the middle layer of the plasmalemma (nm)
127 (10)
59 (10)
133 (10)
57 (10)
135 (21)
56 (18)
110 (32)
33 (34)
-
40 (8)
Freeze-fracturea Northcote and Lewis t968, Fig. 1 Branton and Deamer 1972, Fig. 17 Witlison 1976, Fig. 2 Thin sections after fixation with tannic acid The present study Thin sections after conventional fixation The present study
a These figures represent the margin or base of the collar to which the ring appears connected on the outer surface, not the ring structure proper
sections. Presumably, this difference shows that, in freeze-fracturing, the outer leaflet of the plasmalemma tends to break off just outside the neck constriction where it diverges away from the desmotubule and continues into the collar; it is exactly at this point that the plasmalemma fracture face changes from the closely packed, micellar condition (particle clusters) to a relatively smooth face, with few and scattered particles found in the collar and non-plasmodesmatal regions (Willison, 1976). In thin cross sections, however, the bright, middle layer is observed by a perpendicular superposition throughout the section thickness of the (presumably) closely packed subunits in the cylindrical plasmalemma, in the neck region. Therefore, the diameter of this structure will
The effect of tannic acid during fixation is based on its effective precipitation of proteins; the resultant precipitates are cross-linked by glutaraldehyde and show a high affinity to heavy metals (e.g. osmium). The protein-tannic acid-heavy metal complexes induce strong electron-scattering, especially around protein macromolecules which stand out in clear, negative contrast (Futaesaku et al., 1972; Tilney et al., 1973). The formation of a shield of tannic acid-heavy metal complexes around subunit particles brings about a stabilizing effect on the native protein configuration and some avoidance of dehydration shrinkage; in chloroplast thylakoid membranes a quantitative analysis of the relative sizes and distribution of membrane-associated particles, made visible by tannic acid (Olesen, 1978), gave a similar size distribution as that obtained with particles visible after freeze-fracturing.
2. Visualization of the Ring Structure A few micrographs of plasmodesmata, after addition of tannic acid to the primary fixative, were published by Robards (1976) and the negative staining effects of tannic acid, with respect to the desmotubule, were mentioned briefly. But, although the published micrographs showed the presence of a conspicuous ring structure in the neck region, no comments were made on this subject. Careful inspection of the micrographs published in the pioneer work by Robards (1968) reveals some evidence for a ring structure in the outermost neck region, i.e. in parts of sections including the plasmalemma collar (Robards, 1968, Figs. 2 and 4). Despite the numerous papers illustrating plasmodesmatal details that have been published since the work of Robards (1968), none of them equals it in clarity of the plasmalemma canal and the subunit structure of the desmotubule. These circumstances strongly suggest that Robards' material (cambial tissue of Salixfragilis) was to some degree influenced by the presence of a tannic acid-like substance during fixation. This suggestion is supported by the wellknown presence of high concentrations of tannins or tanniferous substances in the genus Salix. Finally, in the same tissue, Robards (1969) found granules of about 24 nm just outside the plasmalemma of differentiating cells; in the present study, similar parti-
P. Olesen: Neck Constrictionin Plasmodesmata cles (20 30 nm and showing medium electron-density) were found after fixation with tannic acid but not after conventional fixation. The crucial role of tanniferous substances in the observation of the ring structure is further stressed by the demonstration of conspicuous ring structures in Epilobium (Figs. 5, 10). In ovules of Quercus (Mogensen, 1973), it was showed that plasmodesmata in the outer integument had an associated rim of osmiophilic material on either end of the plasmodesmatal canal. These cells were strongly influenced by tannins (Mogensen, 1973, Figs. 9, 10), and, no doubt, the osmiophilic rims were equivalent to the ring structures or sphincters demonstrated in the present study.
3. Possible Nature of the Ring Structure (Sphincter) Virtually nothing is known about the chemical composition of the ring structure, but its visibility by the combined effects of tannic acid and lead indicates some proteinaceous components (Olesen, 1978). The reality of the ring structure is strengthened by the demonstration of similar structures in plasmodesmata prepared by cryo-ultramicrotomy (Vian and Rougier, 1974); here, apparent evaginations of the plasmalemma occur at the base of the collar. Robards (1976) enumerated reports on different appearance or composition of the cell wall immediately surrounding plasmodesmata (presence of callose, general ultrastructural appearance, changed carbohydrate deposition, and resistance to cellulase enzymes). Studies on enzyme cytochemistry and ion localization (cf. Van Steveninck, 1976 and references therein) have quite consistently shown high activities of ATPase and high concentrations of C1- -ions along plasmodesmata and, especially, around the collar and the neck region (i.e. corresponding to the localization of the ring structure). Both techniques, however, are based on the precipitation of reaction products, including heavy metal ions (silver and lead), and serious doubts have been raised on the specific localizations obtained with these techniques (cf. Van Steveninck, 1976). Early light microscopical techniques made use of silver impregnation to demonstrate plasmodesmata, and electron microscopical work has shown that both silver ions (AgNO3, Brown et al., 1962) and lead ions (motor traffic exhaust gases, Ophus and Gullvgg, 1974) are precipitated as electron-opaque deposits in and around plasmodesmata before or during fixation. These findings indicate that the precipitation of heavy elements is caused possibly by reducing substances associated with the plasmodesmata. In the present context, it suffices to say that the neck region and its associated ring structure constitutes a very active region of plasmodesmata, whether this activity re-
357 flects true enzyme activity (e.g. ATPase), or is due to the presence of some ill-defined reducing substances.
4. Functional Implications for the Sphincter Current considerations of the function of plasmodesmata in intercellular symplastic transport emphasize the possibility of some transport capacity in the annulus between the desmotubule and plasmalemma (Gunning, 1976) instead of being restricted to the desmotubule (Olesen, 1975). With this in mind, Gunning (1976) stressed that intercellular transport in plants could be regulated by dimensional modulations of plasmodesmata, and suggested that electron microscopists should be looking for ultrastructural equivalents of sphincters. Since then two papers dealing with the ultrastructural localization of hypothetical sphincters in plasmodesmata have been published (Willison, 1976; Evert et al., 1977). Willison considered the possibility that the clustered membrane-associated particles around the plasmodesmatal entrances represent a sphincter between the desmotubule and plasmalemma - this possibility, and the identity of the particles, are discussed below. Evert et al. (1977), on the contrary, demonstrated the presence in Zea mays of some electron-dense structures between the desmotubule and the plasmalemma, which occlude the central cavity (cytoplasmic annulus) adjacent to the neck, and referred to these structures as sphincters. Similar structures have been observed by the present author in maize and two other grass species (Sporobolus rigens and Spartina townsendii), after fixation with tannic acid as well as conventional fixation (Olesen, unpublished); the possibility of such structures representing specialized features of plasmodesmata in grasses is not unlikely, judged by the structural variation found between plasmodesmata from different plant groups (Robards, 1976). In view of the present findings, it seems plausible to conclude that in simple plasmodesmata of dicotyledonous plants [e.g. those in Salsola kali and Epilobium hirsutum representing the basic-type plasmodesmata of Robards (1976)] the ring structure revealed by fixation with tannic acid could be the structural equivalent of the sphincter postulated by Gunning (1976). The demonstration of large, composite particles appearing in approximate nine-fold radial symmetry in this area, which in other respects appears rather active, clearly substantiates such a conclusion.
5. Membrane-Associated Particles Around the Plasmodesmatal Entrance On the basis of their appearance in thin sections, and the correlation in size and localization between
358 freeze-fracture m i c r o g r a p h s (Willison, 1976) a n d thin sections, it is tentatively c o n c l u d e d that the closelypacked s u b u n i t s observed in the bright, middle layer of the p l a s m a l e m m a (Figs. 4, 5, 9) are identical to the particle clusters observed a r o u n d the p l a s m o d e s m a t a l e n t r a n c e in freeze-fracture micrographs. Willison (1976) in a few cases f o u n d these clusters to be more widely spread t h a n in other instances, a n d suggested that one possible f u n c t i o n a l implication for these particle clusters is the c o n t r o l of a sphincter between p l a s m a l e m m a a n d the desmotubule. I n view of the present findings, however, a n d with the ring structure of the neck region being a likely candidate to the structural equivalent of such sphincter (cf. G u n n i n g , 1976), the presence of occasional " o p e n - m o u t h e d " p l a s m o d e s m a t a (Willison, 1976) with spread particle clusters can be readily explained. In the " o p e n " condition, the sphincter (ring structure) w o u l d e x p a n d a n d increase in d i a m e t e r ; if the ring structure is connected to the o u t e r surface o f the p l a s m a l e m m a at the periphery of the collar, then the latter would exp a n d propQrtionately. This is exactly what can be observed i n the m i c r o g r a p h published by Willison (1976, Fig. 5) where the " o p e n - m o u t h e d " plasmodesmos is characterized by a considerably greater diameter a n d a flattened a p p e a r a n c e of the collar. In this way, the particle clusters, which, in the " c l o s e d " condition, are localized in a n d a r o u n d the neck constriction, would be pulled out along the i n n e r rim of the collar. Clues as to the f u n c t i o n a l implications of the clustered particles could possibly be f o u n d in the fact that similar particles are clustered at tight j u n c t i o n s between a n i m a l cells ( M c N u t t a n d Weinstein, 1973), which f u n c t i o n as intercellular seals or low resistance j u n c t i o n s . Since the work of Spanswick a n d C o s t e r t o n (1967), p l a s m o d e s m a t a have been considered as lowresistance j u n c t i o n s which provide direct electrical c o u p l i n g between cells of higher plants, a n d are pres u m a b l y responsible for electrotonic t r a n s m i s s i o n of action potentials (Spanswick, 1976). This study was supported in part by The Danish Natural Science Council (grant no. 5II-6847). The author wishes to thank Lisbeth Thrane Haukrogh for excellent technical assistance.
References Branton, D., Deamer, D.W. : Membrane structure. Protoplasmatologia II/E/1. Wien: Springer 1972 Brown, W.W., Mollenhauer, H., Johnson, C.: An electron microscope study of silver nitrate reduction in leaf cells. Am. J. Bot. 49, 57 63 (1962) Evert, R.F., Eschrich, W., Heyser, W.: Distribution and structure of the plasmodesmata in mesophyll and bundle-sheath cells of Zea mays L. Planta 136, 77 89 (1977) Futaesaku, Y., Mizuhira, V., Nakamura, H. : The new fixation method using tannic acid for electron microscopy and some
P. Olesen: Neck Constriction in Plasmodesmata observations of biological specimens, pp. 155 156. In: Proc. IVth Internatl. Congr. Histochem. Cytochem. 1972 Gunning, B.E.S. : Introduction to plasmodesmata. In: Intercellular communication in plants: Studies on plasmodesmata, pp. 1-13, Gunning, B.E.S., Robards, A.W., eds. Berlin, Heidelberg, New York: Springer 1976 Gunning, B.E.S., Robards, A.W. : Plasmodesmata: Current knowledge and outstanding problems. In: Intercellular communication in plants: Studies on plasmodesmata, pp. 297 311, Gunning, B.E.S., Robards, A.W., eds. Berlin, Heidelberg, New York: Springer 1976 Markham, R., Frey, S., Hills, G.J. : Methods for the enhancement of image detail and accentuation of structure in electron microscopy. Virology 20, 88-102 (1963) McNutt, N.S., Weinstein, R.S. : Membrane ultrastructure at mammalian intercellular junctions. Progr. Biophys. MoI. Biol. 26, 45 101 (1973) Mogensen, H.L.: Some histochemical, ultrastructural, and nutritional aspects of the ovule of Quercus gambelii. Am. J. Bot. 60, 48-54 (1973) Northcote, D.H., Lewis, D.R.: Freeze-etched surfaces of membranes and organelles in the cells of pea root tips. J. Cell Sci. 3, 199-206 (1968) Olesen, P.: Plasmodesmata between mesophyll and bundle sheath ceils in relation to the exchange of C4-acids. Planta 123, 199-202 (1975) Olesen, P.: Structure of chloroplast membranes as revealed by natural and experimental fixation'with tannic acid: Particles in and on the thylakoid membrane. Biochem. Physiol. Pflanzen 172, 319-342 (1978) Ophus, E.M., Gullvgtg,B.M. : Localization of lead within leaf cells of Rhytidiadelphus squarrosus (Hedw.) Warnst. by means of transmission electron microscopy and X-ray microanalysis. Cytobios 10, 45-58 (1974) Osmond, C.B., Smith, F.A.: Symplastic transport of metabolites during C4-photosynthesis. In: Intercellular communication in plants: Studies on plasmodesmata, pp. 22%240, Gunning, B.E.S., Robards, A.W., eds. Berlin, Heidelberg, New York: Springer i976 Robards, A.W.: A new interpretation of plasmodesmatal ultrastructure. Planta 82, 200 210 (1968) Robards, A.W.: Particles associated with developing plant cell walls. Planta 88, 376-379 (1969) Robards, A.W. : Plasmodesmata in higher plants. In: Intercellular communication in plants: Studies on plasmodesmata, pp. 15-57, Gunning. B.E.S., Robards, A.W., eds. Berlin, Heidelberg, New York: Springer 1976 Spanswick, R.M.: Symplasmic transport in tissues. In: Transport in plants II, pp. 35 53, Ltittge, U., Pitman, M.G., eds. Berlin, Heidelberg, New York: Springer 1976 Spanswick, R.M., Costerton, J.W.F.: Plasmodesmata in Nitella translucens: Structure and electrical resistance. J. Cell Sci. 2, 451-464 (1967) Tilney, L.G., Bryan, J., Bush, D.J., Fujiwara, K., Mooseker, M.S., Murphy, D.B., Snyder, D.H.: Microtubules: evidence for 13 protofilaments. J. Cell Biol. 59, 267-275 (1973) Van Steveninck, R.F.M. : Cytochemical evidence for ion transport through plasmodesmata. In: Intercellular communication in plants: Studies on plasmodesmata, pp. 131-147, Gunning, B.E.S., Robards, A.W., eds. Berlin, Heidelberg, New York: Springer 1976 Vian, B., Rougier, M.: Ultrastructure des plasmodesmes apr& cryo-ultramicrotomie. J. Microscopie 20, 307-312 (1974) Willison, J.H.M.: Plasmodesmata: a freeze-fracture view. Can. J. Bot. 54, 2842 2847 (1976) Zee, S.-Y.: Fine structure of the differentiating sieve elements of Viciafaba. Aust. J. Bot. 17, 441-456 (1969) Received 5 August; accepted 9 October 1978