Cytologia 37: 759-768, 1972

Observations on the Developmental Morphology and Fine Structure of Pit Connections in Red

Eva Konrad Hawkins1

Fairleigh Dickinson University, Rutherford, New Jersey, U. S. A.

Received July 22, 1971

Introduction Pits (thin spots) with conspicuous pit connections have been observed since the last century (Klein 1877, early descriptions are reviewed by Falkenberg 1901) in cell walls of . Pit connections are formed between daughter cells of a division (primary pit connections) and between cells that have remained in contact or achieved new contact during development (secondary pit connections). The structure and possible function of pit connections of red algae have been subjects of considerable speculation and controversy. One group of phycologists (Schmitz 1883, Falkenberg 1901, Kohl 1902, Miranda 1930) has held that openings between cells of red algae are plugged by a perforated membrane which, in turn, is bordered by dense plates on both sides. Slender protoplasmic filaments, con necting adjacent protoplasts, would traverse this membrane. Another group (Ambronn 1880, Archer 1876, Kienitz-Gerloff 1902) opposed this conclusion and described the "membrane" as homogeneous. Mangenot (1924) found special cytoplasmic differentiations, "plasmodesms," without any pit closing membrane between adjacent cells of red algae. He viewed these as "synapses" and suggested that they function as sites of an intercellular exchange of nutrients and transmission of stimuli in red algae. Jungers (1933) classified "synapses" into the following types: 1) a densely staining lens-shaped body, set in the central orifice of the crosswall, found in Cera mium and Griffithsia: 2) two densely staining discs separated by a fine membrane, found in Polysiphonia and Delesseria. He did not think "synapses" have a "proto plasmic content" and did not accept their role in "protoplasmic communications." Though this classification was questioned (Muldorf 1937), the structure of pit connections was not clarified (Kylin 1940). Early phycologists postulated the significance of pit connections in the phy logeny and of red algae. However, the resolution of the light microscope severely limited their studies. Light microscopy could not assess the evolutionary (Denison and Caroll 1966) or taxonomic (Dixon 1963) significance of this structure. Knowledge of the fine structure of pit connections may assist not only in classification or in advancing theories of phylogenetic importance. Several para sitic algae establish cytoplasmic contact with cells of their photosynthetic host re 1 Present address: Department of Biology, University of Pennsylvania, Philadelphia, Pa. 19104, U. S. A. 760 E. K. Hawkins Cytologia 37

lative by the formation of pit connections (Martin and Pocock 1953). Phototropic orientations of branches of various red algae to unilateral illuminations are known (Berthold 1882). What regulates these orientations at the cellular level is not under stood. Secondary pit connections are significant in anastomoses of neighboring branches. The highly regular net-like form of some members of the Delesseriaceae is maintained by primary and secondary pit connections (Papenfuss 1937). Through these secondary contacts and reinforcements additional meshes are formed and the gross morphology of entire organisms is altered. Fusion between lateral branches to form a reticulate system of interconnecting cells may be observed in a number of red algal genera (Halodictyon, Haloplegma, Rhododictyon, etc.). In view of the multiple significance of pit connections in the biology of red algae and the insufficient knowledge available to us on their developmental mor phology and fine structure (Myers et al. 1959, Bouck 1962, Peyriere 1963, Bishoff 1965, Bisalputra et al. 1967, Ramus 1969) this study was undertaken to clarify further their structure.

Materials and methods

Ceramium diaphanum and Polysiphonia sp. were collected below low tide level from rocks at Sayville, L. I. and Sea Gate, Coney Island. Callithamnion roseum was obtained from unialgal cultures (Konrad Hawkins 1968). Apical parts

(approx. 1cm long) were fixed in Karnovsky's fixative (1965) for 3 hours. Cal lithamnion and Ceramium were postfixed in 1% sodium cacodylate buffered osmium tetroxide at pH 7.5 for 1hr. Polysiphonia was postfixed in 2% sodium cacodylate buffered osmium tetroxide at pH 8 for 3 hrs. They were embedded in agar prior to dehydration. Dehydration was carried out at 0-4•Ž in a graded

series of acetone/water mixtures. Final embedding was in Epon 812; sections were cut with a diamond knife and stained with uranyl acetate and lead citrate

(Venable and Coggeshall 1965). Micrographs were made with and RCA EMU-3H electron microscope.

Observations

Primary pit connections of varying morphology were found between cortical , segment and axial cells of Ceramium diaphanum. Some suggest biconvex discs , constricted along their equator (Figs. 1-10). Their diameter is 0.3-0.7ƒÊ at the

plane of constriction. A triple-layered membrane (Figs. 1, 2), 80-100A thick, surrounds equatorial furrows facing cell walls. Its relationship to the plasma

membrane (Figs. 7, 8) could not be ascertained, since the latter has not been clearly preserved. The lateral limiting membrane is in contact with microfibrils

of the (Fig. 2). Convex surfaces stain intensely to a depth of 30-50mƒÊ.

Staining intensity gradually decreases at both surfaces to a depth of 100mƒÊ.

Beyond these electron dense regions numerous fibrous thread-like formations traverse the youngest connections (Fig. 7). 1972 Observations on the Developmental Morphology and Fine Structure of Pit 761

Figs. 1-6. Pit connections between cortical cells of Ceramium diaphanum. 1-2, serial longi tudinal sections. Height-to-width ratios of the pit connections indicate a cut near the median in Fig. 1, submedian in Fig. 2. Note attachment of branched microfibrils (mf) of the cell wall (cw) to limiting membrane (lm). 3, longitudinal lateral section through the equatorial furrow. The pit connection appears as two separated discs. 4, oblique lateral section. 5, oblique tangential section. -Microfibrils (mf) of the cell wall show a circular orientation around the equator. 6, near cross section, through equatorial furrow. Scales, 500mƒÊ. 762 E. K. Hawkins Cytologia 37

An attempt was made to correlate occurrence of the biconvex constricted form (Figs. 1-6) with developmental conditions of the linked cells. Dense and vacuolated cells of varying sizes compose the cortex of Ceramium. They are formed by intercalary cell divisions. Biconvex, constricted pit connections were found between cortical cells where one or both of the linked members was intensely

Figs. 7-10. Longitudinal sections of pit connections between segment cells of an apex of Ceramium diaphanum. 7, near median section between second and third segment cell. Fibrous thread-like structures (f) are parallel to the longitudinal axis of the expanding thallus. Plasma membranes

(pm) are not clearly enough defined to allow conclusions about their relation to lateral membranes. 8, longitudinal section between fifth and sixth segment cell. 9, longitudinal lateral section between sixth and seventh segment cell. Note five-layered appearance of adjacent lateral membranes

(arrows) near the innermost edge of the equatorial furrow. 10, oblique lateral section between third and fourth segment cell. Scales, 500mƒÊ. stained and nonvacuolated. This indicated a recent cell division and a newly formed pit connection between daughter cells. Segment cells of Ceramium originate by continuous divisions of dome-shaped apical cells. As their distance from the tip increases, they enlarge into axial cells 1972 Observations on the Developmental Morphology and Fine Structure of Pit 763

surrounded by nodal cortication. Successive segment cells of an apex represent successive developmental stages of one segment cell. Similarly, one may visualize successive pit connections of a single apex as representative stages in the develop ment of one pit connection, with youngest forms occurring nearest to the tip. Pit connections (Figs. 7-10) between segment cells of an apex show similar morphology to those (Figs. 1-6) which contact at least one small nonvacuolated cortical cell of basal regions. This suggests that biconvex, constricted forms re present early developmental stages of pit connections. Morphological similarities to young pit connections of Ceramium diaphanum (Figs. 1-10) were observed also in apices of Callithamnion roseum and Polysiphonia sp. (Fig. 15).

Figs. 11-14. Longitudinal section of pit connections of Ceramium diaphanum. 11, flattened, lens-shaped form between vacuolated cortical cells. Scale 500mƒÊ. 12, pit connection between

axial cells was pulled apart during its preparation for electron microscopy. Note empty, electron transparent space between densely stained opposite halves of the dics. Scale, 1ƒÊ. 13, pit connections between vacuolated axial cells. Scale 10ƒÊ. 14, pit connection of Fig. 13 magnified. Scale, 1ƒÊ.

The proportions of biconvex forms change during growth. Alterations in

proportions are brought about by a disproportionate increase in width of the connections with respect to: 1) depth of the equatorial furrows, 2) height of the connections. Equatorial furrows may not change in depth or may even disappear during growth of the pit connections; yet the connections may increase 5-10 fold

in width during development. Decreased height-to-width ratios of pit connections

(diam>1ƒÊ) mark the development of flat, lens-shaped forms, composed of a granular 764 E. K. Hawkins Cytologla 37

material of high electron density. These were observed between cortical (Fig. 11) and dense axial cells (Fig. 12 see legends). They probably represent transitional developmental stages between young pit connections (Figs. 1-10) and between mature forms (Figs. 13, 14) linking vacuolated axial cells.

Fig. 15. Pit connection between cells of an apex of Polysiphonia sp. Scale, 500mƒÊ.

Discussion

These electron micrographs show a correlation between morphology and develop mental age of pit connections in Ceramium diaphanum. Small, biconvex constricted forms represent young developmental stages of pit connections. As pit connections mature, they increase mainly in width. Flat, wide, lens-shaped, or more correctly, pulley wheel-shaped forms are made up of a granular material of high electron density. How these developmental variations of the form of pit connections relate to images in the light microscope had not been explored in previous studies. An examination of this question may clarify earlier interpretations regarding structure and classification of pit connections.

Pit connections of Ceramium should exemplify one major type found in higher red algae: a single lens-shaped body (Jungers 1933). Curiously, Fig. 1 and Fig. 3 show, in Ceramium, images of both "types" of pit connections described by Jungers

(a lens-shaped body and two separated discs). These images were found between cortical cells of similar developmental stages in a single organism. Figure 1 represents a median longitudinal cut; Fig. 3 shows a lateral longitudinal section through the region of the equatorial furrow. The furrow has an approx. depth of

0.1ƒÊ; i.e. it cannot be resolved with a light microscope. Thus, early investigators could not trace the relationship of two separated discs to a single lens-shaped body, and classified them as distinct "types." Pit connections of Polysiphonia should exemplify the second major type of pit connection found in Florideophycidae: two discs divided by a membrane. Yet, Jungers rarely saw the discs distinctly double in the apex of this alga (1933, p. 12). Having no information as to the varying appearance of pit connections at increasing distances from the median, he attributed the differing appearance of one pit con nection exclusively to different planes of sectioning (p. 12, 1933). He assumed, 1972 Observations on the Developmental Morphology and Fine Structure of Pit 765 erroneously, that small pit connections (which he could not see clearly) have the same morphology as large synapses (p. 12, 1933). In the latter he believed to have seen clearly two discs separated by a membrane . This "membrane", which appeared as a thin, usually nonstaining line of con stant width between "two discs" of large (several ƒÊ wide) pit connections of Poly siphonia and Delesseria, he regarded as a continuation of the middle lamella . It is obvious that this several hundred my thick line is not a membrane. Similar lines observed by this author are believed to result from plasmolytic distortions of pit connections. As shrinking protoplasts are pulled apart during preparation for light or electron microscopy, the thickness of the middle thin line increases to a varying extent depending on the degree of plasmolysis (Fig. 12, see also Fig. 6 of

Bouck). Though this empty space is situated at the same level with a usually electron dense region between adjacent cell walls, it is, of course, unrelated to the middle lamella. Even this electron dense region between adjacent cell walls, which

Jungers described as middle lamella, has been misinterpreted. It usually com prises several layers of microfibrils in addition to the middle lamella. Conden sation of microfibrils in this region is also a plasmolytic change indicating that their attachment to pit connections is stronger than to adjacent plasma membranes.

In view of these preparatory distortions, the drawings of pit connections between axial cells of Polysiphonia fastigiata, Delesseria alata, and Ceramium rubrum

(Figs. 2, 6, 20, Jungers, 1933) do not look significantly different; in fact, they suggest that the classification of pit connections by Jungers into two major types is incorrect. The postulated "Polysiphonia type" pit connection is a misinter pretation of a single body whose morphology varies with development and cannot be clearly resolved in the light microscope. Fundamentally similar pit connections to those of this study were found in Lomentaria baileyana (Bouck 1962), Ceramium echionotum (Peyriere 1963), Laurencia spectabilis (Bisalputra et al, 1967), Pseudo gloiophloea confusa (Ramus, 1969), and Porphyra leucosticta (Lee and Fultz 1970). Though the first electron microscopic study (Myers et al. 1959) of pit con nections of Rhodymenia palmata and Laurencia sp. confirmed the classification of Jungers, preparatory techniques of the late 1950's and the resolution of available electron micrographs left the problem still in doubt. Subsequent work (Dawes et al. 1961) described "a concentration of fine plasmodesmata" between cells of Helmintho cladia. The resolution of these illustrations does not permit a comparison between "plasmodesmata" of Helminthocladia and those of higher plants (Lopez-Saez et al. 1966, Esau et al. 1966, Robards 1968). Ramus (1969) observed a series of flattened vesicles in the aperture of the cell wall of Pseudogloiophloea when pit connections begin to form. He assumed that these membranes originate from the endoplasmic reticulum. As material ac cumulates about them they disappear; perhaps they become incorporated in the pit connection. It seems likely that the fibrous thread-like structures, found in youngest connections of Ceramium (Fig. 7) and Polysiphonia sp. (Rusanowski, personal communication) are vestiges of disappearing flattened vesicles. Should these vesicles secrete the condensing material seen on their surface the question arises how the connections become filled with a granular (Bisalputra et al. 1967, 766 E. K. Hawkins Cytologia 37

Ramus 1969), possibly proteinaceous material (Bisalputra et al. 1967) when vesicles disappear. Is this material synthesized in situ or is it derived from the cytoplasm? The connections are separated from adjoining cytoplasms by plasma mem branes (Bisalputra et al. 1967). How these membranes differ from lateral limiting membranes facing cell walls and why they react differently from lateral limiting mem branes to the same fixative is not clear. Lateral membranes seem to possess an unusual resiliency; they may become turned out during growth of pit connections (see Fig. 7 of Bouck). Perhaps plasma membranes are prone to deform as a result of cellular activities. Bouck (1962) observed small papillae on surface membranes of pit connections of Lomentaria as if material were being discharged into or taken from the connections. Whether the dense peripheral regions of the connections adjacent to plasma membranes result from similar activities is at present unknown. Vesicles near pit connections of Pseudogloiophloea (Ramus 1969) also raise the possibility that the connections are not isolated from adjoining cytoplasms. In higher plant cells vesicles have been observed to move across plasma membranes (Branton and Mohr 1964). Relevant to this question in diverse the cytoplasm extends across the central vacuole from one connection to the next (Fritsch 1945). This is especially pronounced in axial cells of Ceramium diapha num (Fig. 13). In view of these observations it seems plausible that some physiological con tinuity is maintained by pit connections between cells of red algae. The demon stration of such continuity awaits future studies.

This study was supported by a Rockefeller Foundation grant to the Osborn Laboratories of Marine Sciences-New York Aquarium. I thank Dr. M. C. Ledbetter for helpful discussions and Dr. R. F. Nigrelli and the U. S. Naval Applied Science Laboratory for providing facilities for this research.

Summary

Pit connections, biconvex, equatorially constricted discs (0.3-0.7ƒÊ in dia meter), develop between newly formed cortical and segment cells of Ceramium diaphanum. Biconvex, constricted forms represent early developmental stages of pit connections. Similar forms have been found in apices of Callithamnion roseum and Polysiphonia sp. As pit connections mature they increase mainly in width. Equatorial furrows become less pronounced. Flat, wide, lens-shaped forms consist of a granular material of high electron density. Current information on these structures suggests that their classification by Jungers is incorrect. Pit connections of red algae consist of a single body whose morphology varies with development.

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Cytologia Vol. 37, No. 4 pp. (525-768) Issued December 25, 1972 Ausgegeben am 25. Dezember 1972 Paru le 25 decembre 1972