J. Cell Set. S3, 35I-37I (1981) 351 Printed in Great Britain © Company of Biologists Limited 1981 DEVELOPMENT OF THE CELL WALL IN TETRASELMIS: ROLE OF THE GOLGI APPARATUS AND EXTRACELLULAR WALL ASSEMBLY DAVID S. DOMOZYCH,* KENNETH D. STEWART AND KARL R. MATTOX Department of Botany, Miami University, Oxford, Ohio 45056, U.SJl. SUMMARY The green algal flagellate, Tetrasetmis, is a key transition organism in the phylogeny of green algae. It has been proposed that the cell wall of Tetraselmis arose evolutionarily from the fusion of scales and that this event secondarily caused the alteration of some cytoplasmic processes such as mitotic and cytokinetic mechanisms. Ultrastructural and developmental studies of the cell wall were performed with several strains of Tetraselmis. Two major wall types are reported. The wall of type 1 cells consists of a thick inner region covered by a layer of regularly repeating subunits of 26 ran, comparable to the subunits found in the median W2-W6 layer of Chlamy- domonas. The more elaborate type 2 cell wall consists of a thick median wall layer, homologous to the type 1 inner wall, with additional inner and outer strata of hairs, grains and scales. Development of the cell wall begins in the endomembrane system, particularly the Golgi apparatus, where fibrillar tufts and electron-dense droplets are synthesized, modified and transported to the outside. Here, the tufts and droplets are displaced around the protoplast and assemble in several steps to yield the intact wall. Edge-growth assembly of the wall occurs here synchronously with cytoplasmic developments to yield the characteristic anterior flagellar pit. Models explaining various aspects of this development are discussed. When released from the cell, the wall subunits are not completely comparable to stellate scales, but appear to correspond to developmental stages of scales in green flagellates possessing body scales. INTRODUCTION Tetraselmis (Chlorophyta) is an unusual green algal flagellate possessing inter- mediate characteristics between 2 groups often separated at the class level, the Prasino- phyceae and the Chlorophyceae. Like many members of the more primitive Prasino- phyceae such as Pyramimonas (Norris & Pearson, 1975; see also Norris, 1980), Tetraselmis possesses scaly and hair-covered flagella that emerge from a central cellular depression, called the pit. In common with members of the advanced Chlorophyceae (Mattox & Stewart, 1977; Stewart & Mattox, 1978), this organism possesses an early-collapsing telophase spindle during mitosis and phycoplast- mediated cytokinesis (Pickett-Heaps, 1975; Mattox & Stewart, 1977). Prasinophytes with body scales lack such phycoplasts and usually possess persistent interzonal spindles during telophase (Rogers, Mattox & Stewart, 1981; Mattox & Stewart, 1977; • To whom all correspondence and reprint requests should be forwarded at: Section of Plant Biology, Plant Science Building, Cornell University, Ithaca, N.Y. 14853, U.S.A. 352 D. S. Domozych, K. D. Stewart and K. R. Mattox Norris & Pearson, 1975). Therefore, the intermediate position of Tetraselmis makes it crucial to our understanding of green algal phylogeny, in particular the evolution of the Chlorophyceae from the Prasinophyceae. It has been proposed that the early-collapsing telophase spindle and the phycoplast originated in conjunction with the unique evolution of walls from scales in flagellates very much like Tetraselmis (Mattox & Stewart, 1977). According to this hypothesis, the presence of a rigid wall prevents cell elongation during mitosis and spindle elongation (as normally occurs in naked and scale-covered prasinophytes). The phycoplast is interpreted as a microtubular system that ensures the accurate occurrence of cyto- kinesis between daughter nuclei that do not become widely separated in flagellated cells covered by a rigid wall. From such flagellates as Tetraselmis arise the typical volvocalean flagellates and eventually the higher Chlorophyceae (see Domozych, Stewart & Mattox, 1980, for groups included). Manton & Parke (1965) originally reported that the walls of Tetraselmis developed from Golgi apparatus-derived stellate scales, which fuse extracellularly to yield the final wall-like structure. Also, early chemical analyses (Lewin, 1958; Gooday, 1971) have revealed that the wall consists of neutral and acidic polysaccharides associated with certain protein amino acids, unlike the cellulose walls reported in some higher green algae, but similar to the glycoprotein walls recently reported in volvocalean flagellates (Roberts, 1974). From these considerations, we can suppose that the evolution of the cell wall in a Tetraselmis-like organism was of paramount significance in the evolution of the Chlorophyceae. Unfortunately, a complete, detailed structural and developmental study of this cell wall is lacking. Therefore, this study of the cell wall of Tetraselmis was undertaken for the following reasons: (a) to test further the hypothesis of homology and evolutionary affinity between the Tetraselmis wall, prasinophycean scales and the walls of chlorophycean flagellates; and (b) to analyse the developmental sequence of wall formation; i.e. the mechanisms specifically involved in Golgi apparatus-wall precursor development and extracellular wall assembly. MATERIALS AND METHODS Cultures used in this study (CCAP 66/8, Tetraselmis striata; CCAP 161/5, T. impellucida; collection of Drs Luigi Provasoli and Irma Pintner, Pisa 6a, T. comolutae) were routinely maintained on LDM (Starr, 1978) liquid medium in 250 ml Erlenmeyer flasks, under a photo- regime of 16 h light/8 h dark using 1500 lux (m~* cd sr) of cool white fluorescent light at 18 ±2 deg. C. Cultures were transferred to fresh medium every 14 days. Partial synchrony (50 %) of cell division was obtained in cultures after several transfers, usually occurring at the 3-h mark of the dark cycle. To obtain higher synchrony (80-90 %), o-i ml of 10-day-old liquid culture was aseptically spread over the solid surface of 1-3 % agar/LDM medium contained in plastic Petri dishes. After 12 days of growth (in conditions as above), single colonies were aseptically isolated with a Teflon needle and placed in flasks in fresh LDM liquid and main- tained as above. Observation of synchrony was performed using a Zeiss Nomarski optics light microscope and growth stages were monitored at 670 nm and 750 run on a Perkin-Elmer 770 spectrophotometer (1 cm path). Cultures of Pyramimonas inconstans (gift from Dr Luigi Provasoli) and Haematococcus capensis (UTEX-1022) were maintained on enriched ES (Starr, 1978) and modified WHV-o:i (Domozych, Mattox & Stewart, 1981), respectively, under the above conditions. Specimens were prepared for transmission electron microscopy (TEM) examination in the Wall of Tetraselmis: structure and development 353 following manner: log-phase cultures were harvested after 3 h in the dark cycle and centrifuged at 2000 rev./min for 5 min on an International clinical centrifuge (model CL). The resultant supernatant was discarded and the loose pellet was resuspended in 1 ml of fresh LDM medium. To this was added 1 ml of 0-9 % glutaraldehyde (Taab) in o-oi M-cacodylate buffer (pH 7-8). The cells were left in the fixative for 40 min at room temperature, washed in cacodylate buffer (3 times-for 20 min each) and postfixed for 2 h at 15 °C in the dark in 1-2 % OsO4 in o-oi % cacodylate buffer (pH 7'8). The cells were then washed as above, dehydrated through a 10 %- increment acetone series and embedded in Spurr's epoxy resin. Sections were cut on a Sorvall Porter-Blum MT-i ultramicrotome, using a diamond knife. Sections were stained with 1 % uranyl acetate/o-s % lead citrate (5 min each), thoroughly washed with water and observed on an HS-9 Hitachi TEM. To highlight the acidic polysaccharide nature of the large cell wall of T. convolutae, cells were treated as above except for postfixation in 0-5 % ruthenium red/i % OsO4 in o-oi cacodylate buffer (pH 78). Fragment analysis of the T. ttriata wall, Pyramimonas scales and Haematococcus walls were performed as follows: actively growing (log-phase) cells were centrifuged as above and the resultant pellet was resuspended in 3 ml of fresh LDM medium (or cacodylate buffer for the Pyramimonas and Haematococcus). The suspension was sonicated to cavitation for 30 s on a Branson sonifier (60 W). The fragment-containing solution was then centrifuged at 2000 g as above, and the resultant milky-white supernatant was collected and recentrifuged at 10 000 g for 20 min on a Sorvall RC-5 superspeed centrifuge. The resultant pellet was washed and recentri- fuged (3 times) in a 0-05 M-Tris-HCl buffer (pH 8-o), then resuspended in 1 ml of the Tris buffer. Drops of this suspension were placed on Formvar-coated, 150-mesh copper grids. After 5 min, the excess drop was blotted off with filter paper and the grids were stained for TEM as above. Drops of the suspension were also recentrifuged (10000 g as above) and fixed for TEM as above. For shadow-cast specimens, specifically of developing walls of T. striata, actively growing cells were collected at 3 h after the onset of the dark cycle and centrifuged at 2000 rev./min as above. The resultant pellet was then macerated gently with a glass stirring rod and the resultant suspension was placed dropwise on Formvar-coated, 150-mesh copper grids. The grids were then blotted dry and washed in a continuous stream of 0-08 M-Tris-HCl (pH 8-2) buffer. Shadow-cast specimens were observed on the TEM as described above. RESULTS AND