J. Cell Set. H 213-219 (1986) 213Printed in Great Britain © The Company of Biologists Limited 1986

THREE-DIMENSIONAL RECONSTRUCTION OF THECHONDRIOME OF THE UNICELLULAR RED ALGARHODELLA RETICULATA

SHARON BROADWATER AND JOE SCOTTDepartment of Biology, College of William and Mary, Williamsburg, Virginia 23185, USA

SUMMARYThree cells of the unicellular red alga Rhodella reticulata were serially sectioned and photo-

graphed in a transmission electron microscope in order to analyse the organization of themitochondrial system, or chondriome, which, on the basis of cursory examination, appeared toconsist of an interconnected network of one to a few organelles. The chondriome of all three cellswas traced and superimposed on acetate paper and a three-dimensional model using balsa wood wasconstructed of one cell. The chondriome was found to consist primarily of one large, anastomosingmitochondrion located principally at the cell periphery. In addition, it appears that some cells cancontain a few small mitochondria that are not connected to the main body of the chondriome. Thisis the first study to reveal the three-dimensional nature of the chondriome in a red alga.

INTRODUCTION

Studies leading to our present concept of the chondriome have been reviewed byWare & LoPresti (1975) and Pellegrini (1980). In general, investigations haverevealed the great plasticity of mitochondria and have shown that the number andmorphology of the organelle appear to be quite variable from one cell type to another.In addition, it has been shown that the number of mitochondria per cell is oftenmuch less than expected on the basis of examination of electron micrographs.Although a number of phylogenetically diverse organisms have been studied, Beech& Wetherbee (1984) have indicated a paucity of work on the chondriomes in algaethat do not contain chlorophyll b. To our knowledge there has been no detailed studyof the chondriome of any red algal cell. The present work reports some of the resultsof studies on the shape and number of mitochondria in a unicellular, olive-coloured,coccoid red alga described as a new species of the genus Rhodella (Deason et al.1983).

MATERIALS AND METHODS

Cultures of Rhodella reticulata were obtained from the University of Alabama (courtesy ofTemd Deason and Gary Butler), were grown in a 1:1 (v/v) mixture of von Stosch enriched seawater (von Stosch, 1964) and RILA Marine Mix (RILA Products, Teaneck, NJ) and maintainedat 20°C on a 16:8, light:dark, photoregime using cool-white fluorescence bulbs (6—12/iEinsteinm~zs~ ' ) . Cells were prepared for electron microscopy according to procedures used previously in

Key words: chondriome, mitochondria, red algae, Rhodophyta, Rhodella, three-dimensionalreconstruction.

214 S. Broadwater andjf. Scott

this laboratory (Schornstein & Scott, 1982) and were serially sectioned, stained with lead citrateand photographed with a Zeiss EM 9S-2 electron microscope.

Profiles of mitochondria of three cells at a magnification of 60000 were traced directly from aphotographic enlarger onto acetate paper and one of the three cells was traced onto 1/4 inch balsawood as well. The images traced onto acetate paper were superimposed to ensure steric correlation.Errors were due to differences in section thickness (average = 100 nm), tangentially cut profiles andan inability to obtain constant triangulation loci. Various organelles (nucleus, pyrenoid, chloroplastlobes, etc.) were used in aligning sections, but all of the above change somewhat with each sectionand are, therefore, inferior to outside triangulation points, which were not available. The balsawood was cut with a band saw and the profiles were aligned using the acetate sheets prior toconstruction of the model.

RESULTS

Fig. 1 is a section through the central region of a typical cell of R. reticulata.The cells are predominantly uninucleate with the nucleus occupying an eccentricposition. The bulk of the cell is taken up by the plastidome, which consists of a singlechloroplast whose peripheral lobes are connected by narrow isthmuses to a large,central pyrenoid (see Deason et al. 1983, for a detailed ultrastructural description).As seen with the electron microscope, mitochondrial profiles are generally found atthe outer regions of the cell, although occasional branches or small segments maysometimes be found in the cell interior. In many of our fixations the mitochondrialprofiles appeared extremely osmiophilic (Figs 1,2). This particular feature made thetask of recogni2ing and following the mitochondrial network relatively easy.R. reticulata cells vary in si2e from 8 to 30 /im in diameter, with most ranging from8 to 15 /im. Three smaller cells were serially sectioned, resulting in an average of110 sections per cell. In all cases, the greater portion of the chondriome wascomposed of a single, filamentous, highly branched mitochondrion located near thecell periphery. In the reconstructed chondriome there were two small, oval mito-chondria (Figs 3, 4), one measuring 0-3/im X 1-0/im and the other measuring0-5/im X 1-4/im. One of the traced cells had no 'extra' mitochondria; the other hadonly one small mitochondrial segment that was not attached to the rest of thechondriome. In all cases, the small mitochondrial 'pieces' were found near the centralregion of the cell. In the reconstructed chondriome the pieces were located near thenucleus (Figs 1, 3, 4). In one case the orientation of the piece was such that had itcontinued in length, it would have joined with a segment of the main mitochondrionthat extended towards it.

Fig. 1. Medial section through Rhodella reticulata cell used in chondriome recon-struction (shown in Figs 3, 4). The mitochondrial profiles could be mistaken for anumber of single organelles that are either dividing or fusing. The arrowhead denotes aprofile of one of the two small mitochondria that are separate from the primarymitochondrion. The approximate position of this section in the model is marked by thesingle arrowheads in Fig. 4. p, pyrenoid; nu, nucleus. X14 500.

Fig. 2. Different section through same cell as shown in Fig. 1. The reticulate nature ofthe chondriome (arrowheads) is more obvious in this view. The approximate position ofthis section in the model is marked by the double arrowheads in Fig. 4. X14500.

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Reconstruction of Rhodella chondriome 217

Figs 1 and 2 are sections from the cell used as a model for the reconstruction andare correlated with their position on the reconstruction (Figs 3, 4). The mito-chondrion can vary greatly in width, some regions being as small as 50 nm wide. Insome sections, the predominantly closed-loop, reticulate nature of the chondriomeis fairly obvious (Figs 1, 2); in others (not shown), the profiles appear to bear littlerelation to each other.

DISCUSSION

This study and reconstruction represent the first of their kind in a red alga,although primarily single, large, branched mitochondria have been shown in manyother cell types: the yeasts Saccharomyces (Hoffman & Avers, 1973; Stevens, 1977)andPityrosporum (Keddie & Brarjas, 1969, 1972), the algae Chlamydomonas (Grobe& Arnold, 1975), Chlorella (Atkinson et al. 1974; Dempsey et al. 1980), Euglena(Pellegrini, 1980; Pellegrini & Pellegrini, 1976), Pleurochrysis (Beech & Wetherbee,1984) and Polytoma (Gaffal & Kreutzer, 1977), the protozoa Blastocrithidia andTrypanosoma (Paulin, 1975), the flagellate Polytomella (Burton & Moore, 1974) andthe phycomycete Olpidiuim (Lange & Olson, 1976).

The stage of the cell cycle may have an effect on mitochondrial shape and number.Several studies indicate that single, branched mitochondria occur during the growthphase of the cell but several to many ovoid and, or, less-branched mitochondria aretypical of dividing cells and young zygotes (Bromberg, 1974; Calvayrac et al. 1972;Gaffal & Kreutzer, 1977; Grimes et al. 1974; Grobe & Arnold, 1977; Stevens, 1977,1981; Tanaka et al. 1985). Not all cells, however, seem to display gross morpho-logical changes corresponding to the cell cycle (Koukl et al. 1977; Pellegrini, 1980),and the presence or absence of such changes does not appear to have phylogeneticsignificance. Along the same lines, cristae morphology, which is considered by manyto be of great taxonomic importance, appears to bear no relationship to the numberand shape of mitochondria in a cell (Beech & Wetherbee, 1984).

The chondriome of R. reticulata is primarily a single anastomosing mitochon-drion. The indications are that its shape is highly mutable and that segments canbreak off and re-fuse. The latter is inferred from finding a variable number of smallmitochondria in the three serially sectioned and analysed cells, the very narrowwidths seen in several regions of the mitochondrion and from the orientation of oneof the small extra mitochondria that suggested its breakage from the much larger,predominant one. Observations of living Euglena gracilis (Leedale & Buetow, 1970)and serial reconstruction of this species (Pellegrini, 1980) support this view. It maybe only chance that all three of the chondriomes of R. reticulata cells sectioned in this

Fig. 3. Photograph of reconstructed chondriome, dorsal view. In this view, each tierreveals the mitochondrial profiles seen in the respective serial section. The two small,ovoid mitochondria (arrowheads) can be seen. They help to define the region where thenucleus is located (asterisk).Fig. 4. Photograph of reconstructed chondriome, lateral view. The view of the modelhere is at right angles to the plane of sectioning. The single arrowheads indicates theapproximate position of the section shown in Fig. 1, and the double arrowheads indicatethe approximate position of the section shown in Fig. 2.

218 S. Bwadwater and jf. Scott

study were primarily composed of a single mitochondrion. One might expect to findone to three large, branched mitochondria with only a few small ovoid ones. Thispossibility is inferred from the apparent structural plasticity of the chondriome andfrom results of previous studies on other unicells. All of the R. reticulata cellssectioned appeared to be in interphase. No mitotic cells were found, so a comparisonwith cells in other stages of the cell cycle could not be made.

There have probably been more three-dimensional models made of mitochondriathan of all the other organelles combined, and the reconstructions have covered awide variety of organisms. The wealth of information obtained provides an accurateaccount of a cell's chondriome and seems to indicate that the morphology andnumber of mitochondria may be a useful indicator of stages of the cell cycle, but isunfortunately of relatively little use in indicating phylogenetic and taxonomicrelationships.

We thank Jewel Thomas and Bill Saunders for their assistance with this study. We also thankTemd Deason and Gary Butler for providing us with cultures of Rhodella reticulata andinformation on proper culture conditions. Financial support was provided by NSF grant BSR83-07714 to J. Scott.

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(Received 21 January 1986 -Accepted 9 April 1986)