J. Cell Sci. 75, 215-224 (1985)
215
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THYLAKOID FORMATION FROM COILED LAMELLAR
BODIES DURING CARPOSPOROGENESIS IN
FAUCHEOCOLAX ATTENUATA SETCH. (RHODOPHYTA,
RHODYMENIALES)
STYLIANOS G. DELIVOPOULOS* AND PAUL KUGRENS
Department ofBotany, Colorado State University, Fort Collins, Colorado 80523, U.SA.
SUMMARY
Chloroplast development during carposporogenesis in the parasitic red alga Faucheocolax
attenuata Setch. was studied by electron microscopy. Proplastids are usually found in the
peripheral cytoplasm of young carpospores and are characterized by the presence of portions of a
peripheral thylakoid and coiled lamellar bodies that range in size up to 0-5 /im. One type of coiled
lamellar body occurs in the peripheral region of the proplastid and is continuous with the peripheral
thylakoid, while the other type is found in the central portion of the stroma. These coiled lamellae
separate and expand, adding membranes to both thylakoid systems, thereby functioning as
thylakoid-forming bodies. As each coiled lamella unravels, it forms an undulated doublemembraned structure having the same width as a thylakoid. After substantial expansion, the
developing thylakoids begin to straighten and assume a parallel orientation to each other, thus
becoming mature thylakoids. Small coiled lamellae often persist in mature carpospore chloroplasts,
and are utilized in additional thylakoid formation during carpospore germination.
INTRODUCTION
Thylakoid formation in chloroplasts of several plant groups is often preceded by
some kind of thylakoid precursor from which photosynthetic lamellae originate
(Wellburn, 1982). For example, in some caulerpean green algae there is a thylakoidforming body with a coiled lamellar structure (Borowitzka & Larkum, 1974;
Borowitzka, 1976; Calvert & Dawes, 1976). Euglena gracilis Klebs var. bacillaris
Cori (Klein, Schiff & Holowinsky, 1972; Osafune, Klein & Schiff, 1980) and the
chrysophyte Ochromonas danica Pringsheim (Smith-Johannsen & Gibbs, 1972) have
a thylakoid-forming body that is a reticulate system of tubular membranes with
somewhat irregular arrangements.
Only one instance of a specialized thylakoid-forming structure has been reported
for red algae. (Borowitzka, 1978). This structure was found in developing carpospores
of the coralline red alga Lithothrix aspergillum Gray and consists of an irregular,
branched tubular membrane system, made up of aggregated membranous tubules
that are 30-35 nm in diameter, and slightly resembles Euglena's thylakoid-forming
•Present address for correspondence: Botanical Institute, University of Thessaloniki, 54006
Thessaloniki, Greece.
Key words: Faucheocolax, red alga, thylakoid development, ultrastructure.
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S. G. Delivopoulos and P. Kugrens
body (Borowitzka, 1978). The internal thylakoid system is thought to arise from this
structure, although the evidence may be inconclusive (Brawley & Wetherbee, 1981).
In contrast, Batrachospermum moniliforme Roth lacks any distinct thylakoid-forming
centres or bodies (Brown & Weier, 1968). Instead, the photosynthetic lamellae
originate as imaginations of the peripheral thylakoid. Since Batrachospermum is a
primitive member of the Rhodophyta (order Nemaliales) and Uthothrix belongs to a
more advanced order, the Cryptonemiales, Borowitzka (1978) suggested that these
differences in thylakoid development may be phylogenetically significant.
While investigating carpospore development in the parasitic red alga Faucheocolax
attenuata Setch. (Delivopoulos & Kugrens, 1984), we also noted a progressive
increase in thylakoids as carpospores matured. Closer scrutiny revealed a coiled
membrane structure, which apparently gives rise to both peripheral and internal
thylakoids. Since the significance of this membranous body has not been elucidated
previously, its structure and probable involvement in thylakoid formation are
described in this paper.
MATERIALS AND METHODS
Thalli of F. attenuata bearing cystocarps of varying sizes were obtained from dredged material
collected near Smith Island and Salmon Bank, Washington. The thalli were excised from Fauchea
and fixed immediately for light and electron microscopy according to previously described
procedures (Kugrens, 1974). Thin sections were examined with an AEI-6B electron microscope.
OBSERVATIONS
F. attenuata is a parasitic, subtidal red alga, which infects several species of
Fauchea. The slightly pink thalli may be up to 2 mm in size, consisting of short, wartlike branches emanating from a central cushion of cells. Vegetative cells contain
typical red algal chloroplasts; however, developing carpospores possess plastids in a
variety of developmental stages and, generally, these remain less developed than those
Abbreviations used in figures: c, chloroplast; ce, chloroplast envelope; ccl, central coiled
lamellae; cw, cell wall; d, dictyosome; dit, developing internal thylakoid; er, endoplasmic
reticulum;/s, floridean starch; it, internal thylakoid; m, mitochondrion; n, nucleus; nu,
nucleolus; og, osmiophilic granule; p, proplastid; pel, peripheral coiled lamellae; pt,
peripheral thylakoid; v, vacuole.
Fig. 1. Young carpospore containing proplastids with coiled lamellar bodies (arrows).
X22500.
Fig. 2. Proplastid with portions of the peripheral thylakoid and a large central coiled
lamellar body. X69 900.
Fig. 3. Proplastid showing connections between the peripheral coiled lamellar body and
the peripheral thylakoid. X50250.
Fig. 4. Proplastid showing connections between the peripheral coiled lamellae and smaller
central coiled lamellae, perhaps indicating a budding process. X64 950.
Fig. 5. Proplastid exhibiting an expansion and separation of the peripheral coiled lamellae
and contributing to peripheral thylakoid formation. Note the connection between the
peripheral thylakoid and a peripheral coiled lamella (arrow). X72000.
Faucheocolax thylakoid formation
Figs 1-5
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S.G. Delivopoulos and P. Kugrens
of vegetative cells. It is within these developing spores that the full range of thylakoid
development, from a proplastid to a chloroplast, is expressed.
Young carpospores (Fig. 1) are small, undifferentiated cells, containing a large
nucleus with a prominent nucleolus, a few floridean starch grains, some endoplasmic
reticulum and proplastids, which are usually found in the peripheral cytoplasm. The
small proplastids (0-3—0-8/xm) may vary slightly in structure; some having only
portions of a peripheral thylakoid, others possessing a complete peripheral thylakoid,
while a third type has some portions of the internal thylakoid system. The latter is the
rarest and may indicate some precocious development. One feature common to all
early proplastids, however, is the presence of coiled lamellar bodies (Fig. 1). The
coiled lamellae, during early stages of plastid development, remain tightly appressed
and appear to be of different sizes, due to the plane of sectioning. Often the integrity of
individual membranes in the coiled lamellar bodies cannot be resolved, because they
are tightly compacted.
There appear to be two types of coiled lamellar bodies, as determined by their
position in the proplastid. One type is found in the inner portion of the stroma (Fig. 2)
and is considered the central coiled lamellar body, whereas the second type, the
peripheral coiled lamellar body, is continuous with the peripheral thylakoid (Fig. 3).
In early proplastids, both types can be fairly large, ranging in size up to 0-5 (xm, and
they occupy a significant volume of the proplastid. Connections between the
peripheral and central coiled lamellae were sometimes observed (Fig. 4), giving the
impression that the larger one may bud to form smaller lamellar bodies. Furthermore,
the coiled lamellae and the future undulated thylakoids in this region are generally
surrounded by an electron-transparent area, devoid of plastid ribosomes (Figs 4, 6, 7,
8,9).
The peripheral coiled lamellae appear to contribute thylakoid membranes to the
developing peripheral thylakoid by expansion (Fig. 5), as the proplastids undergo
enlargement during this development. It is possible that the entire peripheral
thylakoid originates from these coiled lamellae.
The central coiled lamellae also appear to expand and separate (Fig. 6), forming
expanded lamellae, which we interpret as developing internal thylakoids, because of
the similarity in the membrane width in both lamellae (20 nm). Fig. 6 shows
Fig. 6. Plastid containing expanding coiled lamellae, apparently forming portions of the
developing internal thylakoid system. X106600.
Fig. 7. Proplastid with several central coiled lamellar bodies exhibiting unravelling to
form internal thylakoids. X 54 000.
Fig. 8. Later stage of internal thylakoid formation showing extensive uncoiling and
connections between an expanded thylakoid and a developing internal thylakoid (arrow).
Note the chloroplast bud (double arrow). X49950.
Fig. 9. Plastid with undulated but extended internal thylakoids. X40 000.
Fig. 10. Late stage of chloroplast development in which the plastid contains curved,
developing internal thylakoids, osmiophilic granules and fully formed internal thylakoids.
A peripheral coiled lamellar body is still present in the plastid. The arrow indicates a
plastid DNA region. X49950.
Faucheocolax thylakoid formation
Figs 6-10
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5. G. Delivopoulos and P. Kugrens
expanding coiled lamellae from both surface and side views. One of the developing
internal thylakoids appears to be spreading out and beginning to form a flat thylakoid.
In any given section there are several central coiled lamellar groups per proplastid and
these may be in various stages of expansion (Figs 7, 8). It is possible that these smaller
groups are interconnected. As the thylakoids proceed to develop, several connections
between developing thylakoids and unexpanded coiled lamellae remain evident (Fig.
8). Initially, these differentiating internal thylakoids are undulating and randomly
oriented (Figs 8, 9), lacking the typical, parallel arrangement found in mature
chloroplasts. However, the thylakoids eventually begin to orient, more or less parallel
to each other (Figs 9, 10), as they continue to expand and separate. In Fig. 10 this
orientation is apparent in a portion of the internal thylakoids, whereas the remainder
are still undulating or curved. This final stage of thylakoid unfurling involves a close
association with osmiophilic granules or plastoglobuli (Fig. 10). The coiled lamellae
may not be used up entirely (Fig. 10); perhaps they may act as reserve membranes to
be used either during future thylakoid formation following chloroplast division, since
Fig. 11. Mature carpospore chloroplasts. Note the uneven distribution of internal
thylakoids. X36150.
Fig. 12. Mature vegetative cell with mature chloroplasts. Note the absence of coiled
lamellar bodies in the chloroplasts and the parallel orientation of thylakoids. X21 150.
Faucheocolax thylakoid formation
221
these chloroplasts are capable of dividing or budding (Fig. 8) during any developmental stage, or forming more thylakoids during carpospore germination.
Mature carpospore chloroplasts (Fig. 11) have lower thylakoid numbers than
vegetative cell chloroplasts (Fig. 12), because they do not fully mature in carpospores.
While there is a complete peripheral thylakoid, which follows the contour of the
chloroplast envelope, the internal thylakoids are few in number. Generally there are
4-7 thylakoids per chloroplast, with four or five being the most common number. In
addition, the thylakoids are unevenly distributed, usually toward one side of the
chloroplast, leaving one third to half of the stroma devoid of thylakoids. In vegetative
cell chloroplasts, however, the thylakoid distribution is uniform (Fig. 12). The
significance of this difference is unknown. Furthermore, vegetative cell chloroplasts
lack any coiled membrane structures.
DISCUSSION
We examined fixed material from widely differing localities (California and
Washington) and collected in different years (1973, 1974, 1977, 1981). We used
several glutaraldehyde concentrations (2-S %) in different buffer solutions (phosphate and Na-cacodylate), dehydrated with ethanol, methanol and acetone (including
propylene oxide when it was needed as a transitional solvent), and embedded thalli in
Spurr's Embedding Medium (Spurr, 1969), Epon812and Araldite. Regardless of the
fixation procedures utilized, the same results were obtained in all cases; namely, that
proplastid coiled membranes occurred only in young carpospores.
The size of coiled lamellar bodies decreased as thylakoids increased, again implying
thylakoid formation from these membrane structures. If coiled lamellae were artifacts
produced from thylakoid degeneration, then more and larger coiled lamellar bodies
would be expected in maturing chloroplasts, which contain comparatively more
thylakoids than the proplastids. Mature vegetative cell chloroplasts, in which
thylakoid development is complete, never possessed any coiled lamellae regardless of
the fixation schedule. Mature, vegetative cell chloroplasts lack any coiled membrane
structures, since they are capable of dividing, numerous thylakoids being present.
Thus, coiled membranes are not necessary in vegetative cell chloroplasts, because
their participation occurs only during the earliest stages of thylakoid formation.
Therefore, based on the preceding logic and observations, it is highly unlikely that we
have introduced artifacts during fixation.
To date, thylakoid origin in red algal plastids has been examined critically in four
studies only (Brown & Weier, 1968; Borowitzka, 1978; Tsekos, 1982; Tripodi &
Gargiulo, 1984). In the initial study of thylakoid development in Batrachospermum,
Brown & Weier (1968) found that the internal or photosynthetic lamellae (thylakoids)
arose from invaginations of the peripheral thylakoid. Subsequently, Borowitzka
(1978) described a tubular thylakoid-forming body in Lithothrix carpospores, which
apparently gave rise to the internal thylakoid system. Tsekos (1982) depicted coiled
membranes in proplastids of Gigartina teedii (Roth) Lamour, which appeared to be
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S. G. Delivopoulos and P. Kugrens
similar to those inFaucheocolax. More recently, Tripodi & Gargiulo (1984) reported
on thylakoid formation from the fusion of flattened vesicles produced in the
proplastids of Nitophyllumpunctatum (Stackh.) Grev.
Since Batrachospermum (order Nemaliales) is a relatively primitive member
of the Florideophyceae and Lithothrix belongs to a more advanced order, the
Cryptonemiales, Borowitzka (1978) postulated that the presence of some kind of
thylakoid body may have evolutionary implications. Borowitzka's (1978) prediction
appears to have been correct. Although ours is not the first report of coiled membranes
in red algal plastids (Cole & Sheath, 1980; Tsekos, 1982; Tsekos & Schnepf, 1983;
Tripodi & Gargiulo, 1984), it is the first to interpret the significance and function of
these structures during thylakoid formation. Some of these authors (Tsekos, 1982;
Tsekos & Schnepf, 1983; Tripodi & Gargiulo, 1984) apparently failed to realize the
importance of these coiled lamellae and their function as thylakoid-forming
structures. For instance, Tsekos & Schnepf (1983) observed coiled membranes in
auxiliary cell plastids and mitochondria of G. teedii. These coiled membranes were
identical to those observed in Faucheocolax and one of their figures (fig. 6) even
shows an unravelling of the coiled lamellae. Recently, Tripodi & Gargiulo (1984)
observed coiled membrane bodies in developing plastids from young carpospores of
N. punctatum, similar to the coiled lamellar bodies in developing plastids from young
carpospores of Faucheocolax. Connections between those coiled membrane bodies
and the developing thylakoids were evident in many cases (Tripodi & Gargiulo, 1984,
figs 3, 4, 6). In our opinion, figs 3 and 6 in their paper probably depict unravelling
coiled membrane bodies, thus illustrating the process of thylakoid formation from
these membrane structures. Consequently, thylakoids in Faucheocolax and,
presumably, G. teedii (Tsekos, 1982) and N. punctatum (Tripodi & Gargiulo, 1984)
exist as prepackaged units in coiled lamellae, which upon separation and expansion
will form the respective thylakoids.
The thylakoid-forming structures of Faucheocolax differ significantly from those
found in Lithothrix. The Lithothrix thylakoid-forming body is tubular, with a
reticulate substructure, situated in the central stroma of the proplastid and always
found in association with plastid DNA (Borowitzka, 1978). In contrast, the coiled
lamellae of Faucheocolax are found in both peripheral and central regions of the
proplastid and generally do not appear to be associated with DNA fibrils.
Furthermore, the central coiled lamellae are surrounded by electron-transparent
areas, devoid of plastid ribosomes, which might indicate DNA regions, but fibrils,
usually interpreted as DNA, were seldom seen in these areas. Rather, these electrontransparent areas may be formed by an intense unravelling of the coiled lamellae,
resulting in ribosomes being 'pushed' away from the immediate vicinity of the
developing thylakoids.
Tripodi (1980) found ring-like structures in Pterosiphonia dendroidea (Mont.)
Falk mitochondria and proplastids, and in chloroplasts of Erythrocystis montagnei
(Derb. et Sol.) Silva carpospores and tetraspores. It may be that the ring-like
structures actually represent thylakoid-forming body remnants, but this possibility
was never discussed.
Faucheocolax thylakoid formation
223
There have been several other reports of proplastids and coiled or whorled
membrane associations in algae (Borowitzka & Larkum, 1974; Borowitzka, 1976;
Cal vert & D awes, 1976; Osaiuneetal. 1980), but only one of these is directly involved
in thylakoid formation. For example, certain members of the green algal Order
Caulerpales have a highly structured and persistent coiled thylakoid-forming body
(Borowitzka & Larkum, 1974; Borowitzka, 1976; Calvert & Dawes, 1976), which also
contains some tubular elements. The intimate association of this thylakoid-forming
body and the earliest formed thylakoids suggests that these coiled lamellae are
involved in thylakoid formation. Coiled lamellae of Faucheocolax are less structured,
smaller, more tightly compressed, and are usually completely incorporated into
thylakoids or considerably reduced in size and are not localized toward one end of the
plastid.
Membrane whorls also were found associated with proplastids in dark-grown
Euglena cells (Osafune et al. 1980). In this alga, however, the membrane whorls are
attached to the outer proplastid membrane, often in close proximity to thylakoidforming bodies and crescent-shaped cytoplasmic microbodies. These membrane
whorls apparently participate in the formation of the prolamellar body; but their
subsequent involvement in thylakoid formation remains obscure.
Borowitzka's (1978) assumption that the presence of some kind of thylakoidforming body in red algae is indicative of a higher phylogenetic position, may be
correct. Structures that appear to function as thylakoid-forming bodies have now been
reported for the Cryptonemiales (Uthothrix) (Borowitzka, 1978), the Rhodymeniales
(Faucheocolax), the Gigartinales (Gigartina) (Tsekos, 1982) and the Ceramiales
(Nitophyllum) (Tripodi & Gargiulo, 1984). It is, however, premature to make
definite conclusions based on the observations from four species, and additional
studies are warranted to confirm or refute Borowitzka's (1978) hypothesis.
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(Received 17 July 1984 -Accepted 2 November 1984)