J. Cell Sci. 50, 149-164 (1981)
Printed in Great Britain © Company of Biologists Limited 1981
149
COMPARATIVE ULTRASTRUCTURE OF
EYESPOT MEMBRANES IN GAMETES
AND ZOOSPORES OF THE GREEN ALGA
ULVA LACTUCA (ULVALES)
H. ROBENEK1 AND M.MELKONIAN"
1
Medizinische Cytobiologie and 'Botanisches Institut,
Federal Republic of Germany
Universitat MUnster, Milnster,
SUMMARY
Eyespot membranes in zoospores, and male and female gametes of the green alga Ulva
lactuca, were studied comparatively by the freeze—fracture technique. The plasmalemma and
the outer chloroplast envelope membrane overlying the eyespot lipid globules are specialized
in all 3 types of reproductive cells. In the eyespot region the protoplasmic face (PF) of the
outer chloroplast envelope membrane contains significantly more intramembraneous particles
(IMP) compared to membrane areas outside the eyespot: in female gametes there are 2-5 times
more IMP//*m s , in zoospores 3 and in male gametes about 4. Small size-class IMP (4-6 ran
diameter) are particularly abundant on both fracture faces of the outer chloroplast membrane,
but size-class distribution is not significantly different between membrane areas inside and
outside the eyespot region. The total number of IMP/eyespot on the P F of the outer chloroplast membrane was calculated to be 4900 in male gametes, 5500 in female gametes and 11 200
in zoospores.
The results are discussed in accordance with the view that these membrane specializations
participate in photoreception relating to green algal phototaxis. Evidence is presented that there
is a correlation between IMP numbers per eyespot in the outer chloroplast envelope membrane
and the different phototactic behaviour of gametes compared to zoospores in Ulva.
INTRODUCTION
The characteristic photobehaviour of flagellated cells of green algae is phototaxis,
i.e. the movement of the cell is oriented with respect to the direction of the light
stimulus (see review by Feinleib, 1980). Typically a flagellated green algal cell shows
positive phototaxis at low or moderate light intensities and negative phototaxis at high
intensities (Feinleib & Curry, 1971). Factors in the chemical environment are also
known to influence phototactic orientation in green algae, e.g. ion concentrations
(Halldal, 1959). A peculiar situation with respect to phototaxis exists in those green
algal species that produce different types of motile cells with different types of phototactic orientation in the same environment. Gametes of the marine green alga Ulva
lactuca are usually positive phototactic, while zoospores exhibit a negative phototactic
response (see e.g. Melkonian, 1979). Upon gametic fusion a phototactic reversal is
initiated and the partially fused gamete pairs swim away from the light source
(Melkonian, 1980).
• To whom correspondence should be addressed at: Botanisches Institut, Westfllische
Wilhelms-UniversitSt, Schlossgarten 3, D-4400 MUnster, Federal Republic of Germany.
150
H. Robenek and M. Melkonian
Evidence is now accumulating that green algae use a 2-instant mechanism for
perceiving light (i.e. successive measurements of light intensity are performed by
1 photoreceptor as the cell changes its position in relation to the light source; see
e.g. Boscov & Feinleib, 1979). Little is still known about the structural basis for this
presumed 2-instant mechanism. Recent freeze-fracture studies performed with several
green algae have given evidence that the phototactic apparatus of green algae consists
of 2 closely linked structures. The eyespot or stigma is part of the chloroplast and
consists of one to several layers of closely packed carotenoid-containing lipid globules.
The eyespot membranes are the plasmalemma and the outer chloroplast envelope
membrane overlying the eyespot lipid globules (Melkonian & Robenek, 1980a).
These membranes were found to be specialized in that they contained a greater
number of intramembraneous particles (IMP)//im2 compared to other membrane
areas outside the eyespot region (Melkonian & Robenek, 1979, 1980a, 19806). It was
concluded that eyespot membrane specializations indicate the site of photoreceptor
location in green algal phototaxis, the eyespot acting as an auxiliary structure facilitating the precision of the phototactic orientation.
In the present study we investigated the detailed structure of eyespot membranes
in gametes and zoospores of the green alga Ulva lactuca in an attempt to relate eyespot membrane structure with the observed different phototactic behaviour of gametes
and zoospores.
MATERIALS AND METHODS
Fertile thalli of Ulva lactuca L. were collected on the rocky flats at the west cliff of the island
of Heligoland (FRG) during spring tides in March 1980. Methods for inducing gamete and
zoospore release have been described previously (Melkonian 1979, 1980). Fixation was performed by mixing equal amounts of 2 % glutaraldehyde, made up in sea-water without buffering
but with final pH adjustment (pH 80 with NaOH), with the cell suspension, giving a final
glutaraldehyde concentration of 1 %. The cells were fixed for 30 min at 20 °C. The cells were
then concentrated by low-speed centrifugation and washed extensively with sea-water. For
freeze-fracturing glutaraldehyde-prefixed cells were incubated in 30 % glycerol for approximately 2 h and the specimens were further processed as previously described (Melkonian &
Robenek, 1980a). For thin sections the cells were postfixed in 1 % OsO* (in half-strength
sea-water) for 10 min at 4 °C. Further processing was as previously described (Melkonian,
1975, 1980).
The freeze-fracture terminology adopted by Branton et al. (1975) was used to denote the
different fracture faces of membranes. Counting of intramembraneous particles was only performed on those membrane fracture faces that showed no contamination by ice crystals. For
counting prints from film, strips were projected onto a grid with an enlargement of 105000:1.
Fig. 1. Freeze-fractured zoospore of Ulva lactuca. In the eyespot area cross-fractured
eyespot lipid globules are seen. PF, protoplasmic face of the plasmalemma. EF0,
external face of the outer chloroplast envelope membrane. Arrows, elevations and
depressions on the membrane faces caused by association with a microtubular flagellar
root. /Z, cross-fractured base of flagellum.
Fig. 2. Protoplasmic face of the outer chloroplast envelope membrane (Chl-PF0) in
the boundary region between the eyespot (left) and membrane areas outside the eyespot (right). The Chl-PFO in the eyespot region contains a higher IMP density.
Eyespot membranes in Ulva
152
H. Robenek and M. Melkonian
Eyespot membranes in Ulva
153
The screen was divided into numbered squares of 2 cm length, which corresponded to a surface
area of the membrane of 0-0363 /tin1. The following numbers of randomly distributed squares
were counted for the determination of particle density:
Plasmalemma E face (EF): 121 squares in 32 different cells in female gametes, 29 squares in
9 different cells in male gametes and 24 squares in 8 different cells in zoospores.
Plasmalemma P face (PF): 74 squares in 19 different cells in female gametes, 29 squares in
9 different cells in male gametes and 24 squares in 8 different cells in zoospores.
Outer chloroplast envelope E face (Chl-EF^: 30 squares in 9 different cells in female gametes,
40 squares in 13 different cells in male gametes and 42 squares in 14 different cells in
zoospores.
Outer chloroplast envelope E face of eyespot region (Chl-EF0Ey): 29 squares in 6 different
cells in female gametes, 28 squares in 9 different cells in male gametes and 41 squares in
13 different cells in zoospores.
Outer chloroplast envelope P face (Chl-PFJ: 34 squares in 9 different cells in female gametes,
34 squares in 10 different cells in male gametes and 97 squares in 32 different cells in
zoospores.
Outer chloroplast envelope P face of eyespot region (Chl-PF0Ey): 87 squares in 24 different
cells in female gametes, 33 squares in 11 different cells in male gametes and 40 squares in
13 different cells in zoospores.
The particles touching the upper and right edges of the squares were counted, while those
lying on the lower and left edges were discarded.
Particle sizes were determined from photographs enlarged to x 153000 and viewed through
an 8 x objective lens equipped with micrometer grating. The particle width was calculated by
measuring the width of the shadowed half of the particle at the boundary between the opaque
half of the particle and its shadow. When the boundary at this point was irregular or difficult
to determine because of high particle density, nevertheless a minimum width was always
taken.
Surface areas of eyespots were evaluated with a morphometric counter (MOP KM II,
Kontron). The following numbers of cells were used for determination of eyespot surface
area: female gametes (« = 58), male gametes (ft = 59) and zoospores (n = 43).
RESULTS
Eyespot position in the cell
Freeze-fractured gametes and zoospores of Ulva lactuea reveal large areas of membrane faces representing the plasmalemma and the outer chloroplast envelope membrane (e.g. Figs. 1, 3). In the eyespot region the fracture plane usually leaves the
interior of the plasmalemma or the outer chloroplast envelope membrane and
continues in a non-membraneous compartment exhibiting hexagonally arranged
lipid globules (Figs. 1,3). Due to this cleavage behaviour of eyespot membranes, it is
possible to evaluate the location of the eyespot with respect to cell symmetry and the
plane of beat of the flagella. The eyespot in gametes and zoospores occupies a posterior
Fig. 3. Freeze-fractured female gamete. PF, protoplasmic face of the plasmalemma.
In the eyespot area cross-fractured eyespot lipid globules are visible. Arrow, path of
a microtubular flagellar root, ms, limits of the mating structure of female gametes.
fl, flagellum. Inset (upper right): eyespot area with cross-fractured lipid globules of
male gametes. Inset (lower left): boundary between PF of the plasmalemma outside
the eyespot region (on the left) and EF of the outer chloroplast envelope membrane
in the eyespot region (on the right); arrows, areas of the PF at the edges of the eyespot
where IMP density is greatly increased.
6
CKL
50
H. Robenek and M. Melkonian
1 /am
Eyespot membranes in Ulva
155
position in the cell (Figs. 1, 3). With respect to the plane of beat of the flagella its
position is strictly defined. In zoospores the eyespot lies exactly in the plane of beat
of one of the two opposite pairs offlagella(Fig. 1). The definite position of the eyespot
with respect to theflagellarapparatus is also evident from close association of a microtubular flagellar root (a 4-stranded root of the 4-2-4-2 cruciate flagellar root system;
see also Melkonian, 1979) with one side of the eyespot (Fig. 1). The path of this root
can be traced along elevations or depressions of plasmalemma or outer chloroplast
envelope membrane adjacent to the root (Fig. 1). In gametes the position of the eyespot with respect to the flagellar apparatus is again defined, the eyespot is about 1-5
to 2 /im away from the plane of beat of the 2flagella.Again the eyespot is associated
with a 4-stranded microtubular flagellar root (Fig. 3). Because of the presence of
a single mating type structure in the gametes (see Melkonian, 1980), it is possible to
determine eyespot position even more accurately. The eyespot of gametes is always
associated with that 4-stranded flagellar root, which is adjacent to the mating type
structure, i.e. mating type structure and eyespot are located on the same side of the
cell if one bisects a gamete along the plane of beat of the 2flagella(Fig. 3).
Eyespot ultrastructure in thin sections
The general ultrastructure of zoospores and gametes of Ulva lactuca has been
described previously (Micalef & Gayral, 1972; Melkonian, 1979, 1980). In gametes as
well as zoospores the eyespot consists of a single layer of lipid globules, which show
hexagonal close-packing when sectioned tangentially (Fig. 4). Individual eyespot
globules measure around 90 nm in all 3 cell types. The layer of lipid globules is located
as in most other green algae directly below the 2 chloroplast envelope membranes
(Fig. 5). In the eyespot region the outer chloroplast envelope membrane is closely
linked to the plasmalemma (Fig. 5). The distance between both membranes in the
eyespot area is rather constant and has been determined to be about 14 nm in all 3 cell
types. The eyespot lipid globules in our preparations are only slightly osmiophilic
because of the short OsO*fixationtimes (10 min) (Fig. 5). With this fixation schedule
however electron-dense material is retained in the space between the outer chloroplast
envelope membrane and the plasmalemma in the eyespot region (Fig. 5). This
material is extracted after prolonged OsO4 fixation. It is noticeable that zoospores and
male gametes exhibit a rather dense staining in this area (e.g. Fig. 5), while female
gametes stain only lightly.
Fig. 4. Tangential section through the eyespot area of female gamete. Hexagonal
arrangement of eyespot lipid globules is seen. Arrows, microtubules approaching the
eyespot area.
Fig. 5. Cross-section through the eyespot of male gametes. /, eyespot lipid globules
in 'negative contrast'. Arrows, cross-sections of microtubules adjacent to the eyespot.
c, chloroplast.
6-2
156
H. Robenek and M. Melkonian
Comparative freeze-fractxtre analysis of eyespot membranes
The special cleavage behaviour of eyespot membranes (see above) made it possible
to determine the eyespot area in the 3 different cell types quite accurately. The eyespot in male gametes measures 1-09 + 0-24 fim2 (standard deviation), in female
gametes 1-55 ± 0-34/im2 and in zoospores 2-6 + 0-45/^m2. The overall shape of the
different eyespots is similar, they are all oval-shaped (e.g. Figs. 1, 3 and right inset in
Fig. 3). The long axis of the eyespot corresponds to the long axis of the cell. The eyespot of zoospores is the most elongated (Fig. 1), while the eyespot of male gametes
exhibits rather similar long and short axes (Fig. 3, right inset). Direct counts of total
numbers of eyespot lipid globules/eyespot give about 80 globules in male gametes,
130 globules in female gametes and 200 globules in zoospores.
IMP numbers/Tim1
IVlCIIlUraJlC
face
PF
EF
Chl-PF0
Chl-PFoEy
Chl-EF0
Chl-EFJEy
Female gamete
Male gamete
Zoospore
3i3°±5i4
6461182
I49O ± 342
35601635
1601 ±373
123H237
3698±3S7
36711317
8641174
791 ±201
1021±176
4469 ±57o
9891187
10121240
14021322
4305 ±637
10211314
1098 1 274
Fig. 6. Comparison of IMP density on different membrane fracture faces between
female gametes, male gametes and zoospores. For each IMP number//*mJ the
standard deviation is given. Further details are described in Materials and methods.
The plasmalemma does not usually fracture in the eyespot region (e.g. see Figs. 1,3).
Sometimes it is possible, however, to reveal small areas of the plasmalemma at the
edges of the eyespot that show a high density of intramembraneous particles (IMP)
(Fig. 3, left inset). A detailed analysis of IMP density and sizes is precluded by the
infrequent occurrence of these fractures.
Fig. 6 summarizes the results obtained from analysis of IMP density on different
membrane faces in all 3 cell types. The PF of the outer chloroplast envelope membrane
in all 3 cell types contains significantly more IMP//mi 2 in the eyespot region compared
to regions outside the eyespot (see also Figs. 2, 7). In female gametes there are 2-4
times more IMP/^m 2 , in male gametes 4-4 and in zoospores 3-1 times more IMP//tm 2
compared to membrane areas outside the eyespot. The EF of the outer chloroplast
envelope membrane does not contain more IMP//tm 2 in the eyespot region compared
to regions outside the eyespot (see Fig. 6).
Particle size analysis performed for both fracture faces of the outer chloroplast
envelope membrane in all 3 cell types is shown diagrammatically in Figs. 8 and 9.
Fig. 7. Freeze-fracture of the protoplasmic face of the outer chloroplast envelope
membrane (PF0) in the eyespot region and in regions outside the eyespot. EF, external
face of the plasmalemma.
Eyespot membranes in Ulva
157
H. Robenek and M. Melkonian
Female gamete
473
Chl-PF0
40 i- Chl-PFoEy
559
30
20
10
8
12
16
8
12
16
Male gamete
40 r Chl-PF0Ey
355
Chl-PF,
374
30
20
10
8
40rOil-PFoEy
12
502
16
8
Zoospore
Chl-PFO
12
16
395
o
520
I
12
16
4
Particle diameter (nm)
Fig. 8
8
12
16
Eyespot membranes in Ulva
159
The majority of IMP on both fracture faces of the outer chloroplast envelope membrane are small particles in the range 2-6 nm. No differences are found between
membrane areas inside or outside the eyespot with respect to the PF of the outer
chloroplast envelope membrane. On the EF of the outer chloroplast envelope membrane about 10% more IMP of the small size class, 4-6 nm, were found in the eyespot area compared to areas outside the eyespot for all 3 cell types. In general IMP
size-class distribution is very similar in all 3 cell types (Figs. 8, 9).
The data on eyespot surface area and particle density allow calculation of total IMP
number per eyespot. Male gametes contain approximately 4870 IMP, female gametes
5520 and zoospores 11190 IMP per eyespot on the PF of the outer chloroplast
envelope membrane.
DISCUSSION
Eyespot position
Eyespot position in green algae is strictly defined with respect to the plane of beat
of the flagella (see review by Dodge, 1969; Melkonian & Robenek, 1979). Variation
in eyespot position exists, however, along the longitudinal cell axis, the eyespot being
located either more anteriorly or more posteriorly. Sometimes even 2 eyespots were
observed along this line as in zoospores of Microthamnion (Watson, 1975). In most
green algae the definite position of the eyespot with respect to the plane of beat of the
flagella is related to its association with a flagellar root (see summary by Moestnip,
1978). In all investigated species it is a specialflagellarroot type, containing more than
2 microtubules (the X-root of an X-2-X-2 cruciateflagellarroot system, see Moestrup,
1978), that associates with the edge of the eyespot. Individual root microtubules are
linked to the outer chloroplast envelope membrane in the eyespot region (Melkonian,
1978). Zoospores and gametes of Ulva lactuca conform to this general scheme.
Freeze-fracture has allowed easy recognition of this spatial relationship between the
eyespot and a flagellar root, which would otherwise have required extensive serial
sectioning. In Ulva gametes it has also been possible to show that only one of two
possible locations of the eyespot is realized, i.e. the eyespot is located on the same side
of the cell as the single mating type structure (see also Melkonian, 1980). This gives
gametes of Ulva lactuca a distinctive left-right symmetry. The significance of the
flagellar root-eyespot association is not known; it has been suggested that either the
root plays a role in eyespot morphogenesis to determine the exact position of the
developing eyespot or it may be actively engaged in signal transduction during
phototactic orientation (Melkonian, 1978; Melkonian & Robenek, 19806).
Fig. 8. Histograms showing the size distribution of IMP on the protoplasmic face of
the outer chloroplast envelope membrane in the eyespot region and in regions outside
the eyespot for female gametes, male gametes and zoospores. Chl-PF0 protoplasmic
face of the outer chloroplast envelope membrane outside the eyespot region. ChlPFoEy, protoplasmic face of the outer chloroplast envelope membrane in the eyespot
region. The total number of particles evaluated for each membrane face is indicated
in the upper right of each histogram.
i6o
H. Robenek and M. Melkonian
Female gamete
364
Chl-EFn
40 i- Chl-EF0Ey
259
30
20
10
8
12
16
8
Male gamete
Ctil-EF0Ey482
Chl-EF0
12
16
366
40 -
30
20
10
4
8
12
Chl-EF0Ey 438
16
4
8
Zoospore
Chl-EFO
12
16
395
40
% 30
t
20
3
10
4
8
12
16
4
Particle diameter (nm)
Fig. 9 .
8
12
16
Eyespot membranes in Ulva
161
Eyespot membranes
Recent observations indicate that green algae use a 2-instant mechanism for light
perception in phototaxis (see e.g. Boscov & Feinleib, 1979; Feinleib, 1980). Such
a mechanism can only operate if the cell frequently changes its position with respect
to the light source. The photoreceptor then compares the light intensity at 2 different
time intervals. It is now accepted that motile cells of green algae rotate around their
longitudinal cell axis during swimming (see discussion by Melkonian & Robenek,
1979). The eyespot probably functions as a shading device and increases the sensitivity and precision of the phototactic orientation. The photoreceptor is shaded by
the eyespot during most of the time of the rotation cycle and excitation of the photoreceptor molecules occurs only during a short light flash. Different morphologies of
the eyespot plate (either concave or convex shape) can possibly modify the duration
of the light flash for the photoreceptor molecules. Excitation of the photoreceptors
triggers a signal, which leads to a membrane depolarization (measured as a primary
photoinduced potential difference by Litvin, Sineschekov & Sineschekov, 1978).
Eventually flagellar beat form is altered in an unknown way to elicit an oriented
phototactic turning movement (see discussion by Melkonian & Robenek, 1980^).
Since the photoreceptor of phototaxis is believed to be a flavin or carotenoid bound
to a protein and embedded in a lipid matrix (see review by Nultsch & Hader, 1979),
the most probable site for photoreceptor location would be in one of the membranes
overlying the eyespot lipid globules. Previously, 2 membranes in the eyespot region
have been found to be specialized using the freeze-fracture technique, namely the
plasmalemma and the outer chloroplast envelope membrane (Melkonian & Robenek,
1979, 1980a, b). Both membranes contain greater numbers of IMP compared to
membrane areas outside the eyespot. The results obtained in this study extend the
earlier observations to gametes of green algae and indicate further that zoospores and
gametes have a similar eyespot membrane ultrastructure. The absence of cleavages
through the plasmalemma in the eyespot region is again interpreted as suggesting
a high IMP density in this area (see also Melkonian & Robenek, 1980a). It can be
calculated that the IMP density on the PF of the plasmalemma in the eyespot region
is probably greater than 8000 IMP/^m 2 , since this has been found to be the critical
particle density at which cleavages still occurred in zoospores of Chlorosarcinopsis
(Melkonian & Robenek, 19806).
Comparison of IMP sizes on the PF of the outer chloroplast envelope membrane
in the eyespot area between Ulva and other green algae shows that small size-class
Fig. 9. Histograms showing the size distribution of IMP on the external face of the
outer chloroplast envelope membrane in the eyespot region and in regions outside
the eyespot for female gametes, male gametes and zoospores. Chl-EF0, external face
of the outer chloroplast envelope membrane outside the eyespot region. Chl-EF0Ey,
external face of the outer chloroplast envelope membrane in the eyespot region. The
total number of particles evaluated for each membrane face is indicated in the upper
right of each histogram.
162
H. Robenek and M. Melkonian
particles (4-8 nm) dominate in the eyespot area of Chlamydomonas (Melkonian &
Robenek, 1980a), Tetraselmis (Melkonian & Robenek, 1979) and Viva (this study).
In all 3 genera studied the density of the IMP is significantly greater in the eyespot
area compared to regions outside the eyespot with respect to the PF of the outer
chloroplast envelope membrane.
Positive and negative phototaxis
Under identical external conditions zoospores of Viva lactuca are negatively phototactic and gametes positively phototactic. It has been known for a long time that light
intensity influences the phototactic sign in green algae (see, for example Strasburger,
1878). Usually an organism responds positively at lower and negatively at higher
light intensities (see, for example Feinleib & Curry, 1971). The intensity at which the
positive reaction becomes negative is called the inversion intensity (Nultsch, 1975).
The absolute value of the inversion intensity depends on the species, but also on
external factors (see review by Nultsch, 1977) like the CO2 tension. Certain antipsychotic drugs also cause a light-intensity-dependent reversal of phototaxis (Hirschberg & Hutchinson, 1980). Litvin et al. (1978) have shown that the primary photoinduced electric potential difference is graded, both the amplitude and the rate of the
potential changes rising with the increase of light intensity. It is not known whether
ion fluxes or electrogenic processes are involved in generating this potential difference.
If one assumes that ion fluxes are responsible for potential generation, then higher
light intensities would lead to greater ion fluxes. It is suggested that the total amount
of ions transported is related to the total amount of either photoreceptors or ion
channels in the eyespot membranes. If one relates IMP in eyespot membranes with
proteins responsible for photoreception or sensory transduction, it should be possible
to compare IMP number per eyespot with the observed phototactic behaviour. If one
applies this concept to the phototactic behaviour of gametes and zoospores in Viva,
it is obvious that negative phototactic behaviour in zoosporea correlates with a greater
number of IMP per eyespot in the outer chloroplast envelope membrane compared
to gametes. It is significant in this respect that male gametes, although their eyespot
is one third smaller than that of female gametes, exhibit a higher IMP density//mia
in the eyespot area than female gametes and therefore their eyespot contains roughly
the same number of IMP as in female gametes. On theoretical grounds both types of
gametes should show similar phototactic behaviour if phototaxis is viewed as a mechanism of bringing both gamete types close together to facilitate gametic fusion.
Another interesting speculation is offered by the events occurring during gametic
fusion. Immediately after flagellar agglutination phototactic reversal is signalled
(Melkonian, 1980). It is attractive to relate the permanent switch in the phototactic
behaviour of the fused gamete pairs to the fact that they contain 2 eyespots with a total
number of IMP in the outer chloroplast envelope membranes of around 10400, which
is comparable to that of zoospores (11300). Since both eyespots in gamete pairs are
positioned side by side (see Kornmann & Sahling, 1977 for light microscopy; and our
unpublished observations), they might be cooperating in phototaxis of the young
motile zygotes.
Eyespot membranes in Ulva
163
The authors wish to thank Mrs K. Ott and Mrs B. Berns for excellent technical assistance.
Part of this study was carried out at the Biologische Anstalt Helgoland and one of the authors
(M. M.) would like to express his thanks to the staff of the Biologische Anstalt for permission
to use their facilities.
REFERENCES
Boscov, J. S. & FEINLEIB, M. E. (1979). Phototactic response of Chlamydomonas toflashesof
light. II. Response of individual cells. Photochem. Photobiol. 30, 499—505.
BRANTON, D., BULLIVANT, S., GILULA, N. B., KARNOVSKY, M. J., MOOR, H. MOHLETHALER
K., NORTHCOTE, D. H., PACKER, L., SATIR, B., SATIR, P., SPETH, V., STAEHELIN, L. A.,
STEERE, R. L. & WEINSTEIN, R. S. (1975). Freeze-etching nomenclature. Science, N.Y. 190,
54-56.
DODGE, J.
FEINLEIB,
D. (1969). A review of the fine structure of algal eyespots. Br. phycol. J. 4, 199-210.
M. E. (1980). Photomotile responses in flagellates. In Photoreception and sentory
transduction in aneural organisms (ed. F. Lenci & G. Colombetti), pp. 45-67. New York:
Plenum.
FEINLEIB, M. E. & CURRY, G. M. (1971). The relationship between stimulus intensity and
oriented phototactic response (topotaxis) in Chlamydomonas. Physiologia PI. 25, 346-352.
HALLDAL, P. (1959). Factors affecting light response in phototactic algae. Physiologia PI. 12,
742-752.
HIRSCHBERG, R. & HUTCHINSON, W. (1980). Effect of chlorpromazine on phototactic behavior
in Chlamydomonas. Can. J. Microbiol. 26, 265-267.
P. & SAHLING, P.-H. (1977). Meeresalgen von Helgoland. Helgola'nder wiss.
KORNMANN,
Meeresunters. 29, 1-289.
F. F., SINESCHEKOV, O. A. & SINESCHEKOV, V. A. (1978). Photoreceptor electric
potential in the phototaxis of the alga Haematococcus pluvialis. Nature, Lond. 271, 476-478.
MELKONIAN, M. (1975). The fine structure of the zoospores of Fritschiella tuberosa Iyeng.
(Chaetophorineae, Chlorophyceae) with special reference to the flagellar apparatus. Protoplasma 86, 391-404.
MELKONIAN, M. (1978). Structure and significance of cruciate flagellar root systems in green
algae: comparative investigations in species of Chlorosarcinopsis (Chlorosarcinales). PI. Syst.
Evol. 130, 265-292.
MELKONIAN, M. (1979). Structure and significance of cruciate flagellar root systems in green
algae: zoospores of Ulva lactuca (Ulvales, Chlorophyceae). HelgoMnder tviss. Meeresunters.
33. 425-435MELKONIAN, M. (1980). Flagellar roots, mating structure and gametic fusion in the green
alga Ulva lactuca (Ulvales). J. Cell Sci. 46, 149-169.
MELKONIAN, M. & ROBENEK, H. (1979). The eyespot of the flagellate Tetraselmis cordiformis
Stein (Chlorophyceae): structural specialization of the outer chloroplast membrane and its
possible significance in phototaxis of green algae. Protoplasma 100, 183-197.
MELKONIAN, M. & ROBENEK, H. (1980a). Eyespot membranes of Chlamydomonas reinhardtii:
a freeze-fracture study. J. Ultrastruct. Res. 72, 90-102.
MELKONIAN, M. & ROBENEK, H. (19806). Eyespot membranes in newly released zoospores of
the green alga Chlorosarcinopsis gelatinosa (Chlorosarcinales) and their fate during zoospore
settlement. Protoplasma 104, 129-140.
MICALEF, H. & GAYRAL, P. (1972). Quelques aspects de rinfrastructure des cellules vegetatives
et des cellules reproductrices d'Ulva lactuca L. (Chlorophyceae). J. Microscop. 13, 417-428.
MOESTRUP, 0. (1978). On the phylogenetic validity of the flagellar apparatus in green algae
and other chlorophyll a and b containing plants. BioSystems 10, 117-144.
NULTSCH, W. (1975). Phototaxis and photokinesis. In Primitive sensory and communication
LITVIN,
systems: the taxes and tropisms of microorganisms and cells (ed. M. J. Carlile), pp. 29—90.
London, New York, San Francisco: Academic Press.
W. (1977). Effect of external factors on phototaxis of Chlamydomonas reinhardtii. II.
Carbon dioxide, oxygen and pH. Arch. Microbiol. 112, 179-185.
NULTSCH,
164
H. Robenek and M. Melkonian
NULTSCH, W. & HADER, D.-P. (1979). Photomovement of motile microorganisms. Photochem.
Photobiol. 39, 423-437.
STRASBURGER, E. (1878). Wirkung des Lichtes und der Wdrme auf Schwdrmsporen. Jena: Fischer.
WATSON, M. W. (1975). Flagellar apparatus, eyespot and behavior of Microthamnion kuetzingianum (Chlorophyceae) zoospores. J. Phycol. 11, 439-448.
{Received 4 November 1980)