Plant Cell Physiol. 49(12): 1805–1817 (2008)
doi:10.1093/pcp/pcn162, available online at www.pcp.oxfordjournals.org
ß The Author 2008. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists.
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Accumulation, Activity and Localization of Cell Cycle Regulatory Proteins
and the Chloroplast Division Protein FtsZ in the Alga Scenedesmus quadricauda
under Inhibition of Nuclear DNA Replication
Milada Vı́tová 1, 4, Jana Hendrychová 1, 4, Mária Čı́žková 1, Vladislav Cepák 2, James G. Umen 3,
Vilém Zachleder 1, * and Kateřina Bišová 1
1
Laboratory of Cell Cycles of Algae, Institute of Microbiology, ASCR, 37981 Trˇeboň, Czech Republic
Algological Centre and Research Centre for Bioindication and Revitalization, Institute of Botany, Academy of Sciences
of the Czech Republic, Trˇeboň, Czech Republic
3
Plant Biology Laboratory, The Salk Institute for Biological Studies, La Jolla, CA 92037, USA
2
Synchronized cultures of the green alga Scenedesmus
quadricauda were grown in the absence (untreated cultures)
or in the presence (FdUrd-treated cultures) of 5-fluorodeoxyuridine, the specific inhibitor of nuclear DNA replication.
The attainment of commitment points, at which the cells
become committed to nuclear DNA replication, mitosis and
cellular division, and the course of committed processes
themselves were determined for cell cycle characterization.
FdUrd-treated cultures showed nearly unaffected growth
and attainment of the commitment points, while DNA replication(s), nuclear division(s) and protoplast fission(s) were
blocked. Interestingly, the FdUrd-treated cells possessed a
very high mitotic histone H1 kinase activity in the absence of
any nuclear division(s). Compared with the untreated cultures,
the kinase activity as well as mitotic cyclin B accumulation
increased continuously to high values without any oscillation.
Division of chloroplasts was not blocked but occurred delayed
and over a longer time span than in the untreated culture. The
FtsZ protein level in the FdUrd-treated culture did not exceed
the level in the untreated culture, but rather, in contrast to
the untreated culture, remained elevated. FtsZ structures
were both localized around pyrenoids and spread inside of the
chloroplast in the form of spots and mini-rings. The abundance and localization of the FtsZ protein were comparable
in untreated and FdUrd-treated cells until the end of the
untreated cell cycle. However, in the inhibitor-treated culture,
the signal did not decrease and was localized in intense
spots surrounding the chloroplast/cell perimeter; this was in
agreement with both the elevated protein level and persisting
chloroplast division.
Keywords: Chloroplast fission — Cyclin-dependent kinase
— Fluorodeoxyuridine — FtsZ protein — Mitotic cyclin B
— Scenedesmus quadricauda.
Abbreviations: BSA, bovine serum albumin; CDK,
cyclin-dependent kinase; CP, commitment point; DTT,
dithiothiothreitol; FdUrd, 5-fluorodeoxyuridine; FtsZ,
Z-ring-forming chloroplast protein; GST, glutathione
S-transferase; PBS, phosphate-buffered saline; ptDNA,
chloroplast DNA.
Introduction
Dividing eukaryotes have to coordinate cellular and
organellar divisions. While animal and fungal cells need to
coordinate cell division with the division of mitochondria,
plants and algae need to coordinate the divisions of the cell,
mitochondria and chloroplast(s). Therefore, careful coordination of cell multiplication with plastid multiplication and
partition at cytokinesis is required to maintain the universal
presence of plastids in the photosynthetic organisms
(Heinhorst et al. 1985a, Heinhorst et al. 1985b, Coleman
1999). However, the mechanism underlining the control
remains enigmatic (Miyagishima 2005).
Since higher plants contain higher numbers of chloroplasts (reviewed in Butterfass 1995), algae containing only
one chloroplast are useful models for the study of both
organelle division and the coordination between the division of the cell and the chloroplast. Chloroplasts of green
algae and higher plants arose from a primary endosymbiotic
event between a cyanobacterium and a flagellate protist.
Bacteria divide by binary fusion, directed by a Z-ring that
is formed by FtsZ protein (Bi and Lutkenhaus 1991).
Analogously, the chloroplasts divide by binary fission using
a homolog of FtsZ protein (Lutkenhaus 1998, Margolin
2000). Homologs of bacterial FtsZ were found in many
plant species (reviewed in Stokes et al. 2000) including
the unicellular green algae Nannochloris bacillaris (Koide
et al. 2004) and Chlamydomonas reinhardtii (Wang et al.
2003). Two types of FtsZ (FtsZ1 and FtsZ2) were identified in plants (see overview in Stokes and Osteryoung
2003). Both of them are essential for chloroplast division
4
These authors contributed equally to this work.
*Corresponding author: E-mail, [email protected]; Fax, þ420-384340415.
1805
1806
Chloroplast fission in Scenedesmus
(Osteryoung et al. 1998); they associate tightly to form the
Z-ring inside the inner envelope membrane, determining the
site of division (McAndrew et al. 2001, Kuroiwa et al. 2002,
Maple et al. 2005). In contrast to FtsZ2, the FtsZ1 protein is
believed not to be present in other structures (El-Kafafi
et al. 2005).
Scenedesmus quadricauda is a green monoplastidic alga
(Chlorophyceae) forming coenobia. The cells divide by
multiple fission; they grow during the G1 phase until they
reach a critical cell size for the attainment of the first
commitment point (CP). If the growth continues, the cells
can attain several additional CPs consecutively. At each
of the CPs the processes are triggered, leading, after a certain time interval, to initiation of the round (s) of DNA
replication(s) and later to nuclear division(s) (Šetlı́k et al.
1972, Zachleder and Šetlı́k 1990, Zachleder et al. 1997)
(Fig. 1). Therefore, the cells divide into 2n daughter cells,
where n is the number of CPs attained, and consequently
the number of doublings (Kates et al. 1968, Poyton and
Branton 1972, Šetlı́k and Zachleder 1984).
The eukaryotic cell cycle is regulated by the activity of
cyclin-dependent kinases (CDKs) in a heterodimeric complex with cyclins (for a review, see Norbury and Nurse 1992,
Nigg 1995, Pines 1996, Stern and Nurse 1996, Mironov
et al. 1999). Similarly the S. quadricauda cell cycle is regulated by the activity of CDK-like kinases with histone H1
kinase activity. There are at least two different complexes
expressing CDK-like activity in S. quadricauda; one accompanies the attainment of CP(s), the other complex is able to
interact with suc1 protein and its activity is specific for
mitosis(es) (Bišová et al. 2000).
Scenedesmus quadricauda has been extensively studied
in an attempt to unravel the relationship between cell and
chloroplast divisions. It has been shown that chloroplast,
similarly to the nucleocytosolic compartment, undergoes
chloroplast DNA (ptDNA) replication, nucleoid division
and chloroplast fission (Zachleder and Cepák 1987b). The
processes of ptDNA replication and nucleoid division can
be uncoupled from nuclear DNA replication and division
by application of 5-fluorodeoxyuridine (FdUrd), an inhibitor of thymidylate synthase that in S. quadricauda specifically inhibits the nuclear DNA replication and without
affecting the ptDNA replication (Zachleder 1994).
Here, we have blocked nuclear DNA replication
by FdUrd in order to uncouple nuclear and chloroplast
division cycles. We showed that chloroplast division could
be performed in the absence of nuclear and cellular division, but it was delayed, slowed down and the number of
daughter chloroplasts often did not agree with 2n number
rule of multiple fissions (see above). Interestingly, treatment
with FdUrd caused extremely elevated steady-state levels
and activities of the cell cycle-regulating proteins mitotic
CDK-like kinase and cyclin B.
Classical cell cycle model
A
S
G1
G2
M
Scenedesmus binary fission
B
G1
pS
S
G2
M
G3
C
C.P.
Scenedesmus multiple fission
C
pS S
G1
G2
C.P.
M
G3
pS S
G1
G2
C
M
G3 C
C.P.
Fig. 1 Diagrams showing different types of cell cycle phases
including the classical cell cycle model (A) for binary fission
(Howard and Pelc 1953) and those found in the alga Scenedesmus
that divide into two (B) and four (C) daughter cells. Individual bars
show the sequence of cell cycle phases during which growth and
reproductive processes take place. Whereas only one sequence of
events leading to the duplication of cell structures occurs during
the cell cycle of cells dividing into two daughter cells (A, B), two
partially overlapping sequences of growth and reproductive events
occur within a single cycle in cells dividing by multiple fission into
four daughter cells (C). Two bars (C) illustrate the simultaneous
course of different phases from two consecutively started
sequences of growth and reproductive events. Schematic pictures
of the cells indicate their size during the cell cycle, and the black
spots inside illustrate the size and number of nuclei. Large black
spots indicate the doubling of the DNA. G1: the phase during
which the threshold size of the cell is attained. It can be called a
pre-commitment period because it is terminated when the
commitment point is reached. CP: the stage in the cell cycle at
which the cell becomes committed to triggering and terminating
the sequence of processes leading to the duplication of reproductive structures. pS: the pre-replication phase between the CP and
the beginning of DNA replication. The processes required for the
initiation of DNA replication are assumed to happen during this
phase. S: the phase during which DNA replication takes place. G2:
the phase between the termination of DNA replication and the start
of mitosis. Processes leading to the initiation of mitosis are assumed
to take place during this phase. M: the phase during which nuclear
division occurs. G3: the phase between nuclear division and cell
division. The processes leading to cellular division are assumed to
take place during this phase. C: the phase during which cell
cleavage occurs. Modified after Zachleder et al. (1997).
Results
Cell cycle progress
Synchronized cultures of S. quadricauda were cultivated
in either the presence or absence of FdUrd. The growth
in both variants was stopped by transfer of the cultures into
the dark after 12 h of the cell cycle. The increase in the cell
size during the light period was the same in both cultures
(Fig. 2). Also the total RNA level in the FdUrd-treated
5
750
40
4
600
30
3
20
2
10
1
0
0
6
12
18
24
450
300
Cell size (µm3)
50
DNA (pg/cell)
RNA (pg/cell)
Chloroplast fission in Scenedesmus
150
0
Time (h)
Fig. 2 The course of changes in cell size (diamonds), amount of
total RNA (circles) and DNA (triangles) in a synchronized
population of Scenedesmus quadricauda grown in the absence
(open symbols) or presence of FdUrd (25 mg l–1) (filled symbols).
Light and dark periods are separated by a vertical line and marked
by stripes above the panel.
culture was increasing at a similar rate to that in untreated
cells and was around seven times higher than at the beginning of the cell cycle (Fig. 2). The DNA level in untreated
cells increased about 7-fold (Fig. 2), while its amount in
FdUrd-treated cells did not change significantly, due to
inhibition of nuclear DNA replication.
CP attainments refer to the progress of growth
processes and readiness of cells to trigger division processes
(Nurse and Bissett 1981, Bišová et al. 2000, Vı́tová and
Zachleder 2005). During the light period (growth phase), all
cells in the untreated synchronized population attained
consecutively two CPs (Fig. 3A, thin solid line, open circles
and squares) and most of them (86%) the third CP. Consequently, most of the cells performed three rounds of DNA
replication (Fig. 2), three rounds of nuclear divisions and
protoplast fissions and released 8-celled daughter coenobia
at the end of their cell cycle. The remaining cells (14%)
divided into 4-celled coenobia (Fig. 3A).
The growth characteristics in FdUrd-treated cells were
the same as those of untreated cells (compare RNA levels
and cell sizes in Fig. 2). The 7-fold increase in RNA is
in agreement with the untreated cultures where it corresponded to three consecutive CPs obtained by most of the
cells (86%) in the population.
Neither DNA replication nor division of nuclei and
cells (protoplasts) occurred in FdUrd-treated cells; however,
the cells attained the CP to divide in spite of the fact that
committed processes could not be performed (for details of
the procedure, see Materials and Methods). These cells
rescued at least two committed sequences of division events
(committed to division into four daughter cells) and their
course was about the same as in untreated culture (Fig. 3A,
thin solid lines, filled circles and squares, respectively).
1807
After 6 h of growth, at the time when the first nuclear
division started in untreated culture, the block of DNA
replication in FdUrd-treated cells became irreversible and
the third CP to division into eight daughter cells could no
longer be rescued (Fig. 3A).
Chloroplast cycle progress
In contrast to the untreated cell cycle that was
completed by protoplast fission (cytokinesis) within 14 h
(Fig. 3A) and where daughter coenobia were released after
about 16 h (Figs. 3A, 4C), FdUrd-treated cells did not
divide at all. Nevertheless, the chloroplast fission started in
these cells after 10 h (Fig. 3B), delayed for 2 h compared
with protoplast fission in untreated cells (compare also
Fig. 4C and H). The commitments to divide the chloroplast
were attained concomitantly with commitments to divide
the protoplasts in untreated cells. While no cell division
events were performed in the presence of FdUrd even if
committed, the committed chloroplast fission was realized
even in the presence of FdUrd (Fig. 3B). However, the
chloroplast fission was delayed by about 2 h and was slower
and less synchronous than in untreated culture (the division
into two chloroplasts in 80% of cells took 9 h, while division
into two protoplasts in 100% of cells in untreated culture
took only 5 h) (Fig. 3B). Moreover, the chloroplasts kept
dividing until 36 h, reaching from eight up to 16 chloroplasts per cell (Fig. 4I, L).
We observed chloroplast fission in FdUrd-treated cells
by fluorescence microscopy, employing chlorophyll autofluorescence (Fig. 4K, L) and by way of electron microscopy (Fig. 5B). Spherical chloroplasts within a ‘mother’ cell
were also observable in transmitted light (Fig. 4I). A single
nucleus with one nucleolus remained in the cell (Fig. 5B). In
spite of the fact that no DNA replication occurred in
FdUrd-treated cells (Fig. 2), the nucleus is about double the
size of the nucleus in untreated cells with the same amount
of DNA (Fig. 5B). The progress of chloroplast fission in
FdUrd-treated cultures was significantly slower than fission
of untreated protoplasts (compare Fig. 3A and B).
Moreover, the number of daughter chloroplasts often did
not agree with the 2n number rule of multiple fissions.
Cyclin B and FtsZ quantification
To detect cyclin B, we used anti-cyclin B1 antibody
raised against recombinant hamster cyclin B1 (Sigma
C8831). Due to the evolutionary distance between green
algae and mammals, we tested the specificity of the antibody.
Since there is only limited information on protein sequences
in S. quadricauda, we took advantage of the recently completed genome sequence of the green alga Chlamydomonas
reinhardtii. Scenedesmus quadricauda is a member of the
order Chlorococcales, and C. reinhardtii is a member of
the evolutionary ancestral order Volvocales within the
1808
Chloroplast fission in Scenedesmus
Cell fraction (cumulative %)
100
Chlorophyta division (van den Hoek et al. 1995). Sequence
comparison of hamster cyclin B1 protein sequence with
sequences of C. reinhardtii A1, B1, D2 and D3 cyclins shows
that there is high sequence similarity among all the sequences
within the cyclin box (Fig. 6A, underlined); hamster cyclin
B1 and C. reinhardtii cyclins A1 and B1 also contain
destruction boxes (Fig. 6A, underlined). There is higher
similarity between hamster cyclin B1 and C. reinhardtii
cyclins A1 and B1 as compared with C. reinhardtii cyclins D2
and D3; the highest similarity is between the two B-type
cyclins (Fig. 6A).
In order to test the antibody specificity more directly,
we produced recombinant glutathione S-transferase (GST)tagged versions of C. reinhardtii A1, B1, D2 and D3 cyclins.
The antibody cross-reacted only with recombinant GST–
CrCYCB1 and not with any other cyclins (Fig. 6B). It
recognized both the full-length version of GST–CrCYCB1
of predicted molecular weight 72 kDa and truncated versions of the recombinant protein of approximately 40 and
35 kDa (Fig. 6B). In S. quadricauda protein extracts, the
antibody cross-reacted with a major band of apparent
molecular weight 66 kDa (Fig. 6C).
FtsZ antibody was raised against FtsZ1 protein of
the unicellular green alga N. bacillaris (Koide et al. 2004).
A
50
0
100
B
50
0
Kinase activity (r.u.)
200
C
150
100
50
0
Cyclin amount (r.u.)
D
30
20
10
FtsZ1 amount (r.u.)
0
E
100
80
60
40
20
0
0
6
12
18
24
30
36
Time (h)
Fig. 3 Time courses of the attainment of CPs to division events
and division events themselves (mitoses, protoplast fission,
daughter cell release) (A), the attainment of CPs to chloroplast
fissions and chloroplast fissions themselves (B), mitotic (suc1bound) CDK activity (C), amount of cyclin B (D), and amount of
chloroplast FtsZ protein (E) in a synchronized population of
Scenedesmus quadricauda grown in the absence (open symbols)
or presence of FdUrd (25 mg l–1) (filled symbols). (A) Solid thin
lines: percentage of untreated (open symbols) and FdUrd-treated
(filled symbols) cells that attained the commitment to divide into
two (circles), four (squares) and eight (triangles) daughter cells;
solid thick lines, dotted symbols: percentage of untreated cells in
which nuclear divisions in two (circles), four (squares) and eight
(triangles) nuclei were completed; dashed lines, open symbols:
percentage of untreated cells in which protoplast fissions in two
(circles), four (squares) and eight (triangles) protoplasts were
completed; dotted lines (crosses): percentage of untreated cells
that released daughter coenobia. No nuclear division, protoplast
fission and daughter cell release occurred in FdUrd-treated cells.
The potential of these cells to divide (commitment point) was
determined in cells in which FdUrd was removed by rinsing and
replaced by an excess of thymidine prior to transfer to darkness. (B)
Solid lines: percentage of FdUrd-treated cells that attained the
commitment to divide their chloroplast (in the presence of FdUrd)
into two (circles), four (squares) and eight (triangles) daughter
chloroplasts. Dashed lines: percentage of FdUrd-treated cells
that contained at least two (circles), four (squares) and eight or
more (triangles) chloroplasts. (C) Mitosis-specific CDK-like kinase
activity of kinases purified by affinity chromatography on suc1
protein determined by phosphorylation of externally supplemented
histone H1 as substrate. A representative autoradiogram showing
the variation of mitotic histone H1 kinase activity for untreated
(upper stripe) and FdUrd-treated (lower stripe) cultures is shown
as an insert. (D) The amount of cyclin B protein detected by cyclin
B-specific antibody (for specificity of the antibody see Fig. 6A–C).
A representative immunoblot with the antigen (66 kDa) variation
in untreated (upper stripe) and FdUrd-treated (lower stripe) cultures
is shown as an insert. (E) The amount of chloroplast FtsZ protein
detected by FtsZ-specific antibody (for specificity of the antibody
see Fig. 6D). A representative immunoblot with the antigen
(49 kDa) variation in untreated (upper stripe) and FdUrd-treated
(lower stripe) cultures is shown as an insert. Light and dark
periods are separated by a vertical line and marked by stripes
above the top panel.
Chloroplast fission in Scenedesmus
untreated culture
1809
A
FdUrd-treated culture
B
Fig. 4 Photomicrographs of Scenedesmus quadricauda eightcelled coenobia at different stages of the cell cycle in untreated
cultures (A–F) and in the presence of FdUrd (25 mg l–1) (G–L).
Coenobia in transmitted light (A–C, G–I) and their chlorophyll
autofluorescence (D–F, J–I) were observed. Numerals in the upper
right-hand corner indicate hours of the cell cycle. The cell cycle of
untreated cells ended at 14 h when the protoplast fissions were
nearly completed and liberation of daughter cells was in progress
(see Fig. 3A). Chloroplast fission in FdUrd-treated cells started
at 14 h with about a 2 h delay compared with untreated cells
(G and J), being completed at about 24 h (I and L). Bar ¼ 10 mm
(all photomicrographs).
In S. quadricauda protein extracts, the antibody crossreacted with a single band of apparent molecular weight
49 kDa (Fig. 6D).
CDK activity and accumulation of cyclin B
To ascertain whether the lack of nuclear division was
accompanied by changes of the cell cycle regulatory
proteins, we determined the mitotic CDK-like kinase
activity (monitored as the activity of suc1-bound H1
kinases) and cyclin B accumulation, analyzed by immunoblotting. In the untreated culture, kinase activity peaked at
13 h of the cell cycle (Fig. 3C). Expression of a mitotic cyclin
was detected as a specific band of about 66 kDa (see inserts
in Fig. 3D) in samples from 10 to 17 h, being most intense in
the 13 h sample (Fig. 3D). Both the kinase activity and
cyclin decreased to a minimum at the end of the cell cycle.
In FdUrd-treated cells, an increase in both kinase
activity and cyclin B amount started similarly to that in
Fig. 5 Electron microphotographs of chloroplast fission in a
synchronized population of Scenedesmus quadricauda grown in
the absence (A) and presence of FdUrd (25 mg l–1) (B). (A) Untreated
cells with divided protoplasts occurring at about 14 h, just before
formation and release of daughter cells; eight divided protoplasts
forming into daughter cells are seen inside of the mother cell wall,
each of them possessing one chloroplast (C) and one nucleus (N).
The mother cell before protoplast fission contained eight nuclei
and one chloroplast. Chloroplast fission was concomitant with
protoplast fission in untreated cells. (B) FdUrd-treated cells with
divided chloroplasts. No nuclear division and protoplast fission
occurred. The cell remained uninuclear (N), but the chloroplast
divided (c). Numerous, large starch granules (S) are characteristic
of FdUrd-treated cells. Bars in A ¼ 2 mm, in B ¼ 5 mm.
untreated cultures. However, they never decreased and were
still increasing several hours after the decrease in the
untreated culture (Fig. 3C, D). In both variants, the antigen
recognized by the anti-cyclin B antibody was found to
accumulate in nuclei (Fig. 7).
FtsZ protein
To analyze whether chloroplast division was affected
at the molecular level, we analyzed the abundance and
1810
A
Chloroplast fission in Scenedesmus
1
50
100
MaCYCB1
CrCYCB1
CrCYCA1
CrCYCD2
CrCYCD3
MaCYCB1
CrCYCB1
CrCYCA1
CrCYCD2
CrCYCD3
MaCYCB1
CrCYCB1
CrCYCA1
CrCYCD2
CrCYCD3
MaCYCB1
CrCYCB1
CrCYCA1
CrCYCD2
CrCYCD3
MaCYCB1
CrCYCB1
CrCYCA1
CrCYCD2
CrCYCD3
B
D
1
2
3
4
5
11
untreated
12
13
175
175
115
93
65
57
115
93
65
57
36
31
36
D
untreated
12 14 16 20 24
FdUrd-treated
12 14 16 20 24 28 32
170
130
95
72
55
43
Fig. 6 Sequence comparison of hamster cyclin B1 (MaCYCB1) with A-, B- and D-type cyclins of the green alga Chlamydomonas
reinhardtii (CrCYCA1, CrCYCB1, CrCYCD2 and CrCYCD3) (A), immunoblots of cross-reactivity of anti cyclin B antibody with recombinant
GST–CrCYCA1, B1, D2 and D2 (B), and with Scenedesmus quadricauda protein extract (C), and immunoblots of cross-reactivity of anti
FtsZ antibody with Scenedesmus quadricauda protein extract (D). (A) The intensity of shading corresponds to the degree of residue
conservation among sequences; the sequences of destruction and cyclin boxes are underlined. (B) Crude protein extracts of bacteria
expressing 1, GST–CrCYCA1; 2, GST–CrCYCB1; 3, GST–CrCYCD2; 4, GST–CrCYCD3; and 5, uninduced control were detected by anticyclin B antibody; specific bands are depicted by arrows and non-specific bands present in non-induced control are depicted by asterisks.
(C) Protein extracts of untreated and of FdUrd-treated cultures at specified hours of the cell cycle were detected by anti-cyclin B antibody;
the arrow indicates a specific band of approximately 66 kDa, and asterisks indicate non-specific bands detected by secondary antibody
only. (D) Protein extracts of untreated culture at specified hours of the cell cycle were detected by anti-FtsZ antibody; the arrow indicates a
specific band of approximately 49 kDa.
Chloroplast fission in Scenedesmus
1811
FtsZ did not exceed the level in untreated cultures; it
increased more slowly and did not decrease (Fig. 3E).
The intracellular localization of FtsZ was examined
by indirect immunofluorescence staining on cross-sections.
The pattern of the fluorescence signal differed remarkably
among samples from different time periods. At the beginning of the cell cycle, we observed spots and tiny rings
(Fig. 8A).
Later in the growth phase, at 7 h, at around the time
when the second nuclear division occurred in the untreated
culture, FtsZ was arranged in circles beside spots (one per
cell) in both cultures (Fig. 8B, E, arrowheads). Immunogold
labeling showed localization of the antigen at the pyrenoid
edge (Fig. 9); therefore, the circle observed in cross-sections
by immunofluorescence could represent the FtsZ structures
surrounding the pyrenoid and not real rings.
At 12 h in both the untreated and FdUrd-treated cell
cycle, the circles occurred only rarely and the small rings
became diffuse (Fig. 8C). Two hours later, the signal in
untreated cells became weak and FtsZ was localized in
dispersed fine spots (Fig. 8D), In contrast, in FdUrd-treated
cells, the signal stayed strong until 24 h, in agreement with
the continuously high protein level of FtsZ (Fig. 3E). FtsZ
was localized to intense spots surrounding the chloroplast/
cell perimeter (Fig. 8G, H).
Discussion
Fig. 7 Immunolocalization of cyclin B in Scenedesmus quadricauda in untreated (A) and FdUrd- (25 mg l–1) treated (B) culture.
The position of the antigen in semi-thin sections was detected by
anti-cyclin B antibody, recognizing a specific band of approximately 66 kDa, and visualized by Cy3-conjugated secondary
antibody. Arrows indicate the localization of mitotic cyclin in
nuclei. Bar ¼ 10 mm.
localization of the FtsZ protein, which is known to be
involved in chloroplast division. The amount of protein
synthesized during the cell cycle was analyzed by immunoblotting in both the untreated and FdUrd-treated cells. The
amount of FtsZ protein increased during the growth phase.
It reached its maximum after 12 h and was decreasing
after 16 h in untreated cultures. In FdUrd-treated cultures,
Processes related to the growth of the cells
The FdUrd-treated cells in our experiments grew
comparably (Fig. 3A) with untreated cells and performed
chloroplast division, whilst the DNA replication division
sequences in the nucleocytosolic compartment were not
performed (Fig. 3B). If the cells had been grown under
continuous illumination in the presence of FdUrd, they
would have attained a giant size (Zachleder et al. 1996). In
the present experiments, the growth to abnormal size was
prevented by putting the culture in darkness after 12 h of
growth in light, just at the time when liberation of daughter
cells in the untreated culture started (Fig. 3A). The size of
nuclei in FdUrd-treated cells increased during the growth
so that, at the time when untreated cell divided giving rise
to eight daughter protoplasts with eight nuclei, their size
was about double that of untreated nuclei (Fig. 5). However, the cell cycle positions of the nuclei differed. While the
untreated nuclei were at the beginning of G1 phase, the
FdUrd-treated nucleus was at the stage just before entry
into S phase which was prevented due to DNA replication
block. The nuclei in FdUrd-treated cells were in a state of
very intensive transcriptional activity since transcription
from a single genome had to ensure a level of transcription
ensured in untreated cells of similar size by 4–8 nuclei. The
size increase of nuclei during G1 phase prior to S phase
1812
Chloroplast fission in Scenedesmus
untreated culture
FdUrd-treated culture
Fig. 8 Immunolocalization of FtsZ antigen in Scenedesmus quadricauda at different stages of the cell cycle in untreated cultures (A–D)
and in the presence of FdUrd (25 mg l–1) (E–H). The position of antigen in semi-thin sections was detected by anti-FtsZ antibody,
recognizing a specific band of approximately 49 kDa, and visualized by Cy3-conjugated secondary antibody. Numerals in the upper righthand corner indicate the hour of the cell cycle. No substantial difference was observable within the first 12 h of the cell cycle between the
untreated and FdUrd-treated cultures. After 14 h the untreated cells divided their protoplasts including chloroplasts, and the FtsZ structures
changed into small spots or tiny rings (D). No division occurred in FdUrd-treated cells, the signal did not decrease and FtsZ was localized
to intense spots surrounding the chloroplast/cell perimeter (G and H). Arrowheads indicate minicircles surrounding pyrenoids. Bar ¼ 10 mm
(all photomicrographs).
has also been described by other authors (for a review, see
Yen and Pardee, 1979).
CDK kinase activity and accumulation of cyclin B
Incompletely replicated or damaged DNA activates in
yeasts, mammals and higher plants a DNA damage/
replication checkpoint, which prevents mitosis by inhibition
of CDK activity (Pelayo et al. 2001, Bartek and Lukas 2007,
Calonge and O’Connell 2008). A delay in the mitotic kinase
activity after DNA damage was also reported in the unicellular green alga C. reinhardtii (Slaninová et al. 2003). The
finding that the CDK activity of S. quadricauda was not
inhibited but, on the contrary, increased to a very high level
(Fig. 3C) indicated that in the presence of FdUrd the DNA
replication did not start at all and the cell therefore showed
neither incompletely replicated nor damaged DNA. The
checkpoint response to these events therefore appears to
have been intact. Indeed, it has been shown in budding
yeast that in the absence of stalled replication forks, the
completion of DNA replication prior to mitosis is not under
the surveillance of known checkpoints (Torres-Rosell et al.
2007). FdUrd blocks the synthesis of thymidine, the substrate for DNA replication, but does not affect the structure
and function of DNA itself. Removing the inhibitor by
rinsing and subsequent addition of thymidine allowed
the normal progress and completion of the cell cycle of
S. quadricauda; this phenomenon was used for determination of CPs in FdUrd-treated cultures (see Materials and
Methods and Fig, 3A). Moreover, the synchronous culture,
to which FdUrd was added at the beginning of the cell cycle
together with thymidine, went through the cell cycle as
an untreated culture (unpublished results), implying that the
cell cycle progression was undisturbed without activated
checkpoints.
Mitotic CDK–cyclin complex activity in cells is inactivated during mitosis due to degradation of cyclin through
ubiquitin-mediated proteolysis (Murray et al. 1989, Glotzer
et al. 1991, Murray 1995, Murray 2004). While this is a
plausible explanation for the decrease of both mitotic kinase
activity and cyclin B amount after mitosis in untreated cells,
neither a decrease of kinase activity nor a decrease of cyclin
B abundance occurred in FdUrd-treated cultures (Fig. 3D).
On the contrary, an abnormal increase in the CDK–cyclin
complex activity was observed, indicating that the mitotic
checkpoint checking degradation of cyclin after mitosis was
intact and activated (Pines 2006). This was confirmed by
Chloroplast fission in Scenedesmus
Fig. 9 Electron microphotograph of FtsZ localization around a
pyrenoid of Scenedesmus quadricauda. (A) Negative control (no
primary antibody added), (B) immunogold labeling of FtsZ-specific
antigen on semi-thin sections detected by specific anti-FtsZ
antibody. Arrows indicate the localization of FtsZ proteins (black
spots), around pyrenoid (P) surrounded by starch (S) (white spots);
other starch granules (S) are located among thylakoids of the
chloroplast (C). Bar ¼ 1 mm (both photomicrographs).
the finding that the high CDK activity was accompanied
by accumulation of cyclin B, implying that cyclin B could
be a part of the kinase complex. It is in line with localization
of cyclin B being found in the nucleus. Cyclin B is known
to be a mitotic cyclin with specific nuclear localization
in both animal and higher plant systems (Mironov et al.
1999, Murray 2004); our data show that it is also true for
algal cells.
Chloroplast fission
We observed that initiation of chloroplast fission in
FdUrd-treated cells was delayed about 2 h compared with
the protoplast fission in the untreated cells. Although the
timing of chloroplast division seemed to be unaffected by
1813
FdUrd treatment, the division itself was perturbed (see
below). Under physiological conditions, chloroplast and
protoplast fissions tend to occur simultaneously (Zachleder
et al. 1996) since cell and chloroplast division(s) need to
be coordinated to ensure the correct numbers of daughter
chloroplasts for daughter cells. It was proposed previously
that the coordination of chloroplast reproductive processes
and those in the nucleocytosolic compartment was governed
by mutual metabolic dependence (Zachleder et al. 1995) and
that chloroplast reproduction was controlled by mechanisms distinct from the reproductive processes in the nucleocytosolic compartment of the alga (Zachleder et al. 1996).
However, in the presence of inhibitors, the chloroplast
fission could occur in the absence of protoplast fission in
tobacco (Heinhorst et al. 1985a, Heinhorst et al. 1985b) and
in S. quadricauda (Zachleder 1994, Zachleder et al. 1996).
Also, the timing of preceding reproductive processes in
chloroplasts (ptDNA replication and nucleoid division) was
not coupled with reproductive processes of nuclei, relying
rather on growth conditions (Zachleder and Cepák 1987a,
Zachleder and Cepák 1987b, Kuroiwa et al. 1989, Zachleder
et al. 1995).
While the mechanism of mutual control between the
nucleocytosolic and chloroplast compartments, particularly
at the molecular level, is not known, this control ensures
that divided chloroplasts are tightly coupled in time, size
and number to dividing protoplasts. However, removed
from this control by FdUrd treatment, the chloroplasts
behave as autonomous organelles, which can initiate and
complete their division cycle independently of the nucleus.
If the feedback control of the nucleus was missing, the
chloroplast division processes would not be so well organized and the chloroplasts would divide later, more slowly,
into differently sized daughter chloroplasts, and often not
following the expected 2n rule.
FtsZ protein
The level of FtsZ protein during the uncoupling
experiments changed differently in FdUrd-treated and
untreated cultures (compare upper and lower panel in
Fig. 3). In untreated cells, maximum levels of FtsZ preceded
the beginning of cytokinesis, during which it then decreased.
This finding is consistent with observations that a Z-ring is
formed before division and disassembles during chloroplast
constriction (Miyagishima et al. 2001, Kuroiwa et al. 2002).
In the presence of FdUrd, FtsZ was increasing more slowly
and after 23 h did not decrease, which might correspond
to the slow and continuous chloroplast fission during this
period. Immunofluorescence analysis shows FtsZ to be
localized in a single circle and small spots during the growth
phase and, in later stages (at 12 h) of FdUrd-treated cells,
as frequent small spots and rings (Fig. 8F). As the circles
disappeared several hours before division (Fig. 8C, D),
1814
Chloroplast fission in Scenedesmus
they are probably not real Z-rings. As shown in Fig. 9, FtsZ
protein is accumulated around the pyrenoid, thus the large
rings could be a projection of an actual spherical arrangement of FtsZ proteins. It is also in line with the observed
dissolution of the pyrenoids just at the time when the large
single rings disappeared. Similarly, FtsZ proteins that were
not involved in the Z-ring were previously detected in the
red alga Cyanidioschyzon merolae (Miyagishima et al. 2001).
Small spots and rings (‘mini FtsZ-rings’) were recently also
observed inside chloroplasts of Arabidopsis thaliana and
were suggested not to be associated with the chloroplast
membrane (Maple et al. 2005).
Materials and Methods
Experimental organism, culture growth conditions and cell cycle
synchronization
The chlorococcal alga S. quadricauda (Turp.) Bréb., strain
Greifswald/15 was obtained from the Culture Collection of
Autotrophic Microorganisms kept at the Institute of Botany,
Třeboň, Czech Republic. The cultures were synchronized by at
least two cycles of alternating light and dark periods (14 h : 10 h).
The suspensions of synchronous cells (106 cells ml–1) were grown at
308C in the inorganic nutrient medium described by Zachleder and
Šetlı́k (1982), aerated with air containing 2% (v/v) CO2 and
illuminated by OSRAM L36/41fluorescent tubes; the light intensity
at the surface of culture vessels was 490 mmol m–2 s–1. To inhibit
DNA replication, FdUrd, the inhibitor of thymidylate synthase,
was added at the beginning of the experiments after re-illumination
of the previously synchronized cultures to the final concentration
of 25 mg l–1.
Determination of total DNA and RNA amount
Total nucleic acids were extracted according to Wanka
(1962), as modified by Lukavský et al. (1973). The DNA assay
was carried out as described by Decallonne and Weyns (1976), with
the modifications of Zachleder (1984); see also Zachleder (1995).
Assessment of cell cycle curves
Commitment point curves. Samples were taken from the
synchronous culture hourly and incubated under aeration at 308C
in the dark. At the end of the cell cycle, the percentages of
binuclear daughter cells, 4- and 8-celled daughter coenobia, and
undivided mother cells were assessed and plotted against the time
of the transfer in darkness. The ‘commitment curves’ illustrated
the course of attainment of individual CPs during the cell cycle
in synchronous culture (John 1984, Šetlı́k and Zachleder 1984,
Zachleder et al. 1997). Because of blocked nuclear DNA replication in FdUrd-treated cells, in the presence of FdUrd it was only
possible to determine commitment to chloroplast division. This
was carried out as described for the commitment to nuclear and
cellular division in untreated cells.
To follow the potential for cell division even in FdUrd-treated
cells, the FdUrd was washed off before transfer to darkness and
excess thymidine (250 mg l–1) was added to the fresh medium to
make thymidylate synthase potentially damaged by FdUrd treatment dispensable.
Nuclear divisions. Nuclei were stained by SYBR Green I dye
(Molecular Probes, Eugene, OR, USA) and observed through
a fluorescence microscope using the technique introduced by
Vı́tová et al. (2005). The percentages of mono-, bi-, tetra- and
octonuclear cells were estimated and plotted on the same graph
with the other cell cycle curves.
Cell division curves. Samples were fixed in 0.25% glutaraldehyde. The percentage of undivided mother cells, mother cells
divided into two, four or eight protoplasts, and daughter coenobia
were determined. Protoplast fission curves and daughter cell
release curves were obtained by plotting the percentages as a
function of time.
Protein extraction
Samples of cell suspensions were centrifuged and the pellets
were washed with SCE buffer [100 mM sodium citrate, 2.7 mM
EDTA-Na2, pH 7 (citric acid)]. The pellets were frozen and stored
at –808C. Protein extracts were prepared as described previously
(Bišová et al. 2003).
Affinity chromatography on immobilized suc1 protein
A specific binding partner of CDK, the suc1 protein (cyclindependent kinase subunit, CKS) (Brizuela et al. 1987) was used for
isolation of the mitotic CDK complexes (Labbe et al. 1989, Bišová
et al. 2000). Affinity chromatography on the immobilized suc1
protein was performed according to the method described previously (Bišová et al. 2000).
Histone H1 kinase activity assay
Using histone H1 as a substrate, kinase activity bound to suc1
protein was assessed employing the procedure described by
Moreno et al. (1989) as modified in our laboratory (Zachleder
et al. 1997, Bišová et al. 2000). Algal proteins bound to suc1–
Sepharose were incubated with 10 ml of the assay buffer [20 mM
HEPES pH 7.5, 15 mM MgCl2, 5 mM EGTA, 1 mM dithiothreitol
(DTT), 0.1% (w/v) histone H1, 0.1 mM ATP, 3.7 106 Bq ml–1 of
[-32P]ATP (specific activity 1.5 1014 Bq mmol–1)] for 30 min at
308C. After separation by 12% SDS–PAGE, phosphorylated
histone H1 bands were visualized by autoradiography. Autoradiograms were scanned and evaluated using SigmaGel software
(Jandel Scientific, San Rafael, CA, USA).
Electrophoretic separation of proteins
For the separation of proteins, SDS–PAGE was performed
using the Laemmli system (Laemmli 1970) in the Mini Protean 3
Apparatus (BioRad Laboratories, Hercules, CA, USA).
Production of recombinant proteins in bacteria
cDNA coding for the C. reinhardtii A-, B- and D-type cyclins
(CrCYCA1, CrCYCB1, CrCYCD2 and CrCYCD3) (Bisova et al.
2005) were amplified from oligo(dT)-primed cDNA by PCR with
primers (CYCA gat for 50 -CACCATGAGCTCTCGCGTCGGCT
Cttc-30 , CYCA gat rev 50 -TCACGAGCGGTGAATGCCGC-30 ,
CYCB gat for 50 -CACCATGGCTCTTCGTGCGGTTCAGCC
C-30 , CYCB gat rev 50 -TTAAGCCACTGGCTGCGCGGGC-30 ,
CYCD2 gat for 50 -CACCATGGAGTGGCTGCTGCTGGAGA
CG-30 , CYCD2 gat rev 50 -TCACCGCTGAGCGGACGGC-30 ,
CYCD3 gat for 50 -CACCATGACGCTGGCGGTGAAGGT
CG-30 , CYCD3 gat rev 50 -CTACTGCTGCACCGGCTGCG
AC-30 ) and cloned into pENTR-SD/D-TOPO vector (Invitrogen,
Carlsbad, CA, USA). The sequences were verified and the cyclin
fragment recombined into pDEST15 (Invitrogen, Carlsbad, CA,
USA), yielding pDEST15-CrCYCA1, B1, D2 or D3, respectively.
The constructs were transformed into BL21-CodonPlus(DE3)-RIL
cells (Stratagene, La Jolla, CA, USA). Cells were grown at 378C
Chloroplast fission in Scenedesmus
until an OD600 of 0.4, then transferred to 208C for 30 min and
induced with 0.5 mM isopropyl-b-D-thiogalactoside for 4 h. The
bacterial pellets were resuspended in 1 SDS–PAGE sample buffer
(62.5 mM Tris–HCl, pH 6.8, 20% glycerol, 2% SDS, 5%
b-mercaptoethanol) and separated by 12% SDS–PAGE.
Sequence analyses
Sequence comparison was done using Vector NTI software
(Invitrogen, Carlsbad, CA, USA). The GenBank numbers of the
sequences are: MaCYCB1, P37882; CrCYCA1, XP_001693167;
CrCYCB1, XP_001701288; CrCYCD2, XP_001695520; and
CrCYCD3, XP_001695469.
Immunoblotting
Separated proteins were transferred by semi-dry blotting
(Semi-dry blotter, Sigma-Aldrich, Prague, Czech Rep.) from the
gel onto a nitrocellulose membrane (pore size 0.2 mm, Protran,
Schleicher and Schuell, Kent, UK) (Towbin et al. 1979) at 1 mA
cm–2 for 1.5 h. The dried nitrocellulose membrane was blocked in
goat serum, 5% (v/v) solution in TBS-T buffer (20 mM Tris pH
7.5, 0.5 M NaCl, 0.05% Tween-20), overnight at 48C. Then it
was incubated with the primary antibody diluted with the
serum solution, rinsed three times with TBS-T, incubated with
the secondary antibody, and washed six times for 10 min
with TBS-T. Immunoreactive bands were detected by enhanced
chemiluminescence (ECL; Amersham, Munich, Germany). The
following primary antibodies were used: anti-FtsZ1 rabbit
antiserum (diluted 1 : 1,000) raised against the FtsZ1 protein
from the unicellular green alga Nannochloris bacillaris (Koide
et al. 2004), and the anti-cyclin IgG fraction of rabbit antiserum
(diluted 1 : 1,000) raised against cyclin B1 (Sigma C8831); for
antibody specificities see Results. The secondary antibody
was peroxidase-conjugated goat anti-rabbit IgG (Sigma A9169)
(diluted 1 : 100,000).
Microscopy
Observations in transmitted light and fluorescence microscopy were carried out using an Olympus BX51 microscope
equipped with a CCD camera (F-ViewII). A U-MWIBA2 filter
block (Ex/Em: 460–490/510–550 nm) was used for SYBR Green I
fluorescence and a U-MWIG2 filter block (Ex/Em: 520–550/
580 nm) for Cy3 and chlorophyll fluorescence.
Immunofluorescence
Cells were fixed with 4% formaldehyde freshly prepared
from paraformaldehyde in PEM buffer (50 mM PIPES, 5 mM
EGTA, 1 mM MgSO47H2O, pH 6.8), dehydrated by ethanol and
embedded in LR-White resin. Semi-thin sections were attached to
the slide.
Slides were incubated for 10 min in phosphate-bufferred
saline (PBS; 0.14 M NaCl, 2.7 mM KCl, 1.5 mM KH2PO4, 6.5 mM
Na2HPO412 H2O, pH 7.4) and blocked in 1% bovine serum
albumin (BSA)/PBS in a wet chamber for 1 h at 378C. They were
then incubated with primary antibody (anti-FtsZ1 or anti-cyclin B)
diluted 1 : 500 in 1% BSA/PBS for 1 h at 378C, washed three times
for 5 min with PBS, incubated with secondary antibody (Cy3conjugated goat anti-rabbit IgG; Jackson Immunoresearch,
Suffolk, UK) diluted 1 : 200 in 1% BSA/PBS for 1.5 h at 378C,
and washed three times for 5 min with PBS. Finally, the slides were
mounted with one drop of Vectashield Hard Set Mounting
Medium (Vector Laboratories, Peterborough, UK) and observed
using an Olympus BX51 microscope.
1815
Immunogold staining
Cells were fixed in 2.5% (v/v) glutaraldehyde in phosphate
buffer (160 mM Na2HPO4, 40 mM KH2PO4, pH 7.2), dehydrated
and embedded in LR White’s resin. Ultrathin sections picked up on
nickel grids were treated for 1.5 h with block solution [10% (v/v)
goat serum, 2% (w/v) BSA, 0.2% (w/v) glycine, 0.2% (w/v) NH4Cl
and 0.05% (v/v) Tween-20 in PBS buffer] and then incubated for
1.5 h with primary antibody against the FtsZ diluted 1 : 10 in PBS.
After washing six times for 2 min with PBS containing 0.05% (v/v)
Tween-20, the sections were incubated for 1 h with colloidal gold
10 nm particles conjugated with goat anti-rabbit antibody diluted
1 : 50 in PBS and washed six times for 2 min with PBS containing
0.05% (v/v) Tween-20. After 10 min fixation in 2.5% (v/v) glutaraldehyde in PBS and washing three times for 2 min with PBS and
three times for 2 min with H2O, the sections were dried at room
temperature overnight, stained with uranyl acetate for 30 min and
rinsed three times with 30% (v/v) ethanol. Dried sections were
stained with lead citrate (0.03 g dissolved in 20 ml of H2O and
0.2 ml of 10 N NaOH) for 20 min, rinsed with H2O and dried.
Finally, the sections were stabilized by carbon coating and examined using an electron microscope TEM JEOL 1010.
Funding
The Grant Agency of the Academy of Sciences of the
Czech Republic (grant Nos. B5020305, A500200614 and
A600200701); the Grant Agency of the Czech Republic
(grant No. 204/06/0102]; the Institutional Research Concepts (Nos. AV0Z50200510 and AV0Z60050516) funded by
the Academy of Sciences of the Czech Republic.
Acknowledgments
We are obliged to Professor S. Kawano (Tokyo University,
Kashiwa) for providing antibody raised against FtsZ protein
and to Dr. J. Nebesářová (South Bohemian University České
Budějovice) for her help with the electron microphotographs.
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(Received September 5, 2008; Accepted October 26, 2008)