P h ysiological and Transcriptomic Evidence for a C lose
C ou p lin g b etw een C hloroplast O ntogeny and C ell
Cycle Progression in the Pennate D iatom
Seminavis robusta1[c][w][OA]
Jeroen Gillard, Valerie D evos, Marie J.J. H uysm an, Lieven D e Veylder, Sofie D 'H ondt, Cindy Martens,
Pieter Vanorm elingen, Katrijn Vannerum, Koen Sabbe, Victor A. Chepurnov2, D irk Inzé*,
M arnik V uylsteke, and Wim Vyverman
L aboratory of P rotistology a n d A quatic Ecology, D epartm ent of Biology, G hent U niversity, B-9000 Gent,
Belgium (J.G., V.D.,
S.D., P.V., K.V., K.S., V.A.C., W.V.); a n d D ep artm en t of P lant System s Biology,
F landers In stitu te for Biotechnology, a n d D ep artm en t of M olecular Genetics, G hent U niversity,
B-9052 G ent, Belgium (J.G., V.D., M.J.J.H., L.D.V., C.M., K.V., D.I., M.V.)
Despite the growing interest in diatom genomics, detailed tim e series of gene expression in relation to key cellular processes are still
lacking. Here, w e investigated the relationships betw een the cell cycle an d chloroplast developm ent in the pennate diatom Seminavis
robusta. This diatom possesses tw o chloroplasts w ith a well-orchestrated developm ental cycle, com m on to m any pennate diatoms.
By assessing the effects of induced cell cycle arrest w ith microscopy an d flow cytometry, w e found that division and reorganization
of the chloroplasts are initiated only after S-phase progression. Next, w e quantified the expression of the S. robusta FtsZ hom olog to
address the division status of chloroplasts during synchronized grow th and m onitored microscopically their dynam ics in relation to
nuclear division and silicon deposition. We show that chloroplasts divide an d relocate during the S/G 2 phase, after w hich a girdle
b an d is deposited to accom modate cell growth. Synchronized cultures of two genotypes w ere subsequently used for a cDNAam plified fragm ent length polym orphism -based genom e-wide transcript profiling, in w hich 917 reproducibly m odulated
transcripts w ere identified. We observed that genes involved in pigm ent biosynthesis an d coding for light-harvesting proteins
w ere up-regulated during G 2/M phase and cell separation. Light and cell cycle progression w ere both found to affect fucoxanthinchlorophyll a/c-binding protein expression and accum ulation of fucoxanthin cell content. Because chloroplasts elongate at the stage
of cytokinesis, cell cycle-modulated photosynthetic gene expression and synthesis of pigm ents in concert w ith cell division might
balance chloroplast growth, w hich confirms that chloroplast biogenesis in S. robusta is tightly regulated.
D iatom s, an extraordinarily diverse g roup of heterokontophyte m icroalgae (Kooistra et al., 2003), dom i1 This w ork was supported by the European Union Framework
Program 6 Diatomics project (grant no. LSHG-CT-2004-512035), the
Research Fund of Ghent University (Geconcerteerde Onderzoeksac­
ties grant no. 12050398), the Belgian Coordinated Collections of
Microorganisms Culture Collection project (grant no. C 3/00/14;
Belgian Federal Science Policy), Research Foundation-Flanders
(postdoctoral fellowship to F.D.V.), and the Institute for the Promo­
tion of Innovation through Science and Technology in Flanders
(predoctoral fellowships to J.G., V.D., M.J.J.H., C.M., and K.V.).
2 Present address: SBAE Industries NV, Oostmoer 22A, 9950
Waarschoot, Belgium.
* Corresponding author; e-mail dirk.inze@psb.ugent.be.
The authors responsible for distribution of materials integral to
the findings presented in this article in accordance w ith the policy
described in the Instructions for Authors (www.plantphysiol.org)
are: Wim Vyverman (wim.vyverman@ugent.be) and Jeroen Gillard
(jeroen. gillard@ugent.be).
[CI Some figures in this article are displayed in color online b u t in
black and white in the print edition.
[w] jp g on[¡ne version 0f
article contains Web-only data.
[oaj Qpen Access articles can be viewed online w ithout a sub­
scription.
www.plantphysiol.org/cgi/doi/10.1104/pp.108.122176
1394
nate the p rim ary p roduction of m any m arine and
freshw ater ecosystem s (G ranum et al., 2005). Based on
the shape an d structure of their unique siliceous cell
walls, tw o m ajor architectural types are recognized:
"centric" diatom s, a paraphyletic group w ith radially
p attern ed valves, an d "pennate" diatom s, a m onophyletic group characterized b y a feather-like valve struc­
ture. W hole-genom e sequencing of Thalassiosira
pseudonana (A rm brust et al., 2004) an d Phaeodactylum
tricornutum (Bowler et al., 2008), representatives of
centric an d pennate diatom s, respectively, has re­
vealed that they com bine plant- an d anim al-like char­
acteristics an d possess m any genes that are com pletely
un k n o w n in other organism s. These genom e studies
enable the unraveling of the genetic basis of the unique
properties underlying the ecological an d evolutionary
success of diatom s. D iatom s have long been know n for
their extrem ely hig h photosynthetic efficiency: com ­
p ared w ith other photosynthetic eukaryotic unicells of
com parable size, diatom s have the highest grow th
rates (Banse, 1982; R aven and Geider, 1988; Sarthou
et al., 2005) an d an unusually high photosynthetic
flexibility that is essential for coping w ith large habitatintrinsic fluctuations in irradiance (Lavaud et al.,
Plant Physiology, N ovem ber 2008, Vol. 148, p p . 1394-1411, w w w .plan tp h y sio l.o rg © 2008 A m erican Society of P lan t Biologists
The Cell a n d C hlo ro p last Cycle of Seminavis robusta
2004). Their hig h grow th rates m ight be attributed to
their h igh Rubisco carboxylase efficiency an d the
p u tativ e presence of an alternative C 4-photosynthetic
pathw ay, enabling tem porary storage of carbon for
use at tim es of low light irradiance (Riebesell, 2000;
W ilhelm et al., 2006; Roberts et al., 2007). The photo­
synthetic flexibility of diatom s is related to their high
capacity for energy dissipation through n o nphoto­
chem ical quenching, w hich can reach a 5-fold higher
level th an th at in plants (Ruban et al., 2004).
As in other groups of heterokontophyte algae, d ia­
tom chloroplasts originate from a secondary endosym biosis, probably the engulfm ent of a red alga b y a
heterotrophic eukaryote (M cFadden, 2001; Falkowski
et al., 2004). As a result, diatom plastids are su rro u n d ed
b y four m em branes instead of the usual tw o, typical for
plan ts an d green algae. M oreover, the outerm ost p air of
m em branes is connected w ith the nuclear envelope in a
n u m b er of diatom s (Stoerm er et al., 1965; Daw son,
1973; Flashim oto, 2005). These characteristics have
im plications for chloroplast protein targeting pathw ays
(A pt et al., 2002; Kilian an d Kroth, 2005) an d possibly
for its division m echanism an d transm ission to d au g h ­
ter cells (Flashim oto, 2005). W hile the chloroplast u l­
trastru ctu re ap pears to be fairly uniform in diatom s,
there is a w id e variation in chloroplast m orphology as
w ell as in the n u m b er of chloroplasts an d their arrange­
m ent w ith in the cells. The chloroplasts of polyplastidic
diatom s, w hich com prise m ost centric diatom s b u t also
several genera of p ennate diatom s, have a very sim ple
m orphology (for review, see M ann, 1996). In contrast,
chloroplasts of m ono-, di-, and tetraplastidic diatom s
often h ave m ore elaborate shapes an d can undergo
com plex changes in m orphology an d arrangem ent
before a n d /o r after cytokinesis. C hloroplast division
involves constriction of the p aren t chloroplasts into
tw o m ore or less equally sized d au g h ter chloroplasts.
Two types of chloroplast division are recognized
am ong diatom s, referred to as autonom ous an d im ­
p o sed division (M ann, 1996). In the autonom ous type,
believed to be the prim itive condition, the chloroplast
constricts into tw o w ith o u t the obvious involvem ent of
any other organelle. In im posed division, w hich ap ­
pears to h ave evolved several tim es in different line­
ages of p en n ate diatom s, chloroplast division occurs
synchronically w ith the form ation of the cleavage fur­
row. In p lants and som e other eukaryotes, chloroplast
division is orchestrated b y a prokaryote-derived divi­
sion m achinery, w hose regulation is not fully u n d er­
stood (O steryoung an d N unnari, 2003; M argolin, 2005;
A dam s et aí., 2008) b u t should som ehow be coordi­
n ated w ith the regulation of the cell cycle m achinery
th at paces the cell division rate on the su rro u n d in g
environm ents an d coordinates critical processes such
as cell grow th, karyokinesis, an d cytokinesis (De
Veylder et al., 2007).
Environm ental factors m ostly im pinge on cell cycle
control th ro u g h cell cycle checkpoints at the G l /S or
G 2 /M cell cycle phase transitions. M any universal
core cell cycle genes that regulate these transitions
P la n t P h y sio l. Vol. 148, 2008
have been found in the tw o sequenced diatom ge­
nom es (A rm brust et al., 2004; Bowler et al., 2008), b u t
diatom -specific genes an d gene regulations m ay con­
tribute to the unique features that distinguish diatom s
from other algae (M ontsant et al., 2007; M.J.J. Fluysm an,
A. De M artino, C. M artens, E. Rayko, J. Gillard, M.
Fleijde, B. M athieu, A. M eichenin, A. M ontsant,
K. V andepoele, Y. Van de Peer, L. De Veylder, D.
Inzé, C. Bowler, an d W. Vyverm an, u n p ublished data).
C hloroplast developm ent is difficult to address in
polyplastidic cells, such as centric diatom s an d cells
of higher plants (Pyke, 1999). Flowever, the observed
coordination of cbloroplast developm ent w ith cell
cycle progression in m any p ennate diatom s creates
an oppo rtu n ity to address m ore easily chloroplast
developm ent in relation to other cellular processes,
because if chloroplast ontogeny is regulated b y the cell
cycle, the controlled progression of chloroplasts can be
stu d ied in synchronized cultures.
Tiere, w e com bined physiological experim ents, cytological observations, an d a cD N A -am plified frag­
m ent length polym orphism (AFLP)-based genome-wide
transcriptom e analysis to identify cell cycle-dependent
checkpoints in chloroplast developm ent an d gene
expression d u rin g synchronized grow th. As a m odel
species, w e used Seminavis robusta (Danielidis and
M ann, 2002), a representative of the diverse an d eco­
logically im portant group of p ennate N aviculaceae. As
in m ost genera of this family, the tw o chloroplasts in S.
robusta divide in an autonom ous m anner an d m ove
from the girdle to the valves an d back, once d u rin g
each cell cycle (C hepum ov et al., 2002; Supplem ental
Fig. SI). S. robusta is particularly w ell suited for cytological studies because of its large size (up to 100 /rm)
and, being a benthic species adh erin g to surfaces
(Thom pson et al., 2008), the easy an d nonintrusive
observation of cellular behavior. M oreover, sexual
reproduction can be controlled, resulting in the first
diatom pedigree th at currently com prises approxi­
m ately 110 clones (C hepum ov et al., 2008). Therefore,
this species offers interesting perspectives for func­
tional genom ics studies as w ell as for forw ard genetics
to investigate key life history traits, including the
dynam ics of their photosynthetic ap p aratu s an d its
interaction w ith other cellular processes an d the envi­
ronm ent.
RESULTS
Cell and Chloroplast D ivision Are Both Arrested in
Response to Darkness and Cell Cycle
Inhibitor Treatments
U sing light deprivation an d tw o cell cycle inhibitors,
w e investigated w hether a relationship exists betw een
the cell cycle checkpoints and the chloroplast division
cycle. For light deprivation, exponentially grow ing
cultures w ere transferred to com plete darkness for 24
h an d com pared w ith light-grow n control cultures by
1395
G illard et al.
flow cytom etry (Fig. IA). The tetraploid p eak (G2+M
phase) presen t in the light-grow n cultures w as absent
in the 24-h dark-treated cultures, indicating that in
the latter the m ajority of cells w ere arrested in the G1
p h ase of the cell cycle. Microscopically, the lightgrow n cultures consisted of b o th dividing an d no n d i­
v id in g cells (Fig. IB). In contrast, the dark-arrested
cultures only contained n ondividing cells (Fig. 1C).
These cells have their tw o chloroplasts u n d iv id ed and
located at the girdle sides of the cell (Fig. 1, D-G ). In
m ost cases, the ventral chloroplast h ad four subcentral
lobes that, across the valves, reached the dorsal side of
the cell (Fig. 1, F an d G). Occasionally, som e postcytokinetic do u b let cells could also be found in the darkarrested cultures. These doublet cells are at a stage
after cytokinesis b u t before cell separation, d u rin g
w hich the cell is devoted to frustule form ation
(Coombs et al., 1967a). Microscopically, the doublet
cells in dark-arrested cultures contained the G1 type of
girdle-located u n d iv id ed chloroplasts.
Next, w e stu d ied chloroplast behavior u p o n treat­
m ent w ith chem ical cell cycle inhibitors. First, hydroxy­
urea (HU) w as ap plied to exponentially grow ing
cultures, thereby inhibiting S-phase progression by
^
Before
treatment
B
1
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Cytological Changes during Cell Cycle Progression in
Synchronized Cultures
.
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d a rk n e s s I
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DNA
Figure 1 . R esponse of 5. robusta to dark treatm ents. A, Flow cytom etric
DNA c o n te n t histogram s of an ex p o n en tially grow ing cu ltu re before
transfer to d ark n ess (top), after 2 4 h of dark incubation (m iddle), an d
after 24 h of light (bottom ). B to D an d F, Light m icro sco p ic p hotographs
of th e 24 -h light-treated cu ltu re (strain F,-8B; B), th e 24-h d ark-treated
cu ltu re (strain F,-8B; C), a 24-h dark-arrested cell in girdle view with
focus on th e ventral ch lo ro p last (strain F2-31B; D), a n d a 24-h darkarrested cell in valve view (strain F2-31B; F). The ch loroplasts a p p e a r
brow nish, a n d th e cell w all is visible d u e to its high contrast. E an d G,
C onfocal flu o rescen ce m icroscopic p h o tographs of cells from darkarrested cu ltu res (strain F2-31B) in w h ich th e ch lo ro p lastid ic signal is
red (autofluorescence), th e n u clear signal is green (SYBR Safe), an d th e
cell w all signal is b lu e (PDM PO; Lysosensor). C hloroplasts of darkarrested cu ltu res are u n d ivided an d pressed against the girdle. The
ventral ch lo ro p last has four subcentral lobes th at ex ten d acro ss th e
valve to w ard th e dorsal sid e of th e cell. Bars = 10 fim .
1396
depleting the cell of d eox y nucleoside triphosphates
(Young and H odas, 1964). A com plete S-phase arrest of
cell division b y H U w as confirm ed b y flow cytom etry
after 72 h of treatm ent (Supplem ental Fig. S2), w hile
the cultures rem ained healthy, because cell grow th
restarted after the inhibitor h ad been w ashed aw ay
(data no t show n). The cells in these S-phase-arrested
cultures all contained girdle-located u n d iv id ed chlo­
roplasts as in dark-arrested G l-p h ase cells, b u t the
subcentral lobes w ere not observed (Fig. 2A). From
these observations, w e conclude th at chloroplast divi­
sion an d m ovem ents do not take place before the S
phase. How ever, these results should be interpreted
w ith caution: because H U depletes the cell's deoxynucleoside triphosphate pool, chloroplast DNA repli­
cation m ight be affected b y the d ru g and, thus,
inhibition of plastokinesis m ight be a direct effect of
the chloroplast's inability to replicate its DNA. To
validate these findings, aphidicolin, another cell cycle
S-phase inhibitor that blocks the nucleus-specific DNA
polym erase a (Ikegam i et al., 1978), w as applied on
dark-arrested cultures that w ere reillum inated im m e­
diately after addition of the drug. M icroscopic analysis
after 24 h revealed th at treated cells d id not divide and
contained u n d iv id ed chloroplasts located at the girdle
sides (Fig. 2, B-D), w hile control cultures div id ed
normally. In contrast w ith H U -arrested cells, ventral
chloroplast lobes w ere observed d u rin g aphidicolin
arrest (Fig. 2D).
Based on the uniform cell cycle arrest in darkarrested cultures, w e established a synchronization
procedure to stu d y cell cycle-m odulated processes. To
this end, exponentially grow ing cultures (12 h:12 h
lighfldark) w ere transferred into darkness for a period
of 24 h an d reillum inated for 12 h. Two m onoclonal
strains, designated Fj-SB an d FX-9A, w ere synchro­
nized in this m anner an d w ere used for the transcriptom e analysis described below. Reillum ination
resulted in the synchronous reactivation of cell cycle
progression, starting from the G1 phase of the darkarrested cells. Synchrony w as evaluated b y estim ating
the am ount of dividing cells at h o u rly intervals,
com plem ented b y observations of chloroplast dy n am ­
ics (Fig. 3). D ividing cells w ere operationally defined
to include only doublet cells, characterized b y the
presence of a cleavage furrow, w hich is visible as an
area of high contrast separating the chloroplast pairs
(see S upplem ental Fig. S3 for an illustration ot the
counting procedure). Im m ediately u p o n reillum ina­
tion, all cells w ere in the G1 phase, containing u n d i­
v ided chloroplasts as previously show n. A fter 4 h, few
cells w ith div id ed chloroplasts ap peared in the cul­
tures, an d after 6 h, som e cells w ere dividing. From 6 h
onw ard, the cultures contained S-, G2-, an d M -phase
cells, in w hich the p roportion of dividing cells p ro ­
gressively increased until 50% at the 9-h sam pling
P la n t P h y sio l. Vol. 148, 2008
The Cell a n d C hloroplast Cycle of Seminavis robusta
D
C
f
♦
%
Figure 2. 5. robusta cells in cell cycle inhibitor-treated cultures. A an d B, L ight-m icroscopic photographs. C an d D, F luorescence
m icro sco p ic photographs. A, H U -treated cell (strain F,-31 B) arrested during S phase. B to D, A p hidicolin-treated cell (strain F,8B) in girdle view (B) w ith focus on th e ventral ch lo ro p last (C) a n d on th e dorsal ch lo ro p last (D). In ea c h case, both chloroplasts
are u n d iv id ed an d lo cated against th e valves. The difference in cell m orphology b etw een cells in A a n d B to D is d u e to the
different av erage cell sizes of th e strains used. Bars = 10 juni.
point. At 9 h, the am ount of div id in g cells decreased as
a consequence of d au g h ter cell separation an d initia­
tion of the second cell division cycle.
The phenotypic cell cycle events d u rin g synchroni­
zation of the cultures w ere further characterized w ith
confocal laser-scanning m icroscopy an d an ap p ro p ri­
ate set of stains for the nucleus (SYBR Safe) an d the cell
w all (2-(4-pyridyl)-5-((4-(2-dimethylaminoethylaminocarbamoyl)methoxy)phenyl)oxazole [PDMPO]). Besides
fluorescent cell w all labeling in diatom s (Shimizu
et al., 2001; Leblanc an d H utchins, 2005), PDM PO
visualizes acidic organelles (D iw u et al., 1999) and
m onitors the location of new ly incorporated silica.
As sh o w n p reviously b y C h epum ov et al. (2002),
chloroplast division occurs b y central constriction (Fig.
4A), an d u p o n com pletion of constriction of the dorsal
(Fig. 4B) an d v entral (Fig. 4C) chloroplasts, the d au g h ­
ter chloroplast p airs m ove bodily in opposite direc­
tions, describing a circular m otion along the p eriphery
of the cell, until they position against the valves (Fig. 4,
D an d E; see also S upplem ental Fig. SI). By com paring
these cells w ith the dark-arrested cells in valve and
girdle view s (com pare Fig. 1G w ith Fig. 4D an d Fig. IE
w ith Fig. 4E), the difference in location betw een u n d i­
v id ed an d div id ed chloroplasts could easily be seen.
D uring this "reorganized chloroplast configuration"
stage, the nucleus, positioned in the center of the cyto­
plasm , w as still u n d iv id ed (Fig. 4E). Taking into con­
sideration th at the chloroplasts divide after S-phase
initiation, this show s that plastokinesis an d subse­
q u en t chloroplast relocation occur d u rin g S / G2 phase
an d are fully established before M phase.
Before signs of m itosis appeared, b u t after chloro­
plast rearrangem ent, the first PDM PO signal w as
detected as an elliptically shaped thin b an d (Fig. 4F).
This signal probably represents a girdle b an d (Sup­
plem ental Fig. S4) th at is ad d ed to the girdle as a result
of cell grow th. C hloroplast reorganization w as fol­
low ed b y karyokinesis (Fig. 4G) an d cytokinesis (Fig.
4, H -K ). Early on d u rin g cytokinesis, PDM PO fluo­
rescence w as initially detected inside tw o "vacuole­
like" com partm ents located against the division p lane
P la n t P h y sio l. Vol. 148, 2008
ru n n in g from pole to pole (Fig. 4H), w hile a PDM POstained cell w all w as no t yet visible. Since the fluores­
cence em ission an d intensity of PDM PO typically shift
w h en the intracom partm ental silica concentration in ­
creases above 3.2 rriM (Shim izu et al., 2001; H azelaar
et al., 2005), the observed fluorescence of PDM PO in
these com partm ents could be paralleled w ith silicon
influx in p reparation for silica deposition d u rin g the
subsequent form ation of the frustule. The deposition
of cell w all m aterial w as observed as a straight line
th at ran betw een the tw o d au g h ter nuclei an d located
60
FT8B
FT9A
50
G2/M
40
30
20
M/G1
10
0
0
1
2
3
4
S
6
r
8
9
10
11
12
t im e (h )
Figure 3. Evaluation of th e synchrony of cell a n d ch lo ro p last division in
5. robusta sy n c h ro n ized strains F,-8B an d F,-9A. The propo rtio n of
dividing cells is presen ted a s a function of tim e, during reillum in atio n of
th e 24-h dark-arrested cultures. D ata are m ean s ± se of six m icro sco p ic
fields (0.09 m m 2). The p red o m in an t m orphological stages in ea c h cell
cy cle p h ase are represented from light to dark gray in this order:
undivided chloroplasts, dividing chloroplasts, dividing cells, an d sep ­
arating cells. G1 co rre sp o n d s to th e p h ase in w h ich cells w ith u n d i­
v ided g ird le-lo cated ch loroplasts are present. The asterisk d en o tes th e
first observ ed cells w ith divided ch loroplasts an d also ap p ro x im ates th e
m o m en t of S -phase initiation, acco rd in g to th e transcriptional in d u c­
tion of th e MCM5 prereplication factor (Supplem ental Fig. S8). G2/M
co rre sp o n d s to the p h ase in w h ich dividing cells w e re increasingly
represented w ith a m axim um in th e a m o u n t of dividing cells at 9 h.
M /G l co rre sp o n d s to th e stage of cell separation.
1397
G illard et al.
Cell Cycle-Modulated Expression of the S. robusta
Chloroplast D ivision Protein FtsZ
Figure 4 . C ytological ev en ts durin g sy n c h ro n ized grow th of 5. robusta
o b serv ed by co n fo cal flu o rescen ce m icroscopy. The chloroplastidic
signal is red (autofluorescence), th e n u clear signal is green (SYBRSafe),
a n d th e silica tracer signal is b lu e (PD M PO; Lysosensor). In p h o to ­
graphs A, C, D, a n d H, th e transm ission light ch an n el w as perm itted in
o rd er to v isu alize th e o u tlin e of th e cell. B to D, C ells show n in valve
view. A, E to j, a n d L, C ells show n in girdle view. A, Cell w ith focus on a
dividing dorsal ch lo ro p last, displaying th e central constriction (arrow).
B, Cell w ith th e dorsal ch lo ro p last divided. C, Cell w ith both c h lo ro ­
plasts d iv id ed . D a n d E, Cell w ith fully rotated, valv e-lo cated c h lo ro ­
plasts an d co n tain in g an u ndivided nucleus. F, Cell w ith valve-located
ch lo ro p lasts an d sh o w in g a b lu e b an d in th e m id d le of th e cell,
ex ten d in g from p o le to p o le. This b an d p robably represents a girdle
b an d (Supplem ental Fig. S4). G, Cell ju st after karyokinesis. Each
d au g h ter n u cleu s is situ ated on a different sid e of th e future division
p lan e. H, Cell durin g cytokinesis. The division of th e protoplast is
a p p a re n t by th e cleav ag e furrow, visible as a line of higher contrast
ru nning from p o le to p o le an d in b etw een both d au g h ter nuclei. The
PDM PO signal is confined to tw o "vacuole-like" com partm ents; as such,
it ap p ears that deposition of silica at th e site of the cleavage furrow has
not yet begun. I, Dividing cell during frustule form ation. D eposition of
silica into th e frustule is visible as a line running from pole to pole and
situated betw een th e chloroplast pairs a n d daughter nuclei. J, Dividing
cell w ith chloroplasts m oving back from th e valves tow ard the girdle. K,
S eparating dau g h ter cells in slightly tilt valve view w ith focus on the
dorsal chloroplasts, w hich are com pletely covering th e girdle area. From
th e ventral chloroplasts, only th e subcentral lobes are visible. L, A new ly
divided cell w ith th e new ly form ed valve stained w ith PDPM O. The
o th er valve is inherited from the m other cell. Bar = 10 juni.
The m icroscopically derived indications of cell cycle
p h ase-dependent chloroplast division w ere validated
b y transcript quantification of the bacterial cell divi­
sion hom olog F tsZ in synchronized S. robusta cultures.
Therefore, a F tsZ gene fragm ent w as am plified w ith a
set of nested degenerate prim ers, cloned, an d se­
quenced, enabling the design of a specific p rim er
p air suited for real-tim e (RT) quantitative (Q) PCR
(Supplem ental Fig. S5). Three constitutively expressed
genes w ere used for d ata norm alization (see "M ate­
rials and M ethods"), an d RT-Q-PCR of the S. robusta
F tsZ ortholog w as done on tw o replicated synchroni­
zations of strain Fj-813 (Fig. 5). Both show ed that FtsZ
expression is m odulated d u rin g synchronization. Its
expression 5 to 8 h after reillum ination increased 2.5to 4-fold w hen com pared w ith its average expression
betw een 0 to 4 h. In bo th replicates, FtsZ w as m axi­
m ally expressed at 7 h after darkness, being 2 h before
m ost cells w ere dividing.
cDNA-AFLP Expression Profiling during Cell
Cycle Progression
In parallel w ith the above-described sam pling (Fig.
3) for cytological observations, sam ples w ere taken at
1-h intervals to conduct a cDNA-AFLP-based genom ew ide transcript profiling to identify m odulated gene
expression profiles correlated w ith the cytological
changes d u rin g the cell cycle. The com plete d ata set,
including annotations an d expression data, is avail­
able online (Supplem ental Table SI).
The cDNA-AFLP analysis resulted in 2,908 transcriptderived fragm ents (TDFs) that w ere scored quantita­
tively. A considerable am ount of expression variation
betw een the tw o strains originated from genotypedependent DNA polym orphism s in the transcripts, re­
sulting in the absence / presence of cDNA-AFLP fragments
35.0
30.0
•2
ui
25.0
£
20.0
♦
*
FtsZ expression - replicate
FtsZ expression - replicate
Proportion of dividing cells
Proportion of dividing cells
1
2
- replicate 1
- replicate 2
O
c
<n
1£0
ÜJ
u
at the site of the previously observed cleavage furrow
(Fig. 41). O nly after this initial deposition of silica,
w hile b o th cells w ere still attached to each other, the
chloroplasts m oved back from the valves to the ventral
an d dorsal girdle sides (Fig. 4J). D uring this last
relocation event, each chloroplast elongated until it
com pletely covered the girdle area. Cell division w as
finalized w h en d au g h ter cells separated (Fig. 4K),
creating tw o n ew cells containing one unstained valve,
inherited from its m other, and one new ly b u ilt valve
stained w ith PDM PO (Fig. 4L).
1398
o' mo
5.0
0.0
0
1
2
3
4
5
6
7
8
9
10
11
12
tim e (h)
Figure 5. Expression o f th e S. robusta FtsZ o rth o lo g as a functio n of
tim e after reillum ination durin g tw o rep licate sy n c h ro n izatio n s o f strain
F.,-8B. T he proportion of dividing cells is p resen ted as a function of tim e
d uring reillum ination to g eth er w ith th e relative expression valu es of
FtsZ. RT-PCR d a ta w e re n o rm aliz ed ag ain st th re e cD N A -A FLP-acquired
referen ce g enes. [See o n lin e article for c o lo r version of this figure.]
P la n t P h y sio l. Vol. 148, 2008
The Cell a n d C hlo ro p last Cycle of Seminavis robusta
(see Supplemental Fig. S6 for an example of a cDNA-AFLP
electropherogram; Vuylsteke et al., 2006). These expression
profiles are of no value for expression analysis and were
removed from the data set; only expression profiles that
w ere highly reproducible (Pearson's correlation, P < 0.05)
across the two strains were kept for further analysis. In this
manner, 955 TDFs were selected and used for adaptive
quality-based clustering (De Smet et al., 2002). With highstringency settings (see "Materials and M ethods"), 917
TDFs w ere g ro u p ed in nine expression clusters (desig­
n ated C1-C9; S upplem ental Fig. S7). Figure 6 show s the
expression p atterns of all these TDFs d u rin g the cell
cycle of b o th genotypes b y hierarchical clustering
(Eisen et al., 1998). TDFs in clusters C l an d C2 h ad
their m axim al expression d u rin g darkness (0 h), and
the level of expression decreased drastically w ithin the
first few h o u rs after darkness. Expression of TDFs in
clusters C3 an d C4 w as induced after illum ination,
p eaking at 2 an d 3 h, respectively, after darkness.
Average p eak expression in cluster C5 occurred at 4 to
6 h after darkness, corresponding w ith the initiation of
chloroplast division an d the appearance of the first
d iv id in g cells. This cluster contained all identified
TDFs w ith nucleotide-binding regions (Table I). Two
of these w ere the universal S-phase-specific genes,
histone FI3 an d MCM5. Flistone FI3 (Sr025; Kapros
et al., 1992; M enges et al., 2002) w as m axim ally ex­
p ressed at 7 h (Supplem ental Fig. S8). The universal
DN A replication licensing factor MCM5 (Sr027) h ad a
discrete expression at 4 h. This latter gene is specifically
involved in the initiation of DNA synthesis an d u n ­
w in d in g of the D N A at the replication forks an d has
b een show n to be tem porally expressed at the G l/S
transition (Tsuruga et al., 1997; Takisaw a et al., 2000).
Therefore, the expression of the MCM5 ortholog rep­
resents a good m arker for S-phase initiation an d w as
u sed to dem arcate the G1 an d S phases d u rin g syn­
chronization. W hile genes in cluster C6 w ere discretely
expressed at 7 h, genes in cluster C7 h ad a broader
expression range. They w ere induced at 6 h, in corre­
spondence w ith the first occurrences of dividing cells,
until the 12th h at the m om ent of cell separation. Genes
in this cluster w ere expressed sim ultaneously w ith the
enrichm ent of G 2-phase cells an d dividing cells. They
consisted of an early an d late expression subgroup,
indicated as C7-Early (C7-E), containing genes induced
at 6 h, an d C7-Late (C7-L), containing genes induced at
8 h. C lusters C8 an d C9 com prised a rather sm all
n u m b er of expression profiles, peaking at three (3, 7,
an d 10-12 h) an d tw o (0 an d 12 h) nonconsecutive tim e
points, respectively.
In total, 378 TDFs (41%) w ith reproducible expres­
sion profiles across the tw o genotypes w ere selected
for sequencing. Since w e w ere interested p rim arily
in the progression of the cell cycle, proportionally
m ore TDFs w ere selected from clusters C5 an d C7, in
w hich expression w as up-regulated after reillum ina­
tion (Supplem ental Table S2). G ood-quality sequences
w ere found in 322 TDFs (85%), of w hich 100 h ad
significant (E-value < IO-3) sim ilarity (Table I) w ith
P la n t P h y sio l. Vol. 148, 2008
genes in the in-house-constructed database (see "M a­
terials an d M ethods"). Expression patterns of these
annotated TDFs are presented in Supplem ental Figure
S9. Seventy-two TDFs show ed hom ology w ith a gene
w ith a putative allocated function, 18 w ere hom olo­
gous w ith sequences w ith o u t an allocated function
(hypothetical proteins), an d 10 TDFs m atched w ith
diatom orp h an genes, for w hich no hom ologous coun­
terparts exist (Table I). These latter genes w ere all
confined to the late phases of the cell cycle, indicating
th at diatom -specific genes m ight be functionally asso­
ciated w ith m itosis an d cell separation. Based on the
gene hom ology an d the m ap p in g of Gene O ntology
(GO) labels an d InterPro dom ains (see "M aterials and
M ethods"; S upplem ental Tables S3 an d S4), the TDFs
w ith annotated functions w ere classified into 11 dis­
tinct functional groups (Table I).
Modulated Expression of Genes Implicated in
Cell Metabolism
The m ajority of the GO-labeled TDFs w ere active
in cellular an d m etabolic processes (Supplem ental
Fig. SIO). TDFs involved in protein biosynthesis w ere
d om inant in S-phase cluster C5. Two genes w ere found
to be involved in am ino acid synthesis, nam ely
N -acc ty 1- y-gl u ta m y 1-ph osph a te reductase (Sr029) and
the bifunctional ATP sulturylase-adenosine 5 '-p h o sphosulfate kinase (Sr033). Various com ponents of the
translation m achinery dom inated this cluster: three
TDFs encoded ribosom al proteins (Sr030, Sr031, and
Sr032) an d one encoded an aspartyl-tRN A synthetase
(Sr034). A translation initiation factor 3 (Sr050) w as
induced shortly after these genes, in cluster C7-E. In
addition, nine posttranslational m odification proteins
w ere identified (SrOOl, Sr002, Sr009, SrOlO, Sr028,
Sr047, Sr048, Sr049, an d Sr073). Four of these genes
w ere expressed in cluster C7-E b u t tw o of them , a ClpX
hom olog (SrOOl; W eart et al., 2005) an d a ubiquitinconjugating enzym e (Sr002), w ere highly expressed in
the G l-p h ase cluster C2 w hen cells w ere released
from the d ark an d w ere ra p id ly dow n-regulated after
the first h o u r of light. A series of hypothetical proteins
w ith undefined function displayed an identical regu­
lation.
Several genes of the chloroplast-localized fatty acid
biosynthetic pathw ays (Ohlrogge an d Browse, 1995)
w ere m odulated d u rin g different phases of the cell
cycle. /3-H y d ro X y a c y 1- A C P - d eh y d ra ta s e (Sr023) w as
identified in cluster C5, w hile tw o fatty acid desaturases (Sr044 an d Sr045) w ere expressed som e tim e later
in cluster C7-E. Transcription of glycerol-3-phosphate
dehydrogenase (Sr091), also referred to as dihydroxyacetone p h osphate reductase (Gee et al., 1988), w as
activated d u rin g cell separation in cluster C9, starting
10 h after reillum ination.
To drive the anabolic pathw ays of protein an d fatty
acid synthesis, the cell d epends on an acetyl-CoA pool,
w hich can be pro d u ced b y the aerobic oxidation of
carbohydrates (Fernie et al., 2004). In this respect, the
1399
G illard et al.
glycolytic enzym e enolase (Sr017) w as expressed in
cluster C5 together w ith the m itochondrion-localized
enzym e isocitrate dehydrogenase (Sr015) an d a m i­
tochondrial phosphate-carrier protein (Sr016), b oth
needed d u rin g active citric acid cycling.
Two TDFs (Sr051 and Sr095), a subunit of the vacuolar
(V)-type H +-ATPase and a V-type H +-pyrophosphatase,
are know n to play a role in vacuolar transport. M ore
particularly, they are responsible for the acidification
or m aintenance of the acidity of organelles (M aeshim a,
2001). Sr051 w as activated d u rin g G 2 /M phase in
cluster C7-E, w hile Sr095 w as initially expressed
slightly d u rin g darkness an d again ab u ndantly d u rin g
cell separation (cluster C9).
Modulated Expression of Photosynthesis-Related Genes
and Genes Involved in Chloroplast Movement
F1-8B
F l -9A
Figure 6 . H ierarchical clu ste red expression profiles of 917 TDFs
rep ro d u cib ly m o d u lated during th e cell cy cle of sy n c h ro n ized cultures
of strain F,-8B an d F,-9A . C lustering w as perform ed using TMEV
softw are (Saeed e t al., 2003) an d the hierarchical clustering algorithm
(Eisen et al., 1998). Each row represents a tag w ith th e relative transcript
accu m u latio n p atterns sh o w n over 12 co n secu tiv e tim e points (col­
um ns) after reillu m in atio n of dark-arrested cultures. Yellow an d blue
c o lo r in tensities reflect up- an d dow n-regulation of g e n e expression
relative to a range of +3 to - 3 , respectively; gray represents m issing
d ata. C luster n am es (in a c c o rd a n c e w ith ad ap tiv e qu ality -b ased clus-
1400
Fifteen TDFs w ere assigned to the functional cate­
gory of photosynthesis (Table I). U sing a y2 correlation
test (Supplem ental Table S5), this functional category
w as found to be dom inantly expressed (P < 0.001) in
cluster C7-L: one TDF w as found in C7-E an d tw o in
C9. N ine copies of the chrom ophyte-specific fucoxanthin-chlorophyll a/c-binding proteins (FCPs; G reen
an d D um ford, 1996) w ere identified, an d they w ere
com plem ented w ith four TDFs involved in the bio­
synthesis of their b o u n d photosynthetic pigm ents:
porphobilinogen synthase (Sr063), p rotoporphy rin o ­
gen IX oxidase (Sr068), an d proto p o rp h y rin IX m ag­
nesium chelatase subunit FI (Sr046). They all encoded
enzym es from the chlorophyll a/c biosynthetic p a th ­
w ay (von W ettstein et al., 1995). In addition, a
¿(-carotene desaturase (Sr062) w as identified, being
an interm ediate enzym e in the biosynthetic path w ay
of /1-carotene and other carotenoids, such as fucoxanthin (Wilhelm et al., 2006). Several Calvin cycle enzymes
w ere also expressed in C7-L and C9: phosphoribulokinase (Sr070), transketolase (Sr093), and glyceraldehyde3-phosphate dehydrogenase (Sr089). Flowever, because
glyceraldehyde-3-phosphate dehydrogenase is also an
enzym e involved in glycolysis, it w as assigned to the
functional category of carbohydrate m etabolism . Four
genes operating in reactive oxygen m etabolism w ere
found in cluster C7-L: one TDF (Sr074) w as highly
sim ilar to m anganese superoxide dism utase (SOD;
W olfe-Simon et al., 2006), an d three TDFs (Sr075,
Sr076, an d Sr077) w ere highly sim ilar to the peroxi­
som al m em brane p rotein M PV17/PM P22, w hich is
know n to up-regulate the activity of SODs in anim al
cells (lida et al., 2003). These genes probably relate to
photosynthetic reactive oxygen species generation,
w hich is an inevitable p a rt of oxygenic photosynthesis
(Florton et al., 1996; A pel an d Flirt, 2004).
tering) a n d cell cy cle phases are in d icated at right an d left, respectively.
The arrow c o rre sp o n d s w ith the expression profile of th e DNA repli­
catio n licensing factor M CM5.
P la n t P h y sio l. Vol. 148, 2008
The Cell a n d C hlo ro p last Cycle of Seminavis robusta
Table 1. Id e n tifie d c e ll c y c le -m o d u la te d genes w ith s ig n ific a n t E value (< 0 .0 0 1 )
No.
Cluster
Protein Name
SrOOl
C2
CIpX, ATPase regulatory subunit
Sr002
C2
U b iquitin -co n ju g atin g en zy m e
Sr003
C2
Rab6 protein
Sr004
Sr005
Sr006
Sr007
Sr008
Sr009
C2
C2
C2
C2
C2
C4
H ypothetical protein
H ypothetical protein
H ypothetical protein
H ypothetical protein
Protein involved in flagellar biosynthesis
U biquitin
SrOlO
C4
N -E thylm aleim ide-sensitive factor
SrO ll
C4
Sr012
C4
Sr013
C4
Sr014
SrOl 5
C4
C5
SrOl 6
C5
H ypothetical ch lo ro p last protein
PhtrCpOl 6
H ypothetical ch lo ro p last protein
PhtrCpOl 6
H ypothetical ch lo ro p last protein
PhtrCpOl 6
H ypothetical protein w ith D U F1349
Isocitrate d eh y d ro g en ase NADPd e p e n d e n t, m o n o m eric type
M itochondrial p h o sp h a te carrier protein
SrOl 7
C5
SrOl 8
SrOl 9
Sr020
Sr021
Sr022
Sr023
Sr024
Sr025
Sr026
Sr027
C5
C5
C5
C5
C5
C5
C5
C5
C5
C5
Sr028
C5
Sr029
Sr030
Sr031
Sr032
Sr033
C5
C5
C5
C5
C5
Sr034
Sr035
C5
C5
Sr036
Sr037
Sr038
C5
C5
C5
Sr039
C6
Sr040
C6
Sr041
C6
Functional
Category
E Value
Percent
Identity
Length (Amino
Acids)
112 1 7 (Phatr v2.0)
5.0E-26
86.3
67
CAL55210
2.0E -17
41.1
175
2 1 535 (Phatr v2.0)
6.0E-18
64.4
96
Z P _00949502
C A F23694
49931 (Phatr v2.0)
49931 (Phatr v2.0)
Y P_001100159.1
3 4 1 4 (Thaps v3.0)
7.0E-09
2.0E -08
5.0E-09
2.0E -07
3.0E -57
4.0E -06
69
31.5
3 0 .4
30.2
61.7
40.2
67
111
111
101
179
392
126 2 0 (Phatr v2.0)
6.0E-12
72.9
83
YP 874373.1
4.0E -06
52.3
48
U nknow n
YP 874373.1
2.0E-19
77.3
67
U nknow n
YP 874373.1
2.0E-23
72.3
66
3 6 9 4 0 (Phatr v2.0)
4 5 0 1 7 (Phatr v2.0)
3.0E -50
2.0E-12
48.5
69.5
198
83
2 7 2 7 6 (Thaps v3.0)
4.0E-45
82.4
116
40391 (Thaps v3.0)
1.0E-58
85.6
137
48381 (Phatr v2.0)
2 1 122 (Phatr v2.0)
5 1 7 1 4 (Phatr v2.0)
4 3 1 0 6 (Phatr v2.0)
19141 (Thaps v3.0)
5 5 1 5 7 (Phatr v2.0)
2 1 3 8 8 (Phatr v2.0)
3183 (Thaps v3.0)
3 1 5 8 (Thaps v3.0)
114 9 0 (Phatr v2.0)
3.0E -14
2.0E -30
8.0E-12
4.0E -07
3.0E -30
3.0E -48
3.0E-25
4.0E -34
1.0E-11
7.0E-28
52.1
80.4
42.1
57.1
73.5
84.1
68.4
100
4 4 .8
60
106
105
122
65
211
114
91
80
112
116
3 3 432 (Thaps v3.0)
9.0E-46
72.6
158
3 6 913
13361
13361
21659
19901
v2.0)
v2.0)
v2.0)
v2.0)
v2.0)
2.0E-23
1 .OE-43
2.0E-35
2.0E-15
5.0E-17
78.2
87.7
89
70.4
61.9
75
107
88
110
75
42051 (Phatr v2.0)
Z P_01060962.1
1.0E-53
8.0E-07
81.1
63
123
129
2 1 905 (Thaps v3.0)
20641 (Thaps v3.0)
2 6 8 6 1 0 (Thaps v3.0)
2.0E-23
6.0E-07
3.0E -17
75.3
35.5
77.7
158
111
72
5595 (Thaps v3.0)
1 .0E-06
4 1 .4
155
3 6 2 7 0 (Phatr v2.0)
1.0E-10
88.2
36
3.0E-22
4 1 .8
117
Posttranslational
m odification
Posttranslational
m odification
Signal transduction
m echanism s
U nknow n
U nknow n
U nknow n
U nknow n
U nknow n
Posttranslational
m odification
Posttranslational
m odification
U nknow n
U nknow n
C arbohydrate
m etabolism
C arbohydrate
m etabolism
Enolase
C arbohydrate
m etabolism
W D 4 0 -re p e a t protein
C ytoskeleton
/3-Tubulin
C ytoskeleton
A ldo/keto red u ctase
Electron transport
Thioredoxin fam ily protein
Electron transport
Flavodoxin
Electron transport
/3-Hydroxyacyl-ACP d eh y d ratase precursor Fatty acid m etabolism
RNA o r DNA helicase
N ucleo tid e binding
H istone H3
N ucleo tid e binding
Protein w ith h elicase region
N ucleo tid e binding
M in ichrom osom e m ain ten an ce-d eficien t
N ucleo tid e binding
protein 5 (MCM 5)
C h a p ero n in -co n tain in g TCP1, su b u n it 2
Posttranslational
(CCT2)
m odification
N -A cetyl-y-glutam yl-phosphate reductase Protein synthesis
60S ribosom al protein L3
Protein synthesis
60S ribosom al protein L3
Protein synthesis
Ribosom al protein L32
Protein synthesis
Bifunctional ATP sulfurylase/adenosine
Protein synthesis
5 '-p h o sp h o su lfa te kinase
Protein synthesis
Aspartyl-tRNA synthetase
T onB -dependent receptor
Signal transduction
m echanism s
H ypothetical protein, conserved
U nknow n
H ypothetical protein
U nknow n
M em b ran e-sp an n in g protein (nonaspanin
U nknow n
family)
Leu-rich repeat-like protein
Signal transduction
m echanism s
R eceptor guanylyl cy clase GC-II
Signal transduction
m echanism s
O rp h a n g en e
U nknow n
Accession
No.
(Phatr
(Phatr
(Phatr
(Phatr
(Phatr
4 8 715 (Phatr v2.0)
(Table con tinues on fo llo w in g page.)
P la n t P h y sio l. Vol. 148, 2008
1401
G illard et al.
Table 1. (C o n tin u e d fro m p re v io u s page.)
No.
Cluster
Protein Name
Sr042
C7-E
G D P-M an 4 ,6 -d eh y d ratase
Sr043
Sr044
Sr045
Sr046
C7-E
C7-E
C7-E
C7-E
Sr047
C7-E
G lu tath io n e 5-transferase
C5 sterol desatu rase
5 -9 -D esaturase
P rotoporphyrin IX m agnesium chelatase,
su b u n it H
26S p ro tease regulatory su b u n it
Sr048
C7-E
M etallo proteinase
Sr049
C7-E
P roly lcarboxypeptidase
Sr050
Sr051
Sr052
Sr053
Sr054
Sr055
Sr056
Sr057
C7-E
C7-E
C7-E
C7-E
C7-E
C7-E
C7-E
C7-L
Sr058
Sr059
Sr060
Sr061
C7-L
C7-L
C7-L
C7-L
Sr062
Sr063
Sr064
C7-L
C7-L
C7-L
Sr065
Sr066
Sr067
C7-L
C7-L
C7-L
Sr068
Sr069
C7-L
C7-L
Sr070
Sr071
C7-L
C7-L
Sr072
C7-L
Sr073
C7-L
Eukaryotic translation initiation factor 3
V-type H+-ATPase su b u n it DVA41
H y pothetical protein
H y pothetical protein
H y pothetical protein
O rp h an g en e
H y pothetical protein
S u ccin ate/fu m arate m itochondrial
tran sp orter
A lcohol o x id ase-related protein
C y to ch rom e P450
M yoinositol deh y d ro g en ase
F ucoxanthin-chlorophyll a/c-binding
protein
f-C a ro te n e desatu rase (ZDS)
P o rphobilinogen synthase
F ucoxanthin-chlorophyll a/c-binding
protein
Light-harvesting protein
Light-harvesting protein
F ucoxanthin-chlorophyll a/c-binding
protein
P rotoporphyrinogen IX oxidase
F ucoxanthin-chlorophyll a/c-binding
protein
P h o sp h oribulokinase
F ucoxanthin-chlorophyll a/c-binding
protein
F ucoxanthin-chlorophyll a/c-binding
protein
Peptidylprolyl cis/trans-isom erase
Sr074
C7-L
S u p ero x ide dism u tase 1 (SOD1)
Sr075
C7-L
Peroxisom al m em b ran e protein
M PV17/PM P22
Sr076
C7-L
Peroxisom al m em b ran e protein
M PV17/PM P22
Sr077
C7-L
Peroxisom al m em b ran e protein
M PV17/PM P22
Sr078
C7-L
Flagellin-sensing 2-like protein
Functional
Category
Accession
No.
E Value
Percent
Identity
Length (Amino
Adds)
C arbohydrate
m etabolism
Electron transport
Fatty acid m etabolism
Fatty acid m etabolism
Photosynthesis
2 5 4 1 7 (Phatr v2.0)
1.0E-11
61.1
60
N P J 04774.1
37371 (Phatr v2.0)
2 8 7 9 7 (Phatr v2.0)
13265 (Phatr v2.0)
5.0E-09
1.0E-13
5.0E-06
3.0E -20
37 .6
61.1
52.5
95.9
114
67
65
54
Posttranslational
m odification
Posttranslational
m odification
Posttranslational
m odification
Protein synthesis
Transport protein
U nknow n
U nknow n
U nknow n
U nknow n
U nknow n
C arbohydrate
m etabolism
Electron transport
Electron transport
Electron transport
Photosynthesis
2 6 4 2 0 6 (Thaps v3.0)
7.0E-66
93.8
145
ZP_01 984772
3.0E -16
40
154
2 9 1 6 6 (Thaps v3.0)
7.0E-13
48.1
121
3 59 3 3 (Thaps v3.0)
2 1 0 3 0 (Phatr v2.0)
10781 (Phatr v2.0)
1 2 740 (Phatr v2.0)
4 5 6 7 8 (Phatr v2.0)
3 29 6 2 (Phatr v2.0)
EAU80690.1
2 3 7 0 9 (Phatr v2.0)
9.0E-16
6.0E-24
3.0E -16
1.0E-16
6.0E -11
9.0E-16
3.0E -04
2.0E-69
42.1
86.6
56.8
58.8
84.2
97.4
28.9
79.7
120
61
111
94
91
45
135
161
4 8 2 0 4 (Phatr v2.0)
4 3 4 6 7 (Phatr v2.0)
1049 (Thaps v3.0)
CAA04401
1.0E-13
6.0E-05
7.0E-76
1.0E-28
50
44
73.6
85.2
110
107
188
108
Photosynthesis
Photosynthesis
Photosynthesis
9 0 4 0 (Phatr v2.0)
YP 984596.1
CAA 05142.1
1.0E-37
3.0E -47
4.0E -11
92.6
89.7
83.3
84
108
39
Photosynthesis
Photosynthesis
Photosynthesis
2 7 0 0 9 2 (Thaps v3.0)
1 4 648 (Phatr v2.0)
CAA04401
4.0E -06
2.0E -24
3.0E-32
85
76
83
69
102
107
Photosynthesis
Photosynthesis
264901 (Thaps v3.0)
CAA04401
6.0E-46
1.0E-32
64.1
85.3
186
107
Photosynthesis
Photosynthesis
1 0 208 (Phatr v2.0)
C A A 04401.1
3.0E-43
6.0E-57
87.6
69.2
126
194
Photosynthesis
2 23 9 5 (Phatr v2.0)
5.0E-33
81.8
113
Posttranslational
m odification
R eactive oxygen
species
m etabolism
R eactive oxygen
species
m etabolism
R eactive oxygen
species
m etabolism
R eactive oxygen
species
m etabolism
Signal transduction
m echanism s
1989 (Thaps v3.0)
5.0E-10
48
67
12583 (Phatr v2.0)
3.0E-45
91
89
1 2379 (Phatr v2.0)
3.0E -16
88.8
116
1 2379 (Phatr v2.0)
1.0E-15
95.1
98
1 2379 (Phatr v2.0)
1 .0E-06
89.6
54
N P _001043191.1
2.0E -06
40.2
77
(Tabie con tinues on fo iio w in g page.)
1402
P la n t P h y sio l. Vol. 148, 2008
The Cell a n d C hlo ro p last Cycle of Seminavis robusta
Table 1. (C o n tin u e d fro m p re v io u s page.)
No.
Cluster
Sr079
C7-L
Sr080
Sr081
Sr082
Sr083
Sr084
Sr085
Sr086
Sr087
C7-L
C7-L
C7-L
C7-L
C7-L
C7-L
C7-L
C8
Sr088
Sr089
C8
C9
Sr090
Sr091
Sr092
C9
C9
C9
Sr093
Sr094
C9
C9
Sr095
Sr096
C9
C9
Sr097
Sr098
Sr099
Sri 00
C9
C9
C9
C9
Protein Name
Ser/Thr protein kinase an d signal
transduction histidine kinase (STHK)
w ith GAF sensor
H ypothetical protein
H ypothetical protein
O rp h a n g en e
O rp h a n g en e
H ypothetical protein
O rp h a n g en e
O rp h a n g en e
5 '-A denylylsulfate reductase,
ch lo ro p last precursor
H ypothetical protein
G ly cerald eh y d e-3 -p h o sp h ate
d eh y d ro g en ase
C y tochrom e P450 3A10
G ly cero l-3 -p h o sp h ate deh y d ro g en ase
F u coxanthin-chlorophyll a/c-binding
protein
Transketolase
Leu-rich rep eat recep to r protein
V-type H+-p y ro p h o sp h atase
S o d ium /calcium ex ch an g e r
fam ily protein
O rp h a n g en e
O rp h a n g en e
O rp h a n g en e
O rp h a n g en e
Functional
Category
E Value
Percent
Identity
Length (Amino
Acids)
Signal transduction
m echanism s
4 8 535 (Phatr v2.0)
1.0E-26
35.2
274
U nknow n
U nknow n
U nknow n
U nknow n
U nknow n
U nknow n
U nknow n
Electron transport
2 4 1 5 0 (Thaps v3.0)
32401 (Phatr v2.0)
2 2 1 1 7 (Thaps v3.0)
4 8 0 7 (Thaps v3.0)
50021 (Phatr v2.0)
2 0 812 (Thaps v3.0)
3345 (Thaps v3.0)
105 1 8 (Phatr v2.0)
1 .0E-08
4.0E-21
7.0E-28
3.0E -28
5.0E-07
1 .0E-04
7.0E-04
1.0E-41
46
49.1
53.9
51.3
509
90.4
3 6 .8
68.8
168
162
112
127
55
36
81
125
U nknow n
C arbohydrate
m etabolism
Electron transport
Fatty acid m etabolism
Photosynthesis
15801 (Phatr v2.0)
2 2 122 (Phatr v2.0)
8.0E-23
6.0E-17
35.5
93.2
178
46
4 3 4 6 7 (Phatr v2.0)
4 0 073 (Thaps v3.0)
2 2 395 (Phatr v2.0)
5.0E-05
5.0E-51
8.0E-33
44
75
52.7
98
135
176
Photosynthesis
Signal transduction
m echanism s
Transport protein
Transport protein
2 9 2 6 0 (Phatr v2.0)
4 5 913 (Phatr v2.0)
2.0E-39
7.0E-15
63.1
40
126
167
CAL50249.1
3 1 322 (Phatr v2.0)
2.0E-11
6.0E-44
55.9
74.4
174
154
U nknow n
U nknow n
U nknow n
U nknow n
4 9 2 8 6 (Phatr v2.0)
4 9 2 8 6 (Phatr v2.0)
2 6 8 3 0 4 (Thaps v3.0)
3 7 765 (Phatr v2.0)
1.0E-13
3.0E-23
4.0E -10
5.0E-31
80.9
86.4
4 4 .7
40.5
59
64
115
201
/3-Tubulin (Sr019), w hich plays a fundam ental role
in cytoskeleton-based cellular m ovem ents, w as highly
in d u ced 4 h an d m axim ally expressed 6 h after d ark ­
ness. This induction (clustered w ithin C5) slightly
p reced ed chloroplast reorganization. A nother TDF
w ith sim ilarity to a W D40-repeat p rotein (Sr018), p u tatively involved in cytoskeleton assembly, w as anal­
ogously expressed; bo th genes w ere dow n-regulated
6 h after darkness b u t /3-tubulin w as again slightly
in d u ced at 8 h, in parallel w ith cell division (Supple­
m ental Fig. S ll). The involvem ent of m icro tubulebased cytoskeleton dynam ics d u rin g chloroplast
m ovem ent w as expected an d further validated by
treatm ent of synchronized cultures w ith the m icrotu­
bule inhibitor nocodazole. N ine hours after treatm ent,
chloroplast division an d reorganization w ere im paired
w h en com pared w ith untreated control cultures. Only
after 26 h d id chloroplast division an d reorganization
proceed, arresting the cells at G2+M phase (data not
shown).
Cell Cycle versus Light Regulation of Photosynthesis and
Pigment Biosynthesis
The m icroscopic observations (Fig. 4) show ed that
chloroplasts elongated after cell division initiation
w h en they relocated to the girdle sides of the cell. To
investigate w h eth er this elongation is paralleled w ith
P la n t P h y sio l. Vol. 148, 2008
Accession
No.
chloroplast grow th, w e determ ined fucoxanthin p ig ­
m ent turnover an d validated the FCP expression w ith
RT-Q-PCR in synchronized cultures. In addition, these
d ata w ere com pared w ith those from synchronized
cultures to w hich the cell cycle inhibitor aphidicolin
h ad been ad d e d shortly before reillum ination (Fig. 7).
This w as done to assess the dependence of pigm ent
turnover an d FCP expression on cell cycle progression
versus light regulation.
U pon reillum ination, fucoxanthin content p er cell
increased u n d e r bo th conditions, b u t the tren d w as
m ore pronounced in the dividing cultures than in the
S -phase-arrested cultures. This fucoxanthin accum u­
lation in the dividing cultures w as obvious after 5 h of
light an d decreased after 10 h, corresponding w ith cell
separation (Fig. 3). In the S-phase-arrested cultures,
the accum ulation curve w as m ore gradual, w ith o u t a
clear p eak an d w ith a sm aller m axim um . A lthough the
difference betw een bo th profiles w as obvious, a lon­
g itudinal ANOVA test w as unable to prove its signif­
icance, w hich could be d u e to the sm all n u m b er of
replicates.
In parallel, in response to reillum ination in b oth
cultures, FCP expression w as activated an d subse­
quently dow n-regulated after 5 h in the arrested
cultures, w hereas in cell cycle-progressing cultures,
its expression w as m aintained throughout the 13-h
tim e course (Fig. 7). These findings suggest that
1403
G illard et al.
Figure 7. C om p ariso n of fucoxanthin pigm ent tu rn ­
o ver an d FCP exp ressio n b etw een light/dark-synchronized cu ltu res a n d lig h t/d ark-synchronized cultures
to w h ich th e cell cy cle inhibitor ap h id ico lin w as
a d d e d shortly before reillu m ination. A difference in
fu co x an th in pig m en t (/¿g m g -1 dry w eight) a c c u m u ­
lation is a p p a re n t after 5 h of reillum ination. In the
S -p h ase-arrested cultures, th e bu ild u p is less pro­
n o u n ced . In parallel, FCP expression is up-regulated
b etw een 1 a n d 5 h after reillum ination in both
dividing a n d arrested cultures, but its expression is
d o w n -reg u lated after th e initial increase in arrested
cu ltu res w h ile it is m ain tain ed in th e dividing c u l­
tures. [See o n lin e article for c o lo r version of this
figure.]
—
F C P e x p re s s io n in ap h id ico lin -tre ated cu ltu re s a fte r re-illum m ation
F u co x an th in c o n te n t in ap h id ico lin -tre ated c u ltu re s afte r re-illum inatton
c ^ 3 F C P e x p re s s io n in light/dark-synchronized c u ltu re s
F u co x an th in co n te n t in light/dark-synchronized c u ltu re s
* <w
£■
6
tim e (h)
chloroplast gro w th occurs in advance of chloroplast
elongation at cytokinesis, w hile chloroplast lighth arvesting com plexes are regulated by bo th the cell
cycle an d light.
DISCUSSION
Regulation of Chloroplast Dynamics w ith Respect to Cell
Cycle Progression and Frustule Silicification
In diatom s, chloroplast division an d developm ent
have n o t yet been considered in relation to cell cycle
regulation. For exam ple, darkness w as show n to arrest
the cell cycle in several diatom species (Vaulot et al.,
1986; Brzezinski et al., 1990), b u t m icroscopic obser­
vations of the effect on chloroplasts are, to our know l­
edge, com pletely lacking. A putative reason is that
m ostly polyplastidic centric diatom s have been stu d ­
ied in w hich the sm all chloroplasts are stochastically
p artitio n ed u p o n cell division and, hence, have not
such an obvious developm ental cycle. In addition, the
fact that flow cytom etry is traditionally preferred to
investigate cell cycle effects m ight have contributed to
the general neglect of m orphology an d cytology.
As show n here for the p ennate diatom S. robusta,
chloroplast division an d developm ent are intim ately
linked w ith cell cycle progression. First, conditions
that activate the G l-to-S cell cycle checkpoint (i.e. light
deprivation, FIU, or aphidicolin) arrest the develop­
m ent of chloroplasts at the plastokinesis stage w ithout
inducing aberrant cell m orphologies. Second, both
d iv id ed chloroplasts rotate from the girdle to the
valves before karyokinesis is initiated. A nd, at last,
the chloroplast division hom olog F tsZ is expressed in
concert w ith chloroplast division in a cell cycle phased ep en d en t m anner, d u rin g the S /G 2 phases. We con­
clude th at the G l-to-S-checkpoint controls chloroplast
division an d relocation an d enables their synchronous
division in S. robusta cultures. Previous observations of
chloroplast dynam ics d u rin g the cell cycle in S. robusta
1404
h ad already show n th at chloroplasts an d the nucleus
divide in a coordinated m anner (C hepum ov et al.,
2002). Now, w e observe th at both also dep en d on each
other an d that com m on m echanism s to regulate their
division should be involved. Similarly, in the red alga
Cyanidioschyzon merolae, chloroplast an d m itochon­
d rio n divisions are regulated at distinct cell cycle
checkpoints (N ishida et al., 2005). How ever, in contrast
w ith aphidicolin-arrested S. robusta cells, in C. merolae
the m itochondrial division is restrained w hile the
chloroplast divides m ultiple tim es (Itoh et al., 1996;
N ishida et al., 2005).
Since its initial discovery in A rabidopsis (Arabidopsis
thaliam; O steryoung an d Vierling, 1995), hom ologs of
the bacterial cell division protein FtsZ are held re­
sponsible for organelle division in m any other eukary­
otes (Beech an d Gilson, 2000; Takahara et al., 2000;
Kiefel et al., 2004) that occurs by FtsZ assem bling into
the bacterial cytokinetic apparatus, called the Z-ring
(O steryoung an d N unnari, 2003; M argolin, 2005). Ac­
cordingly, FtsZ expression is m odulated d u rin g syn­
ch ronized grow th of tobacco (Nicotiana tabacum BY2;
El-Shami et al., 2002) an d C. merolae (Takahara et al.,
2000; N ishida et al., 2005), in a circadian rhyth m in
Chlamydomonas reinhardtii (H u et al., 2008), an d in
lig h t/d ark -sy n ch ro n ized cultures of S. robusta as well.
Its expression is up-regulated after S-phase progres­
sion an d p rio r to cell division, in agreem ent w ith
m icroscopic observations. This show s that the chloro­
plast developm ental cycle can be m onitored un m is­
takably through synchronization of cell division in S.
robusta.
C ontrol of chloroplast division is governed b y con­
served cell cycle regulators of bacterial origin (Adam s
et al., 2008). One of these, the chaperone ClpX, iden­
tified here, w as dow n-regulated u p o n reillum ination
w h en cells reentered the cell cycle. ClpX has been
found to inhibit Z-ring form ation in Bacillus subtilis
(W eart et al., 2005). It is tem pting, therefore, to suggest
that the activation of ClpX in a dark-arrested cell
ensures the inhibition of plastokinesis in S. robusta.
P la n t P h y sio l. Vol. 148, 2008
The Cell a n d C hlo ro p last Cycle of Seminavis robusta
A nother resem blance can be found w ith the regulation
of plastokinesis at the G1 / S transition in A rabidopsis
(R aynaud et al., 2005), in w hich chloroplast division
w as regulated by the activity of the prereplication
factor CDT1. In S. robusta, chloroplast division w as
paralleled b y activation of the S-phase-specific gene
M C M 5 , w hich is also a universal m em ber of the
prereplication com plex. Furtherm ore, our observa­
tions also suggest th at "retrograde" m echanism s
(Koussevitzky et al., 2007), w hich involve signaling
from the chloroplast to the nucleus, m ight be present
as ano th er w ay to coordinate the chloroplast and
nuclear division cycle. R etrograde signaling w as, for
exam ple, rep o rted in response to m em brane tension as
a possible w ay to regulate shape, size, an d division of
chloroplasts in A rabidopsis (H asw ell an d M eyerow itz,
2006). A nalogous feedback m echanism s m ight exist
w ith in diatom s, for exam ple, to control the G 2/M
transition in response to the chloroplast developm en­
tal status, ensu rin g high-quality cbloroplast segrega­
tion of d au g h ter chloroplasts to the new ly divided
cells. In any case, control m echanism s m ay not be as
strict in centric polyplastidic diatom s because, sim ilar
to w h at is know n for land plants, a stochastic p arti­
tioning of chloroplasts u p o n cell division w ill h ard ly
result in cells w ith o u t any chloroplast (C olem an and
N erozzi, 1999).
The m olecular m echanism of chloroplast m ovem ent
in diatom s is still unclear. In polyplastidic diatom s, the
red alga C. merolae, and plants, chloroplast m ovem ents
are know n to be actin dep en d en t (de Francisco and
Roth, 1977; N ishida et al., 2005; Krzeszow iec et al.,
2007). The transcriptional induction of /1-tubulin in S.
robusta suggests the involvem ent of m icrotubules d u r­
ing chloroplast rearrangem ent b u t does no t exclude
the possibility th at actin m ay also be involved.
The intriguing question rem ains w h y p ennate d ia­
tom s display this som etim es com plicated cycle of
repositioning the chloroplast d u rin g cell cycle p ro ­
gression. O ne possibility is th at the position of the
chloroplasts at the valves m ight create a disadvantage
for cell m ovem ent needed tor adequate reaction to
environm ental factors, such as light an d nutrients
(Cohn an d D isparti, 1992). Because cells glide across
the surface b y m ucilage secretion from a valvar slit
(called the raphe; Floagland et al., 1993; W etherbee
et al., 1998; Chiovitti et al., 2006), the underlying actinm yosin system th at is held responsible for the rap h e's
functionality could be im paired b y a valvar location of
the chloropiasts. In cultures of S. robusta, cells w ith a
reorganized chloroplast configuration have indeed
n ever b een observed m oving. This reasoning w as first
introduced in M ereschkow sky's "law " on diatom
chloroplasts, w hich states th at chloroplasts of diatom s
h ave a tendency to leave the raphe as m uch as possible
uncovered (M ereschkowsky, 1904). This is achieved in
n u m ero u s diatom s (Brebissonia spp., Cymbella spp.,
Gomphonema spp., Dickieia ulvacea, Didymosphenia spp.,
an d Lyrella spp.) b y plastid invaginations b eneath the
rap h e (M ann, 1996), w hereas in other diatom s, such as
P la n t P h y sio l. Vol. 148, 2008
S. robusta, it is achieved b y positioning of the chloro­
plasts at the valves only d u rin g a short tim e of the cell
cycle. As observed here, this positioning at the valves
is necessary only to free the girdle area for the ad d ition
of girdle bands, cytokinesis, an d frustule form ation.
The identified girdle b an d s w ere indeed observed late
du rin g the cell cycle, in cells w ith a reorganized
chloroplast configuration. As such, the cyclic m ove­
m ent of chloroplasts could create a large enough tim e
fram e for a cell to position itself optim ally w ith in the
environm ent, m axim ally favoring cell grow th, before
cell division is com m itted. It is an interesting specu­
lation that because centric diatom s are planktonic, lack
a raphe, an d are thus unable to m ove, they d id not
evolve such a w ell-orchestrated chloroplast develop­
m ent cycle. Also, variations in the chloroplast cycle in
diatom s m ight explain the differences in the tim ing of
girdle b an d deposition am ong species (Coombs et al.,
1967b; C hiappino an d Volcani, 1977; Kroger and
W etherbee, 2000; Flildebrand et al., 2007). For exam ple,
T. pseudonana, w hich is polyplastidic, w as show n re­
cently to synthesize its girdle b ands already d u rin g G1
phase (Flildebrand et al., 2007).
G irdle b ands accom m odate cell grow th and, like
frustules, are form ed b y polym erization of silica in a
silica deposition vesicle (SDV) th at is exocytosed
(Pickett-H eaps et al., 1990; K roger an d W etherbee,
2000; Zurzolo an d Bowler, 2001). N ot m uch is know n
about the origin of the SDV, b u t it probably originates
from other internal vesicle com partm ents, such as
the endoplasm ic reticulum , Golgi, an d lysosom e (Lee
an d Li, 1992). In this regard, the TDF Sr051, identified
as a su b u n it of the V-type H +-ATPase, is of p ar­
ticular interest because sucb energy-consum ing proton
tran sp o rt proteins have a know n functionality in the
acidification of organelles (M aeshim a, 2001). It w as
m axim ally expressed 1 h before the initiation of cell
separation (at 8 h after darkness), w hen the two "vacuole­
like" com partm ents also w ere visible b y PDM PO
accum ulation. Because diatom silicification occurs in ­
side an acidic SDV (M ayam a an d K uriyam a, 2002), the
V-type FI+-ATPase couid be related to girdle or valve
form ation at the stage of SDV acidification. A P-type
ATPase w as recently also hypothesized to be involved
in SDV form ation, d u rin g expansion of its plasm a
m em brane in T. pseudonana (Frigeri et al., 2006). Pos­
sibly, bo th identified V-type an d P-type ATPases are
required in parallel d u rin g valve form ation in dia­
toms.
Cell Cycle Synchronization of S. robusta
In m any studies using phytoplankton cultures (for
review, see Pirson an d Lorenzen, 1966; Tamiya, 1966;
K rupinska an d Flum beck, 1994), the endogenous cell
cycle control m echanism s th at respond to n atu rally
ph ased lig h t/d a rk conditions are exploited to stu d y
cell division in synchronous cultures (Otero an d Goto,
2005). D iatom s are know n to be capable of sustaining
long periods of darkness an d retain the ability to start
1405
G illard et al.
grow ing rap id ly u p o n reillum ination (Furusato et al.,
2004). The synchronization protocol established here
for S. robusta relies on this apparently fast release from
the dark-arrested G1 phase. As a result of the identi­
fied com m on regulatory m echanism s in chloroplast
division an d cell cycle progression, this procedure w as
found successful for studying b o th processes sim ulta­
neously.
The uniform ly d ark-induced G l-p h ase arrest in S.
robusta ap p ears u n u su al w h en com pared w ith other
diatom s. The centric diatom Thalassiosira weissflogii
accum ulates, besides G l-p h ase cells, 60% G2+Mph ase cells (Olson et al., 1986; Vaulot et al., 1986;
Brzezinski et al., 1990), an d other centric diatom s
(Chaetoceros muellerii, T. pseudonana, Chaetoceros simplex,
an d M inutocellus polymorhus) plus one p ennate diatom
(Cylindrotheca fusiform is) all accum ulate G l-p h ase cells
together w ith a sm aller fraction (approxim ately 10%)
of G2+M -phase cells (Brzezinski et al., 1990). The
p en n ate diatom P. tricornutum is the only diatom
reported w ith o u t a dark-sensitive G2+M phase, and
its facultative silicon requirem ent has been p u t
forw ard as the m ain reason for this observation
(Brzezinski et al., 1990). H owever, in diatom s, the G2+
M -phase fraction in flow cytom etric signatures in­
cludes, besides G2- an d M -phase cells, also postcytokinetic doublet cells, w hich have com pleted cell w all
form ation b u t have no t yet separated. Therefore, the
"G 2+M -phase" fraction in the dark-arrest experim ent
b y Brzezinski et al. (1990) m ight have been postcytokinetic doublet cells. In S. robusta, the occasionally
detected G2+M -phase cells at 24 h of d ark arrest w ere
m icroscopically observed to be all postcytokinetic
doublet cells containing girdle-located chloroplasts,
typical for G l-p h ase cells. A pparently, only the p ro ­
cess of cell separation an d not cytokinesis is ham pered
in these cells b y the absence of light. Therefore, w e
suggest th at it m ight be useful to address cell separa­
tion in diatom s analogously as in yeast cells, w here it is
defined as a postm itotic phase an d know n to be
m olecularly distinct from the sep tu m form ation p ro ­
cess d u rin g M p h ase (Yeong, 2005; Sipiczki, 2007).
Based on the expression of the prereplication factor
MCM5 an d the occurrence of dividing chloroplasts,
the d u ratio n of the G1 phase in S. robusta can be
estim ated to last for 4 h (33% of the total division time)
d u rin g synchronization u n d e r the applied culture
conditions. Since the last 3 h w ere dom inated by
separating cells, 5 h (41% of the total division time)
are left to fulfill the S, G2, an d M phases. O ur results
suggest th at cell division d u rin g synch ronization oc­
curred faster (<12 h) than d u rin g norm al exponential
grow th, even w h en com pared w ith the highest re­
corded m axim um division rate for S. robusta, being
16.6 h p er division (approxim ately 1.5 divisions p er
d ay u n d er continuous light at 200 pY). As suggested
(Olson et al., 1986; Vaulot et al., 1986), p a rt of the G1
p h ase m ight alread y be accom plished d u rin g dark
arrest. Taking into account this com m ent, o u r m icro­
scopically estim ated cell cycle stage durations of S.
1406
robusta correspond roughly w ith the flow cytom etric
estim ations from P. tricornutum , reported to spend half
its cell division tim e in G1 phase (Brzezinski et al.,
1990).
Gene Expression in Relation to Chloroplast Functioning
and Photosynthesis
The overall results from the cDNA-AFLP experi­
m ent confirm the hypothesis th at several biological
processes are u n d e r tem poral transcriptional control
d u rin g the cell cycle of diatom s, as show n previously
in sim ilar studies of higher plants an d anim als (Cho
et al., 2001; M enges et al., 2002; Brey ne et al., 2002).
O ne of the m ost distinct patterns in gene m odulation
concerns those genes encoding chloroplast proteins,
associated w ith the functioning of chloroplasts d u rin g
photosynthesis, including light-harvesting proteins,
biosynthetic pigm ent enzym es, an d genes coping
w ith oxidative dam age. N early all of these genes
w ere expressed d u rin g G 2/M -phase progression and
clustered into the expression cluster C7-L. M ost n o ta­
ble w as the expression of FCP, a chrom ophyte-specific
type of light-harvesting protein (Bhaya an d G rossm an,
1993; E ppard an d Rhiel, 1998, 2000; Guglielm i et al.,
2005), in concert w ith cell division.
R egulation of FCP expression is know n to m odulate
light harvesting p rim arily d u rin g photoacclim ation
in d ep en d en t of developm ental processes (Falkowski
an d LaRoche, 1991; D u m ford an d Falkowski, 1997;
Leblanc et al., 1999; Oeltjen et al., 2002, 2004; Siaut
et al., 2007). N evertheless, the cell an d life cycle
dependence of photosynthesis has been show n in
several algae, such as Scenedesmus spp. (Post et al.,
1985; Kaftan et al., 1999; Tukaj et al., 2003; Setlikova
et al., 2005), Euglena gracilis (W inter an d B randt, 1986),
Chlorella fusca (Butko an d Szalay, 1985), an d C. fusifor­
mis (C laquin et al., 2004).
In S. robusta, FCP expression w as found to be light
activated in the first place, confirm ing previous results
in centric (Leblanc et al., 1999; O eltjen et al., 2002,2004)
an d pennate (Siaut et al., 2007) diatom s. But after 5 h,
its expression becam e cell cycle d ependent, because it
w as m aintained in dividing cultures w hile being re­
pressed in S-phase-arrested cultures. A possible m o­
lecular m echanism to explain this difference relates to
the retrograde inhibition of light-harvesting proteins
know n in C. reinhardtii an d A rabidopsis. In these
organism s, light-induced expression of light-harvesting
proteins is repressed b y accum ulation of the chloro­
phyll precursor M g-protoporphirin IX (Johanningm eier
an d H ow ell, 1984; N ott et al., 2006). Its accum ulation,
leakage from the chloroplast, an d nuclear repression of
photosynthetic genes are tho u g h t to occur d u rin g
reduced chloroplast function to coordinate pigm ent
biosynthesis w ith the expression of light-harvesting
proteins (N ott et al., 2006). In an analogous way, if
chloroplast developm ent is im paired in S. robusta, a
retrograde signal m ight repress FCP. The fact that
fucoxanthin does no t decrease together w ith FCP
P la n t P h y sio l. Vol. 148, 2008
The Cell a n d C hlo ro p last Cycle of Seminavis robusta
could be a result of the low turnover rates of diatom
pigm ents, ranging from days to w eeks (Riper et al.,
1979; Goericke an d W elschmeyer, 1992), com pared
w ith the low stability of mRNA.
A recent stu d y b y Ragni an d Ribera d'A lcalà (2007)
on th e circadian v ariations in p ig m en t content in
P. tricornutum showed that pigm ent synthesis follows the
som atic gro w th p receding cell division. Furtherm ore,
they observed th at a pronounced circadian p attern of
pigm ent synthesis w as lost w hen cultures w ere con­
tinuously illum inated an d becam e asynchronous. This
stu d y substantiates our finding th at fucoxanthin con­
ten t increases in p rep aratio n of cell division, until it is
d istrib u ted into d au g h ter cells.
Taking together our m icroscopic analysis, cDNAAFLP results, an d pigm ent accum ulation patterns, w e
suggest th at cell cycle m odulation of photosynthetic
light harvesting occurs in concert w ith chloroplast
gro w th to p rep are for its elongation at cytokinesis.
Because chloroplasts in S. robusta elongate at the
stage of cytokinesis, regulated synthesis of m ore func­
tional light-harvesting com plexes w o u ld be expected
to pro v id e a balanced grow th of chloroplasts at this
stage, thereby optim izing the cell's photosynthetic
capacity d u rin g the subsequent G1 phase. Together
w ith light acclim ation an d circadian regulation, cell
cycle-m odulated biosynthesis of the large am ount of
pigm ents th at are contained w ithin diatom s (Chan,
1978,1980; Falkowski et al., 1985; Tang, 1996) could as
such coordinate cellular investm ents an d im prove cell
fitness.
MATERIALS AND METHODS
Cell Culture and Image Acquisition
The Seminavis robusta strain s F1-8B an d F1-9A w ere selected from a first
g eneratio n of siblings o btained b y crossing the w ild -ty p e clones 75 a n d 80
(C h e p u m o v et a lv 2002). Fj-813 an d FX-9A b elong to o p p o site m a tin g types
(C h e p u m o v et a lv 2008) a n d h a d at the tim e of stu d y an average apical cell
len g th of 22.7 ±1.6 a n d 18.9 ±1.2 /xm, resp ectiv ely C u rre n tly they are
m a in ta in e d cry o p reserv ed in the cu ltu re collection of th e L aboratory of
Protistology a n d A quatic Ecology (h ttp ://w w w .p a e .u g e n t.b e /c o lle ctio n .
htm ). Cell cultures w ere g ro w n in F /2 m e d iu m (G uillard, 1975) m a d e w ith
filtered (G F /C g rad e m icrofiber filter; W hatm an) au to clav ed seaw ater col­
lected from th e N o rth Sea. F /2 n u trien ts w e re sterilized th ro u g h 0.2-/xm filters
a n d a d d e d to the filtered au to clav ed seaw ater. N a 2S i0 3 w as a d d e d at a
concen tratio n of 30 m g L-1 m ed iu m . C u ltu res w ere cu ltiv ated a t 18°C w ith a
12:12-h light: d a rk p e rio d a n d a p p ro x im ately 85 /xmol p h o to n s m -2 s -1 from
cool-w hite fluorescent lights. Stock cultures w ere rein o cu lated w eekly by
tran sferrin g sm all aliquots of cell su sp en sio n into fresh m ed iu m . E xperim en­
tal cultures w ere p re p a re d from stock cu ltu res b y in o cu latin g an aliquot of
cells from cell su sp en sio n s created b y scrap in g th e cells from the surface (cell
scrapers; Sarstedt) o r b y d e tach in g the cells b y pip ettin g . O bservations an d
cell cu ltu re p h o to g ra p h y w e re do n e w ith a Zeiss A xiovert 40 lig h t m icroscope
a n d a d ig ital cam era (P ow ershot G3; C anon). Fluorescence m icroscopy of cells
w as p erfo rm ed u sin g Zeiss A x io p lan 200, A xiocam M Rm for im age capturing,
a n d Zeiss filter set 14. C onfocal fluorescence im ages w ere tak en w ith a laserscann in g confocal m icroscope (Zeiss Confocal LSM 510) eq u ip p e d w ith
softw are p ack ag e LSM510 v ersio n 3.2 (Zeiss) a n d e q u ip p e d w ith a 63X
w ater-co rrected objective (num erical a p ertu re 1.2). C h lo ro p last fluorescence
w as v isu alized w ith h e liu m -n eo n laser illu m in atio n at 543 run. N u clear D N A
w as stain ed w ith SYBR Safe (M olecular Probes) th a t w as v isu alized w ith
a rg o n laser illu m in atio n at 488 ru n an d a 500- to 530-run b a n d em ission filter.
Cell w alls w ere stain ed w ith th e L ysosensor Y ellow /B lue DND -160 probe
P la n t P h y sio l. Vol. 148, 2008
(PDM PO; M olecular Probes) as described b y L eblanc a n d H utchins (2005) and
visu alized b y illum ination a t 351 run. E m ission fluorescence w as c ap tu re d in
the line-scanning m o d e, an d for tra nsm ission light im ages, differential inter­
ference contrast optics w ere used.
Cell Cycle Progression Assays and Flow Cytometry
The effect of d arkness o n cell m o rphology w as tested on exponentially
g ro w in g cultures of strain F ^ B (C h e p u m o v et al., 2008) th a t w ere inoculated
a t 1,000 cells m L -1 in tissue cu ltu re flasks (Cellstar; 175-cm2 g ro w th surface
w ith filter screw cap; G reiner Bio-One) w ith 200 m L of g ro w th m edium . A fter
2 d of grow th, the old m e d iu m w as d ecan ted from the flask-attached cells and
the flask w as refilled w ith 200 m L of fresh m e d iu m before tran sferrin g the
cultures to com plete d ark n ess to ensure th a t n o n u trie n t lim itation occurred
d u rin g d a rk incubation. H U (Fluka) w as tested at a final concentration of
6.5 iravi, a d d e d to 100 m L of exponentially gro w in g cultures of strain Fr 31B.
A fter a ddition, the cultures w ere in cu b ated in continuous light d u rin g 72 h
a n d co m p ared w ith blan k cultures to w hich an eq u al v olum e of d im ethyl
sulfoxide w as a d d ed . The effect of aphidicolin (Sigm a-A ldrich; 0.5 /xg m L -1)
w as tested in triplicate o n F -^B cultures containing ap p ro x im ately 5,000 darkarrested cells in 10 m L of m e d iu m a n d c om pared after 24 h of light w ith
dim ethyl sulfoxide-treated control cultures.
D N A content w as m e a su re d o n intact fixed cells. Therefore, a t least 10 m L
of a culture w as centrifuged for 10 m in at 1,500g, a n d the cells w ere fixed in
10 m L of ice-cold m eth an o l in the d a rk a t 4°C (Vaulot et al., 1986). Fixed cells
w ere rinsed tw o to three tim es w ith 4 m L of TE (10 iravi Tris a n d 1 iravi EDTA,
p H 8.0) b y c entrifugation for 10 m in at l,500g a n d re su sp e n d e d in 1 m L of TE.
Sam ples w ere in cu b ated for 40 m in a t 37°C w ith 10 /xL of 30.5 m g m L -1 RN ase
A (R4642; Sigm a-A ldrich) a n d th e n p la c ed o n ice in the dark. To each sam ple,
1 /xL of 4,6-diam idino-2-phenylindole from a stock of 1 m g m L -1 w as a d d ed ,
stain ed for a t least 10 m in o n ice, a n d filtered over a 50-/xm n y lo n m esh
(CellTrics; Partee). The stained cell suspensions w ere analyzed w ith a CyFlow
flow cytom eter a n d FloM ax softw are (Partee).
Synchronization of S. robusta Cells and Sampling
of Material
For the synchronization, each S. robusta strain w as g ro w n in tw o tissue
cu ltu re flasks. Because of the difference in cell size, F -^B a n d F ^ A culture
flasks w ere inoculated w ith 7.5 X IO5 a n d 1 X IO6 cells, respectively, into 200
m L of m ed iu m . Cell den sity estim ates of stock cultures w ere obtained by
m icroscopically c ounting cells in a 100-/xL aliquot of a s u sp e n d e d culture o n a
96-w ell TC plate. C ultures inoculated for synchronization w ere g ro w n for 2 d
in a 12:12-h lig h b d a rk regim e. A t the end of d a y 2, the d a rk p e rio d w as
ex tended for a n o th e r 12 h, arresting the cell culture a t the G1 phase. A first
sam ple w as taken ju st before the light w as sw itched on, follow ed b y 12
sam ples tak en every h o u r after reillum ination. Just before sam pling of each
culture, six p h o to g ra p h s w ere tak en w ith the A xiovert 40 m icroscope a n d a
connected digital cam era (C anon P ow ershot G3). Im ages w ere u sed to count
different cell types u sin g the open-source softw are Im ageJ (h ttp ://r s b .in f o .
n ih .g o v /ij/in d e x .h tm l) an d the cell counter plug-in. M -phase cells w ere easily
identified a n d d istin g u ish ed from interphase cells b y the p resence of a new ly
b u ilt cell w all, situ ated b etw een the tw o valve-located chloroplasts. A s soon as
d a u g h te r cells w ere separating, they w ere counted as n o n d iv id in g cells. In
p re p a ra tio n for cell cu ltu re harvesting, the cells w ere concentrated b y red u c­
in g the m e d iu m of the first flask to less th a n 50 m L b y a spiration w ith a w a ter
p u m p an d attach ed P a steu r p ipette. A fter susp en sio n th ro u g h scraping
(Sarstedt), the cells w ere a d d e d to the second bottle, from w h ic h the com plete
m e d iu m w as aspirated. The collection of su sp e n d e d cells from the tw o bottles
w as tran sferred to a 50-mL Falcon tube a n d centrifuged for 6 m in a t 1,500g.
The su p e rn a ta n t w as p o u re d off, a n d the tu b e c ontaining the p ellet w as frozen
in liq u id n itro g en a n d stored a t —80°C u n til R N A p reparation.
cDNA-AFLP-Based Transcript Profiling
Total R N A w as extracted from each cell sam ple u sin g the R N easy Plant
M ini Kit (Qiagen). Cell lysis w as achieved b y m echanical d isru p tio n in 600 /xL
of R N easy Lysis buffer (Qiagen) b y hig h e st sp ee d a gitation w ith g la ss /
zirconium b eads (0.1 m m d iam eter; Biospec) o n a b ead m ill (Retsch). A ll o ther
steps for R N A extraction w ere d o n e according to the m a n u fa c tu re r's instruc­
tions. R N A concentration a n d p u rity w ere assessed b y sp ectrophotom etry
(N anodrop ND-1000 spectrophotom eter) a n d d e n atu rin g agarose gel electro­
1407
G illard et al.
phoresis. First- a n d seco n d -stran d cD N A synthesis a n d cD NA -A FLP analysis,
w ith BsfYI a n d M sel as restrictio n enzym es, w ere carried o u t according to
V uylsteke et al. (2007), startin g from 2 /xg of total RNA. The final selection of
the cD NA-AFLP am plification p ro d u c ts w as do n e w ith three selective n ucle­
otides, resu ltin g in 128 p rim e r com binations. Expression profiles across the 13
tim e p o in ts for each g en o ty p e w e re q u an tified w ith A FL P-Q uantarPro soft­
w are (Keygene). The raw exp ressio n values w ith in each g en o ty p e w ere
n o rm alized p e r gene b y su b tractin g the average exp ressio n v alu e of each gene
from each d a ta p o in t in the tim e series a n d d iv id in g it b y the s d . R ep roduc­
ibility of exp ressio n profiles b e tw ee n strains Fj-813 an d F -^ A w as estim ated
for every gene b y calculating a P earso n correlation b e tw ee n n o rm alized
expression data. Expression profiles sh o w in g a p o sitiv e a n d significant (P <
0.05) correlation across the tw o strains w ere su b m itte d to a d ap tiv e qualityb a se d clu sterin g (De Sm et et a l, 2002). The m in im al n u m b e r of genes in a
cluster w as set to 10, a n d th e m in im al p ro b ab ility of genes b elo n g in g to a
cluster w as set to 0.95. H ierarchical clu sterin g (Eisen et a l , 1998) w as
p erfo rm ed w ith TMEV softw are (Saeed et al., 2003).
Identification of Differentially Expressed Genes
TDFs w e re p u rified from the gel, follow ed b y am plification an d subse­
q u e n t sequencing as d escrib ed b y V uylsteke et al. (2007). The sequence q uality
w as checked th ro u g h in sp ectio n of th e electrophoretic peaks. W ith th e goodq u ality sequences, a sim ilarity search w as d o n e w ith BLASTN a n d BLASTX
sequence alig n m en ts (A ltschul et al., 1997) a g ain st n u cleo tid e a n d p ro tein
sequences in the pub licly available G enBank d atabases, to w h ic h th e latest
versions of the m o re recently released genom es of Phytophthora soja (U.S.
D ep artm en t of Energy Joint G enom e In stitu te [JGI], v l.l) , Phytophthora
ramorum (JGI, v l.l) , Phaeodactylum tricornutum (JGI, v2.0), a n d Thalassiosira
pseudonana (JGI, v3.0) w ere a d d ed . In case a BLASTN alig n m en t of m o re th a n
30 nucleo tid es w ith 80% coverage b etw een th e q u e ry a n d the h it alig nm ent
w as achieved, the h it sequence w as u sed as a q u e ry for sim ilarity searching
w ith BLASTX. Based o n the h o m o lo g y (E-value cutoff, 1 X IO-3; low est
percentag e identification, 29) an d th e id e n tity of fu n ctio n al GO d o m ain s and
InterP ro dom ains, the TDFs w ere functionally an n o ta ted an d classified into 11
functional g roups. TDFs w ith o u t functional an n o tatio n w ere classified as
u n k now n. G O a n d InterP ro an n o tatio n s w ere p erfo rm ed w ith the online
versio n of th e Blast2GO v l.7 .2 p ro g ram (w w w .B last2G O .de; C onesa et al.,
2005). The p ro g ram extracts th e GO term s associated w ith hom ologies
identified w ith th e N atio n al C enter for Biotechnology In fo rm atio n 's QBLAST
an d re tu rn s a list of GO an n o tatio n s rep resen ted as h ierarchical categories of
increasing specificity. Because the d iato m genom e sequences are n o t in cluded
yet in GenBank, w e u sed the com plete sequences of th eir co rresp onding
d iato m genes as in p u t sequences. For the c o n stru ctio n of S u p p lem en tal F igure
SIO, the d a ta w ere an aly zed at level 2 a n d GO classes w ere set to rep resen t at
least five d ifferen t TDFs.
sequence of the in sert w as 92% identical to th a t of the P. tricornutum FtsZ
p ro te in 14995. Two specific prim ers (Sr FtsZ-FP a n d Sr FtsZ-RP) w ere
de sig n e d w ith Roche P robeF inder o n the cloned sequence for RT-Q-PCR.
RT-Q-PCR Assay
R N A w as isolated a n d q uantified as before. Isolated R N A w as tre a te d w ith
D N asel (G E-H ealthcare) to rem ove all c o n tam inating genom ic D N A . A n
aliquot of 0.5 /xg of total R N A from each sam ple w as u sed for cD N A synthesis.
The reverse transcription w as carried out in a total volum e of 20 /xL w ith oligo(dT)
p rim ers a n d the S u p e rsc rip t II kit (Invitrogen) according to the m anufac­
tu re r's instructions. A 0.5-/xL aliquot of a 5-fold d ilu tio n of the cD N A (2.5 ng)
served as tem plate for each RT-Q-PCR. Prim ers for FCP quantification and
norm alizatio n w ere d e signed usin g the Beacon D esigner 7.0 (P rem ier Biosoft
International; S upplem ental Fig. S5) together w ith a strin g en t set of prim er
d e sig n criteria, inclu d in g p re d ic te d m eltin g tem p eratu res of 58.0°C ± 2.0°C,
p rim er lengths of 17 to 22 nucleotides, a n d am plicon lengths of 75 to 150 bp.
P rim er p airs w ere tested b y RT-Q-PCR o n a p o oled cD N A sam ple u n d e r
the sam e conditions as d escribed below. P rim er reliability w as confirm ed by
the a ppearance of a single p e ak in the m eltin g curve analysis p erfo rm ed b y the
PCR m achine after com pletion of the am plification reaction.
E ight constitutively a n d m o d e ra te ly expressed cD NA -A FLP TDFs w ere
selected as c an d id ate genes to serve for intern al reference d u rin g RT-Q-PCR.
These TDFs w ere excised from the cD NA-AFLP gels, ream plified, and
sequenced, a n d p rim e r p a irs w ere d e signed as before. The e xpression stability
(M) of the p u ta tiv e norm alizatio n genes w as analyzed w ith the geN ORM
program : genes w ith the low est M valu e are the m o st stably expressed and
w ere selected as norm alizatio n genes (V andesom pele et al., 2002). TDFs
srl3af_M 281.1 a n d sr07ae_M536.3 w ere identified b y geN O R M to be the tw o
m o st stably expressed genes (M = 0.426), follow ed b y srl4ah_M 385.6 (M =
0.540). The value for the pair-w ise va ria tio n be tw ee n tw o sequential n o rm al­
ization factors (geN O RM factor V2/3) w as 0.146 d u rin g synchronization,
w hich is sm aller th a n the cutoff valu e of 0.15 p ro p o se d b y V andesom pele et al.
(2002) for reliable d a ta norm alization. Therefore, all three genes w ere u sed for
d a ta norm alizatio n in the su b seq u en t RT-Q-PCR analyses.
RT-Q-PCR w as p erfo rm ed on the Lightcycler 480 (Roche) platform . Each
sam ple w as assayed in triplicate u n d e r the follow ing conditions: 2.5 n g of
tem plate cD NA , 2.5 /xL of the Lightcycler 480 SYBR G reen I M aster M ix (Roche
A p p lie d Science), an d 2 /xL of p rim ers at concentration of 0.5 /x m . The cycling
conditions com prised 10 m in of p rein cu b atio n a t 95°C an d 45 cycles of 10 s at
95°C, 15 s at 58°C, a n d 15 s at 72°C. A m plicon dissociation curves (i.e. m elting
curves) w ere recorded b y h e atin g a t 95°C for 5 s an d at 65°C for 1 m in. Sam ples
w ere cooled at 40°C for 10 s. The relative com parison AACt m e th o d (Pfaffi,
2001) w as u sed to evaluate expression levels of the selected genes relative to
the expression of the norm alizatio n genes in the sam e sam ple. D ata w ere
analyzed a n d n o rm a liz e d w ith qBase (H ellem ans e t al., 2007) for expression
profile generation.
Cloning of Putative FtsZ cDNA from S. robusta
HPLC Pigment Analysis
Based on co n serv ed am in o acid regions of k n o w n FtsZ genes in P.
tricornutum (JGI, v2.0 p ro te in identifiers 14995 a n d 14426) a n d T. pseudonana
(JGI, v3.0 p ro te in identifiers 35728, 269655, an d 15398), 12 oligonucleotide
p rim ers w ere d e sig n e d w ith C O D EH O P (CDH; Rose et al., 1998) a n d by
m a n u a l (m) d esig n (Supplem ental Fig. S5). These p rim ers w e re tested in p a ir­
w ise com binations, a n d th ree (m _FPl, m _R P lb, an d CDH_FP4) w e re suc­
cessfully u sed to am p lify FtsZ cD NA . First, PCR w ith m _ F P l an d m _R P lb
p rim ers co rresp o n d in g to am in o acid sequences W (A /S )(I/L /V )N T D A Q A
an d (I/V )N V D FA D , respectively, w as carried o u t u sin g h o t-sta rt PCR
(AmpliTaq G old D N A polym erase; A pplied Biosystems) in five steps: 9 m in at
95°C; four cycles of 1 m in at 94°C, 1 m in at 35°C, an d 1 m in a t 72°C; fo u r cycles
of 1 m in at 94°C, 1 m in a t 45°C, an d 1 m in a t 72°C; 30 cycles of 1 m in at 94°C,
1 m in a t 50°C, a n d 1 m in a t 72°C; a n d one cycle of 10 m in a t 72°C. A 20-foldd ilu te d , p o o led cD N A sam p le c o n stitu tin g all cell cycle p h ases d u rin g
synchronizatio n w as u sed as tem plate, an d 20 p m o l of each p rim e r w as
u sed in th e 25-/xL reaction m ixture. The PCR p ro d u c ts w ere an aly zed o n a
1.5% agarose gel, a n d a b a n d of the correct size (approxim ately 500 b p) w as
isolated from the gel a n d extracted w ith N u c leo sp in Extract II (M achereyN agel). This extract serv ed as a tem p late in the su b seq u en t PCR w ith nested
p rim er CDH_FP4, c o rresp o n d in g to am in o acid sequence VGIVTKPF, a n d
p rim er m _ R P lb u n d e r the follow ing conditions: 9 m in a t 95°C; 35 cycles of 45 s
a t 94°C, 1 m in at 52°C, a n d 1 m in a t 72°C; follow ed b y one cycle of 10 m in at
72°C. The resu ltin g D N A frag m en t of correct size (approxim ately 300 b p) w as
ligated into a pC R 4 TOPO vector (Invitrogen) a n d sequenced. The D N A
1408
S. robusta p igm ents w ere sam p led from lig h t/d a rk -sy n c h ro n iz e d cultures
(see above) b y filtering 25 m L of su sp e n d e d c ulture over a p re w e ig h ed 25-m m
W h atm an glass fiber filter. Filters w ere w ra p p e d in a lu m in u m foil, frozen in
liquid nitrogen, an d stored a t —80°C. Before analysis, the filters w ere lyophilized for 8 h an d w eig h ed (0.1-mg accuracy) to calculate the d ry w e ig h t of
each sam ple. P igm ents w ere extracted from the filters in 90% acetone by
m eans of sonication (tip sonicator; 40 W for 30 s). Extracts w ere filtered over
a 0.2-/xm A lltech n y lo n syringe filter to rem ove particles a n d injected into a
A gilent 1100 series H PLC system (C hem Station softw are) e q u ip p e d w ith a
M acherey-N agel reverse-phase C18 colum n (N ucleodur C18 p y ra m id ; 5 /xm
particle size). P igm ents w ere analyzed according to the m e th o d of W right and
Jeffrey (1997) usin g a g ra d ie n t of three solvents: 80% m ethanol-20% am m o­
n iu m acetate, 90% acetonitrile, a n d ethyl acetate. Two detectors w ere con­
nected to the H PLC system : a n A gilent sta n d a rd fluorescence detector to
m e a su re fluorescence of chlorophylls a n d th eir derivatives a n d a n A gilent
d io d e arra y detector to m e a su re absorbance of each p e ak at 436 an d 665 run
a n d absorbance spectra over a 400- to 700-run range. F ucoxanthin w as
identified b y com parison of re tention tim es a n d absorbance spectra and
q uantified b y calculating response factors usin g p u re p ig m e n t stan d a rd s
(su p p lied b y DHI).
Sequence d a ta from this article can be fo u n d in the G enB ank/E M B L d ata
libraries u n d e r accession n u m b e rs FJ388875 a n d GE343005 to GE343326.
P la n t P h y sio l. Vol. 148, 2008
The Cell a n d C hlo ro p last Cycle of Seminavis robusta
Supplemental Data
The follow ing m aterial is available in th e online v ersio n of this article.
Supplem ental Figure SI. Schem atic p re sen ta tio n of th e chloroplastidic
events d u rin g the cell cycle of S. robusta.
Supplem ental Figure S2. Flow cytom etric D N A content h isto gram s for
assessing the S-phase cell cycle arrest b y HU.
Supplem ental Figure S3. Estim ation m e th o d for percen tag e of cells in
d ifferen t cell cycle p h a se s b a se d o n p h o to g ra p h s of the sy nchronized
culture.
Supplem ental Figure S4. L aser-scanning confocal p h o to g ra p h s of a S.
robusta cell to illu strate th e a d d itio n of one girdle ban d .
Supplem ental Figure S5. Identification of a S. robusta FtsZ o rtholog an d
d e sig n of specific RT-Q-PCR p rim ers for FtsZ a n d FCP quantification.
Supplem ental Figure S6. Section of a n electro p h ero g ram of the cD NA AFLP fin g erp rin t of the tw o S. robusta strains.
Supplem ental Figure S7. The n in e clusters identified b y ad ap tiv e qualityb a se d clu sterin g (De Sm et et a lv 2002) of 917 exp ressio n profiles that
w ere h ig h ly reproducible.
Supplem ental Figure S8. Expression of the S. robusta M CM 5 a n d histone
H3 o rth o lo g as a fu n ctio n of tim e after reillum ination.
Supplem ental Figure S9. C lu sterin g of the exp ressio n profiles of the cell
cycle-m odulated, an n o ta ted TDFs.
Supplem ental Figure SIO. Blast2GO p ie ch art rep resen tin g the d istrib u ­
tio n of fu n ctio n al GO labels for biological processes.
Supplem ental Figure S ll . Expression of the S. robusta orthologs of
/3-tubulin a n d th e W D 40-repeat p ro te in as a fu n ctio n of tim e after
reillum ination.
Supplem ental Table SI. R epresentation of th e cD NA -A FLP analysis,
in clu d in g all q u an tified TDFs, an n o tatio n s, a n d expression data.
Supplem ental Table S2. D istrib u tio n of selected genes w ith respect to their
expression cluster.
Supplem ental Table S3. M ap p in g of GO labels to the an n o ta ted TDFs,
to g eth er w ith their assigned functional categories.
Supplem ental Table S4. M ap p in g of InterP ro d o m ain s to the a n notated
TDFs.
Supplem ental Table S5. C alculation of y2 correlation b etw een p h o to sy n ­
thetic genes a n d expression cluster C7-L.
ACKNOWLEDGMENTS
We th a n k Bart V anelslander for h elp w ith the sam pling, D ebbie R om baut
for help w ith th e cDNA-AFLP, a n d M artin e De C ock for h elp in p re p a rin g the
m anu scrip t.
Received A p ril 29, 2008; accepted Septem ber 18, 2008; p u b lish e d Septem ber
26, 2008.
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