Developmentally regulated association of plastid division
protein FtsZ1 with thylakoid membranes in Arabidopsis
thaliana.
El-Sayed El-Kafafi, Mohamed Karamoko, Isabelle Pignot-Paintrand, Didier
Grunwald, Paul Mandaron, Silva Lerbs-Mache, Denis Falconet
To cite this version:
El-Sayed El-Kafafi, Mohamed Karamoko, Isabelle Pignot-Paintrand, Didier Grunwald, Paul Mandaron, et al.. Developmentally regulated association of plastid division protein FtsZ1 with thylakoid
membranes in Arabidopsis thaliana.. Biochemical Journal, Portland Press, 2008, 409 (1), pp.87-94.
10.1042/BJ20070543. inserm-00413580
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Biochem. J. (2008) 409, 87–94 (Printed in Great Britain)
87
doi:10.1042/BJ20070543
Developmentally regulated association of plastid division protein FtsZ1
with thylakoid membranes in Arabidopsis thaliana
El-Sayed EL-KAFAFI*1,2 , Mohamed KARAMOKO*1 , Isabelle PIGNOT-PAINTRAND†, Didier GRUNWALD‡, Paul MANDARON*,
Silva LERBS-MACHE* and Denis FALCONET*3
FtsZ is a key protein involved in bacterial and organellar division.
Bacteria have only one ftsZ gene, while chlorophytes (higher
plants and green alga) have two distinct FtsZ gene families,
named FtsZ1 and FtsZ2. This raises the question of why chloroplasts in these organisms need distinct FtsZ proteins to divide. In
order to unravel new functions associated with FtsZ proteins, we
have identified and characterized an Arabidopsis thaliana FtsZ1
loss-of-function mutant. ftsZ1-knockout mutants are impeded
in chloroplast division, and division is restored when FtsZ1 is
expressed at a low level. FtsZ1-overexpressing plants show a
drastic inhibition of chloroplast division. Chloroplast morphology
is altered in ftsZ1, with chloroplasts having abnormalities in the
thylakoid membrane network. Overexpression of FtsZ1 also in-
INTRODUCTION
FtsZ plays an essential role in bacterial and archaeal division [1–
3], in chloroplast division [4–6] and, in some lower organisms
(algae and slime moulds), in mitochondrial division [4,7]. FtsZ,
the most conserved of the bacterial cell division proteins, shares
structural homology with the eukaryotic tubulin element and may
be a progenitor of tubulin [8]. Bacterial FtsZ polymerizes like
eukaryotic cytoskeletal proteins and forms the so-called Z ring,
which forms a scaffold for assembly of at least ten other division
proteins [9]. The Z ring adheres to the inside of the bacterial
membrane and constricts to form a preseptal ingrowth, which then
invaginates further to mediate division. Proteins involved in the
cell division complex or divisome are cytoplasmic, periplasmic
and membrane-embedded proteins. No outer membrane protein
has so far been linked to cell division.
While most bacteria (including cyanobacteria) have only one
FtsZ protein, plastid FtsZ protein sequences form three clades,
including green-plastid FtsZ1, green-plastid FtsZ2 and the red
and chromophyte algal group [10,11]. Phylogenetic analysis
suggests that ftsZ gene duplication occurred subsequent to the
endosymbiotic event. In chlorophytes, the gene duplication giving
rise to the FtsZ1 and FtsZ2 gene families occurred before green
algae branched from the ancestor of land plants [10–12]. The
duplication event raises the question of why plastids in these
organisms need two different proteins in order to divide when the
ancestral bacteria use only one protein.
The major difference between FtsZ1 and FtsZ2 in both chlorophytes and non-chlorophytes lies in their C-terminal sequences.
duced defects in thylakoid organization with an increased network
of twisting thylakoids and larger grana. We show that FtsZ1,
in addition to being present in the stroma, is tightly associated
with the thylakoid fraction. This association is developmentally
regulated since FtsZ1 is found in the thylakoid fraction of young
developing plant leaves but not in mature and old plant leaves.
Our results suggest that plastid division protein FtsZ1 may have a
function during leaf development in thylakoid organization, thus
highlighting new functions for green plastid FtsZ.
Key words: Arabidopsis thaliana, chloroplast division, FtsZ gene
family, plastid division, plastid localization, thylakoid membrane.
The C-terminal conserved motif, present in bacterial FtsZ, is
required for direct interaction with ZipA and FtsA in Escherichia
coli [13,14]. This domain is present in FtsZ2, but not in FtsZ1,
in chlorophytes and in related sequences in non-chlorophytes.
Homologues of ZipA and FtsA have not been identified in eukaryotic genomes, but the protein ARC6 (accumulation and replication
of chloroplasts 6) in Arabidopsis shares homology with the
cyanobacterial protein Ftn2 [15] and interacts with FtsZ2 via
the C-terminal core domain [16], providing in planta evidence
for a functional difference between the two FtsZ protein families
in plants. The conserved N-terminal sequence is sufficient for
bacterial FtsZ polymerization [17]. The major difference between
FtsZ1 and FtsZ2 in higher plants concerns a single amino acid
change in the conserved ‘tubulin signature motif’ [18].
In addition, FtsZ1 and FtsZ2 differ in their biochemical
properties and subplastidial localization [19]. Expression of
FtsZ1 and FtsZ2 in E. coli differentially affects division, and
these different effects are related to the FtsZ2 C-terminal
sequence. Only FtsZ1 is able to polymerize in vitro and forms
GTP-dependent rod-shaped polymers and rings similar to the
bacterial structures, but FtsZ2 can promote GTP-independent
FtsZ1 polymerization. These results, together with other results
showing the interaction of only FtsZ2 with ARC6 and an earlier
expression of FtsZ2 during the cell cycle in BY2 cells [18], suggest
that FtsZ2 and FtsZ1 fulfil different functions during chloroplast
division [19].
In addition to being a key element in the chloroplast division
machinery, plant FtsZ proteins may be involved either directly or
indirectly in the co-ordination of cell division and plastid division.
Abbreviations used: ARC6, accumulation and replication of chloroplasts 6; FST, flanking sequence tag; GC1, giant chloroplasts 1; IEP37, inner envelope
protein of 37 kDa; KARI, ketol-acid reducto-isomerase; OEP21, an outer envelope protein of 21 kDa; PSII, Photosystem II; SAM, shoot apical meristem;
T-DNA, transfer DNA; TEM, transmission electron microscopy; WT, wild-type.
1
These authors contributed equally to this work.
2
Present address: IBIP (Institut de Biologie Intégrative des Plantes), UMR 5004 Agro-M/CNRS/INRA/UM2, Place Viala, 34060 Montpellier Cedex 1,
France.
3
To whom correspondence should be addressed (email denis.falconet@ujf-grenoble.fr).
c The Authors Journal compilation
c 2008 Biochemical Society
Biochemical Journal
*Laboratoire Plastes et Différenciation Cellulaire, Université Joseph Fourier and CNRS, BP 53, F-38041 Grenoble Cedex 9, France, †CERMAV, UPR 5301-CNRS, BP 53, F-38041
Grenoble Cedex 9, France, and ‡Département de Réponse et Dynamique Cellulaire, CEA, F-38054 Grenoble Cedex 9, France
88
E.-S. El-Kafafi and others
An FtsZ1 isoform in the moss Physcomitrella patens is localized
both in chloroplasts and in the cytoplasm, assembling into rings
in both cell compartments, and transfected cells expressing high
amounts of the protein were impeded in cell division [20]. In
higher plants, expression of both FtsZ1 and FtsZ2 genes seems
to be cell-cycle-regulated [18], and in Arabidopsis, the prereplication factor AtCDT1 is involved not only in nuclear DNA
replication but also in plastid division by means of an interaction
with ARC6 [21].
Here, we describe the characterization of an Arabidopsis
ftsZ1 mutant in which chloroplast division is strongly impeded
but restored when FtsZ1 is expressed at a low level. FtsZ1
overexpression in both backgrounds [ftsZ1 and WT (wildtype)] induces a strong phenotype with a drastic effect on
chloroplast morphology and division. Both null and FtsZ1-overexpressing plants have an accelerated development. ftsZ1deficient plants have an altered thylakoid membrane network,
whereas FtsZ1 overexpression results in an increased network of
twisting thylakoids and larger grana. We show that FtsZ1, in
addition to being present in the stroma, is also found tightly
associated with the thylakoid membranes and that this association
is developmentally regulated.
EXPERIMENTAL
Plant material, transformation and growth
Arabidopsis, Wassilewskija ecotype (Ws), was used as WT plants
for all experiments. The FtsZ1 T-DNA (transfer DNA) insertion
mutant FST (flanking sequence tag) number 128A08 was obtained
from the FLAGdb/FST initiative (http://193.51.165.9/projects/
FLAGdb++/HTML/index.shtml). Genomic DNA was amplified
with the Tag6 (5′ -CTTTCATCTACGGCAATGTACCAGC-3′ )
and Tag7 (5′ -GTCGATAAGAAAAGGCAATTTGTAG-3′ ) specific primers to detect the T-DNA. To follow the presence of the
endogenous copy of the FtsZ1 gene and identify homozygous
lines, genomic DNA was PCR-amplified with specific couples
of primers: FtsZ1/D-FtsZ1/R and FtsZ1/D-Tag6 (FtsZ1/D: 5′ -ATGGCGATAATTCCGTTAGCACA-3′ ; FtsZ1/R: 5′ -AGGGGCATCTGAAAAGAAGAT-3′ ). Homozygous lines were propagated
by repeated self-pollination and the absence of FtsZ1 expression
was assayed by immunoblotting with polyclonal anti-FtsZ1
antibodies [18]. In order to exclude the presence of more than one
T-DNA, Southern-blot analysis was performed. Nuclear DNA
of the selected lines was digested with EcoRI, a restriction
enzyme with a unique site in FtsZ1 (within intron 1) and in
the T-DNA. DNA fragments were fractionated on 0.8 % agarose
gels, denatured and transferred on to nitrocellulose membrane.
The membrane was hybridized, under standard high-stringency,
with a T-DNA internal probe. For complementation analysis, the
Arabidopsis FtsZ1 cDNA was ligated into the pFP101 vector that
allows for the selection of transgenic Arabidopsis seeds via the
GFP (green fluorescent protein) expression driven by the At2S3
seed-specific promoter [22]. The construct was expressed in the
FST number 128A08 mutant as well as in the WT plant. T1
plants were analysed by immunoblotting with polyclonal antiFtsZ1 antibodies and by microscopy.
For plant growth, seeds were stratified at 4 ◦C for 2 days before
growth on soil at 23 ◦C with a 16 h/8 h light/dark photoperiod at
a light intensity of 60 µmol · m−2 · s−1 .
Protein isolation, chloroplast protein fractionation, treatments and
immunoblotting analyses
Total plant protein extracts were obtained following the Tanaka
method [23]. Stromal, thylakoid and envelope proteins purified
c The Authors Journal compilation
c 2008 Biochemical Society
from lysed chloroplasts were separated on a step gradient of 0.93
and 0.6 M sucrose in 10 mM Mops (pH 7.6) and 1 mM MgCl2
by centrifugation as described in [24]. For high-salt, alkaline and
ETDA washes, the purified thylakoid fraction (30 µg of protein)
was incubated for 30 min at 0 ◦C in 1.0 M NaCl, 0.1 M Na2 CO3
(pH 11.5), 0.1 N NaOH or 5 mM EDTA. The mixtures were then
centrifuged and the pellets were resuspended in SDS loading
buffer. For protease treatment, thylakoids were incubated for
30 min at 22 ◦C with 100 µg · ml−1 thermolysin (Sigma) in the
absence or presence of 5 mM EGTA. Proteolysis was stopped by
supplementing the assays with 20 mM EGTA; thylakoids were
re-isolated by centrifugation, washed once and suspended in SDS
loading buffer.
Protein concentrations were determined by using the Bio-Rad
DC protein and BSA as the standard. When necessary, Coomassiestained gels were used to calibrate loading. For immunoblot
analyses, the proteins were separated by SDS/PAGE, blotted
on to Immobilon-P membranes (Millipore) and incubated with
antibodies as described previously [19].
Pigment analyses and chlorophyll fluorescence
For pigment analyses, leaf samples (70 mg fresh weight)
were frozen in liquid nitrogen prior to grinding in DMF
(dimethylformamide). The resulting extracts, after bubbling with
nitrogen, were stored in the dark for 48 h at 4 ◦C. Extracts were
centrifuged at 12 000 g for 5 min and pigment analyses were performed by HPLC using a 5 mm reverse-phase C30 column
(250 mm × 4 mm) coupled with a 20 mm × 4.6 mm C30 guard
(YMC) and a ProStar 330 photodiode array detector (Varian
Analytical Instruments), as described previously [25]. Peak areas
of the standards were determined using the Varian Poly View 2000
software supplied.
The maximal quantum yield of PSII (Photosystem II)
photochemistry (F) with an F v (variable fluorescence)/F max
(maximum fluorescence) ratio was determined on attached leaves
at room temperature (23 ◦C) by chlorophyll fluorimetry using a
PAM-2000 modulated fluorimeter (Waltz, Effeltrich, Germany)
as described in [26].
Confocal microscopy, TEM (transmission electron microscopy) and
immunogold electron microscopy
Chloroplasts in mesophyll and shoot cells were analysed by
imaging the chlorophyll fluorescence by confocal laser scanning
microscopy with a Leica TCS-SP2 operating system (Leica,
Heidelberg, Germany). Chlorophyll was excited at 633 nm and
the emitted fluorescence was detected between 643 and 720 nm.
For TEM and immunolocalization, leaf samples of 3-weekold plants were fixed in 2.5 % (v/v) glutaraldehyde in 0.1 M
cacodylate buffer for 2 h at room temperature. The samples were
subsequently washed and post-fixed with 1 % osmium tetraoxide
in water for 1 h at 4 ◦C and then dehydrated in ethanol and
infiltrated with an ethanol/resin mixture (2/3–1/3, 1/3–2/3 for
Epon and 80–20, 60–40, 40–60, 20–80, 100, 100 last infiltration
overnight for LR-White). Tissues were embedded in Epon
for structural study and in LR-White for immunolocalization.
Ultrathin sections (60 nm) were prepared with a diamond knife
on an UC6 Leica ultramicrotome and collected on Formvar-coated
200 µm mesh nickel grids. Ultrathin sections were post-stained
with 5 % uranyl acetate in water and lead citrate before examining
on a Philips CM200 electron microscope (Philips, Eindhoven, The
Netherlands).
For immunolocalization, the sections were first incubated with
5 % (w/v) BSA in PBS for 1 h at room temperature and then
with anti-FtsZ1 antibodies (dilution of 1:4 in 1 % BSA in
Association of plastid division protein FtsZ1 with thylakoids
Figure 1
89
ftsZ1 characterization
(A) Gene structure of FtsZ1. Black boxes, exons; lines, introns. The position of the T-DNA insertion in exon 4 is indicated together with the primer locations. (B, C) Identification of homozygous
lines. PCR amplification with the indicated primers on genomic DNA extracted from WT and selected lines (1–4). ‘M’ indicates DNA size marker. (D) T-DNA insertion number in the mutant lines as
analysed by Southern blot with the T-DNA internal probe. (E) Expression of FtsZ1 in the selected plants as analysed by immunoblotting with FtsZ1 antiserum.
PBS) overnight at 4 ◦C. Samples were then incubated with goat
anti-rabbit IgG conjugated to 10 nm gold particles at a dilution of
1:20 in 1 % BSA in PBS for 1 h at room temperature. Controls
were performed by excluding the primary antibody. Finally,
sections were post-stained with 5 % uranyl acetate in water and
lead citrate.
RESULTS
Phenotypic characterization of ftsZ1-deficient and
FtsZ1-overexpressing plants
The Arabidopsis genome encodes a single FtsZ1 plastid division
protein on chromosome 5 at the locus At5g55280. One
Arabidopsis line containing a T-DNA insertion in exon 4 was
identified by the Genoplante FLAGdb/FST initiative (Figure 1A).
Plants homozygous for the mutant alleles were identified by PCR
analysis of segregating plants (Figures 1B and 1C). Segregation
values observed for kanamycin resistance (three-quarters resistant
and one-quarter sensitive individuals after self-pollination of
heterozygous plants) indicated that the mutant lines contained
only one insertion of the T-DNA in their nuclear genome. The
presence of only one T-DNA was also confirmed by Southernblot analysis (Figure 1D). Only the 4.8 kbp EcoRI fragment,
expected from the restriction data, was detected. Western-blot
analysis confirmed the absence of FtsZ1 protein in the selected
mutant lines (Figure 1E). Western blots showed FtsZ1 protein
expression in WT plants and in the heterozygous line 1, but
no expression in the homozygous lines 2, 3 and 4.
Mature ftsZ1 mesophyll and stem cells contained fewer, but
larger chloroplasts than the corresponding cells in WT, although
the cell sizes between the two plants were similar (Figures 2A,
top panels, and 2B). The average chloroplast number at the
equatorial plane decreased from 16 in WT mesophyll cells and
14 in WT stem cells to 7 and 4 respectively in ftsZ1 cells.
The mutant phenotype was rescued by low-level expression
of the FtsZ1 cDNA as shown by Western blotting, which did
not alter the WT phenotype when expressed in WT plants
(Figure 2A, middle panels). However, overexpression of FtsZ1
had a drastic effect on chloroplast division in both the WT
and ftsZ1 backgrounds, with cells harbouring only few enlarged
chloroplasts (Figure 2A, bottom panels). This corroborates
previous results showing altered stoichiometry of FtsZ affecting
chloroplast division [27]. As illustrated with the stem cells
(Figure 2B), all cells of a given tissue were strongly affected by the
absence or overexpression of the FtsZ1 protein. In all the mutant
cells examined, the shape of the large chloroplasts was irregular,
having lost the characteristic globular structure as observed in
the WT plants. Compared with WT plants, ftsZ1 and FtsZ1overexpressing lines exhibited accelerated growth (Figure 2C)
and began flowering approx. 1 week earlier, on average, than
WT plants (2 weeks after germination for the mutant plants
compared with 3 weeks for the WT plants). ftsZ1 and 35S::FtsZ1
plants flowered with the same number of rosette leaves (an
c The Authors Journal compilation
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90
Figure 2
E.-S. El-Kafafi and others
Phenotype of ftsZ1 and FtsZ1-overexpressing plants
(A) Top panels: confocal micrographs of chloroplasts of WT (left column) and ftsZ1 (right column) plant leaves. Middle and bottom panels: WT and ftsZ1 plants expressing FtsZ1 under the control
of the 35S promoter. Relative FtsZ1 expression levels are shown by immonoblots on the left side of each panel. Scale bar, 20 µm. (B) Confocal micrographs of hypocotyl cells from WT, ftsZ1 and
overexpressing (35S::FtsZ1) plants. Scale bar, 40 µm. (C) Phenotype of 4-week-old WT, ftsZ1 and 35S::FtsZ1 plants. (D) Chlorophyll fluorescence was measured as described in the Experimental
section. Error bars represent the S.D. for three measurements. (E) Accumulation of pigments indicated on the horizontal axis was measured as described in the Experimental section. Error bars
represent the S.D. for three measurements.
average of 8 leaves) when the WT plants flowered with an
average of 14 larger rosette leaves. After 6 weeks of growth, the
size of the mutant plants was greatly reduced compared with
the WT plants. Both mutant lines were able to produce viable
seeds. Photosynthesis was investigated using pulse-amplitudemodulated chlorophyll fluorescence analysis and pigment content
on 3-week-old plantlets. The F v /F max ratio was identical between
the mutant plants and the WT plants (Figure 2D). This indicates
that the PSII reaction centre was properly assembled and
photochemically competent in both mutant plants. Equally, the
amount of photosynthetic pigments was unaltered in both mutant
plants (Figure 2E).
Chloroplast morphology and ultrastructure is affected in ftsZ1 and
35S::FtsZ1 plants
Three-week-old rosette leaves were fixed for TEM and observed
at different levels of magnification. In WT plants, individual
ellipsoidal chloroplasts, aligned along the cytoplasmic membrane,
c The Authors Journal compilation
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were observed at the cellular level (Figure 3A). These chloroplasts
contained starch granules, and their internal structures, including
evenly stacked granal thylakoids connected by stromal lamellae,
were completely developed (Figures 3D and 3G). These lamellae
are oriented along the convex side of the chloroplast facing the
interior of the cell. In contrast, abnormally long chloroplasts were
observed in the leaves of ftsZ1 (Figure 3B) and 35S::FtsZ1 plants
(Figure 3C). Although the long chloroplasts in the null plant were
aligned along the plasma membrane, as was observed in the
WT plant, the chloroplasts were detached from the plasma
membrane at several points in the FtsZ1-overexpressing plant.
Changes in thylakoid orientation were often observed in the
null plant (Figure 3E; see also Supplementary Figure S1 at
http://www.BiochemJ.org/bj/409/bj4090087add.htm), and membranes appeared less appressed (Figure 3H). Overexpression of
FtsZ1 induced an increased network of twisting thylakoids (Figure 3F) and an increased number of thylakoids per grana stack
(Figure 3I). An increase in starch granule number was observed in
the null mutants, but no starch granules were observed in FtsZ1overexpressing plants.
Association of plastid division protein FtsZ1 with thylakoids
Figure 3
91
FtsZ1 affects chloroplast morphology and ultrastructure
Electron micrographs of rosette leaf mesophyll chloroplasts from 3-week-old WT, FtsZ1 and 35S::FtsZ1 plants. (A–C) Overviews showing the chloroplast number and morphology within the cells
(scale bar, 5 µm). (D–F) Inside chloroplast views showing the thylakoid arrangement (scale bar, 1 µm). (G–I) Higher magnification showing the grana stacking (scale bar, 200 nm).
FtsZ1 is localized within the stroma, but is also found associated
with the thylakoids
The changes in thylakoid organization in ftsZ1-null and ftsZ1overexpressing plants indicated a possible role of FtsZ1 in
thylakoid integrity and therefore suggested an association or at
least some contact between FtsZ1 and thylakoid membranes.
In order to test this hypothesis, we investigated the localization
of FtsZ1 proteins in WT Arabidopsis plants by suborganellar
fractionation and Western blotting, using an anti-FtsZ1 antibody
previously shown to react with proteins from purified chloroplast
extracts [28] (Figure 4A). The stromal, thylakoid and envelope
fractions were purified from lysed chloroplasts by sucrosedensity-gradient centrifugation [24]. Blots were first probed with
the anti-FtsZ1 antibody and subsequently probed with antisera
against (i) KARI (ketol-acid reducto-isomerase), a stromal protein
involved in the amino acid biosynthetic pathway [29], (ii) PsbB
(CP47), a thylakoid-integrated subunit of the PSII reaction centre,
(iii) IEP37, an inner envelope protein of 37 kDa [30], and (iv)
OEP21, an outer envelope protein of 21 kDa [31]. An FtsZ1
signal co-localized with the stromal and thylakoid controls, but
not with the envelope proteins, suggesting that FtsZ1 localizes
to thylakoid membranes (Figure 4A). Purified thylakoids were
next washed with 1 M NaCl, 0.1 M Na2 CO3 (pH 11.5) or 0.1 M
NaOH to further characterize the association of FtsZ1 with the
thylakoid membranes (Figure 4B). FtsZ1 was not released from
the thylakoids by ionic extraction but was solubilized by alkaline
extractions, indicating that it is peripherally associated with the
membrane. The localization with the thylakoid membranes is
independent of magnesium concentration because in the presence
of EDTA, FtsZ1 is still detected in the thylakoid fraction
(Figure 4B). As a control, release of PsbB from membranes was
tested under identical conditions, but none was observed. It is
therefore possible that FtsZ1 is associated with one leaflet of the
membrane via lipid interaction.
To determine FtsZ1 topology, thermolysin proteolysis was
performed with isolated thylakoids (Figure 4C). Under such
a treatment, proteins on the stromal side of the thylakoid
membrane are degradable, while lumen proteins, not accessible to
thermolysin, are protected. FtsZ1 was accessible to thermolysin
in the absence of EGTA, while the addition of EGTA inhibited
the cleavage reaction, suggesting that FtsZ1 is located on the
stromal-facing side of the thylakoid membranes.
To confirm the localization of FtsZ1 visually, immunoelectron
microscopy was performed with 3-week-old Arabidopsis WT
and FtsZ1-overexpressing plants (35S::FtsZ1). Small clusters and
large clusters of gold particles in the WT and 35S::FtsZ1 plants
respectively were observed in close association with the thylakoid
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E.-S. El-Kafafi and others
Figure 5 Immunolocalization of the FtsZ1 protein in association with
Arabidopsis thylakoids
Ultrathin sections of chloroplasts from WT and overexpressing (35S::FtsZ1) plants were
incubated successively with anti-FtsZ1 antibodies and goat anti-(rabbit IgG) secondary antibody
conjugated to colloidal gold. Gold clusters in association with the thylakoids are indicated by
the arrow in both panels (scale bar, 100 nm).
Figure 4
FtsZ1 is localized in the stroma and thylakoid membranes
(A) Immunoblots of protein fractions (30 µg of each) isolated from chloroplasts, stroma,
thylakoids and envelopes of 4-week-old plants were analysed with FtsZ1, KARI, PsbB (Agrisera,
Vännäs, Sweden), IEP37 and OEP21 antisera. The faint signal in the thylakoid fraction observed
with the envelope markers indicated a very low level of contamination of the thylakoids with
envelope membranes. (B) Purified thylakoids (30 µg of proteins) were incubated for 30 min in
the indicated solutions, and the proteins present after centrifugation in the pellet were analysed
by immunoblotting with either the anti-FtsZ1 or anti-PsbB antibodies. (C) Thylakoid fractions
were incubated with thermolysin (100 µg · ml−1 ) in the absence (–EGTA) or presence (+EGTA)
of EGTA. The control (no thermolysin) proteins (thylakoid) and treated thylakoid proteins were
analysed by immunoblotting with the anti-FtsZ1 antibody.
network (Figure 5). The large number of gold particles in the
35S::FtsZ1 plant are in agreement with the results observed by
immunoblot analysis (Figure 2A).
Association of FtsZ1 with the thylakoids is developmentally
regulated
FtsZ1 was reported previously to be a soluble protein found only
in the stromal fraction [19,32], while our results suggest that FtsZ1
is also associated with thylakoid membranes. To understand this
discrepancy, we investigated whether FtsZ1 association with the
thylakoids differed at various points during plant development.
To test this hypothesis, we examined developmentally associated
changes in FtsZ1 protein amount and localization by Western
blotting. Proteins from subplastidial fractions were obtained from
plants grown for 22, 35 and 43 days. A strong FtsZ1 signal
was observed in the thylakoid fraction as well as in the stromal
fraction of plants grown for 22 days (Figure 6). After 35 days of
growth, only a faint signal was observed in the thylakoid fraction, while a strong signal was observed in the stromal fraction.
FtsZ1 is no longer detected in the thylakoid fraction, but is still
c The Authors Journal compilation
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Figure 6
regulated
FtsZ1 association with the thylakoids is developmentally
Immunoblot analysis of protein fractions (30 µg of each) isolated from chloroplasts, stroma and
thylakoids of 22-, 35- and 43-day-old plants respectively with FtsZ1, KARI and PsbB antisera.
strongly present in the stroma in plants grown for 43 days. As
expected, KARI was present only in the stroma, whereas PsbB
was found exclusively in thylakoids, indicating the purity of
the two fractions. The correlation between the level of FtsZ1
in the thylakoid fraction and the developmental stage of the
tissue demonstrated the regulation of FtsZ1 localization within
the chloroplast.
DISCUSSION
In order to highlight new plant-specific functions for FtsZ1,
we have characterized an ftsZ1-null mutant of Arabidopsis. As
expected from previous reports using an antisense strategy [27],
ftsZ1 mutant cells contained fewer but larger chloroplasts than WT
plants. Inhibition of chloroplast division is rescued by expressing
FtsZ1 in these mutant plants, but the complementation is dosedependent, since weak expression restores the WT phenotype,
while a strong expression causes inhibition of chloroplast division
and changes in chloroplast morphology, as previously shown [33].
Such a dose-dependent effect also occurs when FtsZ1 is expressed
in E. coli, mimicking the expression of other bacterial ftsZ
genes in E. coli [19]. These results confirm that a strict stoichiometry between subunits of the chloroplast division machinery is
necessary for proper division. The overall life cycle of both ftsZ1
plants and FtsZ1-overexpressing lines is not affected, confirming
that impaired plastid division has no significant effect on plant
growth and development [34], although these plants are smaller
than WT controls, flower earlier and contain a reduced number
Association of plastid division protein FtsZ1 with thylakoids
of rosette leaves. This phenotype is not a direct consequence of
smaller cells and/or diminished photosynthetic yield, since the
cell size and photosynthetic capacity, as determined by measuring
the maximal photochemical efficiency of PSII and quantifying the
photosynthetic pigments, are unaltered in both transgenic lines.
Developmental variation in the rate of leaf initiation has been
suggested to result from change in the rate of cell division in
the SAM (shoot apical meristem) [35]. The phenotype observed
in the mutant plants might be driven by changes in cell division in
the SAM, thus connecting cell division and plastid division as
suggested by a number of studies [18,20,21,36].
Mesophyll chloroplast ultrastructure is affected differently in
the null and FtsZ1-overexpressing plants. FtsZ1 chloroplasts
have frequent changes in their thylakoid orientation and
fewer thylakoids per granal stack, while FtsZ1-overexpressing
chloroplasts display a highly disturbed thylakoid network with
an increase in granal stacking. Organizational changes of the
thylakoid membranes within some, but not all, chloroplast
division mutants have also been described. Granal stacks in
GC1 (giant chloroplasts 1)-deficient giant chloroplasts are more
densely packed than in WT plants [37]. Thylakoids in arc5 and
arc6 chloroplasts are also highly stacked when compared with WT
chloroplasts and show increased stacking when plants are grown
under high light [38]. Under the same conditions, a decrease
is observed with WT plants. Interestingly, FtsZ1-overexpressing
plants show an increased network of twisting thylakoids similar to
those observed in arc3 [38]. These results demonstrate that some
proteins involved in plastid division also affect chloroplast internal
structures. GC1 is plastid-localized and is anchored to the stromal
surface of the chloroplast inner envelope [37]. Arc5 encodes a
cytosolic dynamin-like protein and forms a cytosolic ring structure
on the outside of the chloroplast [39]. Arc6, which encodes a Jdomain protein and is a homologue of the cyanobacterial cell
division protein Ftn2 [15], has been shown to be an integral inner
envelope membrane protein. Arc3, a fusion of FtsZ and PIP5K
(phosphatidylinositol-4-phosphate 5-kinase) [40], has recently
been shown to be located in the stroma [41]. None of these
proteins have been found associated with thylakoids, suggesting
that the effect on the thylakoid network in these mutants is
indirect. The twisting thylakoids observed in arc3, resembling
those observed in the FtsZ1-overexpressing plants, might result
from the misplacement of FtsZ1. As a matter of fact, Arc3 has
been shown to interact specifically with FtsZ1, acting in division
site placement [41].
The localization of FtsZ proteins in higher plants is more
complex. Both FtsZ1 and FtsZ2 have been found in the chloroplast
stromal compartment in Arabidopsis chloroplasts [32], but
localization in the thylakoid fraction has not been addressed
previously. We have shown that FtsZ1 and FtsZ2 are localized
within the stroma in chloroplasts from mature spinach (Spinacia
oleracea) leaves, but that FtsZ2 is also found associated with
the chloroplast envelopes [19]. In the present study, we show
that FtsZ1 is present in the stromal and thylakoid fractions from
the chloroplasts of young Arabidopsis seedlings. Moreover, we
demonstrate that the levels of FtsZ1 associated with the thylakoids
decreases with the age of the plants. This result shows that FtsZ1
localization within the chloroplast is developmentally regulated
and suggests that the protein fulfils specific functions at different
stages of plant development. It is not known yet whether the
difference in localization between spinach and Arabidopsis is a
consequence of the different developmental stage and/or culture
conditions of the analysed plants. In the previous experiments,
spinach leaves, purchased from a local market, were grown
under external conditions, while Arabidopsis plants in the present
study were grown under strict controlled temperature and light
93
conditions. It is not known so far whether FtsZ1 association
with the thylakoids is dependent on these growth conditions.
Alternatively, it is possible that the discrepancy reflects species
difference.
The thylakoid organization phenotypes associated with the
absence or overexpression of FtsZ1 in the mutant plants
corroborate the hypothesis that FtsZ1 is involved in determining
thylakoid morphology. It is therefore conceivable that the organizational changes of the thylakoid membranes within the arc
chloroplasts reflect an increased localization of FtsZ1 within the
thylakoids in the absence of the ARC proteins. Interestingly,
dynamin-like proteins, which are involved in plastid division
[39], have also been shown to be a determinant in thylakoid
morphology [42,43]. In Arabidopsis, FZL, which is related to
FZO, a dynamin protein involved in mitochondrial morphology
in fungi and animals, is distributed between the thylakoid and
envelope membranes. Fzl mutants have abnormalities in the
morphology and distribution of granal and stromal thylakoids
[43]. The results presented in this paper suggest that the dynamic
duo of FtsZ and dynamin, which has been shown to be involved in
plastid division, participates in the remodelling of the thylakoid
membranes. FtsZ1 and FZL are not involved in the early steps of
thylakoid biogenesis since both ftsZ1 and fzo chloroplasts have
a preformed thylakoid network. Other proteins such as VIPP1
(vesicle-inducing protein in plastids 1) [44] and Thf1 (thylakoid
formation 1) [45] have been implicated in chloroplast vesicle
trafficking and have been shown to affect thylakoid formation. The
association of FtsZ1 with the thylakoids during leaf expansion
suggests that FtsZ1 might be involved in thylakoid membrane
partitioning to daughter plastids during division and in the rearrangement of the internal membrane structures when new
thylakoid membranes are synthesized, thus highlighting specific
functions for green plastid FtsZ.
We thank Catherine Albrieux (Physiologie Cellulaire Végétale, Grenoble, France) for help
with the thylakoid purification; Maryse Block (Physiologie Cellulaire Végétale, Grenoble,
France) for helpful comments on the chloroplast membrane system; Hélène Pesey, JeanPierre Alcaraz, Eliane Charpentier, Abder Lahroussi and Damien Nissou for technical
assistance; Marcel Kuntz for help with the pigment analysis; Renaud Dumas (Physiologie
Cellulaire Végétale, Grenoble, France) for the gift of the anti-KARI antibody; and Maryse
Block (Physiologie Cellulaire Végétale, Grenoble, France) for anti-IEP37 and anti-OEP21
antibodies. We thank Livia Merendino and Stéphane Lobreaux for a critical reading of
this paper prior to submission, and Thomas Bollenbach (Cornell University) for advice
on improving the English in this paper. This work was supported by a grant [ACIDRAB (Action Concertée Incitative-Dynamique et Réactivité des Assemblages Biologiques)
03/41, number 03 5 90 to D. F. and number 03 5 92 to I. P.-P.] from the CNRS and the
MEN (Ministère de l’Education Nationale), by the Egyptian government (fellowship to
E.-S. E.-K.), and by the Ivory Coast government (fellowship to M. K.).
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