Download Chloroplast Targeting, Distribution and Transcriptional Fluctuation of

Plant Cell Physiol. 41(10): 1119–1128 (2000)
JSPP © 2000
Chloroplast Targeting, Distribution and Transcriptional Fluctuation of
AtMinD1, a Eubacteria-Type Factor Critical for Chloroplast Division
Kengo Kanamaru, Makoto Fujiwara 1, Meesoon Kim, Akitomo Nagashima, Emi Nakazato, Kan Tanaka
and Hideo Takahashi 2
Laboratory of Molecular Genetics and Breeding, Institute of Molecular and Cellular Biosciences, The University of Tokyo (UT), Yayoi 1-1-1,
Bunkyo-ku, Tokyo, 113-0032 Japan
;
In Arabidopsis thaliana, a mature mesophyll cell contains approximately 100 chloroplasts. Although 12 arc mutants (accumulation and replication of chloroplasts) and
two chloroplast division genes homologous to eubacterial
ftsZ have been isolated from A. thaliana, the molecular
mechanism underlying the chloroplast division is still unclear. We characterized AtMinD1, a eubacterial minD homolog, for chloroplast division in A. thaliana. AtMinD1green fluorescent protein targeted to the chloroplasts and
possibly associated with the envelope membranes in vivo.
During the seed germination, the AtMinD1 transcripts were
accumulated twice, just after release from cold treatment
and at the beginning of rapid greening, in similar fashion to
AtFtsZs. Furthermore the transcript level in a severest chloroplast division mutant, arc6, was 3–5-fold higher than that
in wild-type.
Key words: Arabidopsis — Chloroplast division — Eubacterial minD.
The nucleotide sequence reported in this paper has been submitted to the DDBJ, EMBL and GenBank under accession number
AB030278.
Introduction
The chloroplast in a plant is a symbiotic resultant derived
from an ancestral cyanobacterium (Douglas 1998, Gray 1988).
During the evolutionary process, higher plants developed the
unique proplastids that can differentiate to not only the
chloroplast but also other organelles like amyloplasts, leucoplasts, etioplasts and chromoplasts (Goldschmidt-Clermont
1998). The plastids in higher plants still maintain approximate
150 kb DNA encoding about 120 genes. However, the plastid
DNA (ptDNA) itself does not keep any genes for ptDNA replication and plastid division (Shinozaki et al. 1986, Sugiura
1995, Martin et al. 1998, Sato et al. 1999, Abdallah et al.
2000). Therefore, these processes are absolutely governed by
the host nucleus. Transcription of the ptDNA for the chloroplast functions is also mediated by the nuclear-encoded
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eubacteria-type I factors (Isono et al. 1997, Tanaka et al. 1997,
Kanamaru et al. 1999). Thus, higher plant plastids themselves
have lost autonomy to keep their continuity (Abdallah et al.
2000). Then it is suggested such a ‘eubacteria-type system’ also
controls the chloroplast division through nuclear-encoded regulatory factors.
In Arabidopsis leaves, proplastids increase in number to
some extent through the mitotic cell division. Subsequently, a
few rounds of post-mitotic plastid divisions occur to develop
approximately 100 chloroplasts in mature mesophyll cells
(Pyke and Leech 1992). Young wheat chloroplasts containing
thylakoid membranes and small granal stacks divide when they
are expanded up to about half the size of the final volume. The
process is progressed in order by central constriction, twisting
around the isthmus, and production of two daughter chloroplasts (Ellis and Leech 1985). To analyze genetic control of the
chloroplast division, 12 arc (accumulation and replication of
chloroplasts) recessive mutants have been isolated from Arabidopsis thaliana (Marrison et al. 1999, Pyke 1999). The nuclear
ARC genes are involved in several steps in the chloroplast division. Particularly, the ARC6 gene product is likely to initiate
both proplastid and chloroplast division. The arc6 mutant has
only two giant chloroplasts per leaf cell (Robertson et al.
1995). However, the molecular substances of any ARC genes
have not been reported yet.
Recent accumulation of the expressed sequence tag (EST)
database and progress of the genome sequence project in various plants have provided good opportunities to find out conserved eubacteria-type genes. Then two nuclear-encoded ftsZ
genes (AtFtsZ1-1 and AtFtsZ2-1) were identified and demonstrated to be critical for chloroplast division (Osteryoung and
Vierling 1995, Osteryoung et al. 1998). Eubacterial FtsZ is well
known as a structural homolog and a possible evolutionary progenitor of the eukaryotic tubulins (Erickson 1997, Löwe and
Amos 1999). The FtsZ protein plays a central role in eubacterial cell division by forming the cytoskeletal framework of the
cytokinetic ring at the midcell (Jacobs and Shapiro 1999, Rothfield et al. 1999). In A. thaliana, transgenic antisense plants of
each AtFtsZ gene reveal not only drastic reduction of chloroplast number but also expansion of the volume in mature leaf
cells. In Physcomitrella patens, disruption of the ftsZ homolog
Present address: Plant Functions Laboratory, Institute of Physical and Chemical Research (RIKEN), Wako-shi, Saitama, 351-0198 Japan.
Corresponding author: E-mail, htakaha@imcbns.iam.u-tokyo.ac.jp; Fax, +81-3-5841-8476; Phone, +81-3-5841-7825.
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Localization and transcription of AtMinD1
(PpftsZ) also specifically inhibits chloroplast division but not
mitochondrial division (Strepp et al. 1998). A pea chloroplast
FtsZ forms multimers in vitro and complements an Escherichia
coli temperature-sensitive ftsZ mutant in vivo (Gaikwad et al.
2000).
In addition to FtsZ, E.coli cell division requires MinC,
MinD, and MinE. The individual cell has three potential division sites (PDS) located at the midcell and adjacent to the cell
poles, but only the central site is available for cell division.
These proteins are determinants for the correct division site
where the FtsZ ring is assembled (de Boer et al. 1989, Cha and
Stewart 1997). The minD homologs are encoded on chloroplast DNAs of Chlorella vulgaris (Wakasugi et al. 1997) and
two earliest-diverging green plants discovered to date, Nephroselmis olivacea (Turmel et al. 1999) and Mesostigma viride
(Lemieux et al. 2000). Although higher plant ptDNAs no longer maintain the minD gene (Shinozaki et al. 1986, Sato et al.
1999), we have registered a nuclear-encoded Arabidopsis minD
homolog gene in the database (DDBJ/EMBL/GenBank accession no. AB030278), and Colletti et al. (2000) reported very recently that the homolog (AtMinD1) mediates placement of the
chloroplast division apparatus. These findings indicate that
chloroplast division utilizes the eubacteria-type molecular system, even if not in all aspects.
In the present study, we investigated the chloroplast targeting and distribution of AtMinD1-green fluorescent protein
(GFP) in vivo, transcriptional fluctuation of AtMinD1 during the
seed germination, and accumulation of the transcripts in arc6.
Materials and Methods
Plant materials and growth conditions
Imbibed seeds of A. thaliana were sown on Jiffy 7 (AS Jiffy Products Ltd, Norway), MS medium containing 0.4% gelrite (Murashige
and Skoog 1962), or triple paper filters (Whatman #540 for top and #2
for middle and bottom, 70 mm) soaked in MS medium. The seeds
were placed at 4C under dark conditions for 24 h and then cultivated
at 23C under continuous white light (70–100 mol m–2 s–1). The seeds
of Col (Columbia), WS (Wassilewskija), Ler (Landsberg erecta), arc5
and arc6 were distributed by the Nottingham Arabidopsis Stock Centre
(NASC) in the U.K. Nicotiana tabacum (BY4) was aseptically cultivated on MS medium containing 3% sucrose and 0.4% gelrite at 25–
27C.
DNA and RNA techniques
Total DNA from adult Arabidopsis leaves was purified as described (Edwards et al. 1991). Total RNA was purified from Arabidopsis young seedlings as described (Carpenter and Simon 1998). To purify enough RNA, approximate 2 ml (WS for Fig. 7) and 1 ml (WS, Ler,
arc5 and arc6 for Fig. 8) of the dried seeds were imbibed and sown on
triple paper filters soaked in MS medium. Restriction enzymes, modification enzymes, DNA polymerases, and kits were purchased from
TaKaRa Shuzo, Japan. UltraClean 15 DNA Purification Kit was purchased from MO BIO Laboratories Inc., U.S.A. Positively charged nylon membrane, CSPD ready-to-use, and DIG DNA labeling mixture
were purchased from Boehringer Mannheim, Germany.
Isolation of AtMinD1 genomic DNA clone
A P1 clone MZF18, containing the entire genomic AtMinD1, was
kindly provided by Dr. S. Tabata (Kazusa DNA Research Institute, Japan). A total of 2.4 kb of the BamHI–NruI region eluted from MZF18
was subcloned into the BamHI–EcoRV sites of pBluescript to yield
pminDg.
Isolation of AtMinD1 cDNA clone
Oligonucleotides MIND-F 5-CCGGATCCATGGCGTCTCTGAGATTG-3 and MIND-R 5-CCGAGCTCGCTAGCCGCCAAAGAAAGAGA-3 were used for amplification of AtMinD1 by pfu DNA
polymerase from an Arabidopsis cDNA library. The PCR products
were digested with BamHI and SacI and were subcloned into pBluescript. The product, pminDc, was subjected to sequencing performed
by a LI-COR Model 4000 DNA sequencer (Li-COR Inc., U.S.A.).
Phylogenetic analysis of MinD family proteins
Amino acid sequences of MinD family proteins were obtained
from DDBJ/EMBL/GenBank databases, except for the homolog in
Synechocystis sp. PCC6803 from CyanoBase (http://www.kazusa.or.jp/
cyano/). The sequences were aligned using the CLUSTAL W v.1.7
(Thompson et al. 1994). Amino acid positions of ambiguous alignment were omitted from subsequent phylogenetic analyses. A phylogenetic tree was constructed by the neighbor-joining method (Saitou and
Nei 1987) with the PHYLIP package v.3.5c (J. Felsenstein, University
of Washington, DC). Bootstrap analyses for 100 replicates were performed to provide confidence estimates for their tree topologies.
Low stringent Southern hybridization analysis
Purified 3 g of total Arabidopsis DNA was digested with appropriate restriction enzymes and was electrophoresed on a 0.8% agarose
gel. Then the DNAs were alkali-transferred onto a positively charged
nylon membrane in 0.2 M NaOH. Two DNA probes specific for the 5half and 3-half of the AtMinD1 gene were prepared by DIG-PCR. The
PCR reactions were performed with pminDc and two sets of primers,
BS-Sac/RI(F) 5-CTCGAGCTCGAATTCCTGCAG-3 and AtMinDCl(R) 5-CCATCGATTCCTGCAGGAC-3 for the 5-half probe and
AtMinD-Cl(F) 5-GCCGGATTCATAACCGCC-3 and AtMinDend(R) 5-CGCTCTAGACGCCAAAGAAAGAG-3 for the 3-half
probe. Hybridization in high SDS hybridization buffer (7% SDS, 50%
de-ionized formamide, 5 SSC, 2% blocking reagent, 50 mM sodium
phosphate buffer pH 7, 0.1% N-lauroylsarcosine sodium salt and
0.005% yeast RNAs) was performed for 12 h at 42C. The membrane
was washed with 2 SSC, 0.1% SDS and with 0.2 SSC, 0.1% SDS at
room temperature. Finally, CSPD ready-to-use was spread on the hybridized membrane before exposure of a Fuji X-ray film for 30 min.
Plasmid constructions
To study the subcellular localization of AtMinD1 protein, GFP
was used as the reporter (Chalfie et al. 1994, Sheen et al. 1995). The
GFP was fused with the whole AtMinD1 protein (326 amino acid residues) or N-terminally deleted AtMinD1N (249 amino acid residues,
lacking the N-terminal 77 amino acid residues). Two PCR fragments
were amplified from pminDg by MD-SalI 5-TGTGTCGACAGCTGAAGAAGCAGCAG-3 and MD-NcoI 5-TTGCCATGGAGCCGCCAAAGAAAGAGAAG-3 for the entire AtMinD1, and by MD-BamHI 5-CAGGATCCATGGATGTCGGTCTCTCTCTCG-3 and MDNcoI for AtMinD1N. The PCR products were cloned into the SalINcoI sites of the CaMV35S-sGFP(S65T)-NOS vector (Isono et al.
1997) to yield pMinD-GFP and pMinDN-GFP, respectively. To overexpress AtMinD1 under the control of the CaMV35S promoter in A.
thaliana, 1 kb of BamHI-SacI fragments from pminDc were inserted
into the same sites of pBI121. The plasmid was named pBI-35SD.
Localization and transcription of AtMinD1
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Fig. 1 High identity between AtMinD1 and entire rice MinD. The two MinD proteins were aligned with the CLUSTAL W (Thompson et al.
1994). The database accession numbers are as follows: A. thaliana (AB030278); Oryza sativa (Rice) (AP001129, AF149810). A conserved ATP/
GTP-binding motif (P-loop) is underlined. A black arrowhead shows the position of the intron site in rice minD gene.
Transient expression method to detect AtMinD1-GFP signals in vivo
The pMinD-GFP was introduced into tobacco young leaf cells by
particle bombardment method (Kim and Minamikawa 1996). DNAcoated gold particles (1.5–3.0 m in diameter) were bombarded using
a device (Model GIE-III; TANAKA Co. Ltd, Japan). The conditions
were: helium shock pressure, 2 kgf cm–2; vacuum of the chamber,
600 mmHg. The bombarded samples after 20–80 h were analyzed with
a confocal laser scanning microscope (Model TSC4D; Leica Microsystems, Germany). The fluorescent micrographs were taken with
excitation at 488 nm and emission at 530 nm to detect GFP signals and
at over 665 nm to detect chlorophyll autofluorescence. Incorporated
images from the microscopy were processed using Adobe Photoshop
v.4.0 (Adobe Systems Inc., U.S.A.).
Construction and observation of AtMinD1-overexpressing transgenic
Arabidopsis
Agrobacterium strain C58 harboring pBI-35SD was used for
Arabidopsis (WS) transformation (see Plasmid constructions, above).
Vacuum infiltration was performed as described (Bechtold and Pelletier
1998). More than 20 transformants (SMD) were selected on MS-plates
containing 50 g ml–1 of kanamycin. The green tissues of the T2 and
T3 progenies were supplied for the microscopic observations with the
confocal laser scanning microscope (Model TSC4D; Leica Microsystems, Germany).
Northern hybridization analysis
DIG-labeled ssDNA probes were prepared as follows. First,
PCRs were performed with 1 g of total Arabidopsis DNA or 0.1 g
of cDNA library as the template. Following oligonucleotide sets were
used for first PCR, MIND-F and MIND-R (see Isolation of AtMinD1
cDNA clone, above) for amplification of AtMinD1, FTSZ2-F 5-CCG-
GATCCATGCTTAGAGGTGAAGGG-3 and FTSZ2-R 5-CCGAGCTCCCGGGCTTTAGACTCGGGGA-3 for AtFtsZ2-1, RBCL-F 5AGGGATCCATGTCACCACAAACAGAG-3 and RBCL-R 5-TTACTGCAGATCTCTTTCCATACTTCAC-3 for rbcL, and RBCS-F 5GGATCATGGCTTTCCTCTATGCTCT-3 and RBCS-R 5-GAGCTCTTAACCGGTGAAGCTTGG-3 for rbcS. The approximate 1 kb PCR
fragments entirely covering AtMinD1, 1.2 kb fragments entirely covering AtFtsZ2-1 and 1.4 kb fragments almost covering rbcL were purified from 0.8% agarose gel, and 550 bp fragments entirely covering
rbcS were purified from 1.6% agarose gel by UltraClean 15, respectively. An aliquot of 300 ng of each PCR product was supplied for second PCR containing DIG DNA labeling mixture. A sample of 10 pmol
of MIND-R, FTSZ2-R, RBCL-R or RBCS-R was used for specific
amplification (30 cycles) of DIG-labeled antisense strands in 50 l reaction mixture. The DIG-labeled products were stable for at least 1
year when stocked at 20C. An aliquot of 5 g of total RNA was denatured and electrophoresed in RNA gel containing 1.2% agarose before vacuum-transfer onto a positively charged nylon membrane. The
membrane was UV-crosslinked and pre-hybridized in high SDS hybridization buffer (see Southern blot analysis, above) for 2 h. Hybridization with 10 l of DIG-labeled ssDNA probes was performed in
8 ml of fresh high SDS hybridization buffer for 14 h at 50C. The immunological detection was performed according to the manufacturer’s
protocol. Finally, CSPD ready-to-use was spread on the hybridized
membrane before exposure of a Fuji X-ray film for up to 2 h.
Results
Well-conserved AtMinD1 but possessing an N-terminal extension
MinD homologs have been identified in widespread liv-
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Localization and transcription of AtMinD1
Fig. 2 Phylogenetic relevance of widespread MinD proteins. Phylogenetic tree was constructed using the neighbor-joining method (Saito
and Nei 1987). The scale bar represents 0.1 substitutions per amino
acid position. The database accession numbers are as follows: B. subtilis (Z15113); E.coli (J03153); Helicobacter pylori (AE001468);
Aquifex aeolicus (MinD2, AE000712); Synechocystis sp. PCC 6803
sll0289(ORF) (D64005); Guillardia theta (AF041468); C. vulgaris
(AB001684); Prototheca wickerhamii (AJ245645); N. olivacea
(AF137379); A. thaliana (AB030278); Oryza sativa (Rice)
(AP001129,
AF149810);
Archaeoglobus
fulgidus
MinD-1
(AE001056), MinD-2 (AE000970); Methanococcus jannaschii
MJ0547(ORF) (U67504), MJ0169(ORF) (U67474); Chlamydia pneumoniae (AE001661); Chlamydia trachomatis (AE001329); Saccharomyces cerevisiae NBP35 (X95533); Homo sapiens NBP (U01833).
ing cells from bacteria to human. As shown in Fig. 1, the identities at amino acids level are over 70% with entire rice MinD
that is annotated on chromosome VI (accession no.
AB001684). As well as other MinD proteins, AtMinD1 possesses an ATP/GTP binding motif (P-loop) at the position of
Gly-66 to Thr-73 (Saraste et al. 1990) and a ParA-family ATPase domain at the central region from Trp-136 to Asp-245
(Davis et al. 1996). A characteristic feature specific in the
AtMinD1 is the N-terminal extension. The approximate 60
amino acid residues of the N-terminal Ser/Thr-rich extension
was assigned as a possible plastid-targeting signal by the ChloroP and the TargetP programs (Emanuelsson et al. 1999,
Emanuelsson et al. 2000). The phylogenetic tree of 19 MinD
proteins illustrated that AtMinD1 forms a tight group within
those of photosynthetic organisms (Fig. 2).
Fig. 3 Low-stringency Southern hybridization analysis of AtMinD1.
Two specific probes of the AtMinD1 gene were prepared as described
in Materials and Methods. The 5-half and 3-half probes correspond to
the regions encoding Met-1 to Asp-187 and Ala-188 to Gly-326,
respectively. The border is almost consistent with the intron site of rice
minD. Genomic DNA was digested with PstI, HindIII, EcoRI and
ClaI. Disclosed genome sequence including AtMinD1 on the chromosome V MZF18 provided the ‘Estimated size (kb)’ of the signals. DIGlabeled DNA fragments digested with EcoT14I (Boehringer Mannheim) were loaded in lane M.
No closely related sequences with the AtMinD1 gene in the
Arabidopsis nuclear genome
In Fig. 3, Southern hybridization analysis was performed
under low stringency by the use of 5¢-half and 3¢-half probes,
respectively. Both probes were hybridized with a single band in
all lanes. The sizes of hybridized bands were well consistent
with those estimated from the disclosing sequence data, except
for a more than 10 kb signal detected in ClaI-digested DNA
when hybridized with the 3¢-half probe. This result suggests
that the Arabidopsis nuclear genome does not possess closely
related sequences with AtMinD1. In addition, our extended database search has not shown any other AtMinD1 homologs in
the Arabidopsis genome so far.
AtMinD1-GFP targeted to the chloroplast membrane
Eubacterial MinD protein is a membrane-localized protein (de Boer et al. 1991, Marston et al. 1998, Raskin and de
Boer 1999). To clarify the AtMinD1 localization in plant cells,
we constructed a gene to express whole AtMinD1-GFP under
the control of a CaMV35S promoter. The construct was introduced into tobacco young leaf cells by bombardment, and the
transiently expressed GFP signals were observed (Fig. 4). The
green signals of AtMinD1-GFP in tobacco guard cells were
preferably targeted to chloroplasts, scarcely on the cell membrane and cytosol. The green signals at the chloroplasts of each
cell showed either of two different distribution patterns. One is
the frequently observed pattern shown in panel c. The intense
Localization and transcription of AtMinD1
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Fig. 4 Specific targeting of AtMinD1-GFP to chloroplasts. AtMinD1-GFP protein fusion was transiently expressed under the CaMV35S promoter in tobacco leaf cells. The two samples were observed with a confocal laser scanning microscope. Images of chlorophyll (a, f), GFP (b, g),
and DIC (d, i) are shown in red, green, and black and white, respectively. Bar, 20 m.
fluorescent signals were observed as large and small patches on
the chloroplasts. Another occasionally observed pattern is that
the AtMinD1-GFP signals distribute all over the chloroplasts
(Fig. 4h). Next, we expressed the N-terminally deleted
AtMinD1 fused with GFP (AtMinD1,N-GFP) and examined if
the targeting or the distribution pattern was affected by the deletion (Fig. 5). In contrast to the specific targeting of whole
AtMinD1-GFP to the chloroplasts, the signals of AtMinD1,NGFP could no longer specifically target to the chloroplasts.
Rather, the signals were distributed on the cell membrane as
lots of patches. These results suggest that AtMinD1 targets to
chloroplasts and the conserved region in the MinD family has a
potential to associate with membranes in vivo as well as eubacterial MinD proteins.
Severe division inhibition and expansion of the plastids by
overexpression of AtMinD
Eubacterial MinD is a determinant of the cell division site
Fig. 5 Subcellular localization of AtMinD1N-GFP on the tobacco cell membrane. Partial AtMinD1 lacking 77 amino acid residues of its Nterminus was fused with GFP. The AtMinD1N-GFP was also transiently expressed. Chlorophyll and GFP signals were merged on each image.
The z-axis distance between images of left and right is 17 m. Bar, 20 m.
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Localization and transcription of AtMinD1
Fig. 6 Severe division inhibition and expansion of the chloroplasts by AtMinD1-overexpression. SMD is the transgenic Arabidopsis overexpressing AtMinD1 under control of the CaMV35S promoter. Chloroplasts of cotyledons (A), palisades (B), upper hypocotyls (C), and lower
hypocotyls (D) in 9–10-day-old seedlings were observed with a confocal laser scanning microscope. Upper panels show DIC images and lower
panels show chlorophyll autofluorescent images excited by blue light (488 nm). Bar, 20 mm in A and B, 100 mm in C and D.
and plays an inhibitory role in the process. We established
transgenic Arabidopsis lines that overexpress AtMinD1 under
the control of the CaMV35S promoter, named SMD (sense
AtMinD1 overexpressing lines). None of the transgenic plants
showed apparent defects in growth, flowering, fertility or macroscopic appearance (data not shown). However, in a microscopic view, the number and shape of the plastids were severely affected (Fig. 6). In cotyledons, the observed chloroplast
number was reduced by less than 10 compared with more than
30 in wild-type (Fig. 6A). The effect was more severe in rosette
leaves, and the chloroplast number was reduced to only one or
two per cell (Fig. 6B). Simultaneously, the chloroplast size be-
came bigger and the shape became irregular. The fewer and
bigger chloroplasts were also found in upper hypocotyls (Fig.
6C), but not so much in lower hypocotyls (Fig. 6D). These results are basically consistent with recent observations (Colletti
et al. 2000).
Transcriptional fluctuation of AtMinD1 during seed germination
In a unicellular primitive red alga, the ftsZ transcripts specifically accumulate just before plastid division during the cell
and organelle division synchronized by light–dark cycles
(Takahara et al. 2000). In addition, pea ftsZ transcripts accumulated within 24 h after exposure of etiolated seedlings to sun-
Localization and transcription of AtMinD1
Fig. 7 Transcriptional fluctuation of AtMinD1 during the seed germination. Total RNAs were prepared from young seedlings of WS (0–
72 h after release from cold treatment) as described in Materials and
Methods. The length of each transcript was determined by DIGlabeled RNA molecular weight marker I (Boehringer Mannheim).
light, and are relatively abundant in young leaves (Gaikwad et
al. 2000). As shown in Fig. 7, we checked the amounts of both
AtMinD1 and AtFtsZ2-1 transcripts in Arabidopsis seedlings
grown under continuous light for 3 d after release from cold
treatment because we could reproducibly trace the gene expressions necessary for chloroplast division and development in
cotyledons. Under these conditions, the young seedlings appear out of the seed coats after 36 h and begin rapid greening
and expansion of cotyledons around 48 h. Transcripts of both
large and small subunit genes for ribulose-1,5-bisphosphate
carboxylase/oxygenase (Rubisco), plastid-encoded rbcL, and
nuclear-encoded rbcS, were gradually accumulated in a similar
fashion up to 36 h. Subsequently, they were markedly increased
around 48 h (Fig. 7, rbcL and rbcS), whereas AtMinD1 and
AtFtsZ2-1 exhibited a quite different accumulation pattern with
the photosynthetic genes (Fig. 7, AtMinD1 and AtFtsZ2-1).
They had two peaks at 12 h after release from cold treatment
and around 48 h. Each transcript size coincided well with the
deduced length. We also observed that transcripts of AtFtsZ1-1
fluctuated in a similar fashion to AtMinD1 and AtFtsZ2-1, although the signal was very weak (data not shown).
Accumulation of AtMinD1 transcripts in arc6
As shown in Fig. 6 and reported by Colletti et al. (2000),
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Fig. 8 Accumulation of AtMinD1 transcripts in arc6 mutant. Total
RNAs were prepared from young seedlings (2–5 d after release from
cold treatment) as described in Materials and Methods. (A) Transcripts
of AtMinD1 and AtFtsZ2-1 in arc6 and WS. (B) Transcripts of
AtMinD1 in arc5 and Ler.
AtMinD1-overexpressing plants resulted in extreme phenotypes of reduction and expansion of the chloroplasts. Then we
compared the levels of AtMinD1 transcripts between arc6 and
its parental WS between 2 and 5 d after release from cold treatment, because very similar abnormality is also observed in the
chloroplasts of the arc6 mutant (Robertson et al. 1995). Under
these conditions, the first true leaves emerge enough to see on
day 5 (data not shown). In the arc6 mutant, the level of accumulated AtMinD1 transcripts were 3–5-fold higher than WS
(Fig. 8A). The transcriptional fluctuation still remained in arc6.
In contrast, the amount of AtFtsZ2-1 transcripts in arc6 was not
altered compared with WS. We also checked another plastid division mutant, arc5, that reduces chloroplast number to approximately 13 because of an impairment at a later stage of the
chloroplast division than arc6 (Robertson et al. 1996). The level of AtMinD1 transcripts in arc5 was not higher than that in its
parental strain Ler (Fig. 8B).
Discussion
We reported here about the chloroplast targeting in vivo
and transcriptional fluctuation of a higher plant MinD,
AtMinD1. The widespread MinD proteins are conserved well,
and the higher plant MinD homolog is found from not only a
dicot (Arabidopsis) but also a monocot (rice) (Fig. 1, 2).
Transiently expressed AtMinD1-GFP targeted to chloro-
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Localization and transcription of AtMinD1
plasts (Fig. 4, 5). The N-terminus of AtMinD1 is indispensable
for specific targeting. The GFP signals of AtMinD1-GFP were
frequently observed as patches on the chloroplasts (Fig. 4a–e),
otherwise, they were occasionally scattered in/on the chloroplasts (Fig. 4f–j). The signal strength of the latter pattern was
generally weaker than that of the former. This is probably because of the difference in the expression level of the fusion proteins. The cellular localization of eubacterial MinD has been
investigated well in E.coli and Bacillus subtilis. Interestingly,
E.coli MinD protein rapidly oscillates between the cell poles
through an association (to the inner membrane)-dissociation (to
the cytosol) cycle (Raskin and de Boer 1999), whereas B. subtilis MinD always localized at both cell poles (Marston et al.
1998). We have observed neither regular oscillations of
AtMinD1-GFP nor oppositely localized two patches on a single
chloroplast (Fig. 4 and data not shown). It was, however, unclear how many and which chloroplasts in the AtMinD1-GFPexpressing young leaf cells were actually in the division process. To define the relationship between the appearance of the
AtMinD1-GFP patches and plastid division or development,
we are in the process of establishing the stable lines that express various amounts of the fusion proteins for more careful
observation.
Does AtMinD1 work inside, outside, or on both sides of
the chloroplasts? This is very important to elucidate the
AtMinD1 function. In plants and algae, a ubiquitous ring-like
structure, observed as electron-dense bundles, appears at the
central constricted isthmus of a dividing plastid (Mita et al.
1986, Kuroiwa et al. 1998). This is named the PD ring (plastid
division ring). The PD ring consists of an outer ring and an inner ring on the chloroplast membrane (Hashimoto 1986). Behavior of the PD ring as a chloroplast division apparatus is well
defined in Cyanidioschyzon merolae (Miyagishima et al. 1999).
The observations that AtFtsZ1-1 protein was translocated into
the chloroplasts but AtFtsZ2-1 protein localized in the cytosol
(Osteryoung et al. 1998) raise a model that AtFtsZ1-1 and
AtFtsZ2-1 work in the inner and outer rings, respectively. If the
intimate relationship between eubacterial FtsZ and MinD is
also conserved between AtMinD1 and AtFtsZs, then AtMinD1
would work at both sides of the plastid envelopes; otherwise,
there should be another MinD homolog in A. thaliana. It has
been clearly shown that AtMinD1 could be imported into pea
chloroplasts in vitro (Colletti et al. 2000). Alternatively, our results suggest that AtMinD1 targets to chloroplasts in vivo, and
propose a possibility that a part of the membrane-associated
AtMinD1 proteins may localize even outside of the chloroplasts.
In E. coli, restriction of the adjacent potential division
sites (PDS) requires the activity of the MinCD (de Boer et al.
1992). The MinE ring allows FtsZ ring formation at the midcell by suppressing MinCD activity (Raskin and de Boer 1997).
MinC directly binds to both MinD and FtsZ, and MinC can
prevent FtsZ polymerization in vitro (Hu et al. 1999). On the
other hand, B. subtilis utilizes DivIVA for MinCD action in-
stead of MinE. The alternative DivIVA has no significant similarity with MinE (Cha and Stewart 1997). DivIVA retains
MinCD at the cell poles after division in order to select the
midcell division site for subsequent division (Marston and Errington 1999). Interestingly, C. vulgaris retains a minE homolog together with minD on the chloroplast DNA (Wakasugi
et al. 1997), whereas N. olivacea and M. viride have lost the
gene from their chloroplast DNAs (Turmel et al. 1999, Lemieux et al. 2000). Higher plants probably utilize some other
unique nuclear-encoded factors for the chloroplast division besides AtFtsZs and AtMinD1, for example a molecular clamp
between FtsZ and MinD, such as eubacterial MinC.
It is unknown if the chloroplasts set PDS-like positions or
the organelle poles. Eubacterial cells make a tight frame of
peptidoglycan to keep the cell shape and to resist extracellular
circumstances (Wong and Pompliano 1998). The peptidoglycan synthase gene ftsI (pbpB) has been encoded on chloroplast
DNA in the N. olivacea and M. viride but no longer on the
chloroplast DNA in C. vulgaris nor higher plants. It is unlikely
that the higher plant chloroplasts have such a whole frame between the inner and outer membranes to fix the special structures. Rather, actin filaments are the surrounding structures of
the chloroplasts because they possibly contribute to the division process (Kuroiwa et al. 1998).
The transcripts of AtMinD1 and AtFtsZs were accumulated in the same fashion in young seedlings (Fig. 7). They might
be synchronously transcribed in balance to produce adequate
amounts of the proteins to progress the division process. However, the regulation of the AtMinD and AtFtsZ expression
mechanism is probably not quite the same because arc6
affected only total amounts of AtMinD1 but not of AtFtsZ2-1
(Fig. 8). Then, what is the physiological meaning of the two
peaks of AtMinD1 and each AtFtsZ transcript during seed germination? This was not caused by a circadian rhythm because
the interval of the two peaks was around 36 h. In A. thaliana,
the plastids partially initiate the development during embryogenesis and arrest at an intermediate state during the seed maturation and dormancy. After the beginning of the seed germination under light, the plastids develop further to functional
chloroplasts (Mansfield and Briarty 1996). The developed
chloroplasts in the cotyledons contain less extensive thylakoid
membranes than those in true leaves and are structurally close
to the young chloroplasts in the true leaves (Deng and Gruissem 1987). The first peak of AtMinD1 and AtFtsZ transcripts
appeared just after the release from the cold treatment, in other
words, soon after beginning of the irradiation of continuous
light. The first accumulations of the transcripts would be necessary to enter the intermediate chloroplasts the first division cycle(s). Consequently, the drastic expansion and development
occurs to establish functional chloroplasts. The second accumulations of the transcripts around 48 h might be correlated to
the chloroplast expansion and maturation.
In arc6 young seedlings, AtMinD1 transcripts were apparently accumulated more than WS (Fig. 8). ARC6 acts upstream
Localization and transcription of AtMinD1
of ARC3, ARC5, and ARC11 during the initiation of both proplastid division and chloroplast division (Marrison et al. 1999).
Considering that arc6 is a recessive mutant, we have an idea
that ARC6 negatively affects to transcription of AtMinD1, if
not directly.
As well as antisense lines of each AtFtsZ (Osteryoung et
al. 1998) and the arc mutants (Pyke 1999), AtMinD1-overexpressing plants looked almost healthy and did not show apparent defects (Fig. 6) (Colletti et al. 2000). It is admittedly unclear why the higher plant chloroplasts need to divide into
more than 100 per cell. Laboratory conditions for plant cultivation are usually optimized. The physiological significance of
chloroplast division may become clear when harder or checkered circumstances are imposed on the plants. Such approaches will provide clues to understand the molecular mechanisms
of chloroplast division in response to environmental factors,
e.g. light, temperature, etc.
Acknowledgements
K.K. and M.F. contributed equally to this work. We thank Dr. Y.
Niwa for kindly supplying the sGFP vector, and Dr. S. Tabata for the
P1 MZF18. This work was supported by Grants-in-Aid for Scientific
Research (Priority Area Nos 10170207, 09460046, and 11740434)
from the Ministry of Education, Science, Sports and Culture of Japan;
by Special Coordination Funds for Promoting Science and Technology from the Science and Technology Agency; and by the Biodesign
Research Program of RIKEN. M.F. was a Research Fellow of the Japan Society for the Promotion of Science until March, 2000.
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(Received June 26, 2000; Accepted July 24, 2000)