全 文 ：Journal of Genetics and Genomics
(Formerly Acta Genetica Sinica)
March 2007, 34(3): 229-238


Received: 2006-03-02; Accepted: 2006-07-28
This work was supported by the National Natural Science Foundation of China (No. 30470879).
① Corresponding author. E-mail: yhe@mail.cnu.edu.cn; Tel: +86-10-6890 2345; Fax: +86-10-6890 2345
www.jgenetgenomics.org
Research Article
A Nucleus-encoded Topological Specificity Factor PpMinE in
Physcomitrella patens has Conserved Function Similar to Its
Chloroplast-encoded Ancestor
Jiaying Zhu1, Weizhong Liu2, Weiwei Zhou1, Yong Hu 1, Yikun He1,①
1. College of Life Science, Capital Normal University, Beijing 100037, China;
2. College of Life Science, Shanxi Normal University, Linfen 041004, China
Abstract: A nucleus-encoded MinE gene, designated PpMinE, from Physcomitrella patens was identified using RT-PCR. The
presence of both N- and C-terminal extensions in PpMinE protein suggested its cyanobacterial origin. The transient expression of
PpMinE using green fluorescent protein fusion in tobacco (Nicotiana tabacum L.) indicated that the PpMinE was a chloro-
plast-targeted protein. Overexpression of PpMinE in Escherichia coli caused division site misplacement and minicell formation,
suggesting evolutionary functional conservation of MinE during plant phylogenesis. According to the phylogenetic tree, PpMinE
protein has a close relationship with the highland plants, which suggests that the transfer events of MinE gene from plastid to nu-
cleus might have occurred before the origin of the land plants.
Keywords: Physcomitrella patens; plastid division; PpMinE; lateral gene transfer (LGT); evolution; transient expression


Chloroplast division is one of the most critical
cellular processes in plants, because plant cells are
unable to synthesize this organelle de novo[1] and be-
cause the formation of chloroplast is a complex proc-
ess that involves multiple distinct steps, such as ex-
pansion, division site selection, division initiation,
and constriction and scission of chloroplasts, which
are controlled by different nuclear genes. Previous
studies have shown that the division machinery is
conserved in chloroplasts and bacterial cells. In Es-
cherichia coli, correct placement of the division sep-
tum involves selection of the proper midcell division
site. This requires the site-specific inhibition of septa-
tion at potential septation sites that are present at cell
poles[2]. This process is controlled by the products of
the Min system, including MinC, MinD, and MinE[2,3].
MinC acts as an inhibitor of septation that is given
topological specificity by the action of MinE. MinD is
responsible for recruiting MinC and MinE to cell
membrane. MinE is a topological specificity factor
that induces the redistribution of most of the cellular
MinC and MinD into a membrane-associated polar
zone at one end of the cell. This is followed by forma-
tion of the E-ring, a prominent ring-like MinE struc-
ture that is adjacent to the medial edge of the polar
zone near midcell[4]. MinE is dynamic, oscillating
from side to side about the midcell position, sweeping
MinD out of its path[5, 6]. The pole-to-pole oscillation
occurs repeatedly within each division cycle. As a
result, the time-averaged concentration of MinC sep-
tation inhibitor is maintained at a lower level at mid-
cell than elsewhere along the length of the cell, which
230 Journal of Genetics and Genomics 遗传学报 Vol.34 No.3 2007
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ensures division initiation only at midcell[7, 8].
According to the present well-accepted serial
endosymbiosis theory, eukaryotic hosts phagocytize
and retain photosynthesizing prokaryotes. Subse-
quently, these prokaryotic endocytobionts undergo
genetic reduction involving transfer of some chloro-
plast genes to the eukaryotic nucleus and loss of oth-
ers, and are transformed into plastid[9]. Most likely, all
chloroplasts were derived from a single primary en-
dosymbiotic event involving capturing of a cyano-
bacterium [10, 11]. The first evidence of the existence of
MinE in chloroplasts was obtained from sequencing
the chloroplast genome of the green alga Chlorella
vulgaris[12]. The C. vulgaris chloroplast genome con-
tains MinD- and MinE-like open reading frames
(ORFs), which are arranged tandemly in the same
order as in E. coli. The chloroplast genome of the
cryptophyte alga Guillardia theta also encodes MinD
and MinE in the same gene order[13]. But no recog-
nizable MinE homologs have been found in any
chloroplast genome of land plants until a nuclear gene
of Arabidopsis, AtMinE1, which encodes a chloro-
plast protein homologous to MinE, was identified[14].
This suggests that the transfer of MinE has occurred,
from the chloroplast to the nucleus, during the evolu-
tion of plants. But when did this transfer happen?
To investigate the evolution and function of
MinE genes, the existence of a nuclear gene in Phy-
scomitrlla patens that encodes the homologs of MinE
was reported in this study. Overexpression of PpMinE
results in aberrant cell division in E. coli, which sug-
gests that PpMinE has retained some of its ancestral
functions, and can still recognize the cell division site
and take part in the division process. So PpMinE is a
typical MinE gene. Evolutionary analysis suggests
that the transfer of MinE in green plant lineage from
chloroplast to nuclear genome might occur during the
evolution of moss.
1 Materials and Methods
1. 1 Materials
Physcomitrlla patens were conserved in the au-
thors’ lab and was grown on PP-NO3 medium at 23℃
under 16 h light/8 h dark cycles. After 14 days of
growth on medium, P. patens were harvested for RNA
extraction.
1. 2 Cloning and sequence analysis of minE gene
Total RNA was isolated from P. patens for re-
verse transcriptase-PCR (RT-PCR) using TaKaRa
RNA PCR Kit (AMV) Ver.2.1 with gene-specific nu-
cleotide sequence according to the instructions given
by the manufacturer (TaKaRa Technologies, Dalian,
China).
All the expressed sequence site (EST) sequences
available in the NCBI EST database (http://
www.ncbi.nlm.nih.gov/EST/) using the E. coli MinE
protein sequences as the query sequences were probed
by the authors of this study. Some highly significant
EST sequences were found after the query sequences
were being probed. All MinE EST sequences of
P. patens under accession numbers BJ608760,
BJ596983, BJ580680, BJ608760, and BJ596983 were
assembled into a full-length cDNA by BioEdit
(http://mbio.ncsu.edu/BioEdit/bioedit.htlm) and Vec-
tor NTI Suite 6 software package (http://
www.informaxinc.com).
To clone the full-length MinE gene, two pairs of
gene-specific primers were designed according to the
assembled sequences (mossEsens1: 5′-ATTGTTTC-
TATCCGTTGTTATCG-3′; mossEanti2: 5′-GTCTG-
GTACTACGGTCCAGGTCA-3′; mossEsens3: 5′-G-
GCAAGCTGAGTTTCTTGGGATT-3′; mossEanti4:
5′-AACCAAAGGGCTCTGTGCTACAC-3′). PCR am-
plifications were carried out with a denaturation for
1 min at 94℃ followed by 30 cycles of denaturation
for 30 s at 94℃, a 30 s annealing at 60℃, and a 50 s
extension at 72℃ and a 5 min extension at 72℃.
Amplification products were electrophoresed using
Jiaying Zhu et al.: A Nucleus-encoded Topological Specificity Factor PpMinE in Physcomitrella patens has Conserved Function… 231
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1% agarose gel. Bands of the appropriate length were
harvested and the DNA was extracted using a DNA
Gel Extraction kit (Sangong, Shanghai, China). The
extracted PCR products were cloned into pMD18-T
vector (TaKaRa Technologies, Dalian, China), and
multiple copies were sequenced on both strands. The
verified and sequenced gene was designated as
PpMinE. Its sequence can be accessed through Gen-
Bank under accession number DQ011580.
Amino acid sequences of MinE family proteins
were obtained from GenBank/EMBL/DDBJ database.
The sequence alignment was performed using the
program CLUSTALW (http://www.ebi.ac.uk/clustalw/)
with the default parameters[15]. Phylogenetic trees were
constructed using the NJ (Neighbor-Joining) method of
Saitou and Nei with the PHYLIP packages v3.5c
(http://evolution.genetics.washington.edu/phylip/)[16].
Bootstrap analyses for 1,000 replicates were per-
formed to provide estimates of confidence for their
tree topologies.
1. 3 Plasmid construction
Enhanced green fluorescence protein (EGFP)
was used as a reporter to examine the subcellular lo-
calization of PpMinE under the control of the
CaMV35S promoter in tobacco. The cDNA fragment
encoding the PpMinE was amplified by PCR with a
pair of primers set with restriction site (underline)
XbaⅠ 5′-GCATCTAGAATGTGCGTTGTTGCAGT-
G-3′ and SalⅠ 5′-GCAGTCGACGACATTCGGC-
GCTGAACC-3′. The PCR product was digested with
XbaⅠ and SalⅠ, and ligated into XbaⅠ/SalⅠ di-
gested plgz2 vector, which contained EGFP, to yield
pMinE-EGFP. This construct was transformed into
E. coli by electroporation. After digestion by XbaⅠ
and SacⅠ, the fragment PpMinE-EGFP was ligated
into XbaⅠ/SacⅠ digested pBI121 vector to yield the
construct pMEG. This construct was transformed into
Agrobacterium LBA4404 by electroporation.
1. 4 Transient expression and visualization of an
PpMinE:EGFP fusion in living tobacco
cells with agroinoculation
Tobacco (Nicotiana tabacum L.) plants were
grown in pots at 25℃ in a growth chamber under 16
h light/8 h dark photoperiod. Agrobacterium LBA-
4404 harboring pMEG were grown overnight at 28℃
in 3.5 mL of LB medium containing kanamycin (50
μg/mL) and streptomycin (50 μg/mL). 1 mL of the
culture was then diluted with 50 mL of LB medium
containing antibiotics, 10 mmol/L MES (pH 5.6), and
20 μmol/L acetosyringone. The culture was incubated
overnight at 28℃, with shaking at 300 r/min. Agro-
bacterium cells were collected by centrifugation at
3,000 r/min and then resuspended in solution MMA
(10 mmol/L MgCl2, 10 mmol/L MES, pH 5.6, and
100 μmol/L acetosyringone). After adjusting to a final
OD600 of 0.6, the cells were incubated at room tem-
perature for 3 h without shaking and then pressure
infiltrated into a leaf of a N. benthamiana plant using
a 2 mL syringe without needle[17]. After 3 days, leaves
that were agroinculated were examined with a confo-
cal laser-scanning microscope (Leica, Germany). The
EGFP was excited at 488 nm using argon/krypton
laser. Recorded images of EGFP and chlorophyll were
analyzed using Leica Confocal Software (Leica,
Germany).
1. 5 Overexpression of PpMinE in E. coli and
microscopic examination
The plasmid containing PpMinE-EGFP was
transformed into E. coli DH5α for studying the loca-
tion of PpMinE proteins in E. coli. After overnight
growth at 37℃ in LB medium containing ampicillin,
1 mL of the overnight culture was diluted 100 times
in the same medium in the presence of 50 μmol/L
IPTG (isopropyl-beta-D-thiogalactopyranoside) for 3
hours at 37℃ before microscopic examination. The
cells were then mixed with an equal volume of 1%
low melt agarose of 60℃. The mixture solution was
232 Journal of Genetics and Genomics 遗传学报 Vol.34 No.3 2007
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immediately dropped on a glass slide and covered
with a cover glass without fluorescence. Microscopic
and photographic observations were performed under
Leica DMRE microscope (Leica, Germany), and im-
ages were collected using SPOT CCD (Leica, Ger-
many).
2 Results
2. 1 Sequence analysis
On the basis of the assembled PpMinE se-
quences, MinE gene of P. patens was isolated using
RT-PCR. In the entire sequenced chloroplast genome
of P. patens[18], none of sequences was found to be
closely related to MinE, suggesting that PpMinE is a
nucleus gene. The full length of PpMinE cDNA is
1,403 bp. It contains a 774 bp ORF, which encodes a
257 amino acid peptide (Fig. 1). The length of the
PpMinE genomic sequence isolated using PCR was
1,329 bp and only contains a 556 bp intron between
75 bp and 76 bp of the cDNA.

Fig. 1 The nucleotide sequence and the deduced amino acid sequence of PpMinE
The line indicates the TAG (termination codon) before the initiation codon.

Jiaying Zhu et al.: A Nucleus-encoded Topological Specificity Factor PpMinE in Physcomitrella patens has Conserved Function… 233
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The deduced amino acid sequence of the
PpMinE was compared with other known MinE se-
quences (Fig. 2). The MinE protein of E. coli contains
two functional domains: the N-terminal anti-MinD
domain (AMD), and the C-terminal topological speci-
ficity domain (TSD)[17, 19, 20]. The sequence alignment
of MinE proteins revealed that the N-terminal domain
of PpMinE is well conserved corresponding to the E.
coli AMD, but PpMinE has a longer N-extension,
which is predicted to be transit peptide by the PSORT
(http://psort.nibb.ac.jp/form.html). Whereas, the C-
terminal domain corresponding to the E. coli TSD is
poorly conserved. The C-terminal sequence has two
conservative domains, which is denoted by Ⅰ and
Ⅱ in Fig. 2. This is the difference of MinE sequences
between E. coli and plants.

Fig. 2 Alignment of the MinE proteins
Letters on black (gray) background show the residues identical to the consensus. Dashes indicate the gaps in the alignment. The
locations of AMD9 (anti-MinCD domain) and TSD (topological specificity domain) in the Escherichia coli protein are indicated by
double-headed arrows. Highly conserved C-terminal regions in angiosperm Ⅰ and Ⅱ are underlined. The database accession
numbers are as follows: Arabidopsis thaliana (AB046117); Brassica napus (DO118104); Oryza sativa (AY494651); Zea mays
(AB127981); Physcomitrella patens (DQ0110580); Chlorella protothecoides (AJ238631); Chlamydomonas reinhardii (AY176186);
Chlorella vulgaris (AB001684); Guillardia theta (AF041468); and E. coli (AAB59063).
234 Journal of Genetics and Genomics 遗传学报 Vol.34 No.3 2007
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With regard to the alignment of the predicted
amino acid 257 through the PpMinE, PpMinE shared
73% identity with MinE of A. thaliana[21], 11.3%–
25.8% identity with that of the prokaryotic photosyn-
thetic cynobacterium, and 10.2%–15.3% identity with
the other eubacteria.
2.2 Transient expression of PpMinE in N. ben-
thamiana
To examine the localization pattern of PpMinE, a
fusion construct pMEG that comprised the full-length
PpMinE cDNA in frame with EGFP was generated by
the authors of this study. The construct pMEG was
transiently expressed under the CAMV35s promoter
in tobacco leaf by agroinoculation. As a result, tran-
siently expressed chimeric EGFP was exclusively
localized in chloroplasts, whereas nonfused GFP was
observed only in the cytoplasm and cell wall (Fig. 3).
Thus, the PpMinE is a chloroplast-targeted protein.
2. 3 Overexpression of PpMinE caused division
site misplacement and minicell formation in
E. coli
Because plastids arise from an endosymbiotic
event between a primitive eukaryotic cell and a pho-
tosynthetic prokaryote, bacterial cell division has
been used as a paradigm to study plastid division. To
study whether eukaryotic MinE proteins that are in-
volved in plastid division can function even when
they are introduced back into E. coli cells, plasmid
pMinE-EGFP in which PpMinE-EGFP was under the
control of the lac promoter was constructed by the
authors of this study. Microscopic examination of
E. coli cells, both wild type and those overexpressing
EGFP, showed the normal bacterial division charac-
teristic of correct placement of the division site at

Fig. 3 Chloroplast localization of EGFP fused with the PpMinE
The fused protein was transiently expressed under the CaMV35S promoter in tobacco leaf cells. EGFP signals and chlorophyll
autofluorescence were observed using a confocal laser scanning microscope. Fluorescent images of chlorophyll (A, D, G; red),
EGFP (B, E, H; green), and merged images (C and F). A–C: nonfused GFP; D–F: chimeric GFP with PpMinE; G and H: wild type.
Scale bars = 8 μm.
Jiaying Zhu et al.: A Nucleus-encoded Topological Specificity Factor PpMinE in Physcomitrella patens has Conserved Function… 235
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midcell (Fig. 4 A and B; white-colored arrows). In
contrast, the bacterial cells overexpressing PpMinE
showed abnormal cell division characteristic of the
formation of aberrant separation events adjacent to
the cell poles leading to minicell formation (Fig. 4D;
white-colored arrows).
Similar to the cells only expressing PpMinE,
overexpression of PpMinE-EGFP fused protein re-
sulted in formation of minicells (Fig. 4E), which
proved that EGFP fusion did not affect the function of
PpMinE. The EGFP-derived fluorescence showed a
bright ring structure, which was similar to the MinE
ring in E. coli. This indicates that PpMinE still func-
tions in the processes of cell division when it ex-
presses in E. coli.
2. 4 Detecting the period during which lateral
gene transfer of plant MinE occurs
Construction of a phylogenetic tree of 26 MinE
sequences known at present, from wide-ranging
eubacterial, algal chloroplasts, E. coli, and land plants
revealed a close relationship between PpMinE and the
MinE proteins of land plants (Fig. 5). This suggests
that the nucleus-encoded PpMinE has a chloroplast
origin. Table 1 indicates that the PpMinE gene has
successfully transferred from chloroplast to nucleus as
a land plant gene, suggesting that the lateral gene
transfer (LGT) of MinE from plastid to nucleus might
occur during the evolution of moss.


Fig. 4 Overexpression of PpMinE affects the division of Escherichias coli
A: Wide type E. coli (white arrow shows the correct placement of the division site at midcell); B–C: E. coli overexpressing EGFP,
the fluorescence fills the whole cells; D: E. coli overexpressing a PpMinE showing aberrant septation events adjacent to the cell
poles (white arrow) and subsequently minicell formation; E: E. coli overexpressing a PpMinE-EGFP fusion protein. The fluores-
cence representing PpMinE localization shows the bright rings. Scale bar = 2 μm.
Table 1 MinE genes in different eukaryotic species
Species Location Accession No.
Arabidopsis thaliana Nucleus AB046117
Physcomitrella patens Nucleus DQ011580
Mesostigma viride Chloroplast AF166114
Nephroselmis olivacea Chloroplast AF137379
Prototheca wickerhamii Chloroplast AJ245645
Chlorella vulgaris Chloroplast AB001684
Guillardia theta Chloroplast AF041468
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Fig. 5 Phylogenetic tree of widely distributed MinE proteins
The phylogenetic tree was constructed by the Neighbor-Joining method. Bootstrap analyses for 1,000 replicates were performed to
provide confidence estimates for their tree topologies. The scale bar represents 0.1 substitutions per amino acid position.

3 Discussion
MinE protein functions as a regulating factor in
the bacterial cell division. In E. coli, overexpression
of MinE leads to formation of minicells[3]. Sequence
analysis indicates that PpMinE gene is a typical MinE
homolog. Overexpression of PpMinE-EGFP in E. coli
also leading to formation of minicells showed that
MinE of P. patens has functions similar to E. coli
MinE, which suggests that the PpMinE protein has
conserved function when its encoding gene is trans-
ferred from chloroplast to nucleus.
It is generally believed that many of these nu-
cleus-encoded chloroplast-targeted proteins were
originally encoded in the ancestral chloroplast ge-
nome, then were transferred to the host nuclear ge-
nome, where they acquired the porter expression and
targeting signals to allow the encoded proteins to be
synthesized and re-imported into the organelle with
the help of transit peptide during the integration of the
endosymbiosis[22-24]. But there is no data on the
evolutionary process of MinE gene from the plastid to
the nucleus in plants. In cryptophyte alga (Guillardia
theta)[13] and several types of green algae, MinE genes
were found to be located in chloroplast genome [25, 26].
However, the complete chloroplast sequences of the
land plants (including 14 angiosperms, 2 gymno-
sperms, 1 fern, and 3 mosses) were probed by the au-
thors of this study, and the result indicated that no
MinE homolog was found (data not shown). Recently,
the higher plant A. thaliana AtMinE has been found in
the nuclear genome. Overexpression of AtMinE re-
sults in the inhibition of chloroplast division, thereby
resulting in the formation of a few and larger chloro-
plasts in A. thaliana cell[1, 27-30]. This indicates that
MinE gene has similar functions after it was success-
fully transferred from chloroplast to nucleus in high-
Jiaying Zhu et al.: A Nucleus-encoded Topological Specificity Factor PpMinE in Physcomitrella patens has Conserved Function… 237
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land plants. But when did the transfer events occur
during the evolution of green plant lineage? The nu-
cleus-encoded PpMinE gene that was isolated from P.
patens by the authors of this study suggests that the
PpMinE gene was successfully transferred to nucleus.
Therefore, the transfer of MinE from plastid to nu-
cleus might have occurred before the origin of moss.
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小立碗藓叶绿体分裂相关基因 PpMinE的克隆及其功能与进
化分析
朱佳瑛1，刘维仲2，周伟巍1，胡 勇1，何奕昆1
1. 首都师范大学生命科学学院，北京 100037；
2. 山西师范大学生命科学学院，临汾 041004
摘 要：用 RT-PCR 技术从小立碗藓中（Physcomitrella patens）克隆了核编码的 MinE 基因，命名为 PpMinE，并克隆了该
基因的基因组DNA。序列比对显示该基因编码的蛋白质与真细菌和绿藻叶绿体编码的MinE蛋白具有较高的相似性。pMinE-
EGFP 融合蛋白在烟草中的瞬时表达证明该蛋白定位于叶绿体内。在大肠杆菌中过量表达 PpMinE 导致细胞不正常分裂，产
生无染色体的小细胞，这表明 MinE 的功能在进化上是保守的。在系统发育树中，PpMinE 和高等陆生植物有较近的亲缘关
系。在已知的陆生植物的叶绿体基因组中没有找到 MinE 的同源蛋白，这暗示在进化过程中 MinE 从叶绿体到细胞核的水平
转移可能发生在陆生植物发生以前。
关键词：小立碗藓；质体分裂；PpMinE；基因水平转移；进化；瞬时表达
作者简介：朱佳瑛(1978-)，女，四川成都人，硕士，研究方向：分子细胞生物学。E-mail: zhujybj@sohu.com