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Chinese Science Bulletin 2004 Vol. 49 No. 6 567—573
Cloning and expression of
AtPLC6, a gene encoding a
phosphatidylinositol-specific
phospholipase C in
Arabidopsis thaliana
XU Xiaojing1, CAO Zhixiang1, LIU Guoqin1,
Madan K. Bhattacharrya2 & REN Dongtao1
1. State Key Lab of Plant Physiology and Biochemistry, College of Biological Sciences, China Agricultural University, Beijing 100094,
China;
2. Agronomy Department, Iowa State University, Ames, Iowa 500111010, USA
Correspondence should be addressed to Ren Dongtao (e-mail: Ren@
cau.edu.cn)
Abstract A full-length cDNA clone corresponding to a
putative phosphatidylinositol-specific phospholipase C (PIPLC) was isolated from Arabidopsis thaliana by screening a
cDNA library and using RT-PCR strategy. The cDNA, designated AtPLC6, encodes a putative polypeptide of 578 amino
acid residues with a calculated molecular mass of 66251.84 D
and a pI of 7.24. The sequence analysis indicates that the
polypeptide contains X, Y, EF-hand and C2 domains. The
overall structure of putative AtPLC6 protein, like other plant
PI-PLCs, is most similar to that of mammalian PLCδ. The
recombinant AtPLC6 protein expressed in E. coli was able to
hydrolyze phosphatidylinositol 4,5-biophosphate (PIP2) to
generate inositol 1,4,5-trisphate (IP3) and 1,2-diacylglycerol
(DAG). The protein hydrolyzes PIP2 in a Ca2+-dependent
manner and the optimum concentration of Ca2+ is 10 µmol/L.
These results suggested that AtPLC6 gene encodes a genuine
PI-PLC. Northern blot analysis showed that the AtPLC6 gene
is expressed at low level in all examined tissues, such as roots,
stems, leaves, flowers, siliques and seedlings under normal
growth conditions. The gene is strongly induced under low
temperature and weakly induced under various stresses,
such as ABA, high-salt stress and heat. These results suggested that AtPLC6 might be involved in the signal-transduction pathways of cold responses of the plants.
Keywords: Arabidopsis thaliana, AtPLC6, gene cloning, expression
in prokaryotic cell, cold stress.
DOI: 10.1360/03wc0514
Phosphatidylinositol-specific phospholipase C (PIPLC) is a critical enzyme in various signal-transduction
pathways in eukaryotic cells. PI-PLCs can be activated by
the extracellular stimuli such as hormones, growth factors
and biotic or abiotic stress. When it is activated it hydrolyzes phosphatidyl-inositol 4,5-biophosphate (PIP2) to two
second messengers, inositol 1,4,5-trisphosphate (IP3) and
1,2-diacyl-glycerol (DAG). The IP3 mobilizes Ca2+ from
intracellular organelles, thereby regulating Ca2+ and
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Vol. 49 No. 6
March 2004
Ca2+/calmodulin-dependent enzymes and channels, while
the DAG, at least in animal cells, activates protein kinase
C (PKC) which regulates, through phosphorylation, over
100 known proteins, including enzymes, receptors, transport and contractile proteins, and cytoskeletal components[1].
Much evidence shows that in animal cells, PI-PLCs
are involved in a variety of cellular processes such as metabolism, secretion, contraction, sensory mechanism,
reproduction, cell growth[1]. Several years after the animal
PI-PLC was identified, the PI-PLC activities were detected in both soluble and plasma membrane fractions of
plant cells, and purified from different plant species and
tissues[2,3]. The first cDNA clones encoding plant PI-PLCs
were reported in 1995 for Arabidopsis and soybean, and
the recombinant proteins shown the typical PI-PLCδ activities based on the in vitro assay[4,5]. Plant cDNA clones
encoding additional active PI-PLC isozymes have also
been obtained from Arabidopsis, soybean, potato (Solanum tuberosum), wild tobacco (Nicotiana rustica), rape
seed (Brassica napus) and hairy finger-grass[4—9]. During
the last few years, PI-PLC-mediated phosphoinositide
signaling pathway has been shown to perform important
roles in the responses of plant cells to extracellular stimuli,
such as osmotic stress[10], auxin[11], ABA[12—14], light and
gravitational force changes[15,16], pathogen attack[17], and
pollination[18]. Seven genes encoding putative PI-PLC
proteins are found in Arabidopsis genome by data base
searching. Genes corresponding to AtPLC1S, AtPLC1F,
AtPLC1G and AtPLC2 have been cloned and characterized by different groups. Further analysis shows that
AtPLC1S and two genes named as AtPLC5 and AtPLC6
are found tandem located in a 13 kb fragment on Arabidopsis chromosome V. It is still not known how and
where the AtPLC5 and AtPLC6 genes perform their function, or even the two genes are only the pseudo-genes in
genome. Here we describe the isolation of AtPLC6 cDNA
(GenBank accession number: AF434167) and characterization of the recombinant AtPLC6 protein. We report that
the AtPLC6 gene is induced by low temperature.
1
Materials and methods
( ⅰ ) Plant material and treatment. Arabidopsis
thaliana (Columbia ecotype) plants were grown at 22℃
in a growth room with 12-h photoperiod at a photon flux
density of 100 microeinsteins·m−2·s−1. Except for organ-specific expression analysis, 4-week-old plants were
used for experiments. Cold, heat shock, NaCl and ABA
treatments were performed as described by Yamaguchi-Shinozaki[19]. Fully expended leaves of plants were
taken at the indicated times after treatment, quick frozen
in liquid nitrogen, and stored at −80℃ until use.
(ⅱ) Preparation of RNA and DNA. Total RNA
was isolated from samples using Trizol agents (Invitrogen),
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following the manufacturer’s instruction. Genomic DNA
was prepared according to the method of Lin[20].
(ⅲ) Cloning of AtPLC6 3′-region by EST library
screening. According to genomic sequence encoding the
putative AtPLC6, we designed primer pairs to perform
PCR using genomic DNA as template. Primer sequences
were 5′
-CCTTGAGCTACATGCCTAC-3′(forward), and
5′
-TGTTTG GGACGCAACTGCAGCAGAG-3′(backward). The PCR mixture was subjected to 35 cycles of
94℃ for 30 s, 58℃ for 1 min and 72℃ for 2 min. Sequence confirmed DNA fragment was used as probe to
screen a λ-PRL2 cDNA library (ABRC DNA stock number CD4-7). DNA fragment was labeled with [α32P]-dCTP
(Parkin-Elmer) using a random-primed DNA-labeling kit
(Amersham-Pharmacia Biotech). The nucleotide sequences of two positive clones were identical to that of the
PCR fragment. The two cDNA clones were both partial
cDNA fragment contained a stop codon and Poly (A+) tail
at their 3′-terminals. The deduced amino acid sequences
revealed that the cDNA clones were derived from the
same gene and with lower identity to other cloned Arabidopsis PI-PLC. We named this novel gene AtPLC6.
(ⅳ) Reverse transcription-PCR cloning of the fulllength AtPLC6 cDNA. The reverse transcription was
performed using Oligo dT(16) as primer and total RNA
from leaves of Arabidopsis plants as template (M-MLV
reverse transcriptase was purchased from Promeaga). The
full-length AtPLC6 cDNA was PCR amplified using several primer pairs designed according to the computer analyzed result of genomic sequence. The successful PCR
primer pair was AtPLC6 F2 (5′-ATTTAACTAAATTGCAGGAAC-3′) and oligo dT(16), and annealing temperature of 57℃. The full-length cDNA was subcloned
into pGEM-T easy vector (Promeaga) and sequenced. The
full-length cDNA sequence was registered in GenBank
(GenBank accession number: AF434167). According to
the sequencing result, the coding region of the cDNA was
PCR amplified using primer pairs of AtPLC6 C2 with
BamHⅠ site (5′-ggatccATGAAGAGAGATATGGG-3′)
(forward) and AtPLC6 A2 (5′-AAAGAAAGTGAAACCGCATGAGAAG-3′) (backward). The PCR fragment
was subcloned into pGEM-T easy vector and sequence
confirmed.
(ⅴ) Expression of the recombinant AtPLC6 protein
in Escherichia coli. The BamHⅠ/EcoRⅠ fragment of
AtPLC6 coding region was introduced into the BamHⅠ/
EcoRⅠ sites of pET30a in frame and the His tag coding
sequence was fused to AtPLC6 at N-terminal to yield
pET30a-PLC6C2A2.
E. coli BL21 strain was transformed with pET30aPLC6C2A2. Six independent transformants were screened
for protein expression using a small-scale culture. The
transformants (in 3 mL LB liquid media) were grown at
568
37℃ to A600 = 0.6 and were subsequently induced for 2 h
with 0.1 mmol/L isoptopyl-β-D-galactopyranoside (IPTG).
E. coli cells were harvested, washed and suspended in 1×
SDS sample buffer. After heat and spin down, the supernatants were subjected to a SDS-polyacrylamide gel.
A single E. coli BL21 transformant showing high
expression level was selected for a large-scale production
of recombinant AtPLC6. The recombinant AtPLC6 protein
was purified from cultures on column of Ni2+ Chelating
SepharoseTM Fast Flow (Amersham-Pharmacia Biotech)
according to the manufacturer’s instruction.
(ⅵ) Assay of PI-PLC activity. The purified recombinant AtPLC6 proteins were dialysis against 1/4 concentration of enzyme assay buffer (50 mmol/L Tris-maleate,
pH 6.25) at 4℃ over 8 h and the buffer was changed one
time.
The activity assay was performed as described by
Melin[2] with simply modification. The reaction mixture
contained 50 mmol/L Tris-maleate, pH 6.25, 0.2 mmol/L
PIP2 (Sigma), various amounts of Ca2+ (prepared with a
Ca2+/EGTA buffer system), and 0.1 µg/µL recombinant
AtPLC6 protein in a final volume of 50 µL. Boiled protein
served as a control. The reaction was performed at 37℃
for 20 min, and then was stopped by the addition of 1 mL
of chloroform: methanol (2︰1, v/v). Tubes were placed
on ice for 5 min and 250 µL 1 mol/L HCl was added.
Tubes were vortexed for 5 s and centrifuged at 14000 g for
2 min. The upper, aqueous phase was removed and neutralized by the addition of 2 mol/L NaOH. The IP3 was
measured using D-myo-inositol 1,4,5-trisphosphate [3H]
Biotrak Assay System (Amersham Pharmacia Biotech)
according to the manufacturer’s instruction.
( ⅶ ) Western blot. Proteins were separated on
SDS-polyacrylamide gel electrophoresis (SDS-PAGE)
according to Laemmli[21], using a 4.5% stacking gel and
10% separating gel. After SDS-PAGE, the proteins were
electrotransferred to a nitrocellulose membrane. Western
blotting was performed as described by Towbin[22]. The
membrane was incubated with anti-His monoclonal antibody (1︰4000 dilution). Following three times washing
the membrane was incubated with horseradish peroxidase-conjugated goal anti-mouse antibody (1︰10000 dilution). The membrane was visualized by using a LumiLight Western Blotting Substrate Kit (Roche), following
the manufacturer’s instruction.
(ⅷ) Northern blot hybridization. Samples of total
RNA (7.5 µg/lane) were denatured and separated in a 15%
(v/v) formaldehyde-1.2% (w/v) agrose gel and then transferred onto Zeta-Probe membrane (Bio-Rad) using 10×
SSC. After rinsing in 2×SSC for 5 min, the membrane
was UV cross-linked and air-dried.
A 600 bp DNA fragment specific for AtPLC6 was
PCR amplified with primer pairs of AtPLC6-a3 (5′CTChinese Science Bulletin
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CGAGACTGAAACTGGATACCTT-3′) and AtPLC6-a4
(5′
-GACTAGTTTTCCAACATGACTC-3′
) using genomic DNA as template. The PCR fragment was purified
and used as probe. The probe was labeled with [α32P]dCTP (Parkin-Elmer) using a random-primed DNAlabeling kit. Hybridization and washing were described by
Church[23].
2
Results
(ⅰ) Cloning and structural analysis of AtPLC6
cDNA. Using PCR amplified putative At-PLC6 DNA
fragment as probe, we screened an EST cDNA library
constructed from mRNA isolated from Arabidopsis plants.
The first round screening was of 100000 pfu plaques. We
obtained six positive clones. Two of the six positive clones
were hybridized with the DNA probe in the second round
screening. The two clones were selected for sequencing.
The nucleotide sequences revealed that the clones were
derived from the same gene, and they were only the partial
cDNA fragments. The longest one has 800 base pairs with
Poly (A+) tails at the 3′end. The sequences were identical
to that of putative PI-PLC PCR fragment in both the
clones. The GenBank searching results showed that the
sequence of the cDNA shared lower similarity to the former cloned cDNA in Arabidopsis (~51% to AtPLC5
cDNA). Because five genes, as AtPLC1S (accession
number: D38544), AtPLC1F (accession number: U13203),
AtPLC1G (accession number: U76423), AtPLC2 (accession number: D50804) and AtPLC5 (accession number:
AF434168), were already cloned, we named the new
cloned gene AtPLC6.
According to the partial cDNA sequence and genomic sequence of AtPLC6, several primer pairs were
designed for RT-PCR to clone the full-length cDNA and
its open reading frame. The longest PCR product was
1954 bp long containing an open reading frame of 1734
bp. The cDNA sequence upstream of proposed ATG
translation initiation codon contains two stop codons (39
bp and 48 bp sites) within translation frame. The 3′-noncoding region is 126 bp long and contains an AATAAA
putative polyadenylation signal and poly (A+) tail (Fig. 1).
The cDNA of AtPLC6 encodes a putative polypeptide of 578 amino acids with calculated molecular mass of
66251.84 and pI of 7.26. The amino acid sequence of the
predicted protein contains the X (amino acids 113—257),
Y (amino acids 312—430) and C2-like (amino acids 438
—562) domains that has been reported to be conserved in
various PI-PLCs. In addition, AtPLC6 has an EF-hand
motif (amino acids 26—106) in the N-terminal region.
AtPLC6 does not contain a PH domain (Fig. 2(a)). The
overall structure of AtPLC6 protein is similar to mammalian δ-type PI-PLCs (Fig. 2(a)). Analysis on the amino acids sequence of mammalian β-, γ-, ε-, δ-type PI-PLCs and
plants PI-PLCs show that two tobacco PI-PLCs (accession
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nos: CAA65127 and CAA72681) and the Arabidopsis
AtPLC5 share 60.2%, 59.7% and 54.3% identity to
AtPLC6 (Fig. 2(b)).
(ⅱ) A recombinant AtPLC6 protein posseses PI-PLC
activity. To investigate whether the AtPLC6 cDNA encodes a genuine PI-PLC, we analyzed the biochemical
properties of a His-tagged recombinant AtPLC6 protein
expressed in E. coli cells carrying pET30a-PLC6C2A2.
The His-AtPLC6 fusion protein of 71 kD accumulated in
E. coli cells when IPTG was added to the medium (Fig.
3(a), lane 2). Most of the fusion protein was found in the
insoluble fraction and a small amount was detected in the
soluble fraction (Fig. 3(a), lanes 3 and 4). The protein was
purified from the soluble fraction using a column of Ni2+
Chelating Sepharose Fast Flow (Fig. 3(a), lanes 5—7).
The anti-His monoclonal antibody recognized the protein
indicated that it was His-AtPLC6. Proofed molecular mass
of the His-AtPLC6 protein is 71 kD (the molecular mass
of AtPLC6 is 66 kD and His tag is 5 kD) (Fig. 3(b)).
Since an important feature of PI-PLC is Ca2+ dependence of its reaction, the effect of various Ca2+ on the
PIP2-hydrolyzing activity of AtPLC6 was detected using
the His-AtPLC6, as shown in Fig. 4. The PIP2 hydrolysis
of His-AtPLC6 was completely Ca2+-dependent. The
PIP2-hydrolyzing activity was low at lower concentrations
of Ca2+ (< 0.1 µmol/L) and increased gradually as the
concentration of Ca2+ was increased. The optimum concentration of Ca2+ was 10 µmol/L. The PIP2-hydrolyzing
activity decreased more rapidly when the concentration of
Ca2+ was higher than 10 µmol/L. The rates of hydrolysis
of PIP2 by AtPLC6 were 246.7±54.0 pmol·mg−1·min−1
at 0 µmol/L Ca2+, 1113.6±330.7 pmol·mg−1·min−1 at 10
µmol/L Ca2+, and 341.2±92.3 pmol·mg−1·min−1 at 100
µmol/L Ca2+.
(ⅲ) Expression of AtPLC6 gene is induced by low
temperature. To test the tissues-specific expression of
AtPLC6 gene, we performed Northern blot analysis of
total RNA prepared from roots, stems, leaves, flowers,
siliques and seedlings under normal growth conditions.
AtPLC6 mRNA was detected in all examined tissues (Fig.
5). The expression levels in stems, leaves and flowers
were higher than that in other tissues.
To examine the effect of environmental stress on the
expression level of AtPLC6 mRNA, we performed Northern blot analysis of total RNA prepared from leaves of
plants that had been subjected to ABA, NaCl-stress, low
temperature and heat for 12 h. The AtPLC6 mRNA accumulated to a significant level under low temperature,
while the AtPLC6 mRNA accumulated slightly under
ABA, NaCl-stress and heat (Fig. 6(a)). Time course
analyses were performed to reveal the induction of
AtPLC6 mRNA expression under low temperature. As
shown in Fig. 6(b), expression of AtPLC6 mRNA reached
two peaks within 48 h. The first peak appeared 1 h after
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Fig. 1. Nucleotide and deduced amino acid sequence of the Arabidopsis AtPLC6 cDNA. The amino acid sequence of the putative coding region is
shown beneath the nucleotide sequence. Asterisks indicate the in-frame stop codons.
treatment and then decreased to the control level at 6 h.
The second peak appeared 24 h after treatment and then
decreased to control level again at 48 h. The results suggested that expression of AtPLC6 mRNA is possibly involved in the low temperature response of Arabidopsis
plants.
3 Discussion
The completion of Arabidopsis genome sequence has
greatly shorten the time required for new genes cloning,
for example, researchers may explore new genes using the
database information. However, the information from ex570
pression of the predicted genes is even more important to
functional analysis of the genes. The EST cDNA library
screening result shows that AtPLC6 gene is expressed in
Arabidopsis plants. According to the partial cDNA sequence and database predicted AtPLC6 mRNA sequence,
we designed several primer pairs to perform RT-PCR. Finally, the full-length AtPLC6 cDNA was cloned. The sequence alignment results show that predicted mRNA is 50
bp longer than cDNA in the first exon, 48 bp shorter than
cDNA in the junction of exons 4 and 5, 21 bp longer than
cDNA in the junction of exons 6 and 7.
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Fig. 2. Structure comparison of AtPLC6 with other PI-PLCs (a), and phylogenetic analysis of animal and plant PI-PLCs (b).
Fig. 3. SDS-PAGE (a) and Western blot (b) analysis on purification of
the recombinant AtPLC6. 1, Total proteins in noninduced E. coli; 2, total
proteins in induced E. coli; 3, the insoluble protein in induced E. coli; 4,
the soluble protein in induced E. coli; 5—7, the purified recombinant
AtPLC6 protein through an affinity column; M, marker proteins with
molecular masses in kilodaltons.
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March 2004
Fig. 4. Ca2+ dependence of PIP2 hydrolysis by the recombinant
AtPLC6 protein.
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Fig. 5. Northern blot analysis of AtPLC6 gene expression in various
tissues.
Fig. 6. Expression of the AtPLC6 gene in response to various stresses.
(a) Total RNA from samples were taken 12 h after treatment; (b) total
RNA from samples as the indicated time after low temperature treatment.
Animal PI-PLCs have been classified into four distinct subfamilies: PI-PLCβ, γ, δ and ε, on the basis of their
enzymatic characteristics and comparison of their primary
amino acid sequences. Five domains, a PH domain, an
EF-hand domain, an X and a Y domains, and C2 domain,
are conserved in animal and yeast PI-PLC enzymes[24—26].
The five domains constitute the structure of PI-PLCδ. In
animal PI-PLCδ, both X and Y domains constitute the
catalytic domain of enzyme[24], PH domain is required for
interaction with plasma membrane, and involved in the
binding of lipid substrate and in processive catalysis[25].
C2 domain is involved in calcium-triggered phospholipid
binding and EF-hand domain is required for enzyme to be
active[26]. Although PI-PLC has been purified from several
plant species, no amino acid sequence has ever been obtained for any of these purified proteins. Analysis on the
deduced amino acid sequences shows that plant PI-PLC
proteins do not contain a PH domain, this is a distinguishing feature between plant PI-PLC proteins and animal
PI-PLCδ[27]. Except for their Ca2+-dependent manner, the
regulatory mechanisms of plant PI-PLCs are unknown. As
plant PI-PLCs lack a PH domain, their interaction with
plasma membrane and PIP2 must clearly differ from
PI-PLCδ. The evidence, that the C2 domain in plant
PI-PLC was sufficient for Ca2+ and lipid binding[27,28],
suggests that C2 domain in plant PI-PLC may perform the
functions of both C2 and PH domain of PI-PLCδ.
572
EF-hand domain is also essential to preserve the activity
of plant PI-PLCs[27]. AtPLC6, like other plant PI-PLC[29],
contains X, Y, C2 and EF-hand domains and the overall
structure is most closely related to that of animal
PI-PLCδ[30].
Although most of the recombinant plant PI-PLCs
expressed in E. coli catalyze the Ca2+-dependent hydrolysis of PIP2 or PI, there is still some recombinant product,
PsPLC in pea for example which does not have the properties as those reported in some mammalian systems[28].
Our results show that recombinant AtPLC6 hydrolyze the
PIP2 and produce IP3 and DAG. The PIP2 hydrolysis activity in optimum Ca2+ concentration of 10 µmol/L is
about 4 times higher than that in 0.1 µmol/L Ca2+. The
activities are markedly reduced in the range of 0.1—1
mmol/L Ca2+. The Ca2+-dependent and optimum Ca2+
concentration are the same as other plant PI-PLC and
animal PI-PLCδ[31,32]. The alignment result of amino acid
sequence shows that AtPLC6 shares higher homologies
with two tobacco PI-PLC and AtPLC5, especially in conserved X and Y domains (over 90%). The sequence and
enzymatic information suggest that AtPLC6, we cloned
here, encodes a novel PI-PLC isoenzyme.
The stomatal guard cells responsing to abscisic acid
(ABA), aperture regulation and cytosolic Ca2+ oscillations
in tobacco were inhibited by U-73122, a PI-PLC specific
inhibito[12]. Permanent transgenic tobacco plants with very
low levels of PI-PLC in guard cells were only partially
able to regulate their stomatal apertures in response to
ABA[13] and Mills et al.[33] proposed that PI-PLCs were
involved in the events associated with the inhibition of
stomatal opening by ABA, but not in ABA-induced
stomatal closure. Using sense and antisense transgenic
Arabidopsis plant, Sanchez et al. demonstrated that
AtPLC1S is necessary for the inhibition of germination
and seedlings growth by ABA but that overexpression of
AtPLC1S did not result in the induction of ABA-regulated
genes, demonstrating that AtPLC may be involved in secondary ABA responses[14]. Applying of PI-PLC specific
inhibitors affected the osmotic stress-induced calcium
signal and blocked the expression of some dehydration-induced genes[34]. When elicitor and PI-PLC inhibitor
were added to the tobacco cells, the defence responses of
the cells were effectively blocked, implying that PI-PLC
signaling is essential for the activation of plant defense
responses[35]. Promoter region of PsPLC was found to
contain light-responsive cis-element, suggesting that
PI-PLC may be involved in plant light response[28]. Taking
all together, plant PI-PLCs take part in many important
cellular responses and signal transduction pathway.
In Arabidopsis plants, AtPLC1F expression is induced by flowering[7], AtPLC1S and AtPLC1G expressions are induced by stress[4,36], while AtPLC2 gene is
constitutively expressed in all tissues[6]. Induction of
AtPLC6 by low temperature (Fig. 6) suggests the accuChinese Science Bulletin
Vol. 49 No. 6 March 2004
ARTICLES
mulation of AtPLC6 protein in plant cells, which have
been exposed to low temperature. The accumulation of
AtPLC6 probably contributes to the enhancement of the
efficiency of signal transduction under low temperature
and increases the ability of plant cells to adapt to this condition. Analysis of the mutants in which the AtPLC6 gene
is overexpressed or downregulated should give us information of AtPLC6 function.
Acknowledgements We thank Arabidopsis Biollogical Resource Center for the CD4-7 cDNA library. This work was supported by the State
Basic Research Program (Grant No. 2003CB114304), the National
Natural Science Foundation of China (Grant Nos. 30270064, 30370140
and 30170457) and the Excellent Young Teacher Program of MOE,
P.R.C.
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(Received October 24, 2003; accepted January 30, 2004)
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