Plant Physiol. (1998) 116: 239-250
Molecular and Enzymatic Characterization of Three
Phosphoinositide-Specific Phospholipase C Isoforms from
Potato1
Joachim Kopka,
Christophe Pical,
Julie E. Gray, and
Bernd Müller-Röber*
Max-Planck-Institut für Molekulare Pflanzenphysiologie,
Karl-Liebknecht-Strasse 25, Haus 20, D-14476 Golm/Potsdam, Germany
(J.K., B.M.-R.); and Department of Molecular Biology and
Biotechnology, University of Sheffield, P.O. Box 594, Sheffield,
S10 2UH, United Kingdom (C.P., J.E.G.)
 |
ABSTRACT |
Many cellular responses to
stimulation of cell-surface receptors by extracellular signals are
transmitted across the plasma membrane by hydrolysis of
phosphatidylinositol-4,5-bisphosphate (PIP2), which is
cleaved into diacylglycerol and inositol-1,4,5-tris-phosphate by
phosphoinositide-specific phospholipase C (PI-PLC). We present structural, biochemical, and RNA expression data for three distinct PI-PLC isoforms, StPLC1, StPLC2, and StPLC3, which were cloned from a
guard cell-enriched tissue preparation of potato (Solanum tuberosum) leaves. All three enzymes contain the catalytic X
and Y domains, as well as C2-like domains also present in
all PI-PLCs. Analysis of the reaction products obtained from
PIP2 hydrolysis unequivocally identified these enzymes as
genuine PI-PLC isoforms. Recombinant StPLCs showed an optimal
PIP2-hydrolyzing activity at 10 µm
Ca2+ and were inhibited by Al3+ in equimolar
amounts. In contrast to PI-PLC activity in plant plasma membranes,
however, recombinant enzymes could not be activated by
Mg2+. All three stplc genes are expressed in
various tissues of potato, including leaves, flowers, tubers, and
roots, and are affected by drought stress in a gene-specific manner.
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INTRODUCTION |
In animal cells many cellular responses to stimulation of
cell-surface receptors by extracellular signals are transmitted across
the plasma membrane by hydrolysis of PIP2, which
is catalyzed by PI-PLC. This reaction generates the two secondary
messengers: IP3, a compound soluble in the
cytosol that triggers transient increases of the cytosolic
Ca2+ level, and DG, a lipid that stays within the
plasma membrane and activates PKC (Berridge, 1993
). In plants an enzyme
with PKC activity has been partially purified from Brassica
campestris (Nanmori et al., 1994), and evidence has been obtained
for the participation of PKC in the elicitor-induced defense response in potato (Solanum tuberosum; Subramaniam et al., 1997). DG
has been shown to induce both ion pumping in patch-clamped guard cell protoplasts and the opening of intact stomata (Lee and Assmann, 1991).
Furthermore, it is now well established that
IP3-mediated Ca2+ release
occurs in plant cells (Drøbak, 1992, 1993; Coté and Crain, 1993,
1994).
Increasing evidence strongly suggests that
IP3-mediated signal transduction is functional in
guard cells. The phytohormone ABA, which is involved in multiple stress
responses, also triggers stomatal closure and inhibits stomatal
opening. Extracellular application of ABA or injection of this
phytohormone into guard cells of Commelina communis and
Vicia faba results in transient increases in cytosolic
Ca2+ (McAinsh et al., 1990; Schroeder and
Hagiwara, 1990). In addition, Lee et al. (1996) demonstrated that ABA
induces an increase in cytosolic IP3
concentration within seconds after the extracellular application of ABA
to V. faba guard cell protoplasts, which is accompanied by a
rapid turnover of inositol phospholipids. Photolysis of "caged"
IP3 within guard cells of C. communis
(Gilroy et al., 1990) and V. faba (Blatt et al., 1992)
causes an increase of cytosolic Ca2+ and a
subsequent stomata-closing reaction. Finally, increasing concentrations
of both Ca2+ (Schroeder and Hagiwara, 1989) and
IP3 (Blatt, 1992) reduce inward-rectifying K+ currents in plasma membranes of V. faba guard cells. Inward-rectifying K+
channels are believed to be involved in driving the opening of guard
cells.
Current evidence supports a model in which the transduction of the ABA
signal in guard cells utilizes changes in cytosolic concentrations of
IP3 and Ca2+ and ultimately
leads to the inactivation of inward-rectifying K+
channels and stomatal closure. However, in C. communis ABA
can induce stomatal closure by a second,
Ca2+-independent pathway (Allan et al., 1994). In
addition, both cyclic ADP-Rib and IP3
independently are able to trigger Ca2+ release
from plant vacuoles (Allen et al., 1995). Therefore, it appears that
guard cells operate different, and in part complementary, signal
transduction pathways that all lead to stomatal
closure.
To investigate the role of the IP3-mediated
pathway in guard cells from a new angle we decided to clone cDNAs of
PI-PLCs and other enzymes of the phosphoinositide-signaling pathway
expressed in epidermal fragments and possibly guard cells of potato
leaves. Epidermal fragments were used as the source for the isolation of guard cell mRNA, even though it is possible to isolate mRNA from
guard cell protoplasts (Nakajima et al., 1995) or from single plant
cells (Karrer et al., 1995). These latter methods, however, exhibit
inherent analytical problems, as discussed previously (Kopka et al.,
1997b).
Phosphoinositide metabolism in higher plants has recently been
approached by molecular cloning of cDNAs encoding multiple PI-PLC
isoforms from Arabidopsis thaliana (Hirayama et al., 1995, 1997; Yamamoto et al., 1995) and Glycine max (Shi
et al., 1995; GenBank accession nos. U41473, U41474, and U41475). Our laboratory has recently cloned CDS from potato and Arabidopsis (Kopka
et al., 1997a). An eye-specific CDS in Drosophila
melanogaster has been shown to be essential for
IP3-mediated signal transduction during light
perception (Wu et al., 1995), where the enzyme is required for the
regeneration of PIP2 from DG (one of the reaction products of PI-PLC activity) via PA, and thus participates in PI-PLC-signaling cascades. Another enzyme involved in the resynthesis of PIs, DG kinase, has also been cloned recently from higher plants (A. thaliana; Katagiri et al., 1996).
To obtain a probe for heterologous screening of potato PI-PLC cDNAs we
initially cloned a novel PI-PLC homolog from A. thaliana ecotype C24, which was identified by a search of the plant database of
expressed sequence tags (J. Kopka and B. Müller-Röber,
unpublished data). This clone was successfully used to isolate cDNAs
coding for PI-PLCs from Nicotiana rustica (Pical et al.,
1997). Here we report the isolation of cDNAs representing three PI-PLC
isoforms from epidermal fragments of fully expanded potato leaves. We
demonstrate gene expression of the three isoforms in a variety of
tissues and differential gene regulation under different stress
regimes. Using purified recombinant proteins we identified the reaction products produced by the potato PI-PLCs and characterized the cation
requirements of the three isoforms.
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MATERIALS AND METHODS |
Cloning and Sequencing of cDNAs
A novel PI-PLC homolog was cloned from Arabidopsis
thaliana (J. Kopka and B. Müller-Röber, GenBank
accession no. X85973). Two HindIII fragments (0.7 and 0.8 kb) containing most of the coding region of the novel cDNA were used
for heterologous screening of a 
-ZAP II cDNA library (Stratagene),
which was prepared from poly(A+) RNA that was
isolated from epidermal fragments of potato (Solanum tuberosum L. cv Désiree) leaves (Müller-Röber et
al., 1995; Kopka et al., 1997b). DNA fragments were labeled with
[
32P]dCTP using a random-primed DNA-labeling
kit (Boehringer Mannheim). Hybridization of plaque lifts overnight at
42°C in PEG buffer (Amasino, 1986) was followed by washes at 45°C
in 6× SSC and 0.5% SDS for 15 min and in 5× SSC and 0.5% SDS for 15 min. pBluescript II SK plasmids containing target inserts were obtained
from hybridizing phages by in vivo excision according to the
manufacturer's (Stratagene) protocol. The plasmids containing the
longest inserts were manually sequenced (T7 sequencing kit, Pharmacia).
Complete nucleotide sequences of full-length cDNAs representing the
genes stplc1, stplc2, and stplc3 were submitted
to the EMBL nucleotide sequence database. Standard molecular biology
methods were performed as described previously (Sambrook et al., 1989).
Computational Analysis of Predicted Amino Acid Sequences
The computational services and options of the Wisconsin package
(version 8.1, Genetics Computer Group, Madison, WI) were used with
default parameters. Analysis of amino acid sequence homology was
performed with the BLAST program (Altschul et al., 1990). Multisequence
alignments were created with the PILEUP option. The percentage of
sequence identity and similarity was determined by pairwise alignment
using the GAP program. Hydropathy plots were generated according to the
algorithm of Kyte and Doolittle (1982). Relative molecular masses and
pIs were calculated from the deduced amino acid compositions with the
Compute pI/Mw tool available at the ExPASy Molecular Biology Web server
(Geneva, Switzerland). The secondary structure of plant PI-PLCs was
predicted by submitting a multisequence alignment of all known plant
PI-PLCs to the SSPRED Web server at the European Molecular Biology
Laboratory (Heidelberg, Germany). Subcellular sorting was predicted at
the PSORT Web server for analyzing and predicting protein-sorting signals at the Institute for Molecular and Cellular Biology (Osaka, Japan). Analysis of StPLC primary structures for conserved protein domains was performed with the PROFILESCAN program at the Web server of
the ISREC Bioinformatics Group (Lausanne, Switzerland).
Plant Material
Potato plants were obtained through Saatzucht Fritz Lange KG (Bad
Schwartau, Germany). Plants were grown in soil in individual 3-L pots
in a greenhouse under periods of 16 h of light (with additional
illumination giving a total light intensity of approximately 100-200
µmol photons s
1 m2;
22°C) and 8 h of darkness (15°C).
Material for RNA analysis of steady-state mRNA levels in different
tissues (Fig. 3) was harvested from well-watered, flowering potato
plants at approximately 3 pm, 8 h after the beginning
of the light period. Epidermal fragments for this experiment were prepared from source leaves, i.e. fully expanded, nonsenescent leaves
from the fifth node downward (Müller-Röber et al., 1995; Kopka et al., 1997b). Sink leaves were immature leaves (<1 cm in
length) harvested from the first visible node of nonflowering shoots.
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| Figure 3.
RNA analysis of stplc steady-state
transcript levels in various tissues of potato plants. Epidermal
fragments were prepared from source leaves. Total RNA (50 µg per
lane) was probed with the complete cDNAs of stplc1,
stplc2, and stplc3 and with a cDNA encoding potato 25S rRNA (see ``Materials and Methods''). Transcript
sizes are indicated.
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Leaflets of fully expanded leaves used for analysis of the short-term
effect of local wounding on transcript levels (Fig. 4) were crushed
once in vertical orientation to the major vein of the leaflet with a
5-cm clamp used to seal dialysis tubes. Total RNA was prepared from
these leaves 6 h after application of wound stress. Fast wilting
of leaves for RNA analysis of short-term effects on gene expression
(Fig. 4) was achieved by air-drying the root system after gentle
removal of the pot and adhering soil. Wilted leaves were harvested
after 6 h. In this experiment leaf fresh weight was reduced by
approximately 20% compared with untreated plants.
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| Figure 4.
RNA analysis of stplc steady-state
transcript levels in fully expanded leaves of potato plants that were
untreated, locally wounded, or wilted by severe short-term drought.
Leaves for RNA preparation were detached 6 h after application of
stresses at approximately 4:30 pm. RNA preparations (25 µg per lane) were also tested for wound-induced expression of the
proteinase inhibitor II gene (pin2), for drought-induced
expression of the tas-14 gene, and for induction of Suc
synthase (susy) gene expression.
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The effect of slowly developing, long-term drought stress was studied
on leaves of the upper third to fifth node because most leaves of the
lower nodes were subject to fast senescence under the experimental
conditions applied. Plants were not watered for 4 d before the
leaves were collected, whereas control plants were subjected to a
normal watering regime and were kept under otherwise identical growth
conditions. Leaves and epidermal fragments were prepared as described
by Kopka et al. (1997b).
RNA Analysis
RNA from different tissues of potato plants was prepared according
to the method of Logemann et al. (1987), except RNA of epidermal
fragments, which was prepared as described by Kopka et al. (1997b).
Total RNA was quantitated spectrophotometrically at 260 nm. Total RNA
from each sample (25 or 50 µg) was electrophoretically separated in a
denaturing 15% (v/v) formaldehyde-1.5% (w/v) agarose gel and blotted
onto Hybond-N+ membranes (Amersham). Fixation of
RNA to the membrane was achieved by incubation in 50 mm
NaOH for 5 min at room temperature and a subsequent wash in 2× SSC
(Noonberg et al., 1994). Labeling of cDNA fragments and hybridizations
were performed as described for plaque lifts. Washes were performed at
65°C in 3× SSC and 0.5% SDS for 15 min and in 0.2× SSC and 0.5%
SDS for 20 min. Blots were exposed to Kodak X-OMAT AR film.
The following DNA probes were used for hybridizations: the
SpeI/XhoI fragments containing the complete cDNA
inserts of stplc1, stplc2, and stplc3;
the 3.5-kb Asp718/BamHI fragment of plasmid efEST
G56 coding for 25S rRNA of potato (GenBank accession no. R28706); the
2.7-kb Asp718/BamHI fragment of POTSSYN coding for potato Suc synthase (Salanoubat and Belliard, 1987); the 0.6-kb PstI fragment of PINR5 coding for the wound-inducible potato
proteinase inhibitor II (pin2 gene; Peña-Cortés
et al., 1996); and the full-length fragment of a tomato cDNA coding for
the drought-stress-inducible tas-14 gene (Godoy et al.,
1990). Transcript sizes of stplc1, stplc2, and
stplc3 were determined in relation to the electrophoretic mobility of rRNA species with a single RNA blot, which was successively probed.
Analysis of Recombinant StPLC Expressed in Escherichia
coli
Recombinant StPLC1, StPLC2, and StPLC3 proteins were expressed in
E. coli BL21 cells as GST-StPLC fusion proteins using the pGEX-4T-2 vector (Pharmacia), which provides a thrombin cleavage site for removal of the GST part of the fusion proteins. The complete coding regions of stplc1, stplc2, and
stplc3 were amplified by PCR using Taq-DNA
polymerase. The forward primers were specific for each isoform and
introduced BamHI restriction sites, which were used for
in-frame cloning of the PCR fragments into the pGEX-4T-2 vector. The
reverse primer was the T7 primer, which anneals to the T7 promoter
region of pBluescript II SK plasmids. The second restriction site for
cloning of PCR fragments was XhoI, which was the cloning
site that was used for construction of the epidermal fragment -ZAP
II cDNA library (Müller-Röber et al., 1995) and therefore
immediately follows the poly(A+) tail of the cDNA
inserts. The resulting expression vectors were named pGEX-StPLC1,
pGEX-StPLC2, and pGEX-StPLC3.
The cloning strategy preserved the native stop codons of StPLCs but
introduced an N-terminal Gly-Ser dipeptide to thrombin-cleaved, recombinant StPLCs in place of the native Met residue. E. coli BL21 was transformed with pGEX-StPLC1, pGEX-StPLC2, or
pGEX-StPLC3. Five to 10 transformants per isoform, each containing a
vector derived from an independent PCR fragment, were screened for
protein expression using a small-scale purification protocol. E. coli BL21 transformants (3-mL cultures) were grown at 28°C to
A600 = 0.6 and were subsequently induced
for 2 h with 2 mm
isopropyl-
-d-galactopyranoside. Fusion protein was
purified using a glutathione-Sepharose resin according to the
manufacturer's (Pharmacia) instructions, and GST- and
PIP2-hydrolyzing activities were determined in
the affinity-purified fractions. Fusion protein isolated from most
transformants had similar, high ratios of PI-PLC activity compared with
GST activity; only a few did not exhibit PI-PLC activity, which may
have been due to errors induced during DNA amplification or cloning.
A single E. coli BL21 transformant carrying either
pGEX-StPLC1, pGEX-StPLC2, or pGEX-StPLC3 and showing high PI-PLC
activity compared with GST activity was selected for a large-scale
production of recombinant StPLC1, StPLC2, and StPLC3. Thrombin-cleaved
StPLCs and uncleaved fusion proteins were purified from 1-L cultures according to the manufacturer's recommendations using PBS buffer (10.1 mm Na2HPO4, 1.8 mm KH2PO4, 0.14 m NaCl, and 2.7 mm KCl, pH 7.3). Thrombin
cleavage was performed overnight at room temperature with fusion
protein that was bound to a glutathione-Sepharose column.
Purification was monitored by protein determination (Bradford, 1976),
by determination of GST activity with 1-chloro-2,4-dinitrobenzene as
substrate (Habig et al., 1974), by determination of PI-PLC activity
with PIP2 as substrate (see below), and by
SDS-PAGE (Laemmli, 1970). Protein purity and molecular masses were
estimated by densitometry of Coomassie brilliant blue G-250-stained
SDS-PAGE gels. Yield of a representative purification from a 1-L
E. coli culture was 0.38 mg of GST-StPLC fusion protein or
0.15 mg of recombinant StPLC. Purity of recombinant StPLCs was greater
than 90% and GST activity was not detectable.
Biochemical Analysis of Recombinant StPLCs
The PI-PLC assay was performed according to the method of Melin et
al. (1992) and Drøbak et al. (1994). The reaction mixture contained 50 mm Tris/maleate, pH 6.25, 0.2 mm of
3H-head-group-labeled PI or
PIP2 at approximately 5000 dpm
nmol1 (Amersham) and varying amounts of
Ca2+ in a volume of 50 µL. A 0.6 mm
micellar lipid stock solution was prepared by mixing unlabeled (PI,
PIP2; Sigma) and 3H-labeled
lipid in chloroform:methanol (2:1, v/v), evaporation of solvent under a
stream of nitrogen, addition of 166 mm Tris/maleate, pH
6.25, and sonication for 10 min. The reaction was performed at 25°C
for 15 min. Substrate consumption did not exceed 15%. The reaction was
stopped by the addition of 1 mL of chloroform:methanol (2:1, v/v).
Phases were separated by adding 0.25 mL of 1 n HCl, and
liquid scintillation counting of water-soluble reaction products was
carried out as described previously (Melin et al., 1992).
Cations were added to the reaction mixtures as chloride salts. The
standard reaction mixture contained 10 µm
Ca2+. Ca2+ concentrations
10 µm were buffered with 1 mm EGTA (Owen,
1976). Studies of the effect of Al3+ and
Mg2+ on plant PI-PLC activity were performed in
the presence of 10 µm Ca2+.
Recovery of IP3 from standard reaction mixtures
in the presence of Al3+ and
Mg2+ was determined with
[3H]IP3 (5000 dpm
nmol1; Amersham).
Analysis of Plant PI-PLC Reaction Products
Standard reactions were performed in 50 µL with 0.1 µg
µL1 recombinant StPLC, 10 µm
Ca2+, and 0.2 nmol µL1
phosphatidyl[2-3H]inositol-4,5-bisphosphate
(5000 dpm nmol1). Reactions were stopped by
chloroform:methanol (2:1, v/v), and lipophilic and water-soluble
reaction products were separated as described above.
The chloroform phase was concentrated under a stream of nitrogen and
applied quantitatively onto silica gel plates with a concentration zone
(type Si 250 PA, J.T. Baker, Deventer, The Netherlands). TLC was
performed with hexane:diethylether:acetic acid (9:1:0.5, v/v) to
analyze neutral lipids or with chloroform:methanol:acetic acid:water
(25:15:4:2, v/v) for separation of PA. Lipids were visualized by iodine
staining. The standard lipid mixtures contained 10 µg of DG and 10 µg of PA (both from Sigma). PIP2 was immobile in both solvent systems.
The aqueous phase of the chloroform:methanol extraction was neutralized
with 2 n NaOH and concentrated by freeze-drying. The reaction products were analyzed on a 25-cm Partisil SAX HPLC column (Whatman, Maidstone, UK). The chromatographic conditions of Brearley and Hanke (1996) were applied. The columns were eluted at 0.5 mL
min1 applying a linear gradient of 0 to 2.5 m NaH2PO4 with
a slope of 0.416 m min1.
3H was monitored with a flow-through counter.
Recovery and retention time of IP3 was determined
with authentic [3H]IP3
(50,000 dpm per reaction mixture).
[3H]Inositol-bisphosphate was prepared by
limited alkaline phosphatase treatment of
[3H]IP3 (Brearley and
Hanke, 1996). Separation of inositol phosphates on the Partisil SAX
column was possible but limited by severe peak tailing and did not meet
the superior quality of Partisphere SAX columns (Brearley and Hanke,
1996).
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RESULTS |
Cloning and Sequence Analysis
Using the coding region of an A. thaliana cDNA encoding
a novel plant PI-PLC homolog as a molecular probe, we cloned three PI-PLC isoforms from a -ZAP II cDNA library, which was prepared from
poly(A+) RNA isolated from epidermal fragments of
potato leaves (Müller-Röber et al., 1995). The 1981-bp cDNA
of stplc1 codes for a 596-amino acid protein; the cDNA of
stplc2 has 1940 bp with an open reading frame representing
565 amino acids, and stplc3 encodes a 585-amino acid
polypeptide within a cDNA of 2009 bp. All clones were full length, as
was shown by determining transcript sizes corresponding to these
stplc genes (compare with Fig. 3). The primary structures of
StPLC1, StPLC2, and StPLC3 (Fig. 1) were
similar to those of currently known plant PI-PLC homologs. Relative
molecular weights of the potato proteins were between 64,294 and 67,766 and the pIs ranged from 5.27 to 6.19. The homologies between the coding regions of stplc1, stplc2, and stplc3
were 68 to 81%, and the deduced proteins showed 63 to 77% identity at
the amino acid level.
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| Figure 1.
Multisequence alignment of the amino acid
sequences of three PI-PLC isoforms, StPLC1, StPLC2, and StPLC3, as
deduced from corresponding cDNA clones isolated from a potato epidermal
fragment cDNA library. Amino acid residues conserved in at least two of the three sequences are shaded in black; conservative substitutions are
marked in gray. Predicted domains of conserved secondary structure (SS)
are indicated by bars (, -helix; , -sheet). Conserved functional domains are underlined in black (X and Y domain) or gray
(C2-domain; PROSITE database at the ExPASy Molecular
Biology Web server; see Essen et al., 1996). His residues of the
reaction center, which are invariable within the X domains of all known enzymatically active PI-PLCs, are marked by arrows.
Ca2+-binding amino acids within the active site are marked
by asterisks (*). Putative Ca2+-binding amino acids within
the C2-like domain are indicated by arrowheads. Note that
the linking region between X and Y domains of StPLC1 contains a short
sequence repeat (A and B), which is highlighted by frames.
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Analysis of homology between the potato PI-PLC proteins (not shown)
indicated that StPLC2 and StPLC3 are more closely related to each other
than to any other plant PI-PLC. StPLC1 appeared to be as
distantly related to StPLC2 and StPLC3 as to PI-PLCs from other plant
species. In general, homology among plant PI-PLCs was higher than
52.0% (identical amino acids). In addition to high amino acid
homology, plant PI-PLCs also appeared to share common features on the
secondary structure level. Using the SSPRED program for the analysis of
conserved structural elements (see ``Materials and Methods''), we
identified 14 -helix-forming and 19 -sheet-forming domains in a
multisequence alignment based on all presently known plant PI-PLCs. The
positions of these putative structural elements are shown in Figure 1.
StPLC isoforms do not contain obvious subcellular sorting signals or
transmembrane-spanning domains and therefore these proteins are most
likely located in the cytosol and, considering their presumed role, may
be associated with the plasma membrane.
Compared with other PI-PLCs, plant PI-PLCs seem to form a distinct
subgroup. All PI-PLCs identified so far exhibit high homology within
three domains: X and Y, which together constitute the catalytic domain
of these enzymes, and a C2-like domain (Fig. 1).
The C2-like domain of StPLCs was identified by
the C2 domain profile (PS50004) of the PROSITE
database. C2 domains are
Ca2+-dependent protein-phospholipid interaction
domains, which could mediate membrane attachment of the amphipathic
plant PI-PLCs. C2 domains were first identified
in Ca2+-regulated PKC isoforms (Hug and Sarre,
1993) and are present in a variety of mammalian enzymes involved in
transmembrane signaling, such as phospholipase A2
(Clark et al., 1991), PI-PLC (Essen et al., 1996), synaptotagmin (Perin
et al., 1991), and rabphilin-3A (Shirataki et al., 1993).
The C2-like domains of StPLCs align to the
eight-stranded -sandwich structure of the C2
domain in rat PLC
1 (Essen et al., 1996). The predominant
predicted secondary structure of StPLCs within this region is -sheet
conformation (Fig. 1, 14-19). The location of most of these
-sheet domains is in agreement with the two known crystal structures
of C2 domains within rat PLC1 (Essen et al.,
1996) and rat synaptotagmin I (Sutton et al., 1995). Moreover, StPLCs
contain the polybasic core region K-(K,R)-T-K typical for
C2 domains (Fig. 1, -14) and five putative Ca2+-binding residues (Fig. 1, arrowheads), which
correspond to the Ca2+-interacting amino acids
identified in crystallized C2 domains (Sutton et
al., 1995; Essen et al., 1996; Shao et al., 1996).
Hydropathy plots of StPLC isoforms illustrate the pattern of sequence
conservation and diversity. Within both amphipathic X and Y domains, a
large number of conserved hydrophilic and hydrophobic subdomains can be
identified in all three StPLC isoforms (Fig. 2). N termini of StPLC isoforms are also
amphipathic. In this region, StPLC2 and StPLC3 exhibit almost identical
hydropathy patterns, whereas StPLC1 is more variable. The linking
region between the X and Y domains is highly hydrophilic and appears to
differ between isoforms in length and hydrophilicity. A bipartite hydrophilic linking region, which contains a short sequence repeat that
is not present in the single hydrophilic domains of StPLC2 and StPLC3,
is characteristic of StPLC1 (Figs. 1 and 2).
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| Figure 2.
Hydropathy plots of the deduced amino acid
sequences of StPLC1, StPLC2, and StPLC3. The total number of amino
acids (aa) constituting each isoform is listed. The relative positions
of the X and Y domains, the C2 domain, and the sequence
repeat in StPLC1 (A and B) are indicated by bars. The conserved
hydropathy pattern of StPLC isoforms is marked by 20 individually
numbered hydrophilic and hydrophobic domains. Note that significant
differences between isoforms are present at the N terminus and within
the highly hydrophilic linker region between the X and Y domains.
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RNA Expression
Analysis of RNA expression of the three potato PI-PLC isoforms was
performed on various tissues from potato plants (Fig.
3). Steady-state mRNA levels of all
isoforms were high in flower, petiole, and stem. In contrast, fully
expanded source leaves and a preparation of epidermal fragments
contained low amounts of stplc transcripts. Epidermal
fragment RNA is 90% pure guard cell RNA and may be contaminated only
by RNA from trichomes (Kopka et al., 1997b). As judged by
phosphorimaging analysis, intensities of hybridization signals observed
with RNA isolated from epidermal fragments and source leaves were
approximately equal (data not shown). Expression in sink leaves and in
tubers differed between isoforms and, in the case of Stplc1,
was approximately 10-fold higher than in fully expanded source leaves.
Stplc2 and stplc3 transcript levels were also
elevated in sink leaves but showed only an approximately 2- to 3-fold
difference compared with fully expanded leaves (for definitions of leaf
stages, see ``Materials and Methods'').
The Stplc1 transcript was hardly detectable in preparations
of tuber RNA. In contrast, transcript levels of stplc2 and
stplc3 in tubers were equal or slightly higher than in
stems. Cross-hybridization between the three stplc probes
and their corresponding mRNAs in this and the following experiments can
largely be excluded because successive hybridizations of the probes to
a single membrane revealed slightly different transcript sizes (compare
with Fig. 3) or exhibited differential response of stplc
transcript levels to environmental signals (see below; compare with
Fig. 4). Furthermore, no
cross-hybridization between stplc cDNAs was observed in
DNA-blot experiments performed under the same hybridization conditions
as used here for the RNA analysis (not shown).
Gene expression of stplc isoforms in source leaves showed
different short-term responses to local wound stress and air drying. Whereas stplc1 exhibited strong reduction of its transcript
level in response to both stresses, stplc2 mRNA was equally
strongly induced by both treatments (Fig. 4). Local wounding induced
only a minor, short-term alteration in the expression of
stplc3. Analysis of transcript levels in this experiment was
performed 6 h after application of wound or drought stress,
respectively (see ``Materials and Methods''). The effect of the
applied stresses on mRNA composition was verified by monitoring
wound-induced pin2 gene expression (Peña-Cortés et al., 1996) and the drought-induced increase in tas-14
mRNA levels (Godoy et al., 1990; Harms et al., 1995). In addition, application of both wound and drought stress could also be monitored by
an increase in gene expression of Suc synthase (Fig. 4; Kopka et al.,
1997b).
We also compared long-term and short-term drought effects (see
``Materials and Methods'') on stplc transcript levels in
RNA isolated from whole leaves. Under both conditions,
stplc1 and stplc2 showed the same behavior as
described above, whereas stplc3 transcript levels increased only after long-term drought stress. The same observations were made
with RNA extracted from epidermal fragments (not shown). Previously,
Hirayama et al. (1997) reported transient induction by drought of the
AtPLC1S gene in Arabidopsis rosette plants but constitutive
expression of the AtPLC2 gene.
Analysis of Recombinant StPLC Isoenzymes
Recombinant StPLC isoenzymes were expressed in E. coli
BL21 cells as GST-StPLC containing a thrombin recognition site between the N-terminal GST-tag and StPLC. Recombinant fusion proteins were
affinity purified on glutathione-Sepharose 4B and either eluted with
reduced glutathione to yield GST-StPLC fusion proteins or cleaved while
bound to the column with thrombin to prepare recombinant StPLC
proteins. These preparations contained proteins that showed the
expected electrophoretic mobilities for the relative molecular weights
predicted from the cDNA sequences. Recombinant StPLCs were at least
90% pure and, as judged by SDS-PAGE and enzyme assays, contained no
residual GST (not shown). Preparations of StPLC3 contained
approximately equal amounts of two polypeptides, one polypeptide being
smaller than expected. Because of differences in protein degradation,
the specific enzyme activities of recombinant StPLC isoenzymes could
not be compared.
We characterized the nature of the PIP2
hydrolysis products in reaction mixtures incubated under standard
conditions with StPLC1, StPLC2, and StPLC3. All StPLC isoenzymes
produced the same reaction products from PIP2. As
shown for StPLC1, both an unlabeled lipid that co-migrated on TLC
plates with authentic DG (Fig. 5) and a
3H-labeled, water-soluble product that co-eluted
in HPLC chromatograms with authentic IP3 (Fig.
6) accumulated with increasing reaction time. Under the chosen conditions hydrolysis was complete in 15 min.
The amount of product at this time was approximately 10 µg of lipid
(Fig. 5), and recovery of 3H into the aqueous
phase was approximately 90%. Because of severe peak tailing (Fig. 6),
recovery of IP3 from HPLC columns was not determined. Our data indicate complete conversion of the initial amount
of educt PIP2 (Mr = approximately 1020 g mol1) into DG and
IP3.
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| Figure 5.
Analysis of the lipid products of PIP2
hydrolysis catalyzed by recombinant StPLC1. Reactions were performed
under standard reaction conditions (see ``Materials and Methods'') in
50 µL with 0.1 µg µL1 StPLC1, 10 µm
Ca2+, and 0.2 nmol µL1 PIP2
(5000 dpm nmol1). Reactions were started with
PIP2, and lipophilic reaction products were extracted with
chloroform:methanol (2:1, v/v) at 0, 2, 15, and 30 min. Lipids were
concentrated under a stream of nitrogen, applied quantitatively onto
TLC plates, separated by hexane:diethylether:acetic acid (9:1:0.5,
v/v), and visualized by iodine staining. The standard lipid mixtures
contained approximately 10 µg of DG and 10 µg of PA.
PIP2 was immobile in the chosen TLC solvent system
(autoradiogram not shown).
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| Figure 6.
Analysis of the water-soluble products of
PIP2 hydrolysis, which was catalyzed by recombinant StPLC1.
Reactions were performed in 50 µL, as described in ``Materials and Methods''. Reactions were started with PIP2 and stopped at
0, 2, and 15 min by extraction with chloroform:methanol (2:1, v/v).
Reactions were complete at 15 min (see Fig. 5). The aqueous phase of
the chloroform:methanol extractions was neutralized and concentrated by
freeze drying. A, A volume representing one-half of the reaction
products was applied onto a 25-cm HPLC column. Chromatography was
performed as described in ``Materials and Methods''. 3H
was monitored. B, To demonstrate product recovery, authentic [3H]IP3 (50,000 dpm) was added to a reaction
mixture, extracted at 0 min, and analyzed by applying the same
conditions. IP2, Inositol bisphosphate.
|
|
All reaction products analyzed after 0 to 30 min of incubations
contained a minor, nonaccumulating substance that co-migrated with
authentic PA when analyzed by TLC with a solvent system for the
separation of polar lipids, i.e. chloroform:methanol:acetic acid:water
(25:15:4:2, v/v; not shown). The origin and chemical nature of this
lipid was not further investigated. The water-soluble reaction product
contained a minor but accumulating compound that co-eluted with
inositol-1,4,5-bisphosphate in HPLC chromatograms. Given the fact that
PA did not accumulate, inositol-1,4,5-bisphosphate can only result from
the cleavage of IP3. The origin of the underlying phosphatase activity in our preparations of recombinant StPLC was not
investigated.
A comparative study of the cation requirements for activation of
phosphoinositide hydrolysis by StPLC isoenzymes was performed. All
recombinant StPLCs showed a maximum activity of
PIP2 hydrolysis at 10 µm
Ca2+ and a lower activity at higher
Ca2+ concentrations (Fig.
7A). A detailed investigation of the
activation of StPLC by low concentrations of Ca2+
normalized relative to 10 µm Ca2+
showed no significant difference in the activation pattern between StPLC isoenzymes. Only at 0.5 µm Ca2+
did StPLC2 show a tendency for lower activity compared with the other isoforms (Fig. 7C).
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| Figure 7.
PI-PLC activity in preparations of recombinant
StPLC1, StPLC2, and StPLC3 in the presence of varying amounts of free
Ca2+. In all experiments, 2-3H-labeled,
water-soluble product was determined after lipids were removed with
chloroform:methanol (2:1, v/v). A, PI-PLC substrate was
PIP2 (n = 3). B, PI-PLC substrate was
PI (n = 3). C, Activation of PIP2
hydrolysis at low concentrations of Ca2+
(n = 8). Data presented in A and B are specific
enzyme activities, whereas data in C are expressed as percentages of
maximum activity at 10 µm free Ca2+.
|
|
We also investigated the hydrolysis of PI, a metabolic lipid precursor
of PIP2, under standard PI-PLC reaction
conditions in the presence of recombinant StPLCs (Fig. 7B). All StPLC
isoenzymes showed a minor PI-hydrolyzing activity at 10 µm Ca2+, which was substantially
increased at 10 mm Ca2+. At this
Ca2+ concentration preferential cleavage of
PIP2 was lost and the specific activities of all
StPLC isoenzymes were approximately the same with PI or
PIP2 (Fig. 7, A and B). In contrast to StPLC2 and
StPLC3, PI hydrolysis by StPLC1 was already significantly increased at
100 µm Ca2+.
Competition experiments with increasing amounts of
Mg2+ and Al3+ were
performed in the presence of 10 µm
Ca2+. Significant inhibition of
PIP2 hydrolysis starting at equimolar concentrations of Al3+ was observed for all StPLC
isoenzymes. In contrast, within the range of applied concentrations,
Mg2+ did not affect the activity of recombinant
StPLC (Fig. 8). The recovery of
[3H]IP3 from standard
reaction mixtures after extraction with chloroform:methanol (2:1, v/v)
was determined in the presence of 0.01 µg
µL1 recombinant StPLC1 and 0.5, 1.0, 5.0, 10, 50, 100, and 200 µm Mg2+ or
Al3+. Recovery of
[3H]IP3 was approximately
95 ± 2.5% (mean ± sd; n = 3) and,
within the range of applied concentrations, was independent of both the concentration and chemical nature of the cation.
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| Figure 8.
Influence of Mg2+ and Al3+
on PIP2 hydrolysis in preparations of StPLC1, StPLC2, and
StPLC3 in the presence of 10 µm free Ca2+
(n = 2). Mg2+ and Al3+
cations were added as chloride salts. Recovery of authentic
[3H]IP3 from reaction mixtures after
extraction with chloroform:methanol (2:1, v/v) was approximately 95%
and independent of the presence of 0.5 to 200 µm
Mg2+ or Al3+ (data not shown).
|
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| |
DISCUSSION |
The primary structures of all cloned StPLC isoforms show the
typical features of PI-PLC enzymes. Based on amino acid sequence homology, plant PI-PLCs, including StPLCs, form a distinct group within
the family of eukaryotic PI-PLCs (not shown). This group is closely
related to the mammalian PLC subfamily (Irvine, 1996). The X domains
of StPLCs appear to contain the putative active site, including two
conserved His residues. These His residues not only were shown to be
located within the active site of crystallized rat PLC1 (Essen et
al., 1996), but were also essential for enzyme activity, as was
demonstrated by site-directed mutagenesis of PLC1 (Cheng et al.,
1995; Ellis et al., 1995). A similar reaction center with two His
residues was also found in the prokaryotic PI-PLC from Bacillus
cereus (Heinz et al., 1995).
These observations suggest that the StPLCs encoded by the
stplc cDNAs represent enzymatically active PI-PLCs. Previous
investigations of cloned plant PI-PLCs demonstrated specificity for
phosphoinositides (Hirayama et al., 1995; Shi et al., 1995). However,
the fate of the phosphodiester group that links the inositol phosphate
head group to the DG part of the glycerolipid was not analyzed;
therefore, it could not be ruled out that the cloned enzymes might
catalyze a phospholipase D reaction and form PA instead of DG. The
latter would be the expected product of an authentic PI-PLC. Partial complementation of a PI-PLC-deficient yeast mutant by a plant PI-PLC
cDNA as performed by Shi et al. (1995) is important evidence but could
also occur if the PI-PLC activity were only a side reaction catalyzed
by the cloned plant enzyme. To confirm the activity of StPLC, we
demonstrated that recombinant StPLC almost exclusively forms DG and
IP3 from PIP2 (Figs. 5 and
6).
StPLCs, like other plant PI-PLCs (Hirayama et al., 1995; Shi et al.,
1995), are activated by Ca2+ concentrations in
the nanomolar range (Fig. 7). PI-PLCs from soybean (G. max;
Shi et al., 1995) and A. thaliana (Hirayama et al., 1995)
have previously been reported to contain EF-hand motifs, i.e. putative
Ca2+-binding domains, which, however, are absent
from the StPLCs described here. Regulation of plant PI-PLCs by
Ca2+, therefore, most likely involves domains
other than EF-hands. In accordance with this assumption is the
observation that mutating an EF-hand motif in PI-PLC from
Dictyostelium discoideum did not affect the
Ca2+dependence of this enzyme (Drayer et al.,
1995). We suggest that the Ca2+ sensitivity of
StPLCs might be brought about by one of two alternative mechanisms. A
Ca2+ ion could bind to the charged residues in
the reaction center, which are conserved in all PI-PLCs (i.e.
N127, E156,
D158, and E206 of StPLC1;
Fig. 1) and might aid to position the inositol phosphate head group
within the active site. The presence of Ca2+
within the active site of rat PLC1 and interaction with the conserved charged residues (i.e. N312,
E341, D343, and
E390 of PLC1) was shown by a high-resolution
crystal structure of a Ca2+- and
IP3-containing complex (Essen et al., 1996). A
second possibility is that the Ca2+-regulatory
domain in PI-PLC could be the C2-like domain that is present C-terminal to the Y domains of all plant PI-PLCs (Fig. 1).
As suggested by Essen et al. (1996), the C2
domain of PI-PLC could regulate enzyme activity by mediating membrane
interaction of the mainly amphipathic PI-PLC enzymes (Fig. 2) and thus
give access to the phosphoinositide substrate.
Mg2+ ions are known to activate PI-PLC activity
in the presence of 10 µm Ca2+ in
wheat plasma membranes (Pical et al., 1992; Jones and Kochian, 1995).
In contrast, Al3+ is a strong inhibitor of PI-PLC
activity in preparations of plasma membranes from wheat roots (Jones
and Kochian, 1995). We demonstrated that Al3+
inhibited the activity of all recombinant StPLCs and that
Mg2+ was not able to stimulate the recombinant
enzyme preparations (Fig. 8). Therefore, we propose that
Mg2+ does not directly interact with plant PI-PLC
but might stimulate the enzyme via an as-yet-unidentified
PI-PLC-binding component that could be present in preparations of plant
plasma membranes. In contrast, Al3+ appears to
act directly on PIP2 hydrolysis. Two explanations might apply to explain the inhibition of PIP2
hydrolysis: (a) inhibition could be caused by inactivation of the
enzyme because of displacement of an essential
Ca2+ ion from the putative binding sites at the
reaction center or the C2-like domain of the
PI-PLC protein (see above), or (b) an Al3+/PIP2 complex could be
formed that might not be cleavable by PI-PLC. It has been reported that
Al3+ can substitute for
Ca2+ in a liposome complex with a different
phospholipid, i.e. phosphatidylcholine, due to 560-fold higher affinity
of Al3+ for the glycerolipid (Akeson et al.,
1989), and it was proposed that Al3+ binds to the
phosphodiester group of phosphatidylcholine (Hunter and Etherton,
1989).
We found that, as in other plant species, multiple PI-PLC isoforms
exist in potato plants and do not appear to be expressed in a strict
tissue-specific manner (Fig. 3). Although stplc cDNA clones
were isolated from an epidermal fragment cDNA library, we found high
expression for all three genes in several other tissues (cf. Fig. 3),
indicating that phosphoinositide metabolism in guard cells most likely
does not require cell-specific isoforms of PI-PLC or high expression
levels of the corresponding genes. Moreover, expression of three
stplc genes in epidermal fragments under both normal and
environmental stress conditions strongly suggests that multiple PI-PLC
isoforms might be utilized in a single cell type, i.e. guard cells (not
shown). The attempt to prove simultaneous expression of multiple PI-PLC
isoforms in guard cells by promoter studies is part of ongoing
investigations in our group. Whereas we have not yet proven that
multiple isoforms are expressed in guard cells, we have identified one
PI-PLC isoform in potato (J. Kopka and B. Müller-Röber,
unpublished results) and one isoform in N. rustica (Pical et
al., 1997), for which no RNA is detectable in epidermal fragments and
therefore do not appear to be expressed in guard cells.
We performed a comparative analysis at the levels of protein structure,
gene expression, and biochemical properties of the StPLC isoforms
isolated from epidermal fragments. StPLC isoforms show high sequence
homology and conservation of hydropathy patterns within the X and Y
domains (Figs. 1 and 2), which are involved in enzymatic function. In
agreement with this observation we were not able to demonstrate
significant catalytic differences between the recombinant enzymes at
physiological Ca2+ concentrations (10
µm; Figs. 7 and 8). However, specificity toward
PIP2 started to decline at 100 µm
Ca2+ in the case of StPLC1, whereas the other
isoforms lost specificity only at higher Ca2+
concentrations (compare A and B in Fig. 7, 100-10,000 µm
Ca2+). Whether these differences are of
physiological relevance remains an open question.
In contrast to the catalytic domains, StPLC isoforms show rather high
sequence diversity in regions that in mammalian PI-PLC subfamilies are
known to contain regulatory domains, i.e. the linking region between X
and Y domains and the N terminus. We were not able to identify
pleckstrin-homology domains or the src-homology domains SH2
and SH3 in plant PI-PLCs. However, the linking regions of StPLCs
contain a high number of charged amino acids (Fig. 2). The pattern of
hydrophilicity in this region appears to be characteristic for each
StPLC isoform. StPLC1, for example, contains a unique bipartite
hydrophilic domain, each part of which contains a short sequence repeat
(Fig. 2). The structural and biochemical analyses strongly indicate
that StPLC2 and StPLC3 belong to one group, and StPLC1 represents a
less closely related form of plant PI-PLC isoform. This observation was
also substantiated by RNA-expression analysis. Even though there
appears to be no distinct tissue specificity in the expression of these
genes (Fig. 3), the regulation of transcript levels in response to
short-term (Fig. 4) and long-term drought stress (not shown) is
markedly different. After long-term stress, stplc2 and
stplc3 showed an increase in mRNA levels, but, in contrast, stplc1 mRNA level decreased under identical conditions.
Taken together, these results indicate that adaptation to drought might involve changes in the relative levels of PI-PLC isoforms.
In conclusion, in this paper we present the cloning and comparative
analysis of three phosphoinositide-specific StPLC isoforms, which are
expressed in epidermal fragments and at the mRNA level are regulated in
a differential manner. Previously, we reported the isolation of the
first cDNA coding for a plant CDS, which among other tissues appears to
be also expressed in potato epidermal fragments (Kopka et al., 1997a).
The product of the CDS reaction is the initial substrate for the
resynthesis of PI from DG produced in plant plasma membranes (Wissing
et al., 1992). Thus, we are now able to modulate in planta biosynthesis
as well as cleavage of PIP2, which is the lipid
precursor of the second messenger IP3. Transgenic
approaches should allow us to modulate expression of PI-PLC by
attempting guard cell-targeted antisense inhibition studies under the
control of cell-specific promoters (Müller-Röber et al.,
1994).
| |
FOOTNOTES |
1
The European Molecular Biology Organization and
the Biotechnology and Biological Science Research Council are
acknowledged for their support in providing a short-term fellowship to
C.P. and a research grant to J.E.G.
*
Corresponding author; e-mail mueller{at}mpimp-golm.mpg.de; fax
49-331-977-2301.
Received June 11, 1997;
accepted October 5, 1997.
The nucleotide sequence data reported will appear in the EMBL, GenBank,
and DDBJ Nucleotide Sequence Databases under accession numbers
X93564 (stplc1), X94183 (stplc2), and
X94289 (stplc3).
| |
ABBREVIATIONS |
Abbreviations:
CDS, CDP-DG synthase.
DG, diacylglycerol.
GST, glutathione S-transferase.
IP3, inositol-1,4,5-trisphosphate.
PA, phosphatidic acid.
PI, phosphatidylinositol.
PIP2, phosphatidylinositol-4,5-bisphosphate.
PI-PLC, phosphoinositide-specific phospholipase C.
PKC, protein kinase C.
| |
ACKNOWLEDGMENTS |
We wish to thank Prof. Lothar Willmitzer for his support in
recent years. We thank Karsten Harms and Hugo Peña-Cortés
for providing cDNA probes for RNA hybridizations.
| |
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Top
Abstract
Introduction