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Plant Physiol. (1999) 119: 1371-1378
Subcellular Distribution and Tissue Expression of Phospholipase
D
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ABSTRACT |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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Three phospholipase Ds (PLDs; EC
3.1.4.4) have been cloned from Arabidopsis, and they exhibit two
distinct types of activities: polyphosphoinositide-requiring PLD
and PLD
, and
polyphosphoinositide-independent PLD
. In subcellular fractions
of Arabidopsis leaves, PLD and PLD were both present in the
plasma membrane, intracellular membranes, mitochondria, and
clathrin-coated vesicles, but their relative levels differed in these
fractions. In addition, PLD was detected in the nuclear fraction. In
contrast, PLD was not detectable in any of the subcellular
fractions. PLD activity was higher in the metabolically more active
organs such as flowers, siliques, and roots than in dry seeds and
mature leaves, whereas the polyphosphoinositide-dependent PLD activity
was greater in older, senescing leaves than in other organs. PLD
mRNA accumulated at a lower level than the PLD and PLD
transcripts in most organs, and the expression pattern of the PLD
mRNA also differed from that of PLD and PLD in different organs.
Collectively, these data demonstrated that PLD, PLD, and PLD
have different patterns of subcellular distribution and tissue
expression in Arabidopsis. The present study also provides evidence for
the presence of an additional PLD that is structurally more closely
related to PLD than to the other two PLDs.
Activation of PLD (EC 3.1.4.4) has been proposed as an important
step in the signaling of plant responses to ABA, ethylene, and wounding
(Ryu and Wang, 1996 Understanding whether these PLDs are expressed differently in various
organs and/or have different subcellular locations may provide further
insights into the role and regulatory mechanisms of individual PLDs.
The conventional plant PLD, now known as PLD Plant Material
INTRODUCTION
Top
Abstract
Introduction
Methods
Results
Discussion
References
; Fan et al., 1997; Ritchie and Gilroy, 1998). PLD
also has been suggested to play a role in senescence, nutrient
starvation, and plant-pathogen interactions (Young et al., 1996; Lee et
al., 1998). Recently, it was discovered that there are multiple forms
of PLD with distinct regulatory and catalytic properties (Pappan et
al., 1997a, 1997b; Qin et al., 1997). Three PLDs, designated PLD,
PLD, and PLD, have been cloned from Arabidopsis and are encoded
by distinct PLD genes. PLD is the conventional, prevalent form that
is polyphosphoinositide independent when assayed at millimolar
concentrations of Ca2+. In contrast, PLD and
PLD require a polyphosphoinositide cofactor and are most active at
micromolar concentrations of Ca2+. PLD and
PLD hydrolyze phosphatidylserine and
N-acylphosphatidylethanolamine, but PLD does not (Pappan
et al., 1998). The three PLDs all hydrolyze PC, PE, and
phosphatidylglycerol, but the conditions for hydrolysis by PLD are
drastically different from those for hydrolysis by PLD
and PLD. These distinct biochemical properties have led to the
hypothesis that these PLDs in plants are regulated differently and may
have unique cellular functions (Wang, 1997).
, is present in soluble
and membrane-associated fractions, and its relative distribution
between the two fractions varies, depending on the tissues and
developmental stages (Dyer et al., 1994). The conventional PLD has been
found in plasma membrane, microsomal membranes, mitochondrial
membranes, and vacuoles but not in chloroplasts (Brauer et al., 1990;
Xu et al., 1996). However, nothing is known about the intracellular
distribution and expression of the newly identified PLD and PLD.
Therefore, this study was undertaken to compare how these PLDs are
expressed and distributed in different organelles and tissues of
Arabidopsis.
MATERIALS AND METHODS
Top
Abstract
Introduction
Methods
Results
Discussion
References
-suppressed, antisense
Arabidopsis plants were used in this study. The production of the
antisense plants has been described (Fan et al., 1997). Seeds were sown
in soil and cold treated at 4°C overnight. Plants were grown under
14-h/10-h light/dark cycles with cool-white fluorescent light of 100 µmol m
2 s1 at
23°C ± 3°C. Leaves from 6- to 8-week-old plants were
used for all subcellular fractionations. All isolation procedures were performed at 4°C unless indicated otherwise.
Protein Extraction and Assay of PLD Activities
Total protein from Arabidopsis tissues was extracted by grinding with an ice-chilled mortar and pestle with buffer A containing 50 mM Tris-HCl, pH 7.5, 10 mM KCl, 1 mM EDTA, 0.5 mM PMSF, and 2 mM DTT. The homogenate was centrifuged at 10,000g for 10 min at 4°C to remove tissue debris, and the supernatant was centrifuged at 100,000g for 60 min at 4°C. The resulting supernatant was referred to as the soluble fraction, and the pellet, which was referred to as the membrane fraction, was suspended in buffer A and centrifuged again at 100,000g to reduce cytosolic contamination. Protein concentration was determined by the method of Bradford (1976) using a
kit (Bio-Rad).
). Briefly, the
PIP2-independent PLD activity was assayed in the
presence of 100 mM Mes, pH 6.5, 0.5 mM SDS, 1%
(v/v) ethanol, 25 mM CaCl2, 1 mM egg-yolk PC mixed with
dipalmtoylglycerol-3-phospho[methyl-3H]choline,
and 2 to 10 µg of protein in a total volume of 200 µL.
The reaction mixture of the PIP2-dependent assay
included 100 mM Mes, pH 7.0, 100 µM
CaCl2, 2 mM
MgCl2, 80 mM KCl, 0.4 mM lipid vesicles, and 2 to 10 µg of protein at a total volume of 100 µL. The lipid vesicles were made of PE:PIP2:PC
at the ratio of 85:6.5:8.5 mol %. The PLD-mediated hydrolysis of PC
was measured using
dipalmtoylglycerol-3-phospho[methyl-3H]choline.
Release of [3H]choline into the aqueous
phase was quantitated by scintillation counting in both assays.
Antibody Purification and Immunoblotting
Antibodies to PLD and PLD were raised against two 12-amino
acid peptides at their respective C termini, and the antibody to PLD
was raised against a 12-amino acid peptide near its C terminus (Pappan
et al., 1997a, 1997b). The expression of GST-fused PLD was described
previously (Wang et al., 1994), and the PLD and PLD were
expressed in the same way. The GST-fusion proteins of PLD, PLD,
and PLD were extracted and affinity purified using a
glutathione-agarose column according to the manufacturer's
instructions (Pharmacia LKB Biotech). Each PLD protein was separated by
8% SDS-PAGE and transferred onto a PVDF membrane, which was incubated overnight with the respective antiserum at a dilution of 1:250. After
the membrane was washed with 1× PBS buffer, the appropriate membrane
strips corresponding to the respective GST-PLD proteins were cut, and
bound antibodies were eluted for 3 min with 4 mL of a low-pH (2.69)
buffer containing 100 mM Gly, 100 mM NaCl, 0.1% Tween 20, and 0.02% sodium azide. The eluent was neutralized rapidly to pH 7.5 with Tris-HCl buffer, pH 9.0, and the purified antibodies were used immediately for immunoblotting or stored at
80°C until use. For immunoblotting, protein fractions were separated in 8% SDS-PAGE gels and transferred onto PVDF membranes. The
membranes were incubated with crude serum or affinity-purified antibodies against PLD, PLD, or PLD; this was followed by
incubation with a second antibody conjugated with alkaline phosphatase.
The antibody-antigen complex was visualized by the alkaline phosphatase reaction (Dyer et al., 1994).
Total RNA and mRNA Isolation and RNA Blotting
Total RNA was isolated from different organs of Arabidopsis with a cetyltrimethylammonium bromide extraction method (Fan et al., 1997).
Poly(A+) RNA was isolated from total RNA using an
mRNA purification kit according to the manufacturer's instructions
(Pharmacia LKB Biotech). For RNA blotting, 20 µg of total RNA or 1.5 µg of mRNA was separated by denaturing 1% formaldehyde-agarose gel
electrophoresis and transferred to nylon membranes. PLD-, PLD-,
and PLD-specific probes were labeled with
[-32P]dATP by random priming. The
hybridization, washing, and visualization were performed as described
previously (Fan et al., 1997).
Subcellular Fractionation
Plasma and intracellular membrane were prepared by using an aqueous polymer two-phase system (Larsson et al., 1987; Xu et al.,
1996). Total membranes were prepared from fully expanded leaves (20 g)
and loaded onto a solution to give a 60-g phase system with a final
composition of 6.4% (w/w) dextran T500, 6.4% PEG 3350, 0.25 M Suc, and 5 mM potassium phosphate. After
mixing, the two phases were separated by centrifugation. The upper
phase, containing the plasma membrane, was washed twice with
lower-phase polymer, and the lower phase, containing intracellular
membranes, was washed twice with upper-phase polymer to reduce
contamination. The washed upper and lower phases were diluted 5-fold
with a buffer containing 0.25 M Suc and 5 mM
potassium phosphate, and then they were centrifuged at
100,000g for 60 min. The pellets were resuspended in buffer
A.
. Briefly, leaves (5 g) were ground with an ice-chilled mortar
and pestle with a 20-mL grinding buffer, pH 7.5, containing 50 mM Hepes, 0.33 M sorbitol, 1 mM
MgCl2, 1 mM
MnCl2, 2 mM EDTA, 5 mM
ascorbic acid, and 1% BSA. The homogenate was filtered and then
centrifuged in a swinging-bucket rotor at 2000g for 3 min. The resulting pellet was suspended in 5 mL of grinding buffer and
overlaid on a 20-mL Percoll gradient, which contained 7.5 mL of
Percoll, 7.5 mL of 2× grinding buffer, and 1 mg of glutathione, and
was prepared by centrifugation at 5000g for 40 min. The
gradient was centrifuged at 2000g for 15 min. The lower
green band was collected and diluted 3-fold with a buffer containing 50 mM Hepes-KOH, pH 8.0, and 0.33 M sorbitol. Chloroplasts were then pelleted and washed twice with 25 mL of buffer, and the final pellet was suspended in the same buffer.
). Leaves (20 g) were homogenized with a
mortar and pestle in 50 mL of prechilled grinding medium containing 0.3 M mannitol, 50 mM Mes, pH 7.2, 1 mM
EDTA, 1 mM MgCl2, 0.2%
defatted BSA, 0.5% (w/v) PVP-400, 4 mM Cys, and 10 mM -mercaptoethanol. The homogenates were filtered
through four layers of cheesecloth and centrifuged at 3,300g
for 20 min. The pellet was suspended in 5 mL of resuspension medium I
containing 0.3 M mannitol, 20 mM Mes, pH 7.2, 2 mM
potassium phosphate, 1 mM EDTA, 0.1% (w/v)
defatted BSA, 2 mM MgCl2,
and 14 mM -mercaptoethanol. The suspension was
loaded onto a discontinuous gradient composed of 5 mL of 47%, 6 mL of
26%, and 3 mL of 21% (v/v) Percoll. The gradients were centrifuged at
58,500g for 45 min in a swinging-bucket rotor. The
mitochondrial band, located at the interface between the 26% and 47%
Percoll layers, was removed and diluted with an equal volume of
resuspension medium II containing 0.3 M mannitol, 20 mM Mes, pH 7.2, 1 mM
EDTA, 0.1% defatted BSA, 2 mM
MgCl2, and 2 mM DTT. The
diluted mitochondria (5 mL) were loaded onto a second Percoll gradient
composed of 7.5 mL of 47% and 7.5 mL of 26% (v/v) Percoll prepared as
in the first gradient. The gradient was centrifuged at
58,500g for 30 min. The mitochondrial band, located at the interface between the 26% and 47% Percoll layers, was collected and
diluted with 10 volumes of medium II. The mitochondria were pelleted by
centrifugation at 18,800g for 5 min and resuspended in 1 mL
of medium II.
). The
resuspended pellet was loaded onto a Suc step gradient (6 mL of 5% and
4 mL of 30% Suc) and centrifuged in a swinging-bucket rotor at
67,000g for 40 min. The 5% layer was then removed, diluted
with homogenizing medium, and centrifuged in a fixed-angle rotor at
150,000g for 90 min. The pellet was resuspended in the
homogenizing medium.
; Liu and Whittier, 1994), with some modifications. Briefly, about 20 g of leaves was washed, cut into pieces with scissors, and ground in liquid nitrogen. The powder was
suspended in 50 mL of a buffer containing 0.5 M Suc, 1 mM spermidine, 4 mM spermine, 10 mM
EDTA, 10 mM Tris, pH 7.6, and 80 mM KCl. The
homogenate was filtered and then centrifuged at 3000g for 5 min in a swinging-bucket rotor. The nuclear pellet was dispersed gently
in a suspension buffer containing 50 mM Tris-HCl, pH 7.8, 5 mM MgCl2, 10 mM -mercaptoethanol, and 20% glycerol. The
nuclear suspension was loaded onto a discontinuous Percoll gradient
composed of 5 mL of 40%, 60%, and 80% (v/v) Percoll on 5 mL of 2 M Suc cushion. All of the Percoll solutions
contained 0.44 M Suc, 25 mM
Tris-HCl, pH 7.5, and 10 mM
MgCl2. The gradients were centrifuged at
4000g in a swinging-bucket rotor for 30 min. The white
nuclear band appeared in the 80% Percoll layer above the 2 M Suc and was removed with a Pasteur pipette.
After dilution, nuclei were pelleted by centrifugation, washed twice
with the grinding buffer, and resuspended in the nuclear resuspension
buffer.
Assays of Marker Enzymes for Subcellular Fractions
The NADH-Cyt c reductase assay was performed at 25°C in a 3-mL reaction volume containing 100 µL of 50 mM NaCN, 200 µL of 0.45 mM Cyt c, 2.5 mL of 50 mM sodium phosphate buffer, pH 7.5, and 10 µg of protein extract (Briskin et al., 1987). Cyt c oxidase activities were assayed by monitoring the decrease at
A550 using dithionite-reduced horse heart
Cyt c as a substrate at 25°C in 50 mM potassium phosphate buffer, pH 7.2. Fumarase
activities were determined spectrophotometrically by monitoring the
increase at A240, as described by Cooper
and Beevers (1969). Vanadate-sensitive ATPase was assayed in a 1-mL
reaction volume containing 3 mM ATP, 3 mM MgSO4, 30 mM Tris-Mes buffer, 50 mM
KCl, and 10 µg of protein in the presence or absence of 50 µM
Na3VO4. The ATP substrate was present as Tris salt after treatment with Dowex 50-W exchange resin
(H+ form). The assay was performed at 38°C for
30 min, and the released Pi was determined by using ammonium molybdate.
| |
RESULTS |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
|
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Subcellular Localization of PIP2-Dependent and PIP2-Independent PLD Activities in Leaves
Fractions enriched in the plasma membrane, intracellular membranes, chloroplasts, mitochondria, nuclei, and clathrin-coated vesicles were prepared from fully expanded Arabidopsis leaves. The identity and purity of each fraction were determined by assaying activities of appropriate marker enzymes (Table I). The plasma-membrane fraction showed the highest activity of its marker enzyme vanadate-sensitive ATPase but little activity for the other enzymes tested. The intracellular membrane fraction had the highest activity of Cyt c reductase, a marker enzyme of ER, indicating enrichment of ER in this fraction. The mitochondrial fraction displayed high activities of the mitochondrial marker enzymes Cyt c oxidase and fumarase, but it had little ATPase and Cyt c reductase activities. Chloroplasts and nuclei showed very little enzymatic activities that are characteristic of other organelles. The identities of the chloroplast and nuclear fractions were confirmed further by microscopic observation and DNA agarose-gel electrophoresis (data not shown). The purity of the clathrin-coated vesicle fraction was less defined than that of the other fractions because of the lack of appropriate marker enzymes. The presence of ATPase and Cyt c reductase activities indicated that this fraction contained some plasma membrane and ER membranes.
|
Intracellular Association of PLD
Organ Distribution of PLD
Tissue Expression of PLD
Activation of PLD has been linked to various cellular processes,
such as hormonal and developmental signaling, membrane synthesis, remodeling, and lipid degradation (Wang, 1997 Received October 6, 1998;
accepted December 21, 1998.
Abbreviations:
GST, glutathione S-transferase.
PC, phosphatidylcholine.
PE, phosphatidylethanolaminePIP2, phosphatidylinositol 4,5-bisphosphate.
PLD, phospholipase D.
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Molecular cloning and functional analysis of polyphosphoinositide-dependent phospholipase D, PLD
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[Abstract]
but not PLD or PLD (Qin et al., 1997). PLD
activity was detected in the plasma membrane, intracellular membranes,
clathrin-coated vesicles, and mitochondrial fractions (Fig.
1A). The highest specific activity was
obtained from the plasma membrane, whereas no PLD activity was found
in chloroplasts and nuclei (Fig. 1A). The relative distribution of the
PLD activity corroborated well the level of PLD in various fractions (see Fig. 3A), suggesting that the different methods of
fraction isolation did not interfere significantly with the PLD
assay. To confirm that the PIP2-independent,
PC-hydrolyzing activity came from PLD and not from PLD or PLD,
the same subcellular fractions were prepared from PLD antisense,
transgenic Arabidopsis in which the expression of the PLD gene was
suppressed genetically (Pappan et al., 1997a). Almost no PLD
activity was detected in the subcellular fractions prepared from the
PLD-suppressed leaves.
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Figure 1.
Subcellular distribution of
PIP2-independent (A) and -dependent (B) PLD activities in
Arabidopsis leaves. The assay conditions for the two types of PLD
activities were as described in ``Materials and Methods''. Identical
methods were used to isolate the intracellular fractions from fully
expanded leaves of wild-type (WT) and PLD antisense Arabidopsis. PM,
Plasma membrane; Chl, chloroplast; Nu, nucleus; IM, intracellular
membrane; CCV, clathrin-coated vesicle; Mito, mitochondria of wild-type
and PLD antisense plants.
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Figure 3.
Immunoblot of PLD (A) and PLD (B) in various
subcellular fractions. Intracellular fractions were isolated from
wild-type Arabidopsis leaves, and equal amounts of proteins (10 µg
per lane) were loaded and separated by 8% SDS-PAGE. PLD and PLD
were made visible by alkaline phosphatase after blotting with
affinity-purified PLD and PLD antibodies, respectively. See
Figure 1 for abbreviations.
and PLD cloned from Arabidopsis are optimal,
whereas PLD is virtually inactive (Qin et al., 1997; Pappan et al.,
1998). To confirm that PLD did not contribute substantially to this
PLD activity, the PIP2-dependent PLD activity was
assayed in the fractions of the PLD-suppressed transgenic plants.
The PIP2-dependent PLD activity had the same distribution pattern in the PLD-suppressed plants as in wild-type plants. This result also indicates that the suppression of PLD in
the transgenic plant does not alter the subcellular distribution of the
PIP2-dependent PLDs.
, PLD, and PLD in
Leaves
and PLD because of their overlapping
requirements for PIP2 and
Ca2+ (Qin et al., 1997). Thus, the subcellular
association of different PLDs was analyzed further by immunoblotting
with PLD antibodies raised against 12-amino acid peptides of PLD,
PLD, or PLD (Pappan et al., 1997b). The specificity of these
antibodies to their respective antigens was examined by immunoblotting
the purified GST-PLD, GST-PLD, and GST-PLD with antibodies to
PLD, PLD, and PLD. PLD and PLD antibodies reacted only
with their respective GST-fusion proteins, whereas the PLD antibody
cross-reacted with PLD but not with PLD (Fig.
2). Therefore, PLD and PLD
antibodies are specific to their respective target proteins, whereas
the PLD antibody can recognize both PLD and PLD proteins.
These antibodies were affinity purified against the purified, GST-fused
PLD, PLD, and PLD.
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Figure 2.
Antibody specificity against PLD , PLD, and
PLD. Purified GST-PLD, GST-PLD, and GST-PLD (0.1 µg
per lane) were separated by 8% SDS-PAGE. The blots were incubated with
anti-PLD, anti-PLD, or anti-PLD polyclonal antibodies (1:1000
dilution) and then the second antibody. PLD-antibody complexes were
made visible by alkaline phosphatase.
antibodies detected an abundance of PLD
in the plasma membrane, intracellular membranes, clathrin-coated vesicles, and mitochondria, a minute amount in nuclei, and none in
chloroplasts (Fig. 3A). As expected, no
PLD protein was detected in the subcellular fractions isolated from
the PLD-suppressed transgenic plant (data not shown). This
distribution of PLD protein was consistent with that of the
PIP2-independent activity (Fig. 1A). PLD
antibody detected one band in the plasma membrane, intracellular membranes, nuclei, mitochondria, and clathrin-coated vesicles but not
in chloroplasts (Fig. 3B). On the other hand, no PLD protein was
detected in any of the subcellular fractions (data not shown). The
titers of the PLD and PLD antibodies were similar, as estimated
by ELISA against the respective synthetic peptides, and the PLD
antibody was specific and reacted well to bacterially expressed PLD
(Fig. 2). Therefore, the inability to detect PLD could be
attributable to a low level of PLD protein in leaves. This result
also means that the band detected by the PLD antibody can be
considered to be PLD rather than from PLD protein. The relative
levels of PLD and PLD proteins in the fractions differed; the
banding intensity of PLD protein was greater in the plasma membrane
and clathrin-coated vesicle-enriched fractions, whereas the greatest
association of PLD protein was with the intracellular membrane
fraction. The PLD and PLD bands in the clathrin-coated vesicle
fraction migrated more slowly on both blots, and this was found to be
caused by a difference in sample-buffer composition.
, PLD, and PLD
activity in soluble
fractions was high in roots, flowers, and siliques, moderate in stems,
and low in seeds, leaves, and seedlings (Fig. 4A). The membrane-associated PLD
activity was highest in siliques, lowest in dry seeds, and intermediate
and similar in the other organs (Fig. 4B). In contrast, the specific
PIP2-dependent PLD activity was highest in old,
senescing leaves, lowest in dry seeds, and intermediate and similar in
the other organs (Fig. 5). The PIP2-dependent PLD activities were about 2- to
5-fold higher in membrane-associated fractions than in soluble
fractions.
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Figure 4.
Tissue distribution of
PIP2-independent PLD activity in Arabidopsis. A, Soluble
PLD activity from 100,000g supernatant. B,
Membrane-associated PLD activity from the pellet after
centrifugation at 100,000g of the 10,000g
supernatant. St, Stem; Fl, flower; Si, silique; Sd, dry seed; Rt, root;
OL, old leaf (bottom leaves of flowering plants with yellowing at the
tip); YL, young leaf (not fully expanded, top leaves of 2-month-old
plants); Sl, seedling (10 d old).
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Figure 5.
Tissue distribution of PIP2-dependent
PLD activity in Arabidopsis. A and B are soluble and
membrane-associated PIP2-dependent activity, respectively,
assayed from various tissues of 2-month-old plants. The protein samples
and abbreviations were the same as those used in Figure 4 for assays of
PLD activity.
protein, as assessed by immunoblotting, was
essentially the same as that of PIP2-independent
PLD activity in different organs (Fig.
6). More PLD protein was present in
flowers, stems, roots, and siliques than in other organs. As in
subcellular fractions, no PLD band was detected using purified PLD antibody (data not shown). PLD was detected in flowers, stems, roots, and old leaves in soluble fractions (Fig.
7A), whereas weaker signals were found in
membrane fractions of flowers, stems, and siliques (Fig. 7B). In
addition, two protein bands with estimated molecular masses of 85 and
99 kD were detected by the affinity-purified PLD antibody in the
soluble fractions of flowers, stems, and old leaves (Fig. 7A), whereas
only the lower band was detectable in the membrane fraction. This may
suggest the presence of another PLD isoform that is closely related to
PLD.
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Figure 6.
Immunoblot of PLD in soluble (A) and membrane
(B) fractions of different tissues. Equal amounts (10 µg per lane) of
soluble and membrane-associated protein were separated by 8% SDS-PAGE.
The blots were incubated with the PLD antibody. The protein samples
and abbreviations are the same as those in Figure 4.
View larger version (42K):
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Figure 7.
Immunoblot of PLD in soluble (A) and membrane
(B) fractions of different tissues. Equal amounts (10 µg per lane) of
soluble and membrane-associated protein were separated by 8% SDS-PAGE.
PLD bands were immunodetected with incubation of the filters with
affinity-purified PLD antibody. The protein samples and
abbreviations are the same as those in Figure 4.
, PLD, and PLD Genes
, PLD, and PLD genes in
different tissues, RNA-blot analysis was performed using PLD, PLD, and PLD cDNAs as probes (Fig.
8). Previous Southern-blot analysis had
established that these cDNA probes do not cross-hybridize with one
another under highly stringent hybridization conditions (Qin et al.,
1997). The level of PLD transcript was high in roots, stems, and
flowers, moderate in leaves, seedlings, and siliques, and undetectable
in dry seeds (Fig. 8A). In contrast, the level of PLD mRNA was high
in roots and flowers, moderate in stems, leaves, and seedlings, low in
siliques, and undetectable in seeds (Fig. 8B). Additionally, more than
one band was detected on the RNA blot when PLD was used as a probe,
and the lower band corresponded to the size of the cloned
PLD cDNA. No extra bands were detected when PLD and PLD probes
were used, which suggests the presence of another PLD mRNA that is
closely related to PLD.
View larger version (41K):
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Figure 8.
Expression of PLD , PLD, and PLD RNA
levels in different organs. A, Total RNA (20 µg per lane) isolated
from various organs of 2-month-old Arabidopsis was probed with PLD
cDNA. B, Total RNA hybridized with PLD cDNA. Blots in both A and B
were stripped and hybridized with an rRNA probe to indicate the equal
loading of total RNA. C, mRNA (1.5 µg per lane) probed with PLD
(upper), and the blot stripped and probed with a PLD cDNA probe
(lower). St, Stem; Fl, flower; Si, silique; Sd, dry seed; Rt, root; YL,
young leaf; Sl, seedling (10 d old).
and PLD were detected using total RNA.
Under the same conditions and using a PLD cDNA probe with the same
specific radioactivity as the PLD probe, however, PLD mRNA was
not detectable (data not shown). This indicated that the level of the
PLD mRNA was lower than the levels of PLD or PLD mRNA. When
the mRNA from total RNA of leaf, stem, flower, and silique was isolated
for blotting, the PLD transcript became detectable (Fig. 8C). As a
direct comparison, the same mRNA blot was hybridized with PLD (Fig.
8C), and the relative distribution of PLD in different organs was
similar to that on the total RNA blot. The pattern of PLD expression
was different from that of PLD and PLD. Most noticeably, the mRNA
level in siliques relative to the levels in leaves and flowers was much
higher for PLD than for PLD and PLD.
DISCUSSION
Top
Abstract
Introduction
Methods
Results
Discussion
References
). The discovery of
different forms of PLD (Qin et al., 1997) provides some molecular and
biochemical bases for such functional diversity. The present study has
shown that PLD, PLD, and PLD of Arabidopsis also have
different intracellular distribution and expression patterns. At the
PLD activity level, one major difference is that the specific PIP2-dependent activity was higher in
membrane-associated fractions than in soluble fractions in all organs,
but the distribution of PIP2-independent activity
in the two fractions varied from organ to organ. This result is
consistent with a recent report that demonstrated a predominant
localization of PIP2-dependent activity in the
membrane-associated fraction of leaves (Pappan et al., 1997a), and it
also extends the same distribution pattern of
PIP2-dependent activity to the other organs in
Arabidopsis. Another major difference is that the
PIP2-dependent activity showed the highest
activity in older leaves, whereas the
PIP2-independent activity was more active in
metabolically active tissues such as flowers, siliques, and roots. It
is interesting that the levels of both polyphosphoinositides and
phosphatidic acid were also found to increase with senescence
(Borochov et al., 1994). Regulation of the levels of
polyphosphoinositides and the polyphosphoinositide-requiring PLDs may
be coordinated.
-deficient
plants also suggested that the PIP2-independent
PLD is not a direct promoter of senescence because antisense
suppression of PLD did not alter natural plant senescence (Fan et
al., 1997). However, suppression of PLD retarded ABA- and
ethylene-promoted senescence in detached leaves. PLD is believed to
play a role in phytohormone signaling, and thus its deficiency renders
tissues less sensitive to ABA and ethylene treatments (Fan et al.,
1997). Such a signaling role of the conventional plant PLD has also
been suggested in carrot cells (Lee et al., 1998) and barley aleurone
(Richie and Gilroy, 1998).
was found in all organs, PLD was
detectable in some organs, but PLD was undetectable in any
subcellular or tissue fractions. The inability to monitor the PLD
protein indicates that the amount of this protein is much lower than
that of PLD and PLD because the PLD antibody has a titer
similar to that of the PLD antibody and reacted well with
bacterially expressed PLD (Fig. 2). Consistent with the immunoblot
results, RNA blotting showed that the level of PLD mRNA was much
lower than that of PLD and PLD mRNAs, and PLD mRNA could be
detected only in isolated mRNA. This suggests that a low level of
PLD gene expression may be responsible for the small amount of
PLD. In addition, the pattern of PLD mRNA accumulation in
different organs was different from that of PLD and PLD mRNAs.
Another possible reason for the lack of immunodetection of PLD could be proteolytic removal in the cell and/or during isolation of the
PLD C-terminal peptide to which the antibody was raised. However,
nothing is yet known about the posttranslational processing of these
PLDs.
, but not PLD or PLD, was found in the nuclear fraction. This nuclear location is particularly interesting because in yeast PLD1 is present in nuclei and its association with
nuclear membranes is required for completion of meiosis and subsequent
sporulation (Sung et al., 1997). This could mean that PLD may play a
role in cell division and reproduction. The trace amount of PLD
detected in the nuclei was likely the result of contamination from
other fractions (Table I), because a recent immunocytochemical study of
castor bean did not find PLD in the nuclei (Xu et al., 1996).
is responsible for the
PIP2-independent PLD activity, and PLD and
PLD both possess PIP2-dependent activity (Qin
et al., 1997; Pappan et al., 1998). In this study, the levels of PLD
protein and the PIP2-independent PLD activity
correlated well, but the levels of PLD and PLD proteins and the
PIP2-dependent PLD activity did not.
Specifically, high levels of specific
PIP2-dependent activity are associated with
membrane fractions, whereas most PLD protein was detected in the
soluble fractions and PLD was undetectable in any fraction. This
discrepancy could result if the membrane-associated PLD were more
active than the soluble form. The low level of soluble activity might
be caused by the presence of PLD inhibitors and/or the absence of
PLD activators in the cytosol. Thus, the membrane-associated PLD is
the activated form. Another possibility is that other PLD isoforms
contributed significantly to the PIP2-dependent
activities detected in membranes. In fact, two protein bands were
detected by the purified PLD antibody, and two species of mRNA
hybridized specifically to PLD cDNA. These results indicate the
presence of at least one additional PLD, whose sequence is more closely
related to that of PLD than to that of PLD or PLD. Studies are
under way to clone and characterize other PLDs from Arabidopsis.
could be another isoform that contributed to the
high level of membrane-associated, PIP2-dependent
activity. Although the immunoblot and RNA-blot results indicate a much
lower level of expression for PLD than for PLD, a recent study
using PLDs expressed in Escherichia coli has shown that
PLD is much more active toward PC than toward PLD (Pappan et al.,
1998), and the present study used PC as the substrate for measuring PLD activities. Thus, it is probable that, although the low level of PLD
eluded immunodetection, it still gave a portion of the membrane-associated PIP2-dependent activity.
Furthermore, the absence of detection of PLD could result from a
proteolytic removal of its C-terminal peptide to which the PLD
antibody was raised. Recent analysis in this laboratory has
demonstrated that proteolytic deletion of the C-terminal portion does
not affect PLD activity (K. Pappan and X. Wang,
unpublished data). Further studies are warranted to clarify these
possibilities
in this organelle is consistent with its localization in other
plant species (Xu et al., 1996). On the other hand, this study has
shown that substantial amounts of both
PIP2-dependent and -independent PLD activities
and PLD and PLD proteins are associated with the mitochondrial
fractions of Arabidopsis leaves. Immunocytochemical localization did
not find PLD inside the mitochondria of castor bean leaves (Xu et
al., 1996). However, PLD of corn roots was suggested to be associated
with mitochondrial membranes (Brauer et al., 1990). The PLD
observed in the mitochondrial fraction likely is associated with
mitochondrial membranes, but the exact location of these PLDs in this
organelle requires further investigation.
1
This work was supported by grants from the U.S.
Department of Agriculture and the National Science Foundation. This
paper is contribution 99-83-J of the Kansas Agricultural
Experiment Station.
FOOTNOTES
*
Corresponding author; e-mail wangs{at}ksu.edu; fax
1-785-532-7278.
ABBREVIATIONS
LITERATURE CITED
Top
Abstract
Introduction
Methods
Results
Discussion
References
retards abscisic acid- and ethylene-promoted senescence of postharvest Arabidopsis leaves.
Plant Cell
9:
2916-2919
, , and from plants.
Arch Biochem Biophys
353:
131-140
[CrossRef][ISI][Medline], from Arabidopsis.
J Biol Chem
272:
7055-7062
and regulation of plant PLD, - and - by polyphosphoinositides and calcium.
J Biol Chem
272:
28267-28273
Copyright Clearance Center: 0032-0889/99/119//08
© 1999 American Society of Plant Physiologists
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