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J Biol Chem, Vol. 274, Issue 53, 38232-38240, December 31, 1999
From the In animal cells, phosphoinositides are key
components of the inositol 1,4,5-trisphosphate/diacylglycerol-based
signaling pathway, but also have many other cellular functions. These
lipids are also believed to fulfill similar functions in plant cells,
although many details concerning the components of a plant
phosphoinositide system, and their regulation are still missing. Only
recently have the different phosphoinositide isomers been unambiguously identified in plant cells. Another problem that hinders the study of
the function of phosphoinositides and their derivatives, as well as the
regulation of their metabolism, in plant cells is the need for a
homogenous, easily obtainable material, from which the extraction and
purification of phospholipids is relatively easy and quantitatively
reproducible. We present here a thorough characterization of the
phospholipids purified from [32P]orthophosphate-
and myo-[2-3H]inositol-radiolabeled
Arabidopsis thaliana suspension-cultured cells. We then
show that NaCl treatment induces dramatic increases in the levels of
phosphatidylinositol 4,5-bisphosphate and diacylglycerol pyrophosphate
and also affects the turnover of phosphatidylcholine. The increase in
phosphatidylinositol 4,5-bisphosphate was also observed with a
non-ionic hyperosmotic shock. In contrast, the increase in
diacylglycerol pyrophosphate and the turnover of phosphatidylcholine were relatively specific to salt treatments as only minor changes in
the metabolism of these two phospholipids were detected when the cells
were treated with sorbitol instead of NaCl.
Phosphoinositides are quantitatively minor phospholipids that play
an important role in the transduction of physiological signals,
such as hormones, growth factors, and neurotransmitters in animal
cells (1). One of the key early events triggered by these physiological
stimuli is the activation of phosphoinositide-specific phospholipase C (PI-PLC),1
resulting in the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) to the two second messengers,
inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) and
diacylglycerol, which induce Ca2+ release from internal
stores and stimulate protein kinase C, respectively (1, 2). During the
last decade, it has become evident that in addition to serving as
precursors to Ins(1,4,5)P3 and diacylglycerol,
phosphoinositides actively participate in other cellular functions:
they have been shown to regulate the dynamics of the actin cytoskeleton
through the interaction with actin-binding proteins (3, 4), and to
potentiate the activation of protein kinase C (5) and PI-PLC (6, 7). In
addition, phosphoinositides phosphorylated at the
D3-hydroxy group of the inositol headgroup are required for
specific vesicle trafficking steps (8, 9) and are able to activate the
recently identified novel protein kinases Akt/PKB and
phosphoinositide-dependent kinases (10). Recently, a new
3-phosphorylated phosphoinositide, phosphatidylinositol 3,5-bisphosphate (PtdIns(3,5)P2), was identified and shown
to accumulate in yeast cells subjected to hyperosmotic or NaCl stress (11).
Contrasting with the very detailed picture available for the components
of the phosphoinositide system, and their function and regulation in
animal cells, it is still not clear for plant cells whether any
physiological factors act by stimulating PI-PLC-catalyzed PtdIns(4,5)P2 hydrolysis. However, it has been demonstrated
that micro-injected "caged" Ins(1,4,5)P3 can release
Ca2+ from internal stores (12) and is also able to trigger
stomatal closure (13, 14). There is also evidence that
phosphoinositides may participate in the regulation of cytoskeletal
structures in plant cells (15, 16). A number of reports also suggest
that a wide range of signals, such as light, hormones, and stress, may
mediate their effect through phosphoinositide metabolism (17), although
the effects reported were often very limited and the identity of the
lipid or inositol phosphate species affected not always clearly
demonstrated. Interestingly, hyperosmotic stress was recently shown to
induce an increase in PtdIns(3,5)P2 synthesis in
Chlamydomonas moewusii and in some higher plant cells
(18).
With the exception of phosphatidylinositol 5-phosphate (PtdIns(5)P) and
phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P3), all the phosphoinositide isomers found in animal and yeast cells have
also been identified in plant cells (11, 18-21). cDNA clones representing functional enzymes necessary for the synthesis of these
phospholipids, phosphatidylinositol (PtdIns) 4-kinase (22), phosphatidylinositol monophosphate (PtdInsP) kinase (23), PtdIns 3-kinase (24), have also been characterized in plants. Several PI-PLC
isoforms have been cloned from plant species (25-28).
Salinity is a common and detrimental environmental stress imposed on
land plants (29). Salt tolerance is mediated by multiple determinants
(30), a notion reflected in the observation that the expression of
numerous genes is altered in plants subjected to high salt conditions
(31). Knowledge of the mechanisms by which plants perceive and
transduce salinity stress should provide crucial information to
successfully improve salt tolerance in plants through genetic
engineering. In Arabidopsis thaliana, the expression of one
PtdInsP kinase gene (23) and one PI-PLC gene (25) is increased by salt
stress, suggesting that phosphoinositides may participate in the
response to high salinity. There is also evidence that cytosolic
Ca2+ is a likely component of the transduction cascade
(32-34). Here we present results clearly showing that increased
synthesis of PtdIns(4,5)P2 and diacylglycerol pyrophosphate
(DGPP) and also increased turnover of phosphatidylcholine (PtdCho), may
be involved in the response to high salinity in plant cells.
Plant Material and Growth Conditions--
A. thaliana
(ecotype Fi-3) cells were grown in suspension in Murashige and Skoog
(MS) medium as described (35), except that the phosphate concentration
was 50 mg/liter, and subcultured every week. For phospholipid labeling,
the cells were used 2 days after the last subculture.
Chemicals--
[32P]Orthophosphate
(32Pi),
myo-[2-3H]inositol, and
[methyl-14C]choline chloride were purchased
from Amersham Pharmacia Biotech. All cell culture chemicals were of
cell culture grade and purchased from Sigma. Standard lipids were from
Sigma. Monomethylamine (40% aqueous solution) was from Fluka. Standard
[3H]glycerophosphoinositol phosphates
([3H]GroPInsPns) were purified
from [3H]inositol-radiolabeled yeast cells as described
in Ref. 11.
32Pi Labeling and Cell
Treatment--
Cells were collected 2 days after sub-culture, filtered
through a metal mesh and washed with phosphate-free MS medium. They were resuspended to an A600 Myo-[2-3H]inositol Labeling--
Cells were grown
in inositol-free MS medium supplemented with
myo-[2-3H]inositol for 5 days, and subcultured
for another 2 days in the same growth medium. Cells were then collected
and resuspended to an A600 [methyl-14C]Choline Labeling--
Cells were
radiolabeled with [methyl-14C]choline exactly
as described for 32Pi, except that the culture
medium was full MS.
Extraction of Inositol Lipids--
The extraction procedure used
followed that described for yeast cells (36). After extraction, the
washed lower chloroform-rich phases were dried under nitrogen. The
lipid extracts were then analyzed by thin-layer chromatography (TLC) or
HPLC as described below.
Thin Layer Chromatography--
Dried
[32P]-radiolabeled lipids extracted from A. thaliana cells were dissolved in chloroform and spotted on silica
gel 60 TLC plates (Merck) pre-treated with
K+-oxalate/EDTA/methanol (1% (w/v)/1 mM/50%
(v/v)). The TLC was developed either with an alkaline,
CHCl3/CH3OH/15 M
NH4OH/H2O (90/70/4/16; v/v/v/v), or an acidic,
CHCl3/CH3OCH3/CH3OH/HAc/H2O (40/15/14/13/7.5; v/v/v/v/v) solvent system. Radiolabeled lipids were
visualized and quantified using a PhosphorImager (Molecular Dynamics).
HPLC of Deacylated Lipids--
Dried lipids or individual TLC
spots visualized by phosphorimaging were resuspended in deacylation
mixture (monomethylamine/CH3OH/H2O/butanol; 5/4/3/1; v/v/v/v) and incubated at 53 °C for 40 min. The deacylated lipids were cooled on ice for 15 min before drying in a vacuum evaporator. The dried samples were resuspended in 1 ml of
butanol/petroleum ether/ethyl formate (20/4/1; v/v/v) plus 0.95 ml of
H2O and 50 µl of 0.5 M Hepes/KOH, pH 7.5. After centrifugation at 100 × g for 3 min, the lower
phases containing the GroPInsPns were
transferred to Eppendorf tubes while the upper phases were washed once
with 1 ml of H2O. After centrifugation, the new lower phases were combined to the corresponding first lower phases. The
combined lower phases were dried in vacuo. The dried samples were dissolved in H2O and stored at
Samples were applied on a Partisphere 46 mm x 250-mm 5-µm SAX HPLC
column (Whatman) and eluted with the following gradient: H2O for 5 min, up to 150 mM
(NH4)H2PO4 (pH 3.8) in 60 min, 150 mM (NH4)H2PO4 for 20 min, up to 1 M
(NH4)H2PO4 in 25 min, 1 M (NH4)H2PO4 for 10 min, at a flow rate of 1 ml/min. Fractions of 1 ml were collected and
counted in a Beckman LC6000IC scintillator counter.
Polyethyleneimine-Cellulose TLC--
The lipid giving rise to
the deacylation product present in fraction 76 in Fig. 2A
was believed to be DGPP (the deacylation product would therefore be
glyceropyrophosphate). Fraction 76 was hydrolyzed with 4 M
formic acid for 10 min at 100 °C, and then aliquots of fraction 76, both before and after mild acid hydrolysis, were spotted on a
polyethyleneimine-cellulose TLC sheet together with authentic
glycerophosphate (GroP), Pi, and 32Pi. The TLC was then developed and the
unradiolabeled standards stained exactly as described (37).
Separation of 32P-Radiolabeled Phospholipids--
The
pattern of 32P-radiolabeled phospholipids extracted from
suspension-cultured A. thaliana cells and separated by
one-dimensional TLC is presented in Fig.
1. In order to identify some of the
radiolabeled phospholipids, non-radiolabeled commercial phospholipids
were mixed with the purified radiolabeled samples, prior to TLC
separation. As shown in Fig. 1, the addition of excess non-radiolabeled
lipids affected the migration of the corresponding radiolabeled lipids. In this way, and by comparison to the TLC profiles obtained with other
plant species (37), the different radiolabeled phospholipids were
tentatively identified as indicated in Fig. 1. The TLC alkaline solvent
system used here resolved two phosphatidylinositol bisphosphate (PtdInsP2) isomers, PtdIns(3,5)P2 and
PtdIns(4,5)P2, obtained from
32Pi-radiolabeled yeast cells (data not shown),
and has previously been shown to separate
phosphatidylinositol-3,4-bisphosphate (PtdIns(3,4)P2) from
PtdIns(4,5)P2 (20), as well as PtdIns(4,5)P2
from PtdIns(3,5)P2 (18) from plants. However, it does not
permit a clear separation of the different PtdInsP isomers (see,
e.g., Ref. 38).
Identification of [32P]- and
[3H]Inositol-radiolabeled Phospholipids--
In order to
confirm the identity of the different 32P-radiolabeled
phospholipids we analyzed their deacylation products by chromatography on a strong anion-exchange HPLC column. Fig.
2A shows that two glycerophosphoinositol monophosphate isomers,
[3H]glycerophosphoinositol 3-phosphate
([3H]GroPIns(3)P) and
[3H]glycerophosphoinositol 4-phosphate
([3H]GroPIns(4)P), and two glycerophosphoinositol
bisphosphate isomers, [3H]glycerophosphoinositol-3,5-bisphosphate
([3H]GroPIns(3,5)P2) and
[3H]glycerophosphoinositol-4,5-bisphosphate
([3H]GroPIns(4,5)P2), purified from
myo-[2-3H]inositol-radiolabeled yeast cells,
as described (11), were well resolved from each other on the
Partisphere SAX column.
The profile obtained with the water-soluble fraction after deacylation
of phospholipids extracted from
32Pi-radiolabeled A. thaliana cells
comprised peaks eluting from the column exactly as authentic
[3H]GroPIns(3)P, [3H]GroPIns(4)P,
and [3H]GroPIns(4,5)P2, respectively (Fig.
2A). [32P]GroPIns(4)P represented
approximately 90% of the total
[32P]GroPInsPns, which
closely agrees with the levels reported for Spirodela
polyrhiza (39, 40), Commelina communis (19), and
Chlamydomonas eugametes (20). The identity of the A. thaliana derived, 32P-radiolabeled peaks that did not
co-chromatograph with any of the standard
[3H]GroPInsPns was deduced from
similar profiles published for other plant systems (39, 41, 42) and
from the myo-[2-3H]inositol labeling profile
(Fig. 2C), which showed that two peaks detected with
32Pi-labeling did not incorporate
myo-[2-3H]inositol. The
32P-radiolabeled bands tentatively identified as
PtdIns(4,5)P2, PtdInsP, and DGPP in Fig. 1 were
individually scraped from a TLC plate, deacylated, and the resulting
water-soluble products chromatographed individually on the Partisphere
SAX column (Fig. 2B). Approximately 95% of the
radioactivity present in each of the PtdIns(4,5)P2 and
PtdInsP bands was recovered in the water-soluble phase after deacylation, demonstrating that these lipids did not contain
significant amounts of phosphosphingolipids, some of which have been
shown to co-chromatograph with PtdInsP2s on TLC (43, 44).
The water-soluble product obtained from the deacylation of the putative
[32P]PtdIns(4,5)P2 band eluted from the
column as a single peak at the same position as yeast
[3H]GroPIns(4,5)P2 (Fig. 2B). The
deacylation product obtained from the PtdInsP band contained two
radiolabeled compounds, which behaved like yeast
[3H]GroPIns(3)P and [3H]GroPIns(4)P,
respectively (Fig. 2B). The
[32P]phosphatidylinositol 4-phosphate
([32P]PtdIns(4)P)/[32P]phosphatidylinositol
3-phosphate ([32P]PtdIns(3)P) ratio calculated from
the deacylation product of the PtdInsP TLC band was similar to that
deduced from the HPLC profile obtained from total lipid deacylates
(Fig. 2A). Finally, the deacylation product of the putative
[32P]DGPP band eluted as a single peak at 76 ml (Fig.
2B), matching exactly the elution of one of the products
present in the total 32P-labeled lipid deacylate (see Fig.
2A). Mild acid hydrolysis of fraction 76 produced two
radioactive compounds, which, on polyethyleneimine-cellulose TLC
plates, chromatographed exactly as authentic GroP and Pi
(data not shown). This confirmed that the original lipid putatively identified as DGPP indeed was DGPP.
We also examined the incorporation of
myo-[2-3H]inositol into lipids of A. thaliana cells (Fig. 2C). This demonstrated that the
three 32P-radiolabeled GroPInsPn
isomers identified in Fig. 2A as representing
[32P]GroPIns(3)P, [32P]GroPIns(4)P, and
[32P]GroPIns(4,5)P2 also incorporated
myo-[2-3H]inositol, and that the other peaks
observed in Fig. 2A, except for [32P]GroPIns,
were absent in the total lipid deacylate from
myo-[2-3H]inositol-radiolabeled cells. The
[3H]GroPIns(4)P/[3H]GroPIns(3)P ratio was
similar to the one determined for 32P-radiolabeled samples.
PtdIns(4,5)P2 was the only PtdInsP2 isomer we
could detect in both 32Pi- and
myo-[2-3H]inositol-radiolabeled A. thaliana cells, under the conditions tested. In addition, we were
unable to detect any PtdIns(3,4,5)P3, an isomer that has so
far never been detected in plant cells, although the amount of
radioactivity incorporated into polyphosphoinositides would probably
not have allowed us to detect this compound as in animal cells levels
of PtdIns(3,4,5)P3 never exceed 0.1% of the radioactivity
in PtdIns(4)P and PtdIns(4,5)P2 even under stimulated conditions.
Time Course of 32P Incorporation into A. thaliana
Phospholipids--
The incorporation of 32P into most
phospholipids was detectable after a few minutes of labeling (Fig.
3). However, the radiolabeling of the
quantitatively minor lipids (i.e. the polyphosphoinositides, DGPP, and phosphatidic acid (PtdOH)) reached a maximum much earlier than for the other phospholipids. For PtdInsP,
PtdIns(4,5)P2, PtdOH, and DGPP, a maximum was reached
between 15 and 30 min, while the levels of
[32P]phosphatidylglycerol ([32P]PtdGro),
[32P]phosphatidylethanolamine, [32P]PtdIns,
and some additional unidentified lipids continued to increase well
beyond the time scale of the experiment. This obviously reflects the
fact that the quantitatively minor lipids all possess a monoester
phosphate group, which approaches isotopic equilibrium with the added
32Pi much more rapidly than the diester
phosphate group present in the other phospholipids because of the much
higher rate of turnover of phosphomonoesters. Since under these
conditions (i.e. short term radiolabeling) the monoester
phosphates will be at isotopic equilibrium with the
32Pi and the diester phosphates of these
compounds will scarcely be radiolabeled, any change in radioactivity
can be interpreted as a change in mass for the polyphosphoinositides
and DGPP, as long as the monoester phosphates of these compounds can be
shown to have attained isotopic equilibrium. This is most commonly
demonstrated by extracting a fixed mass of cells at different times
during a short term radiolabeling regime and extracting the lipids and quantifying the amount of 32P associated with each lipid,
as in Fig. 3. When the level of 32P in a given compound
reaches a plateau the monoester phosphate of that compound has attained
isotopic equilibrium. Fig. 3 shows that the 32P
incorporation into the quantitatively minor phospholipids reached equilibrium after 30 min of labeling with 32Pi.
However, depending on the experiment, it was sometimes necessary to
label the cells for 90 min in order to reach a stable and reproducible background 32P incorporation into the phospholipids, as
shown, for example in Fig. 1. Therefore, the cells were routinely
radiolabeled with 32Pi for 90 min before lipids
were extracted or before application of any treatments. As previously
observed with plant cells, the steady-state level of
PtdIns(4,5)P2 was much lower than that of PtdInsP (see,
e.g., Fig. 1) (17, 38).
Effect of Salt and Hyperosmotic Stresses on
Phospholipids--
NaCl (0.4 M) induced large
increases in the steady-state levels of several
32P-radiolabeled phospholipids, namely
PtdIns(4,5)P2 and DGPP. The levels of radioactivity also
increased in an additional phospholipid, which, on TLC, chromatographed
exactly like authentic soybean PtdCho (Fig.
4). To confirm that this lipid indeed was
PtdCho, we briefly radiolabeled cells with
[methyl-14C]choline. As shown in Fig.
4D, this lipid incorporated choline, and no other lipid
appeared to be radiolabeled under these conditions. The increased
incorporation of 32P-radiolabel into PtdCho is suggestive
of an increase in turnover of this lipid, but since the diester
phosphate of PtdCho is nowhere near isotopic equilibrium, the increase
in radioactivity does not allow any conclusions about changes in the
steady-state levels of this lipid to be drawn from data gathered under
this experimental regime. The accumulation of
[32P]PtdIns(4,5)P2 was consistently
detectable within minutes of NaCl addition, and the levels rose
8-25-fold after 10-20 min (Figs. 4B and
5). The effect of NaCl on the
incorporation of radiolabel into PtdCho was very similar whether cells
were radiolabeled briefly with 32Pi or
[methyl-14C]choline (Fig. 4, A and
D). The steady-state levels of PtdIns(4,5)P2 remained high for at least 1 h, and returned to levels close to those of control cells after 2-6 h (Figs. 4B and 7). In
contrast, no decrease in [32P]PtdCho was observed 6 h following salt stress (Fig. 4B). After a lag phase of a
few minutes, during which [32P]DGPP was not affected by
the salt treatment, it reached concentrations 10-40 times higher than
in control cells after 15 min, and continued to rise slowly for at
least 1 h (Fig. 4B, 6,
and 7). Thereafter, DGPP remained
elevated between 1 and 2 h of treatment and started to decrease
significantly after 6 h (Fig. 4, A and B).
Of the other phospholipids, only PtdInsPs were affected by the salinity treatment, whereas PtdIns and PtdOH showed no alterations (Fig. 4,
A and C). However, we consistently detected a
sharp drop in PtdOH levels, by approximately 50%, during the first few
minutes following the addition of NaCl (Fig. 4, A and
C). By 15 min, PtdOH concentration had returned to its value
before addition of salt. HPLC analyses of
[3H]inositol-radiolabeled lipids revealed that of the two
PtdInsP isomers, PtdIns(4)P was the one that responded to the salinity stress (Fig. 6). Simultaneously to the large increase in
PtdIns(4,5)P2, PtdIns(4)P decreased by almost 50%, while
PtdIns(3)P was unaffected. This decrease in the levels of PtdIns4P is
exactly what would be predicted to occur if activation of PtdIns(4)P
5-kinase(s) preceded activation of the plant PtdIns 4-kinase(s). To
determine whether the accumulation of PtdIns(4,5)P2, DGPP,
and PtdOH induced by the salt treatment was solely due to the
hyperosmotic shock imposed, or whether it also reflected a
NaCl-specific response, we analyzed the effect of sorbitol at a
concentration equivalent to the osmolarity produced by the NaCl
concentration used. Figs. 6 and 7 show that sorbitol (0.8 M) had similar effects on PtdIns(4,5)P2 and
DGPP to those described above for NaCl; however, these effects were not
as pronounced as with NaCl, especially for DGPP. Interestingly, sorbitol had almost no effect on the incorporation of
32Pi into PtdCho (Fig. 7).
The strongest evidence that a
phosphoinositide/Ca2+ signal transduction system operates
in plant cells has been provided by the identification of homologues of
the components of the classical animal phosphoinositide system, such as
phosphoinositide-metabolizing enzymes (22, 23, 25, 26, 28) and inositol
lipids themselves (19, 21, 38, 45, 46). In the present study, we have identified phosphoinositides as well as other phospholipids in suspension-cultured A. thaliana cells, and provide evidence
that some of these lipids may play an important function in the
response to salt stress, and hyperosmotic stress.
PtdIns(4)P was the major polyphosphoinositide in A. thaliana
cells, representing at least 90% of the total polyphosphoinositides, while PtdIns(4,5)P2 was present in low amounts, in
unperturbed cells. However, PtdIns was by far the most abundant
inositol lipid in these cells. Such a distribution has been observed in
several other plant systems (see, e.g., Refs. 19 and 38). We
were not able to detect PtdIns(5)P, PtdIns(3,4)P2,
PtdIns(3,5)P2 or PtdIns(3,4,5)P3.
PtdIns(3,4)P2 (19-21) and PtdIns(3,5)P2 (11, 18) have been detected in plant cells. In C. communis, the
main PtdInsP2 isomer in guard cells has been identified as
PtdIns(3,4)P2 (19), whereas in mesophyll protoplasts,
PtdIns(4,5)P2 was readily detectable but the presence of
PtdIns(3,4)P2 could not be confirmed (41). In A. thaliana cells, PtdIns(3,5)P2 and
PtdIns(3,4)P2, if present, represent very minor
phosphoinositides. This suggests that in plants, the distribution of
the different phosphoinositide isomers varies greatly between different
species and/or cell types. This could reflect cell-specific functions
of some of the phosphoinositides. It is also possible that the level of
some of these lipids is very low in unperturbed cells but is
specifically increased under certain conditions, as shown here for
PtdIns(4,5)P2. For example, PtdIns(3,5)P2,
present in very low amounts in non-stimulated yeast and plant cells,
was recently shown to be specifically induced by challenging these
cells with hyperosmotic solutions (11, 18).
Unstimulated A. thaliana cells also contained low levels of
DGPP. High levels of a non-identified lipid were detected in
32P-radiolabeled protoplasts from C. communis
(41). This lipid probably represented DGPP, since it eluted from a
Partisphere SAX column at a position very similar to the lipid we
identified as DGPP in A. thaliana cells. The high levels
detected in C. communis protoplasts were probably due to the
high osmolarity (0.45 M mannitol, 20 mM KCl)
conditions, and possibly also the electroporation, used to label the protoplasts.
Osmotic stress can affect phosphoinositide metabolism in plant cells,
but so far most reports have not shown dramatic alterations in the
turnover of any phosphoinositide isomer. For example, Cho et
al. (47) did not see any effect of hyperosmotic shock on PtdInsP2 levels in carrot cells grown in suspension
culture, but observed a 25% decrease in PtdInsP levels. The
unicellular green algae Dunaliella salina responded to
hyperosmotic shock by increasing its PtdInsP2 content by
approximately 30% after 10 min of treatment (48). As mentioned
earlier, hyperosmotic stress was very recently shown to stimulate the
turnover of PtdIns(3,5)P2 in C. moewusii and
higher plant cells (18). This response was maximal at 150-200 mM NaCl. At higher concentrations, no effect on
PtdIns(3,5)P2 levels were observed (18). The turnover of
PtdIns(4,5)P2 in C. moewusii, tomato, pea, and
alfalfa cells was not affected by 150 mM NaCl (18). This is
in contrast to the more dramatic effects on PtdIns(4,5)P2
we observed for A. thaliana cells. PtdIns(4,5)P2 levels increased by 8-25 times in A. thaliana cells
subjected to hyperosmotic stress. The increase in
PtdIns(4,5)P2 did not depend on the chemical nature of the
osmotic agent used; sorbitol and NaCl had the same effect, indicating
that this response was due to hyperosmolarity. Such rapid and large
effects on PtdIns(4,5)P2 have not been identified in plants
previously, and are indicative of a possible involvement of the
classical (4,5)P2-inositide pathway in response to
hyperosmotic stress in plants. In preliminary experiments, in which the
effect of increasing NaCl concentrations were tested, an increase in
PtdIns(4,5)P2 level was detected already at 50 mM NaCl (data not shown); the increase was, however, more
pronounced at 400 mM. Clearly, the effect of hyperosmotic
stress on phosphoinositide turnover varies significantly between plant species.
The expression of a PtdInsP kinase gene can be induced by salt stress
(23). However, this induction was observed only 1 h after salt
stress. The 8-25-fold increase in PtdIns(4,5)P2 occurs within minutes and is therefore almost certainly due to the activation of endogenous PtdIns 4-kinase and PtdIns(4)P 5-kinase activities rather
than to newly synthesized enzymes. On the other hand, the PI-PLC
isoform found to be overexpressed under salt stress (25) may well
contribute to the re-adjustment of PtdIns(4,5)P2 levels seen after 30 min of salt treatment. This hydrolysis of excess PtdIns(4,5)P2 could also represent an initial step in a
cascade leading ultimately to growth adjustment.
It is possible that during the early period of increasing accumulation
of PtdIns(4,5)P2 some of this lipid is hydrolyzed to Ins(1,4,5)P3 and diacylglycerol, which could be involved in
the response to hyperosmolarity/salinity stress. For example, it was recently shown that cytosolic free calcium concentration in whole A. thaliana seedlings increased following a
hyperosmotic/salt shock (34). This increase in cytosolic calcium was
inhibited by U73122, an inhibitor of PI-PLC-dependent
processes in animal cells, thus suggesting that PI-PLC may be involved
in the early response to hyperosmotic/salt stresses. Alternatively,
PtdIns(4,5)P2 may participate in salinity signaling in
plant cells through association with actin-binding proteins and
re-organization of the cytoskeleton. PtdIns(4,5)P2 may also
trigger its effects by regulating various enzymes or cellular functions
such as vesicle trafficking (8), or ion channels (49), an area that is
virtually unexplored in plant systems.
NaCl-specific alterations in the levels of some lipids were also
observed. DGPP also accumulated upon non-saline and NaCl stresses, but
salinity induced significantly stronger responses. An increase in DGPP
can also be triggered by low concentrations of mastoparan (37).
However, the function of this lipid is at present unknown. Since PtdOH
is the direct precursor of DGPP, and we did not detect any decrease in
PtdOH, PtdOH production is probably stimulated during salt stress, for
example from the PI-PLC-catalyzed hydrolysis of
PtdIns(4,5)P2 or through direct DAG kinase stimulation.
In response to salinity, osmotic stress, and other stresses, some
plants, e.g. spinach, barley, and sugar beet, synthesize and
accumulate non-toxic solutes, such as glycine betaine, proline, and
various sugars, thus maintaining turgor and the driving force for water
uptake (50). Glycine betaine is synthesized from choline in a two-step
reaction (51). The free choline used for the synthesis of glycine
betaine is believed to be derived from PtdCho or phosphocholine, although it has been shown that free choline is almost exclusively used
for PtdCho synthesis (52). It is possible that salinity stimulates the
synthesis of a specific pool of PtdCho that is used more efficiently
for the synthesis of glycine betaine.
In conclusion, our results show that, in A. thaliana
suspension-cultured cells, salinity induces quick and dramatic
increases in several phospholipids, PtdIns(4,5)P2, DGPP,
and, to a lesser extent, in the turnover of PtdCho, and that the
alterations in DGPP and PtdCho metabolism are, at least in part,
specific to salinity, whereas the PtdIns(4,5)P2 increase is
truly a response to hypertonicity and not merely to increases in sodium
or chloride. The elucidation of the downstream targets of these lipids
should help understand the molecular mechanisms of salinity and
hopefully direct us toward possible strategies to alleviate its
detrimental effects on plant growth. With the recent findings that
depending on plant species, PtdIns(3,5)P2 (18) or
PtdIns(4,5)P2 (this work) synthesis is affected by
hyperosmotic stress, and that distinct phosphoinositide distributions
exist between cell types (19, 41), it seems now obvious that the
phosphoinositide system in plants is as complex as in animal and yeast
cells. These differences may reflect cell-specific functions of
phosphoinositides, in addition to species differences.
We thank Dr. Irina Staxén for help with
the A. thaliana cell culture. We thank Dr. Teun Munnik
(University of Amsterdam) for advice with TLC, and Prof. Richard Cyr
for the A. thaliana suspension cell culture.
*
This work was supported by European Community Grant
BIO-CT96-0775 (to C. P., C. L., and M. S.) and by grants
from the Swedish Natural Science Research Council (to C. L.), the
Swedish Council for Forestry and Agricultural Research (to M. S.),
and the Swedish Foundation for Strategic Research (to C. L. and
M. S.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
§
To whom correspondence should be addressed. E-mail:
pbio-cpi@pop.net.lu.se.
The abbreviations used are:
PI-PLC, phosphoinositide-specific phospholipase C;
PtdIns(4, 5)P2,
phosphatidylinositol 4,5-bisphosphate;
Ins(1, 4,5)P3,
inositol 1,4,5-trisphosphate;
PtdIns(3, 5)P2,
phosphatidylinositol 3,5-bisphosphate;
PtdIns(5)P, phosphatidylinositol
5-phosphate;
PtdIns(3, 4,5)P3, phosphatidylinositol
3,4,5-trisphosphate;
PtdIns, phosphatidylinositol;
PtdInsP, phosphatidylinositol monophosphate;
DGPP, diacylglycerol pyrophosphate;
PtdCho, phosphatidylcholine;
32Pi, [32P]orthophosphate;
MS, Murashige and Skoog;
GroPInsPn, glycerophosphoinositol phosphate;
TLC, thin-layer chromatography;
GroP, glycerophosphate;
PtdInsP2, phosphatidylinositol bisphosphate;
GroPIns(3)P, glycerophosphoinositol 3-phosphate;
GroPIns(4)P, glycerophosphoinositol 4-phosphate;
GroPIns(3, 5)P,
glycerophosphoinositol 3,5-bisphosphate;
GroPIns(4, 5)P,
glycerophosphoinositol 4,5-bisphosphate;
PtdIns(4)P, phosphatidylinositol 4-phosphate;
PtdIns(3)P, phosphatidylinositol
3-phosphate;
PtdOH, phosphatidic acid;
PtdGro, phosphatidylglycerol;
PtdIns(3, 4)P2, phosphatidylinositol 3,4-bisphosphate.
Salinity and Hyperosmotic Stress Induce Rapid Increases in
Phosphatidylinositol 4,5-Bisphosphate, Diacylglycerol Pyrophosphate,
and Phosphatidylcholine in Arabidopsis
thaliana Cells*
§,,
,, and
Department of Plant Biochemistry, Lund
University, P. O. Box 117, SE-22100 Lund, Sweden and the ¶ Centre
for Clinical Research in Immunology and Signalling, University of
Birmingham, Birmingham B15 2TT, United Kingdom
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1.0 and
incubated at 26 °C and 130 rpm for at least 15 min before addition
of 32Pi (0.1-0.2 mCi/ml). The cells were
further incubated in the same conditions for 90 min, or the specified
time when indicated otherwise, and then treated with NaCl or sorbitol
for 15 min or as otherwise indicated. The treatment was terminated by
addition of two volumes of ice-cold methanol, vortexing, and freezing
in liquid nitrogen.
1.0 and processed
further as indicated above for 32Pi labeling.
20 °C until analyzed.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (61K):
[in a new window]
Fig. 1.
Separation and identification of
phospholipids from 32Pi-radiolabeled A. thaliana cells. Two-day-old A. thaliana
suspension-cultured cells were incubated with phosphate-free MS medium
containing 32Pi (0.1-0.2 mCi/ml) for 30 min.
The lipids were extracted and separated by TLC using an alkaline
(left) and an acidic (right) solvent system, and
visualized with a PhosphorImager. Excess lipid standards were added in
order to identify some of the radiolabeled lipids.
Lane C, no addition; lane
1, plus PtdIns(4)P; lane 2, plus
PtdIns(4,5)P2; lane 3, plus PtdCho.
The position of the added standard lipids is indicated by an
arrow to the left in each lane. The plates were
subsequently stained with iodine vapors. Lane I
represents lane 3 after iodine staining.
View larger version (20K):
[in a new window]
Fig. 2.
HPLC analysis of the water-soluble products
after deacylation of lipids from
32Pi-radiolabeled A. thaliana
cells. A. thaliana cells were
32P-radiolabeled as indicated in Fig. 1 (A and
B), and the extracted lipids were deacylated. A,
the water-soluble products were mixed with
[3H]GroPInsPn of known structure
(purified from [3H]inositol-radiolabeled yeast), and
separated on a Partisphere SAX column. ------, 3H
incorporation (dpm); 
, 32P incorporation (dpm).
B, the bands corresponding to lipids tentatively identified
as PtdIns(4,5)P2, PtdInsP, and DGPP were scraped
individually from a TLC plate, de-acylated and the corresponding
water-soluble products analyzed by HPLC as in (A); ,
PtdIns(4,5)P2; 
, PtdInsP; 
, DGPP. C,
A. thaliana cells were cultured in full MS medium in the
presence of myo-[2-3H]inositol and their
lipids extracted and analyzed by HPLC as in A. All peaks are
identified as the lipids they are derived from.
View larger version (45K):
[in a new window]
Fig. 3.
Time course of 32P incorporation
into phospholipids of A. thaliana cells.
Two-day-old cells were incubated with 32Pi for
the indicated period of time, before their lipids were extracted and
analyzed by TLC as described under "Experimental Procedures." The
incorporation into individual lipids was visualized and quantified with
a PhosphorImager. A, phosphorimage. B,
time-course graphic representation. ,
PtdIns(4,5)P2; , PtdInsP; 
, PtdOH 
, PtdIns; ,
DGPP; 
, PtdCho. The data presented are representative of three
independent experiments.
View larger version (72K):
[in a new window]
Fig. 4.
Effects of NaCl on phospholipids extracted
from 32Pi-radiolabeled or
[methyl-14C]choline-radiolabeled
A. thaliana cells. Two-day-old A. thaliana cells were incubated with phosphate-free MS medium in the
presence of 32Pi (0.1-0.2 mCi/ml)
(A--D), or
[methyl-14C]choline chloride (3.33 µCi/ml)
(D) for 90 min. MS or NaCl (final concentration 0.4 M) was added to the cells. 0.3-1-ml aliquots were removed
at the indicated times, and the lipids were extracted and analyzed by
TLC with the alkaline solvent system, as described under
"Experimental Procedures." The radiolabeled phospholipids were
visualized with a PhosphorImager. The incorporation into the different
phospholipids was quantified from the phosphorimage. A,
phosphorimage (upper panel). An excess of
authentic PtdCho was added to one of the samples and visualized with
iodine vapors (lower panel). B and
C, graphic representation. , PtdIns(4,5)P2;
, PtdInsP; , PtdOH; , PtdIns; , DGPP; , PtdCho.
D, after labeling with 32Pi or
[methyl-14C]choline, cells were treated with
MS or NaCl (0.4 M) for 15 min before their lipids were
extracted and analyzed by TLC. The data presented are representative of
five independent experiments.
View larger version (20K):
[in a new window]
Fig. 5.
Effects of salt on the phospholipids
extracted from
myo-[2-3H]inositol-radiolabeled
A. thaliana cells. Arabidopsis
thaliana cells (5 ml) were radiolabeled with
myo-[2-3H]inositol before the addition of MS
( ) or NaCl (final concentration 0.4 M) (). After 15 min, the lipids were extracted and analyzed by HPLC as described under
"Experimental Procedures." A part of the chromatogram is presented.
The different peaks are identified as the lipids they are derived from.
The data presented are representative of two independent
experiments.
View larger version (18K):
[in a new window]
Fig. 6.
Comparison of the effects of salt and
sorbitol on the phospholipids extracted from
32P-radiolabeled A. thaliana cells as
analyzed by HPLC. Two-day-old A. thaliana cells were
radiolabeled with 32Pi for 90 min before the
addition of MS ( ), NaCl (final concentration 0.4 M)
(), or sorbitol (final concentration 0.8 M) (). After
15 min, the lipids were extracted, deacylated, and the water-soluble
products analyzed by HPLC. A part of the chromatogram is presented. The
different peaks are identified as the lipids they are derived from. The
data presented are representative of three independent
experiments.
View larger version (42K):
[in a new window]
Fig. 7.
Comparison of the effect of NaCl and sorbitol
on phospholipids from 32Pi-radiolabeled
A. thaliana cells as analyzed by TLC. Two-day-old
A. thaliana cells were radiolabeled with
32Pi for 90 min, and then treated with MS, NaCl
(final concentration 0.4 M), or sorbitol (final
concentration 0.8 M) for 15 min or 2 h. The lipids
were extracted and analyzed by TLC (upper part).
The incorporation into the different phospholipids was quantified from
the phosphorimage (lower part). The data
represent the results obtained with one experiment, and from one TLC
plate. Similar results were obtained in an additional independent
experiment.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
ACKNOWLEDGEMENTS
FOOTNOTES
Supported from the Medical Research Council, Royal Society,
and Medical Faculty of the University of Birmingham; Medical Research Council research fellow.
ABBREVIATIONS
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INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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M. E. Williams, J. Torabinejad, E. Cohick, K. Parker, E. J. Drake, J. E. Thompson, M. Hortter, and D. B. DeWald Mutations in the Arabidopsis Phosphoinositide Phosphatase Gene SAC9 Lead to Overaccumulation of PtdIns(4,5)P2 and Constitutive Expression of the Stress-Response Pathway Plant Physiology, June 1, 2005; 138(2): 686 - 700. [Abstract] [Full Text] [PDF]
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R. Zhong, D. H. Burk, C. J. Nairn, A. Wood-Jones, W. H. Morrison III, and Z.-H. Ye Mutation of SAC1, an Arabidopsis SAC Domain Phosphoinositide Phosphatase, Causes Alterations in Cell Morphogenesis, Cell Wall Synthesis, and Actin Organization PLANT CELL, May 1, 2005; 17(5): 1449 - 1466. [Abstract] [Full Text] [PDF]
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R. Zhong and Z.-H. Ye Molecular and Biochemical Characterization of Three WD-Repeat-Domain-containing Inositol Polyphosphate 5-Phosphatases in Arabidopsis thaliana Plant Cell Physiol., November 15, 2004; 45(11): 1720 - 1728. [Abstract] [Full Text] [PDF]
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M. E. Ercetin and G. E. Gillaspy Molecular Characterization of an Arabidopsis Gene Encoding a Phospholipid-Specific Inositol Polyphosphate 5-Phosphatase Plant Physiology, June 1, 2004; 135(2): 938 - 946. [Abstract] [Full Text] [PDF]
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Y. Kobayashi, S. Yamamoto, H. Minami, Y. Kagaya, and T. Hattori Differential Activation of the Rice Sucrose Nonfermenting1-Related Protein Kinase2 Family by Hyperosmotic Stress and Abscisic Acid PLANT CELL, May 1, 2004; 16(5): 1163 - 1177. [Abstract] [Full Text] [PDF]
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L. Zonia and T. Munnik Osmotically Induced Cell Swelling versus Cell Shrinking Elicits Specific Changes in Phospholipid Signals in Tobacco Pollen Tubes Plant Physiology, February 1, 2004; 134(2): 813 - 823. [Abstract] [Full Text] [PDF]
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A. Mazel, Y. Leshem, B. S. Tiwari, and A. Levine Induction of Salt and Osmotic Stress Tolerance by Overexpression of an Intracellular Vesicle Trafficking Protein AtRab7 (AtRabG3e) Plant Physiology, January 1, 2004; 134(1): 118 - 128. [Abstract] [Full Text] [PDF]
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F. Apone, N. Alyeshmerni, K. Wiens, D. Chalmers, M. J. Chrispeels, and G. Colucci The G-Protein-Coupled Receptor GCR1 Regulates DNA Synthesis through Activation of Phosphatidylinositol-Specific Phospholipase C Plant Physiology, October 1, 2003; 133(2): 571 - 579. [Abstract] [Full Text] [PDF]
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R. Zhong and Z.-H. Ye The SAC Domain-Containing Protein Gene Family in Arabidopsis Plant Physiology, June 1, 2003; 132(2): 544 - 555. [Abstract] [Full Text] [PDF]
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D. Sbrissa, O. C. Ikonomov, R. Deeb, and A. Shisheva Phosphatidylinositol 5-Phosphate Biosynthesis Is Linked to PIKfyve and Is Involved in Osmotic Response Pathway in Mammalian Cells J. Biol. Chem., November 27, 2002; 277(49): 47276 - 47284. [Abstract] [Full Text] [PDF]
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E. Ruelland, C. Cantrel, M. Gawer, J.-C. Kader, and A. Zachowski Activation of Phospholipases C and D Is an Early Response to a Cold Exposure in Arabidopsis Suspension Cells Plant Physiology, October 1, 2002; 130(2): 999 - 1007. [Abstract] [Full Text] [PDF]
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L. Zonia, S. Cordeiro, J. Tupy, and J. A. Feijo Oscillatory Chloride Efflux at the Pollen Tube Apex Has a Role in Growth and Cell Volume Regulation and Is Targeted by Inositol 3,4,5,6-Tetrakisphosphate PLANT CELL, September 1, 2002; 14(9): 2233 - 2249. [Abstract] [Full Text] [PDF]
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Z. Mou, X. Wang, Z. Fu, Y. Dai, C. Han, J. Ouyang, F. Bao, Y. Hu, and J. Li Silencing of Phosphoethanolamine N-Methyltransferase Results in Temperature-Sensitive Male Sterility and Salt Hypersensitivity in Arabidopsis PLANT CELL, September 1, 2002; 14(9): 2031 - 2043. [Abstract] [Full Text] [PDF]
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B. Mueller-Roeber and C. Pical Inositol Phospholipid Metabolism in Arabidopsis. Characterized and Putative Isoforms of Inositol Phospholipid Kinase and Phosphoinositide-Specific Phospholipase C Plant Physiology, September 1, 2002; 130(1): 22 - 46. [Abstract] [Full Text] [PDF]
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I. Y. Perera, J. Love, I. Heilmann, W. F. Thompson, and W. F. Boss Up-Regulation of Phosphoinositide Metabolism in Tobacco Cells Constitutively Expressing the Human Type I Inositol Polyphosphate 5-Phosphatase Plant Physiology, August 1, 2002; 129(4): 1795 - 1806. [Abstract] [Full Text] [PDF]
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 C. Nasuhoglu, S. Feng, Y. Mao, I. Shammat, M. Yamamato, S. Earnest, M. Lemmon, and D. W. Hilgemann Modulation of cardiac PIP2 by cardioactive hormones and other physiologically relevant interventions Am J Physiol Cell Physiol, July 1, 2002; 283(1): C223 - C234. [Abstract] [Full Text] [PDF]
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L. Xiong, K. S. Schumaker, and J.-K. Zhu Cell Signaling during Cold, Drought, and Salt Stress PLANT CELL, May 1, 2002; 14(90001): S165 - 183. [Full Text] [PDF]
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 D. W. Hilgemann, S. Feng, and C. Nasuhoglu The Complex and Intriguing Lives of PIP2 with Ion Channels and Transporters Sci. Signal., December 4, 2001; 2001(111): re19 - re19. [Abstract] [Full Text] [PDF]
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I. Heilmann, I. Y. Perera, W. Gross, and W. F. Boss Plasma Membrane Phosphatidylinositol 4,5-Bisphosphate Levels Decrease with Time in Culture Plant Physiology, August 1, 2001; 126(4): 1507 - 1518. [Abstract] [Full Text] [PDF]
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D. B. DeWald, J. Torabinejad, C. A. Jones, J. C. Shope, A. R. Cangelosi, J. E. Thompson, G. D. Prestwich, and H. Hama Rapid Accumulation of Phosphatidylinositol 4,5-Bisphosphate and Inositol 1,4,5-Trisphosphate Correlates with Calcium Mobilization in Salt-Stressed Arabidopsis Plant Physiology, June 1, 2001; 126(2): 759 - 769. [Abstract] [Full Text] [PDF]
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D. E. Monks, K. Aghoram, P. D. Courtney, D. B. DeWald, and R. E. Dewey Hyperosmotic Stress Induces the Rapid Phosphorylation of a Soybean Phosphatidylinositol Transfer Protein Homolog through Activation of the Protein Kinases SPK1 and SPK2 PLANT CELL, May 1, 2001; 13(5): 1205 - 1219. [Abstract] [Full Text]
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S. Takahashi, T. Katagiri, T. Hirayama, K. Yamaguchi-Shinozaki, and K. Shinozaki Hyperosmotic Stress Induces a Rapid and Transient Increase in Inositol 1,4,5-Trisphosphate Independent of Abscisic Acid in Arabidopsis Cell Culture Plant Cell Physiol., February 1, 2001; 42(2): 214 - 222. [Abstract] [Full Text] [PDF]
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 Z. Fan and R. A Neff Susceptibility of ATP-sensitive K+ channels to cell stress through mediation of phosphoinositides as examined by photoirradiation J. Physiol., December 15, 2000; 529(3): 707 - 721. [Abstract] [Full Text] [PDF]
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J. Oshiro, S. Rangaswamy, X. Chen, G.-S. Han, J. E. Quinn, and G. M. Carman Regulation of the DPP1-encoded Diacylglycerol Pyrophosphate (DGPP) Phosphatase by Inositol and Growth Phase. INHIBITION OF DGPP PHOSPHATASE ACTIVITY BY CDP-DIACYLGLYCEROL AND ACTIVATION OF PHOSPHATIDYLSERINE SYNTHASE ACTIVITY BY DGPP J. Biol. Chem., December 22, 2000; 275(52): 40887 - 40896. [Abstract] [Full Text] [PDF]
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O. Pierrugues, C. Brutesco, J. Oshiro, M. Gouy, Y. Deveaux, G. M. Carman, P. Thuriaux, and M. Kazmaier Lipid Phosphate Phosphatases in Arabidopsis. REGULATION OF THE AtLPP1 GENE IN RESPONSE TO STRESS J. Biol. Chem., June 1, 2001; 276(23): 20300 - 20308. [Abstract] [Full Text] [PDF]
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G.-S. Han, C. N. Johnston, X. Chen, K. Athenstaedt, G. Daum, and G. M. Carman Regulation of the Saccharomyces cerevisiae DPP1-encoded Diacylglycerol Pyrophosphate Phosphatase by Zinc J. Biol. Chem., March 23, 2001; 276(13): 10126 - 10133. [Abstract] [Full Text] [PDF]
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