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Published as doi: 10.1096/fj.06-7887com.
 (The FASEB Journal. 2007;21:1958-1967.)
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Stress-inducible and constitutive phosphoinositide pools have distinctive fatty acid patterns in Arabidopsis thaliana

Sabine König, Alina Mosblech and Ingo Heilmann1

Department of Plant Biochemistry, Albrecht-von-Haller-Institute for Plant Sciences, Georg-August-University Göttingen, Göttingen, Germany

1Correspondence: Department of Plant Biochemistry, Albrecht-von-Haller-Institute for Plant Sciences, Georg-August-University Göttingen, Justus-von-Liebig-Weg 11, 37077 Göttingen, Germany. E-mail: iheilma{at}uni-goettingen.de


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Function and development of eukaryotic cells require tight control of diverse physiological processes. Numerous cellular processes are regulated by polyphosphoinositides, which interact with protein partners or mediate release of the second messenger, inositol 1,4,5-trisphosphate (InsP3). Emerging evidence suggests that different regulatory or signaling functions of polyphosphoinositides may be orchestrated by the establishment of distinct subcellular pools; the principles underlying pool-formation are, however, not understood. Arabidopsis plants exhibit transient increases in polyphosphoinositides with hyperosmotic stress, providing a model for comparing constitutive and stress-inducible polyphosphoinositide pools. Using a combination of thin-layer-chromatography and gas-chromatography, phospholipids from stressed and nonstressed Arabidopsis plants were analyzed for their associated fatty acids. Under nonstress conditions structural phospholipids and phosphatidylinositol contained 50–70 mol% polyunsaturated fatty acids (PUFA), whereas polyphosphoinositides were more saturated (10–20 mol% PUFA). With hyperosmotic stress polyphosphoinositides with up to 70 mol% PUFA were formed that differed from constitutive species and coincided with a transient loss in unsaturated phosphatidylinositol. The patterns indicate inducible turnover of an unsaturated phosphatidylinositol pool, which accumulates under standard conditions and is primed for phosphorylation on stimulation. Metabolic analysis of wild-type and transgenic plants disturbed in phosphoinositide metabolism suggests that, in contrast to saturated species, unsaturated polyphosphoinositides are channeled toward InsP3-production.—König, S., Mosblech, A., Heilmann, I. Stress-inducible and constitutive phosphoinositide pools have distinctive fatty acid patterns in Arabidopsis thaliana.


Key Words: hyperosmotic stress • plant • PtdIns (4,5) P2 pools • signal transduction


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
PHOSPHOINOSITIDES ARE CENTRALLY involved in the regulation of a multitude of cellular processes in animal, fungal, and plant systems, and these regulatory functions have been extensively reviewed (1 2 3 4 5) . Phosphoinositide regulation involves the interaction of the lipids with protein partners that may be recruited to target membranes or are modified in their biochemical activity by their phosphoinositide ligands. Examples from both the plant and animal research fields include the regulation of ion-channel or ATPase activity (6 , 7) , cytoskeletal dynamics (8 9 10 11) , and hormonal and stress signaling (1 2 3 4 5) . As yet, the involvement in vesicle trafficking has only been demonstrated with animal cells (8 , 12) . There is an obvious need for tight spatial and temporal regulation of phosphoinositide-protein interactions in all eukaryotes, because a phosphoinositide may bind to various alternative partners, with potentially conflicting effects on physiology. Emerging evidence suggests that phosphoinositide-protein interactions are restricted according to physiological requirements by compartmentation into autonomous phosphoinositide pools. Phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] is the best-studied regulatory phosphoinositide and can, in both plants and animals, perform cellular functions either as an intact lipid ligand for protein partners (13 14 15) or as a substrate for phospholipase C (PLC), which hydrolyzes the lipid into inositol 1,4,5-trisphosphate (InsP3) and diacylglycerol (DAG) (3 , 16) . The list of PtdIns(4,5)P2 functions reported from various eukaryotic systems (1 2 3 4 5) suggests the presence of various physiological PtdIns(4,5)P2 pools compartmentalized along organellar borders or in microdomains within one membrane. Whereas the presence of such independent pools of PtdIns(4,5)P2 has been demonstrated before in plants and animals (17 18 19 20) , previous studies from the animal field have capitalized on the recognition of polyphosphoinositide headgroups by various target proteins (14 , 21) , and so far the lipids constituting distinct phosphoinositide pools have not been characterized regarding differences in fatty acid compositions, which may define distinct molecular lipid species.

The key hypothesis of this study is that phosphoinositides constituting distinct physiological pools may differ in their fatty acid make-up. In this context note that preferences for phosphoinositide substrates with certain fatty acid compositions have been demonstrated in vitro for recombinant enzymes, including phosphatidylinositolphosphate kinase (PIP kinase) and PLC from insect cells (22) and different phosphoinositide phosphatases deficient in patients suffering from Lowe’s oculocerebrorenal syndrome (23) . So far, it is not clear whether such preferences have physiological relevance and how association of phosphoinositides with functional pools correlates with fatty acid patterns of the lipids.

The generation of PtdIns(4,5)P2 species with distinct fatty acid compositions would in part be defined by the biosynthetic enzymes. The parent lipid, PtdIns, is synthesized in the endoplasmic reticulum [ER, (24 , 25) ] from cytidinediphospho-diacylglycerol (CDP-DAG) and myo-inositol by phosphatidylinositol synthase (PI synthase) and is distributed to various cellular locations of plant and animal cells by an unknown mechanism possibly involving targeted vesicle flow or the action of lipid transfer proteins (26 , 27) . PtdIns can be sequentially phosphorylated by phosphatidylinositol kinases (PI kinases) and PIP kinases, which occur in numerous isoforms in Arabidopsis, encoded by multigene families (4) . PI kinase and PIP kinase activities have been found associated with various plant subcellular fractions, including the plasma membrane, the actin cytoskeleton, and endomembrane compartments (4 , 5 , 28) , and it can reasonably be concluded that PtdIns(4,5)P2 can be generated from PtdIns in compartmentalized pools.

PIP kinase activity has been shown to increase transiently after application of various stresses in plants and algae (17 , 29 , 30) , and in animal systems differential activation of PIP kinase isoforms associated with different phosphoinositide pools is being discussed as a means of harmonizing diverse physiological roles of PtdIns(4,5)P2 (31 , 32) . Transient increases in the levels of regulatory phospholipids have previously been reported for Arabidopsis plants challenged by osmotic stimulation, including those for PtdIns(4,5)P2 (33 , 34) and phosphatidic acid (PtdOH) (35) . Based on strong correlative evidence, PtdIns(4,5)P2 transiently increasing in Arabidopsis plants with osmotic stress has been suggested to be a substrate for PLC, generating InsP3 and acting as part of the osmotic signaling cascade leading to Ca2+ release from internal stores (33) . Although downstream effects of phosphoinositide signaling with osmotic stress are not well defined in plants, phosphoinositides transiently increasing with hyperosmotic stress clearly represent a physiological pool functionally different from the majority of constitutive phosphoinositide pools of nonchallenged plants.

In this study, PtdIns(4,5)P2, its precursors, and immediate derivatives were chosen for the targeted biochemical characterization of associated fatty acid compositions, because PtdIns(4,5)P2 is a multifunctional signaling component and is well-characterized regarding its stress-inducible and constitutive dynamics (33 , 34) . Whereas a number of studies have characterized polyphosphoinositide molecular species from nonchallenged animal systems (36 37 38 39) , no such information is available for plants. Here we show that PtdIns species with characteristic fatty acyl moieties can supply different physiological pools of polyphosphoinositides that are defined by their constitutive nature or by their stress-inducible dynamics. The data presented indicate a discrete function of highly unsaturated molecular phosphoinositide species in InsP3 production during osmotic stress.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Plant growth and stress treatment
Arabidopsis ecotype Columbia (col-0) plants were grown on soil under exposure to 140 µmol photons m–2 sec–1 of light in an 8 h light and 16 h dark regime. Rosette leaves were harvested at 6 wk and frozen in liquid nitrogen. Plant material from 6–8 plants was combined. Arabidopsis ecotype Columbia (col-0) plants and col-0 plants expressing the human type I inositolpolyphosphate 5-phosphatase [(40) , expression line 2–8 (41) ] destined for stress treatments were grown under sterile conditions in sealed jars on 0.5% Murashige and Skoog medium including modified vitamins (Duchefa) containing 1% (w/w) sucrose and 0.25% (w/w) Gelrite (Roth). After 14 d plants were transferred to hydroponic cultures in liquid media as described (42) . Hydroponic cultures were exposed to 140 µmol photons m–2 sec–1 of light in an 8 h light and 16 h dark regime and continuously aerated. Eight to ten-week-old plants were treated by adding NaCl or sorbitol in final concentrations of 0.4 M and 0.8 M, respectively, to the hydroponic media. Rosette leaves were harvested before treatment and after various periods of stimulation, as indicated in the results section, and immediately frozen in liquid nitrogen. Care was taken to perform experiments over the same day-time period within the light-dark-regime, and not to cross the light-dark transition.

Lipid extraction and biochemical analyses
Plant material was ground under liquid nitrogen to a fine powder. Polyphosphoinositides were extracted from powdered plant material by using an acidic extraction protocol (43) . Lipids were separated by thin-layer-chromatography (TLC) on silica gel plates (Merck) using developing solvents for optimal resolution: for phosphoinositides and PtdOH, CHCl3:CH3OH:NH4OH:H2O [57:50:4:11(v/v/v/v)] (44) ; for PtdCho and PtdEtn, acetone:toluol:water [91:30:7 (v/v/v)] (45) ; for isolating phosphatidylinositol, CHCl3:methyl acetate:isopropanol:CH3OH:0.25% aqueous potassium chloride [25:25:25:10:9 (v/v/v/v/v)] (46) . Lanes with lipid standards (5 µg) run in parallel to biological samples were cut and lipids were visualized in aqueous 10% (w/w) CuSO4 (Sigma, St. Louis, MO, USA) containing 8% H3PO4 (Sigma) and subsequent heating to 180°C. Unstained lipids were located on the remaining parts of the TLC plates, according to standard migration; scraped; redissolved in their respective developing solvents; and dried under N2 flow. Lipids were transmethylated (47) , fatty acid methyl esters dissolved in acetonitril and analyzed using a GC6890 gas chromatograph with flame-ionization detection (Agilent, Böblingen, Germany) fitted with a 30 m x 250 µm DB-23 capillary column (Agilent). Helium flowed as a carrier gas at 1 ml min–1. Samples were injected at 220°C. After 1 min at 150°C, the oven temperature was raised to 200°C at a rate of 8°C min–1, then to 250°C at 25°C min–1, and then kept at 250°C for 6 min. Fatty acids were identified according to authentic standards and by their characteristic mass spectrometric fragmentation patterns (data not shown), and quantified according to internal tripentadecanoic acid standards of known concentration. Variation in fatty acid patterns obtained with material sampled on different days did not exceed that denoted by SD. Due to limiting material in samples representing isolated minor lipids, fatty acids of low abundance may be absent from fatty acid patterns. InsP3 levels were determined using the [3H]InsP3 receptor binding assay system (GE Healthcare) as described previously (29) .


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Arabidopsis polyphosphoinositides are more saturated than PtdIns
Because to date no information is available on the fatty acid make-up of plant polyphosphoinositides, individual lipids were isolated first from nonstressed Arabidopsis plants in order to investigate the constitutive phosphoinositide pool. Current lipid profiling techniques have not been successful in the analysis of plant phosphoinositide molecular species (39) , partially because of the low abundance of the compounds and because of the presence of numerous interfering secondary metabolites in plant extracts. In the present study, interfering soluble metabolites were removed, the lipids were enriched by thin-layer-chromatography, and the fatty acid composition of Arabidopsis polyphosphoinositides was determined by gas chromatography. When individual phospholipids were analyzed for their associated fatty acids, polyphosphoinositides exhibited fatty acid compositions different from those associated with the structural phospholipid, phosphatidylcholine (PtdCho), or with phosphatidylinositol (PtdIns, Fig. 1 ). PtdCho and PtdIns were highly unsaturated, containing up to 65 mol% of 18:2{Delta}9,12 (where x:y{Delta}z is a fatty acid containing x carbons and y double bonds in position z counting from the carboxyl end) and 18:3{Delta}9,12,15 next to 16:0 and some minor fatty acids (Fig. 1A, B ). The degree of unsaturation in both PtdIns4P and PtdIns(4,5)P2 was substantially lower, the main fatty acids were 16:0 and 18:1{Delta}9, which amounted to 70–85 mol% of the total fatty acids, whereas 18:2{Delta}9,12 and 18:3{Delta}9,12,15 summed up to only 10–20 mol% (Fig. 1C, D ).


Figure 1
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  		Figure 1. Fatty acid composition of phospholipids from Arabidopsis rosette leaves. Total lipid extracts were subjected to thin-layer chromatography. Individual phospholipids were isolated, and fatty acids were transmethylated and analyzed by gas chromatography. A) PtdCho; (B) PtdIns; (C) PtdIns4P; (D) PtdIns(4,5)P2. To compare fatty acid compositions, bars represent mol% of total fatty acids present in each lipid and are the means of three independent experiments ± SD. The asterisk represents a significant difference in the levels of a fatty acid associated with PtdIns4P or PtdIns(4,5)P2 from that of the corresponding fatty acid in PtdIns on a Student’s t test (*P<0.05; **P<0.01). Fatty acids are identified as indicated: white, 16:0; light gray, 16:1{Delta}9; diamonds, 18:0; dark gray, 18:1{Delta}9; diagonal stripes, 18:2{Delta}9,12; black, 18:3{Delta}9,12,15.

Highly unsaturated polyphosphoinositides are formed with stress
Arabidopsis plants respond to hyperosmotic stress with the transient formation of PtdIns4P and PtdIns(4,5)P2 (33 , 34) . To test whether stress-inducible phosphoinositides would exhibit fatty acid patterns different from constitutive phosphoinositides, lipid dynamics and composition were analyzed in plants subjected to hyperosmotic stimulation by 0.4 M NaCl as described before (33 , 34) . Dynamic changes in PtdIns(4,5)P2 were analyzed through GC quantification of fatty acids transmethylated from isolated lipids. Using this technique, increases in PtdIns4P (70±5 to 500±250 pmol g–1 fresh weight) and PtdIns(4,5)P2 (25±4 to 60±15 pmol g–1 fresh weight) were observed within 1 h of stimulation (compare total bars in Fig. 2 E, F). When the fatty acid compositions of phosphoinositide species during the period of their increases was determined (Fig. 2 , contributing fatty acids indicated by the bar segments), it became clear that the observed transient increases in PtdIns4P and PtdIns(4,5)P2 were largely due to the appearance of phosphoinositides containing unsaturated fatty acids, which were present only in minor amounts prior to stimulation (Fig. 2B, C, E, F ; bar segments indicating PUFA are diagonal stripes, 18:2{Delta}9,12; black, 18:3{Delta}9,12,15). After 60 min of stimulation, for instance, more than 2/3 of the PtdIns4P present were attributable to species containing 18:2{Delta}9,12 and 18:3{Delta}9,12,15, which are fatty acids close to absent in PtdIns4P from nonstressed plants (Fig. 2E ). Changes in the degree of unsaturation of fatty acids or in the proportion of polyunsaturated fatty acids associated with PtdIns, PtdIns4P, PtdIns(4,5)P2, and with lipid downstream products of the phosphoinositide pathway are summarized in Tables 1 and 2 , respectively. In yeast and Chlamydomonas moewusii, increases in both PtdIns(4,5)P2 and PtdIns(3,5)P2 with osmotic stimulation have been reported (48 49 50) . However, in Arabidopsis the absence of stress-induced PtdIns(3,5)P2 was demonstrated both with cell cultures (34) and intact plants (33) . To verify the identity of the phosphatidylinositol-bisphosphate detected by the GC-based method, a headgroup-selective receptor-binding assay (51) was used, confirming that PtdIns(4,5)P2 was present prior to stimulation and that the observed increase also consisted of PtdIns(4,5)P2 (data not shown), as has been reported by others (33 , 34) .


Figure 2
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  		Figure 2. Polyphosphoinositide species containing unsaturated fatty acids increase transiently during hyperosmotic stress. Arabidopsis plants were grown in hydroponic culture and exposed to media containing 0.4M NaCl. Rosette leaves were harvested at different time points of stimulation, and individual phospholipids were isolated and the associated fatty acids determined. A–C) GC-traces of fatty acids transmethylated from (A) PtdIns, (B) PtdIns4P, and (C) PtdIns(4,5)P2 isolated from plant tissue stimulated for times indicated on the right (min). Only the range for 18-carbon fatty acids is shown. Peaks correspond to fatty acids as indicated at the bottom. Data are from one representative experiment. Arrows indicate linoleic and linolenic acid signals decreasing in PtdIns and increasing in PtdIns4P and PtdIns(4,5)P2. D–H) Inositol-containing phospholipids or major structural lipids were isolated from leaf tissue at different time points of treatment, and the associated fatty acids were determined by gas chromatography. (D) PtdIns; (E) PtdIns4P; (F) PtdIns(4,5)P2; (G) MGDG; (H) PtdCho. Data represent fatty acids quantified in nmol g–1 fresh weight and are the means of 2–5 independent experiments ± SD. The asterisk represents a significant difference in the sum of PUFAs associated with a lipid after stimulation vs. the sum in the same lipid at time zero on a Student’s t test (*P<0.05; **P<0.01). Molar lipid concentrations can be calculated according to molecular structure. Fatty acids are identified bottom to top: white, 16:0; light gray, 16:1{Delta}9; diamonds, 18:0; dark gray, 18:1{Delta}9; diagonal stripes, 18:2{Delta}9,12; black, 18:3{Delta}9,12,15.


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  		Table 1. Degree of unsaturation of fatty acids in phosphoinositides and derived lipids during hyperosmotic stress.


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  		Table 2. Proportion of polyunsaturated fatty acids (18:2{Delta}9,12 and 18:3{Delta}9,12,15) of the total fatty acids associated with phospholipids during hyperosmotic stress.

Unsaturated PtdIns transiently depletes with hyperosmotic stress
Because the fatty acid composition of PtdIns4P and PtdIns(4,5)P2 formed on hyperosmotic stimulation resembled that of the highly unsaturated PtdIns pool accumulating under nonchallenging conditions, we asked whether the amount and fatty acid composition of PtdIns would change accordingly with application of the stress. Under conditions of hyperosmotic stress, a fast transient decrease in PtdIns was observed (Fig. 2A, D ), consistent with selective turnover of unsaturated PtdIns containing 18:2{Delta}9,12 or 18:3{Delta}9,12,15. The dynamics of PtdIns, PtdIns4P, and PtdIns(4,5)P2 were similar whether plants were stressed by the addition of 0.4 M NaCl or of 0.8 M sorbitol (data not shown), indicating the stress was osmotic rather than a specific effect of the addition of NaCl or sorbitol to the media. At this point we have no data on changes in phosphoinositide fatty acid composition induced by other stresses.

Phosphoinositide changes occur prior to global changes in lipid unsaturation with hyperosmotic stress
To investigate whether global changes in lipid unsaturation after hyperosmotic stress could be observed in parallel with those described for phosphoinositides, two major structural lipids, monogalactosyldiacylglycerol (MGDG) and PtdCho, were also analyzed (Fig. 2G, H ). In both cases hyperosmotic stress resulted in substantial changes in lipid amounts and the degree of lipid unsaturation. The levels of the predominantly plastidial galactolipid MGDG decreased after 30 min of hyperosmotic stress, concomitant with a decreased proportion of associated 18:2{Delta}9,12 and 18:3{Delta}9,12,15 (Fig. 2G , shown as diagonal stripes and black bar segments, respectively). PtdCho levels did not significantly change until 60 min of stimulation, after which a substantial increase in PtdOH was observed (Fig. 2H , shown as diagonal stripes and black bar segments, respectively). The increase in PtdOH was accompanied by an increased proportion of associated 18:2{Delta}9,12 and 18:3{Delta}9,12,15 (Fig. 2H ). Changes in PtdOH levels and fatty acid composition occurred, thus, later than those observed for phosphoinositides in the identical samples.

Stress-induced, unsaturated polyphosphoinositides support InsP3-production
While temporally correlated, the increases in PtdIns4P and PtdIns(4,5)P2 do not balance the decrease in PtdIns (compare scales of Figs. 2D-F ). A possible explanation for the mass imbalance is that pathway intermediates are not statically accumulating but rather are subject to continuous flux toward other downstream metabolites of the phosphoinositide pathway. Because expression of Arabidopsis PLC1 is induced by salt (52) and InsP3 release with osmotic stress has been described for various plant systems (17 , 33 , 53 , 54) , we asked whether some of the unsaturated PtdIns(4,5)P2 formed with osmotic stress would turn toward hydrolysis by PLC, yielding InsP3 and DAG. In plants, DAG is immediately phosphorylated to PtdOH by the enzyme DAG kinase (55) . Formation of the PLC products InsP3, DAG, and PtdOH is shown in Fig. 3 A–C. While DAG levels did not change significantly (Fig. 3B ), PtdOH levels increased temporally correlated to the production of InsP3 after osmotic stress (Fig. 3C ). Importantly, the raised PtdOH levels were due to an increased proportion of unsaturated PtdOH containing 18:2{Delta}9,12 and 18:3{Delta}9,12,15 (shown as diagonal stripes and black bar segments, respectively), consistent with an origin in the hydrolysis of unsaturated PtdIns(4,5)P2 transiently formed with osmotic stress.


Figure 3
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  		Figure 3. Changes in downstream metabolites of the phosphoinositide pathway during hyperosmotic stress. A) Rosette leaves were harvested at different time points of hyperosmotic stimulation and analyzed for the levels of the soluble second messenger InsP3. B–C) Downstream lipid products of the phosphoinositide pathway were isolated and the associated fatty acids determined by gas chromatography. B) DAG; (C) PtdOH. Molar lipid concentrations can be calculated according to molecular structure. Data represent fatty acids quantified in nmol g–1 fresh weight and are the means of three independent experiments ± SD. The asterisk represents a significant difference in the sum of PUFAs associated with a lipid after stimulation vs. the sum in the same lipid at time zero on a Student’s t test (*P<0.05; **P<0.01). Fatty acids are identified bottom to top: white, 16:0; light gray, 16:1{Delta}9; diamonds, 18:0; dark gray, 18:1{Delta} (9) ; diagonal stripes, 18:2{Delta}9,12; black, 18:3{Delta}9,12,15

Forced InsP3 breakdown increases PLC hydrolysis of unsaturated polyphosphoinositides in stressed plants
Because PtdOH could also stem from other sources, and to gain more than merely correlative evidence, we asked whether selective perturbation of the phosphoinositide pathway would affect the predicted flux of the unsaturated lipid backbones toward DAG and PtdOH. Perturbation was achieved by the expression of a human type I inositolpolyphosphate 5-phosphatase [InsP 5-ptase (40) ], which specifically hydrolyzes InsP3. The removal of InsP3 affects upstream phosphoinositide intermediates via a "pull" mechanism (41 , 56) , resulting in reduced levels of PtdIns4P and PtdIns(4,5)P2 (56) , the latter of which is constantly hydrolyzed by PLC. Downstream of the phosphoinositide pathway, increased phosphoinositide turnover should lead to enhanced accumulation of DAG and PtdOH compared to wild type levels. Changes in PtdIns, PtdIns4P, PtdIns(4,5)P2, InsP3, DAG and PtdOH in InsP 5-ptase plants exposed to hyperosmotic stress are shown in Fig. 4 . In the InsP 5-ptase transgenic plants the levels of PtdIns, PtdIns4P, PtdIns(4,5)P2 and InsP3 were all reduced (Fig. 4A-D ), whereas lipid products downstream of PLC were accumulating to levels increased over those seen with wild-type plants (Fig. 4E, F ). As with the wild type plants, the increases in DAG and PtdOH were predominantly due to the formation of unsaturated molecular species of these lipids [diagonal stripes, 18:2{Delta}9,12; black, 18:3{Delta}9,12,15]. Effects of InsP 5-ptase expression on other structural phospholipids have not been described previously, and we observed no change in the levels of PtdCho; whereas the levels of PtdEtn were somewhat reduced compared to those of wild-type plants (data not shown).


Figure 4
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  		Figure 4. Altered phosphoinositide metabolism in Arabidopsis plants expressing the human type I inositolpolyphosphate 5-phosphatase. Transgenic plants were exposed to hyperosmotic stress as described for wild-type plants. Plant material harvested at different times of stimulation was analyzed for levels and composition of inositol-phospholipids (A–C), InsP3 levels (D), and for levels and composition of downstream metabolites of the phosphoinositide pathway (E, F). A) PtdIns; (B) PtdIns4P; (C) PtdIns(4,5)P2; (D) InsP3; (E) DAG; (F) PtdOH. Molar lipid concentrations can be calculated according to molecular structure. Data represent fatty acids quantified in nmol g–1 fresh weight and are the means of two independent experiments ± SD. The asterisk represents a significant difference in the sum of PUFAs associated with a lipid after stimulation vs. the sum in the same lipid at time zero on a Student’s t test (differences in total InsP3 levels in (D); *P<0.05; **P<0.01). In lipid samples, fatty acids are identified bottom to top: white, 16:0; light gray, 16:1{Delta}9; diamonds, 18:0; dark gray, 18:1{Delta}9; diagonal stripes, 18:2{Delta}9,12; black, 18:3{Delta}9,12,15


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Experiments presented in this study were designed to further the understanding how independent functional pools of polyphosphoinositides may be established and maintained. The finding that in nonchallenged Arabidopsis leaves PtdIns exhibited a different fatty acid composition than the polyphosphoinositides, PtdIns4P, and PtdIns(4,5)P2 (Fig. 1) was unexpected at first, because PtdIns gives rise to polyphosphoinositides by headgroup phosphorylation that would not alter the fatty acid moieties of the products. The data indicate that the unsaturated PtdIns accumulating in resting plants was not the precursor of the more saturated polyphosphoinositides present at the same time. Intact PtdIns species from Arabidopsis have been reported to contain 34:2 and 34:3 fatty acid combinations (57) , which is consistent with data presented here (Fig. 1B ) and translates to a composition exclusively of fatty acid combinations of 16:0/18:2 and 16:0/18:3, respectively. Due to the demonstrated promiscuity of PI synthase regarding various CDP-DAG species (58) , however, the fatty acid composition of newly synthesized PtdIns would be expected to reflect that of the cell’s DAG pool and represent a more diverse mixture of molecular species. Thus, on one hand, the PtdIns fatty acid pattern was different from patterns found in DAG or other structural phospholipids, such as PtdCho, where 18:2/18:3 and 18:3/18:3 combinations occur (57) . On the other hand, the PtdIns pattern also differed substantially from the patterns found in PtdIns4P and PtdIns(4,5)P2 (Fig. 1C, D ), which carried more saturated fatty acids. An equivalent pattern has been observed in nonchallenged mouse RAW 264.7 cells, where very long chain polyunsaturated fatty acids were found in PtdIns, but not in derived polyphosphoinositides (38) . A possible explanation for the observed patterns is that a diverse mixture of PtdIns molecular species is drawn on to generate two different pools of polyphosphoinositides, according to fatty acid composition: Under nonchallenging conditions saturated and monounsaturated PtdIns species are used to produce the corresponding species of polyphosphoinositides with constitutive regulatory functions. In this scenario, a pool of highly unsaturated PtdIns may accumulate, which is primed to respond to stress and represents the PtdIns molecular species observed in nonchallenged cells. Between 15 and 30 min of hyperosmotic stress, the primed, highly unsaturated PtdIns is turned over (Fig. 2) , leading to a transient decrease in PtdIns levels and to concomitant formation of highly unsaturated polyphosphoinositides, as is shown in this study (Fig. 2 ; Tables 1 , 2 ). The complex, and at first unintuitive, fatty acid patterns observed may, thus, be explained by the outlined concept of PtdIns partitioning into constitutive and stress-inducible lipid pools. Considering that PtdIns is formed in the ER (24 , 25) , it is possible that PtdIns species with certain fatty acid compositions are retained in the ER, whereas others are delivered to various subcellular locations, including the plasma membrane, the Golgi, or the nucleus, where stress-inducible or developmentally controlled PI kinases and PIP kinases may produce polyphosphoinositides with different regulatory effects. After 60 min of hyperosmotic stress, PtdIns has recovered to starting levels (Fig. 2D ), suggesting increased resynthesis of this lipid or action of polyphosphoinositide phosphatases. As concomitant with the PtdIns increase, at 60 min PtdIns4P accumulates (Fig. 2E ) and only PtdIns(4,5)P2 decreases (Fig. 2F ), phosphatase action appears unlikely. The pattern suggests inactivation of a stress-induced PIP kinase, perhaps via a feedback loop involving termination of the InsP3 signal, and consequential accumulation of the precursor lipids, PtdIns and PtdIns4P.

Hyperosmotic stress results in global changes in lipid unsaturation in eukaryotic cells, and increases in lipid unsaturation are required to maintain membrane integrity (59) . The elevated degree of unsaturation observed for PtdCho after 60 min of hyperosmotic stress (Fig. 2H ) may be part of such a survival mechanism. Note that prior to the changes in PtdCho, MGDG unsaturation in the plastid decreases (Fig. 2G ), which suggests recycling of PUFAs from plastidial MGDG to support unsaturation of PtdCho in the plasma membrane. Changes in phosphoinositide unsaturation with hyperosmotic stress occur earlier than those of other, structural lipids, and, therefore, may not be a result of changes in global lipid unsaturation.

The transient decrease in PtdIns with stress treatment observed in this study is contrasted by a previous report of no PtdIns decrease with stress (57) . Possible explanations include the transient nature of the changes or differences in the stresses applied or the experimental setups. Slight differences in fatty acid composition of PtdIns from nonchallenged plants grown under different conditions (compare Fig. 1B with Fig. 2D ) are likely the result of growth on soil vs. hydroponic culture, which poses a stress to the plants by itself and may cause some turnover of unsaturated PtdIns.

To test whether increased polyphosphoinositide unsaturation (Fig. 2 , Tables 1 , 2 ) during the initial phase of adaptation to hyperosmotic stress was a consequence of fatty acid desaturase action, plant material was harvested over the critical period of osmotic stimulation, total lipid extracts were prepared, and the proportions of the associated fatty acids determined. The amounts of 18:2{Delta}9,12 and 18:3{Delta}9,12,15 transmethylated from the total lipid extracts did not change substantially over 60 min of application of the stress (data not shown), indicating that no net increase in unsaturated fatty acids had occurred. Because the contribution of phosphoinositides to the total phospholipids is only small, specific phosphoinositide desaturation would not be apparent and cannot be ruled out. However, to date no fatty acid desaturases have been reported to have exclusive preference for fatty acids esterified to polyphosphoinositides. The fatty acid composition of total lipid extracts did not substantially change, suggesting fatty acid desaturases were not responsible for the increased degree of polyphosphoinositide unsaturation after osmotic stimulation.

Previous reports of increased phosphorylation of PtdIns and of PtdIns4P with hyperosmotic stress (33 , 34) , as well as the data presented here, suggest that the increase in polyphosphoinositides likely originated in stress-induced activation of PI kinases and PIP kinases, which generate PtdIns4P and PtdIns(4,5)P2, respectively, from primed, unsaturated PtdIns. Preferences of recombinant enzymes toward phosphoinositide substrates with defined fatty acid compositions have been demonstrated in vitro for insect phosphoinositide kinases (22) and for human phosphoinositide phosphatases (23) , suggesting that the fatty acid composition of the phosphoinositide substrate is a modulator of enzymes regulating cellular phosphoinositide production (22) and/or distribution. With respect to physiology, PtdIns pools with different fatty acid compositions may be phosphorylated in response to different endogenous or exogenous cues by different sets of lipid kinases acting on their respective preferred saturated or unsaturated phosphoinositide substrates.

The transient nature of the increases in unsaturated polyphosphoinositides raises the question as to what physiological role the transient phosphoinositide increases may have in stress adaptation. Here we aimed to decide whether transiently formed PtdIns(4,5)P2 would remain intact, serving as a ligand to potential protein partners, or whether the lipid would turn toward hydrolysis by PLC and InsP3 production. Our results with wild-type Arabidopsis and the transgenic plants expressing the human InsP 5-ptase indicate the latter scenario (Figs. 3 , 4) . Arguments in support of this interpretation include: (A) the increase in PtdIns(4,5)P2 was temporally correlated with a concomitant increase in InsP3; (B) the downstream lipid products of PLC action, DAG and PtdOH, accumulated in parallel with InsP3 production; (C) the increases in DAG and PtdOH were largely due to increased formation of highly unsaturated molecular species of these lipids, consistent with their origin in the unsaturated PtdIns(4,5)P2 increasing with osmotic stress; and (D) the effects of osmotic stimulation on DAG and PtdOH formation and composition were increased in the transgenic Arabidopsis plants expressing the human InsP 5-ptase, for which increased phosphoinositide turnover by PLC has been previously reported (41 , 56) . While PtdOH can be formed from all glycerophospholipids by action of phospholipase D (60) , the patterns observed here indicate that PtdOH formed with hyperosmotic stress in Arabidopsis is likely a consequence of combined action of PLC and DAG kinase.

The apparent channeling of unsaturated phosphoinositides through a pathway for InsP3 production leads us to speculate how the unsaturated lipids could be compartmentalized. Despite a generally smaller proportion in glycerophospholipids in sphingolipid- and sterol-enriched lipid microdomains ("lipid rafts") (61) , phosphoinositides are thought to be present in raft-like microdomains in mammalian membranes (62 , 63) . Unsaturated acyl groups associated with a phosphoinositide will compensate the shape of a large, charged headgroup and result in a more cylindrical, bilayer-forming lipid, whereas saturated acyl chains will create an inversely cone-shaped lipid (64 , 65) that generates membrane curvature strain (64 65 66) . Such differences in hydrophobic tails alone have been shown in animal cells to be sufficient for differential lateral distribution and accumulation of lipids in membrane microdomains without action of accessory proteins (67) . The data presented here indicate concerted, stress-inducible turnover of unsaturated phosphoinositides through a pathway defined by PI kinase, PIP kinase, PLC, and DAG kinase. Tobacco homologs of all enzymes of this postulated pathway have been found to be enriched in isolated detergent-insoluble membrane preparations ("lipid-rafts") from tobacco cell plasma membranes resembling microdomains (F. Furt and S. Mongrand, personal communication). Differences in phosphoinositide fatty acid composition, such as those described in this study, may restrict certain phosphoinositide species to raft-like microdomains or preclude others from forming microdomains in Arabidopsis. It remains to be seen whether on hyperosmotic stress the respective enzymes may also be found in the detergent-soluble fraction of the plasma membrane, or whether the pathway is restricted to rafts.

In summary, an attempt was made to disentangle the complex regulatory network of polyphosphoinositides by comparing distinct phosphoinositide pools that are constitutively present in Arabidopsis leaves with those transiently formed during conditions of stress. Constitutive and inducible phosphoinositide pools consist of lipids with distinct fatty acid compositions, and likely differ in their biophysical properties. Tracing of the unsaturated lipid backbones of intermediates derived from stress-induced phosphoinositides indicates PLC-mediated hydrolysis of this signaling pool, leading to production of the second messenger, InsP3. Future refinements in mass-spectrometric analysis may provide tools to monitor some phosphoinositide pools in complex metabolite mixtures based on different molecular composition in order to elucidate physiological functions of individual pools not only for PtdIns(4,5)P2, but also for the multitude of other known polyphosphoinositides.


   ACKNOWLEDGMENTS
 
The transgenic Arabidopsis plants expressing the human type I inositolpolyphosphate 5-phosphatase were kindly provided by Dr. Imara Perera (North Carolina State University, Raleigh, NC, USA). We thank Dr. Ivo Feussner (Georg-August-University Göttingen, Germany) and Dr. Imara Perera and Dr. Wendy Boss (North Carolina State University, Raleigh, NC, USA) for their helpful discussion. We are grateful for generous funding through an Emmy Noether Fellowship from the German Research Foundation (D. F. G., to I.H.).

Received for publication December 15, 2006. Accepted for publication January 25, 2007.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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