Plant Physiol. (1988) 87, 217-2220032-0889/88/87/0217/06/$0l .00/0

Inositol-Containing Lipids in Suspension-Cultured Plant CellsAN ISOTOPIC STUDY

Received for publication August 18, 1987 and in revised form December 18, 1987

BJ0RN K. DR0BAK*l, IAN B. FERGUSON, ALAN P. DAWSON, AND ROBIN F. IRVINEDepartment of Soil, Water and Plant Nutrition, Royal Veterinary-and Agricultural University,Thorvaldsensvej 40, 1871 Copenhagen C., Denmark (B.K.D.); Division of Horticulture and Processing,D.S.I.R., Private Bag, Auckland, New Zealand (I.B.F.); School of Biological Sciences, University of EastAnglia, Norwich NR4 7TJ, Great Britain (A.P.D.); and Institute of Animal Physiology, AFRC,Babraham, Cambridge CB2 4AT, Great Britain (R.F.I.)

ABSTRACT

Polar lipids were extracted from suspension-cultured tomato (Lycoper-sicon esculentum Mill.) cells and analyzed by thin layer chromatography.Four major inositol-containing compounds were found, and incorporationof [32P]orthosphosphate, [2-3lHlglycerol, and myo-[2-3Hlinositol was stud-ied. Results showed that phosphatidylinositol-monophosphate is the phos-pholipid in these cells displaying the most rapid incorporation of[32P]orthophosphate. We suggest that the tracer is incorporated primarilyinto the phosphomonoester group. Two inositol-containing lipids showedchromatographic behavior similar to phosphatidylinositol-4,5-bisphos-phate when using standard thin layer chromatography techniques. Thelabeling pattern of these compounds, however, reveals that it is unlikelythat either of these is identical to phosphatidylinositol-4,5-bisphosphate.Should phosphatidylinositol-bisphosphate be present in suspension cul-tured plant cells, our data indicate chemical abundancies substantiallylower than previously reported.

A role for Ca2+ as a messenger in signal-response coupling inmany cellular events has for a number of years been inferred inboth animal and plant research. It has thus been suggested thatextracellular stimuli upon interaction with the cell surface inducean increase in cytosolic Ca2+ activities and in this way are trans-lated into cellular reactions. The exact mechanisms responsiblefor such changes in the Ca2+ flux have, until recently, largelyremained obscure.

In animal tissues, binding of agonists to membrane-associatedreceptors has been shown to induce phosphodiesteratic cleavageof PI-4,5-P2,2 a diphosphorylated species of PI (2). This givesrise to the production ofDG and Ins-1,4,5-P3. While DG remainsin the membrane matrix and modulates the activity of proteinkinase C, Ins-1,4,5-P3 is released into the cytosol, triggering re-lease of Ca2+ from internal stores. The ensuing increase in cy-tosolic Ca2+ activity leads to a cascade of metabolic events, de-pendent on either the Ca2+ ion itself or on Ca:Ca2+-binding

' Present address: John Innes Institute, Colney Lane, Norwich NR47UH, Great Britain.

2Abbreviations: PI-4,5-P2, phosphatidylinositol-4,5-bisphosphate, PIP,phosphatidylinositol-monophosphate; PI-4-P, phosphatidylinositol-4-monophosphate; PIP2, phosphatidylinositol-bisphosphate; PI, phospha-tidylinositol; Ins-1,4,5-P3, myo-inositol-1,4,5-trisphosphate; DG, 1,2-diacylglycerol; PL, total phospholipid; PE, phosphatidylethanolamine;PC, phosphatidylcholine; PG, phosphatidylglycerol.

protein complexes, such as Ca:calmodulin.The discovery of the phosphoinositide system in animals has

naturally led to the question of whether a similar system mightbe functioning in plants.Some information is available which could suggest at least a

structural presence of such a system in plants. Early work (17)on phytic acid (inositol-hexakisphosphate) biosynthesis has dem-onstrated the presence of inositol phosphates of all phosphoryl-ation steps (i.e. inositol-monophosphate to inositol-hexakis-phosphate) in plant extracts, and more recent results (12, 18)have confirmed the presence of a number of these. In most cases,the isomerism of the extracted inositol phosphates has not beendetermined, and the Ins-1,4,5,-P3 isomer has as yet not beendemonstrated in plant extracts. Work carried out using Ins-1,4,5-P3 isolated from animal tissues nevertheless points towards apotential physiological role of this isomer in plants. Thus, Ins-1,4,5-P3 has been demonstrated to release Ca2+ from microsomesisolated from zucchini hypocotyls (7), corn coleoptiles (21), andoat roots (26) and to affect Ca2 + fluxes across plasma membranesin isolated carrot protoplasts (23). Other intrinsic features of theanimal phosphoinositide system find parallels in plants includinglow cytoplasmic Ca2+ activities, presence of Ca2 +-binding pro-teins with regulatory properties, and Ca2 + and Ca2 + -calmodulin-dependent protein kinases (1). Protein kinases bearing someresemblance to the animal protein kinase C have recently beenreported to be present in various plant tissues (8, 19, 25). How-ever, it remains to be shown that hydrolysis of phosphoinositidesin plants occurs in response to external stimuli.

Agonist-induced hydrolysis of PI-4,5-P2 in animal systems ismediated by a phospholipase(s) C (phosphoinositidase C). Al-though phospholipase C (PLC) has not received much attentionin plant research, three recent reports (6, 11, 14) contain evi-dence for PLC activity in plant cells with defined activity towardsphosphatidylinositol. The presence of PI as a major constituentof plant membranes is well established, and recently the presenceof other inositol-containing phospholipids, tentatively identifiedas PIP and PIP2 has been reported (4, 10, 27). In this study wedescribe the incorporation of various radioactive tracers intoinositol-containing phospholipids in suspension-cultured tomatoleaf cells. The data are interpreted with regard to the turnoverand chemical structure of these lipids.

MATERIALS AND METHODS

Materials. myo-[2-3H]Inositol, [2-3H]glycerol, and [32P]ortho-phosphate were obtained from Amersham, U.K.; Cellulose MN300 HR was from Macherey Nagel, Duren, West Germany; andsilica gel H was from Sigma. PI-4-P and PI-4,5-P2 were ex bovine

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Plant Physiol. Vol. 87, 1988

brain and obtained from Sigma. All chemicals and solvents usedwere of analytical grade.

Labeling of Cells. Tomato (Lycopersicon esculentum Mill.)cells were maintained in solution culture in a standard Murashige& Skoog medium (9) with 9 AM 2,4-dichlorophenoxyacetic acidand 5 AM 2-isopentenyl adenine. Cells were subcultured weeklyand grown in 50 ml of medium in 250 ml conical flasks at 27°Cunder low light (Crompton 40 W cool-white, 50,mol s-I m-2)on a rotary shaker (100 rpm). On day 2 after subculturing,[32P]orthophosphate (total activity 50,Ci) was sterilely addedto each culture together with either myo-[2-3H]inositol (totalactivity 40,Ci) or [2-3H]glycerol (total activity 50,Ci). Cellswere incubated as described above until extraction.

Extraction of Lipids. After various incubation times, approx-

imately 0.2 to 0.5 g fresh weight of cells were subsampled fromcultures under sterile conditions. The culturing medium was re-

moved by rapid filtration on glass fiber filters. The cells were

transferred to a cooled Kondes glass grinder containing 10 ml ofice-cold chloroform/methanol/concentrated HCl (100:150:1,v/v/v). The cells were homogenized for approximately 30 sec,

and 0.6 N HC1 was added to the homogenate to create a two-phase system. The upper acidified phase was removed and dis-carded, and the bottom phase was washed twice with aliquots of4 ml chloroform/methanol/0.6 N HCl, 3:48:47 (v/v/v). The re-

sulting extract was centrifuged at 2000g for 5 min to remove

curd-like material. The lipid-containing phase was recovered andtransferred to 2 ml glass vials after weighing and sampling of 20,ul aliquots for monitoring of radioactivity. The lipid-containingextract was evaporated under a stream of nitrogen. The driedlipid samples were reconstituted in chloroform/methanol/H20(75:25:2, v/v/v) and either used immediately for analysis by TLCor stored at - 25°C until use, after flushing of the vials with N2and sealing of the tubes.

Thin Layer Chromatography. Thin layer chromatography was

performed on TLC plates containing either silica gel H (SH-plates) or a mixture of silica gel H and cellulose (MX-plates,silica gel H/cellulose: 5:2 [w/w]). SH-plates were used for iden-tification purposes exclusively, whereas MX-plates were em-

ployed for both identification and assessment of incorporatedradioactivity. Both layer types were spread on 200 x 200 x 4 mmglass plates to a thickness of 250,um after inclusion of 1% K-oxalate (w/v) and 2 mM EDTA in the silica gel slurry. Plateswere activated at 110°C for 45 min before use. After spottingof the labeled lipid samples, plates were developed in one di-mension in chloroform/acetone/methanol/acetic acid/H20 (40:15:13:12:8; v/v/v/v/v). Lipids were visualized by exposure toI2vapor and radioactive compounds by autoradiography. After de-velopment of autoradiograms, TLC plates were treated with Strip-mix as described by Redgwell et al. (22). Radioactive spots were

identified, cut from the plates, and transferred to scintillationvials. Radioactivity was determined by liquid scintillation spec-

trophotometry (Beckmann LS 2800) using ASC II (Amersham)scintillation fluid.

Presentation of Data. Incorporation data are presented relativeto total incorporation of tracer into total extractable phospho-lipids (PL). We feel that this type of representation best illus-trates the relative turnover rates within the lipid pool.

RESULTS

When expressed on a unit cell fresh weight basis, maximumincorporation of [32P]orthophosphate and [2-3H]glycerol into lipid-extractable compounds was achieved in the time intervals of 24to 48 h and 2 to 24 h, respectively (data not shown). Figure 1shows a typical autoradiogram of phospholipids labeled with[32P]orthophosphate for 1.2 h, extracted as described under "Ma-terials and Methods" and separated on MX-plates. PE, PG, PC,and phosphatidylinositol were identified by co-chromatography

SS-

S

.59-

FIG. 1. Autoradiogram of phospholipids extracted from tomato leafcells labeled for 1.2 h with 32Pi and separated on MX-plates as describedunder "Materials and Methods." The origin is denoted by spot 1. Com-pound 6 is PI, and 7 is PC. PE and PG are contained in spot 8. Spot 2has tentatively been identified as Pi. Inositol-containing compounds otherthan PI are numbered 3 to 5.

with authentic standards, by use of phosphate- and amino group-specific sprays (16) and by reference to published RF values forthese compounds in the solvent system employed.

Initial studies showed, in addition to PI, the presence of threemajor phosphate-containing compounds incorporating myo-[2-3H]inositol. These are indicated as compounds 3, 4, and 5, re-spectively. That no other major compounds incorporating myo-[2-3H]inositol exist in these extracts has been shown by assay ofradioactivity arising from this tracer in sequentially cut/scrapedTLC-lanes and by fluorography of TLC-separations containingmyo-[2-3H]inositol-labeled compounds exclusively (data notshown).

Incorporation of [32P]Orthophosphate. Figures 2 to 5 show therelative incorporation of 32Pi into PI and compounds 3 to 5, asexpressed on the basis of total incorporation into phospholipids.As can be seen (Fig. 2), incorporation of label into PI amountsto 14 to 19% after short labeling times (0.5-2h) followed by asmall drop to about 9% in the period from 24 to 144 h. Compound5 incorporated 32Pi extremely rapidly, as can be seen from Figure3. After 30 min incubation, it was the heaviest labeled of allphospholipids-in some experiments containing as much as 40%of total extracted activity. Similar results were obtained whentracer was added later in the cell cycle (data not shown). Therelative level drops rapidly during the first 24 h of the labelingperiod to around 2% of total PL-32P. In contrast, 32Pi incorpo-ration into compounds 3 and 4 is much slower, as evident from

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INOSITOL-CONTAINING LIPIDS IN CULTURED PLANT CELLS

-I0.

co

0.

0ow0

CL

('4CO)

20 1- __

10 F T-II I U I%v I I I I I II

0.5 1.2 2.0 24 48 72 96 120 144

time (hours)

FIG. 2. Incorporation of 32Pi into PI in tomato leaf cells after varioustimes of incubation. Results are expressed as a percentage of total 32Pincorporated into phospholipids. (Bars represent ± SE, n = 6).

Figures 4 and 5. Compound 4 contains 1.2 to 1.5% of total PL-32P after short labeling times, approaching a steady level of justunder 1% after 24 h. At the earliest sampling time (30 min),large variations were found in label incorporated into compound3 (Fig. 5). The mean is 1.2% and thus comparable to the relativeincorporation figures obtained after longer incubation times.

Incorporation of[2-3H]Glycerol. The incorporation of [2-3H]gly-cerol into PI and compounds 4 and 5 relative to incorporationinto total phospholipid is shown in Figure 6. Data for [2-3H]glycerolincorporation into compound 3 are not shown as very low levelsof label were recovered (<0.03% of total [3H]glycerol-PL). Verylittle change in relative content of [2-3H]glycerol of PI and com-pounds 4 and 5 is observed after the initial incorporation (0-2h). The relative levels of incorporated [2-3H]glycerol after 48 hwere 8 to 10% for PI, 1.1% for compound 5, and 0.04 to 0.06%for compound 4.

Incorporation of myo-[2-3H]inositol. The total incorporation ofmyo-[2-3H]inositol into the inositol-containing lipids was foundto be low after short labeling times when using the specific activityas described under "Materials and Methods." This is likely tobe due to a decrease in specific activity imposed by the largeamounts of unlabeled myo-inositol present in the cell culturemedium (0.56 mM). In all instances where myo-[2-3H]inositollabeling was performed, the cells were incubated for 3 d beforeextraction. Table I shows the relative incorporation of myo-[2-3H]inositol into PI and compounds 3, 4, and 5, together withdata on [32P]Pi:myo-[2-3H]inositol and [32P]Pi:[2-3H]glycerol ra-tios. (Details of labeling and expression of results are given inthe table legend.)

Identification of Putative Phosphoinositides by Co-chromatog-raphy with Authentic Standards. Compound 5 showed exact co-chromatography with authentic PI-4-P standards on both SH-and MX-plates. Authentic PI-4,5-P2 did not show chromato-graphic behavior identical to any of the inositol-containing com-pounds, although it was found to have RF values comparable tocompounds 3 and 4. In many cases, less clear separation of spots3 and 4 was obtained when standard PI-4,5-P2 was chromato-

30 F

-I

0.c.IL00CO

doe

0SU0.%wJ

20 F

10 -

=l~~~~~~~~~~~~~~A0.5 1.2 2.0 24 48 72 96 120 144

time (hours)FIG. 3. Incorporation of 32Pi into compound 5 in tomato leaf cells after

various times of incubation. Results are expressed as a percentage of total32P incorporated into phospholipids. (Bars represent + SE, n = 6).

graphed together with extracted 32Pi-labeled samples, but it wouldappear that the PI-4,5-P2 standards traveled as fast as, or slightlyin front of, spot 4. Authentic LysoPI was found to have a mobilitybetween spot 4 and PIP and did not correspond to any of thelabeled compounds.

DISCUSSION

Incorporation of 32Pi into the diester-phosphate linkage of PIoccurs in the cytidinediphosphate-diacylglycerol biosyntheticpathway. The incorporated phosphate originates from phospha-tidic acid, glycerol-3-phosphate, and ultimately from [y-32P]ATP.It is known that 32Pi is incorporated into ATP very rapidly inplant cells (3), so the rate of incorporation into PI is likely todepend mainly on (a) the rate of PI-turnover and (b) uptake oftracer by the cell. The incorporation of glycerol follows the samebiosynthetic route, initiated by phosphorylation of glycerol toglycerol-3-phosphate. Two mechanisms for myo-inositol incor-poration into PI are known. First, this can be mediated by cy-tidinediphosphate:diglyceride inositol phosphatidate transferaseand second, by a Mn2+-dependent inositol headgroup exchange.Only the first reaction has at present been characterized in detailin vivo. The biosynthesis of the polyphosphoinositides proceedsby a sequential phosphorylation of PI using ATP as phosphatedonor. The incorporation rate of tracer into PI appears to becomparable to that of the other major phospholipids. Calcula-tions based on analysis of Pi in PI indicate chemical levels of 12

I

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Plant Physiol. Vol. 87, 1988

00%

-I

A.

AIL

cm

0CV)0

0

2.0H

1.0 -

I

I I ' X I I I I I II

0.5 1.2 2.0 24 48 72 96 120 144

time (hours)FIG. 4. Incorporation of 32pi into compound 4 in tomato leaf cells after

various times of incubation. Results are expressed as a percentage of total32P incorporated into phospholipids. (Bars represent ± SE, n = 6).

-IILC0

M0o

0

CM

0

2.0

4

1.0 --

I I I I _s~I i I a I I

0.5 1.2 2.0 24 48 72 96 120 144

time (hours)

FIG. 5. Incorporation of 32Pi into compound 3 in tomato leaf cells aftervarious times of incubation. Results are expressed as a percentage of total32P incorporated into phospholipids. (Bars represent + SE, n = 6).

to 14% of total PL equalling approximately 0.4 nmol (mg cellfresh weight) -1 32P-Labeling data after 48 h are consistent withchemical abundancies of PI of around 10 to 13% of total PL,which may suggest that the diesterphosphate has reached isotopicequilibrium by this time. A similar pattern was found for incor-poration of [3H]glycerol into PI. Incorporation after 48 h suggestschemical amounts of 8 to 10% of total PL. After very longlabeling times (i.e. 144 h), the percentage label of incorporated[2-3H]glycerol in PI was found to drop to around 5%. A smallerdrop was registered in relative levels of 32P in PI at similar in-

t

15

IJ0ft

10

5

3

- 74 jA 4z-- > 2

0.5 1.2 2.0 24 48 72 96 120 144

-0.3

-0.2

-0.1

time (hours)

FIG. 6. Incorporation of 3H-glycerol into compounds 4, 5, and PI.Results are expressed as a percentage of total 3H incorporated into totalextractable phospholipids. (U), PI; (A), compound 5; (0). compound4. (Bars represent + SE, n = 4).

cubation times. It is possible that a small decrease in chemicallevels of PI occurs in the late stages of the growth cycle accom-panied by an increase in triacylglycerols-a situation which wouldparallel findings from cultured algal cells (29). PI was, as ex-pected, found to be the major inositol-containing phospholipidin the cells, containing 80% of the incorporated myo-[2-3H]inositolafter incubation for 3 d.Chromatographic data suggest that compound 5 could be PIP.

Several authors have presented evidence for the presence ofinositol-containing phospholipids extracted from various planttissues, which in both one- and two-dimensional TLC-systemsare chromatographically indistinguishable from standard PI-4-Pand PI-4,5-P2 (4, 10, 27). In this study, we found exact co-chro-matography of compound 5 and standard PI-4-P employing bothSH- and MX-plates. Compound 5 was found to incorporate myo-[2-3H]inositol, [32P]orthophosphate, and [2-3H]glycerol in ratioswhich, when isotopic equilibrium had been reached, were con-sistent with a glycerophospholipid structure (Table I). Further-more, fatty acids were identified as being present in compound5, palmitate and linoleate dominating (BK Dr0bak, IB Ferguson,unpublished data). The ratios of 32P:3H-glycerol and 32P:3H-in-ositol, after long incubation times, were 1.8 when expressed onthe basis of a 32P:3H-ratio in PI = 1. This suggests a chemicalratio of 2 mol of phosphate per mol of inositol and glycerol. Thepresented data, when seen in conjunction, establish beyond rea-sonable doubt that compound 5 is PIP.Compounds 3 and 4 both showed chromatographic behavior

similar to authentic PI-4,5-P2, but it seems unlikely that eitherof these is identical with PI-4,5-P2. Both compounds incorporate[2-3H]inositol and [32P]orthophosphate. Incorporation of [2-3H]in-ositol suggests chemical levels of compound 3 comparable to PIP,with compound 4 being around half that quantity if equal num-bers of inositol moieties are assumed. The results in Table Isuggest a phosphate:inositol ratio close to 1:1 for both 3 and 4,rather than 3:1 as would be expected for PIP,. Furthermore,neither compound incorporates [2-3H]glycerol to any major ex-tent, being too low to determine accurately in compound 3 andaround 0.04% of total phospholipid extractable 3H in compound4. The ratio of 32P:3H-glycerol in these compounds is far too highto be compatible with either compounds 3 or 4 being a glycerol-containing phospholipid. The possibility exists that both com-pounds 3 and 4 are inositol-containing phosphosphingolipids.Kaul and Lester (15) have described the presence of at least adozen such compounds in tobacco leaves, many of which still

=

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INOSITOL-CONTAINING LIPIDS IN CULTURED PLANT CELLS

Table I. Percentage of Incorporated [3H]Inositol and Ratios of [32p] to [3Hllnositol and [2'P] to [HI Glycerolin Compounds 3 to 5 and PI

The percentage of incorporated [3H]inositol is calculated on basis of data from six independent experimentswith three replicates using cells incubated for 3 d with the tracer prior to extraction. Ratios of [32P] to [3H]inositolare means of three independent experiments with three replicates from cells incubated with both tracers for3 d prior to extraction. The ratio of [32P] to [3H]inositol is set to = 1 for PI, and other ratios are calculatedrelative to this. Ratios of [32P] to [3H]glycerol are calculated as for [32P] to [3H]inositol and represent fourindependent experiments with three replicates from cells incubated for 2 to 6 d with both tracers. (Data are± SE.)

Compound

3 4 5 PI

[3H]Inositol() 7% 2 3%± 1 9% t 3 80% + 5[32p] to [3H]inositol 0.9 ± 0.3 1.3 ± 0.3 1.8 ± 0.2 1[32p] to [3H]glycerol 47.7 ± 9.1 11.1 ± 2.0 1.8 ± 0.2 1

remain to be further characterized. The small amount of labelderived from [2-3H]glycerol found in the chromatographic regionof compounds 3 and 4 is unlikely to originate from incorporationof secondary metabolites of the tracer. The tritium label of [2-3H]glycerol is lost in the conversion to acetate, so label is notincorporated into fatty acids. A more likely explanation is thata small quantity of PIP2 chromatographs in this region and ismasked by the larger overlying quantities of compounds 3 and4. Evidence supporting this view comes from two sources. Heimand Wagner (10) have presented results obtained from anionexchange chromatographic analysis of transacylated inositol-con-taining lipids which must be interpreted in favor of PIP2 beingpresent in suspension-cultured plant cells, although in very smallquantities. Recent work in our laboratories (BK Dr0bak et al.,unpublished data) using anion exchange chromatography andHPLC analysis of inositolphosphate derivatives and transacy-lated [2-3H]inositol labeled phospholipids (glycerophosphoryli-nositides) isolated from suspension-cultured carrot cells has re-

vealed that if PIP2 is present the amounts are likely not to exceed0.05% of total phospholipids.

Rapid turnover of polyphosphoinositides in animal tissues hasbeen recognized for a number of years. The polyphosphoinosi-tides incorporate 32P very rapidly into their 4 and 5 monoesterphosphates (20) because of the very rapid addition and removalof these phosphates by kinases and phosphomonoesterases (forreview, see Ref. 13). These enzymes make up two futile cycles(Fig. 7) whose function is believed to be to hold these lipids ina state of rapid turnover in readiness for receptor-stimulatedphosphodiesteratic hydrolysis (28).

Recently, evidence for the presence of PI and PIP kinases inplants has emerged. Sandelius and Sommarin (24) demonstratedthat membranes isolated from dark-grown wheat contained PI-and PIP-kinases with activity both toward endogenous and ex-

ogenous substrates. Our results showing a very rapid incorpo-ration of 32P into PIP are in favor of a highly active PI kinasebeing present in plant cells. The very low levels of PIP2, however,suggest that the second futile cycle in Figure 7 (that responsiblefor formation/degradation of PIP2), if it exists, has its equilibriumheavily biased to the left. This can be due to either high phos-phatase activity or low kinase activity. There is abundant evi-dence from animal systems that PIP kinase is stimulated as a

result of receptor activation (5, 28), and it may be that in higher

P1-4,5-P2

FIG. 7. Interconversion between PI, PI-4-P, and PI-4,5-P2 in animalmembranes. PI-4-phosphokinase and PIP-5-phosphokinase are repre-sented by a and b and PIP2-5-phosphomonoesterase and PIP-4-phos-phomonoesterase by c and d.

plants only after such an activation are appreciable levels of PIP,formed.The observations reported in this paper have several impli-

cations for further work on the putative phosphoinositide sig-

nalling system in plants. The presence in plant cells of severalill-characterized, lipid-extractable compounds containing bothinositol and phosphate moieties strongly emphasizes the needfor direct identification whenever conclusions with regard to plantphosphoinositides are drawn. The presented results suggest thatPIP2, if indeed present in cultured plant cells, is a very minorcomponent of the inositide pool. As yet, we have no informationon its turnover rate. However, it is clear from 32P incorporationdata that PIP is metabolized strikingly fast. It remains to beelucidated whether this is directly related to signal response cou-

pling or perhaps reflects a truly futile interconversion betweenPI and PIP.

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