Inositol-Containing Lipids in Suspension-Cultured Plant Cells - NCBI

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Aug 18, 1987 - that either of these is identical to phosphatidylinositol-4,5-bisphosphate. Should phosphatidylinositol-bisphosphate be present in suspension ...
Plant Physiol. (1988) 87, 217-222 0032-0889/88/87/0217/06/$0l . 00/0

Inositol-Containing Lipids in Suspension-Cultured Plant Cells AN 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. IRVINE Department 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 East Anglia, 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 (Lycopersicon esculentum Mill.) cells and analyzed by thin layer chromatography. Four major inositol-containing compounds were found, and incorporation of [32P]orthosphosphate, [2-3lHlglycerol, and myo-[2-3Hlinositol was studied. Results showed that phosphatidylinositol-monophosphate is the phospholipid in these cells displaying the most rapid incorporation of [32P]orthophosphate. We suggest that the tracer is incorporated primarily into the phosphomonoester group. Two inositol-containing lipids showed chromatographic behavior similar to phosphatidylinositol-4,5-bisphosphate when using standard thin layer chromatography techniques. The labeling pattern of these compounds, however, reveals that it is unlikely that either of these is identical to phosphatidylinositol-4,5-bisphosphate. Should phosphatidylinositol-bisphosphate be present in suspension cultured plant cells, our data indicate chemical abundancies substantially lower than previously reported.

A role for Ca2+ as a messenger in signal-response coupling in many cellular events has for a number of years been inferred in both animal and plant research. It has thus been suggested that extracellular stimuli upon interaction with the cell surface induce an increase in cytosolic Ca2+ activities and in this way are translated into cellular reactions. The exact mechanisms responsible for such changes in the Ca2+ flux have, until recently, largely

remained obscure. In animal tissues, binding of agonists to membrane-associated receptors has been shown to induce phosphodiesteratic cleavage of PI-4,5-P2,2 a diphosphorylated species of PI (2). This gives rise to the production of DG and Ins-1,4,5-P3. While DG remains in the membrane matrix and modulates the activity of protein kinase C, Ins-1,4,5-P3 is released into the cytosol, triggering release of Ca2+ from internal stores. The ensuing increase in cytosolic Ca2+ activity leads to a cascade of metabolic events, dependent on either the Ca2+ ion itself or on Ca:Ca2+-binding ' Present address: John Innes Institute, Colney Lane, Norwich NR4 7UH, Great Britain. 2Abbreviations: PI-4,5-P2, phosphatidylinositol-4,5-bisphosphate, PIP, phosphatidylinositol-monophosphate; PI-4-P, phosphatidylinositol-4monophosphate; PIP2, phosphatidylinositol-bisphosphate; PI, phosphatidylinositol; Ins-1,4,5-P3, myo-inositol-1,4,5-trisphosphate; DG, 1,2diacylglycerol; PL, total phospholipid; PE, phosphatidylethanolamine; PC, phosphatidylcholine; PG, phosphatidylglycerol. 217

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 might be 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 demonstrated the presence of inositol phosphates of all phosphorylation steps (i.e. inositol-monophosphate to inositol-hexakisphosphate) 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 been determined, and the Ins-1,4,5,-P3 isomer has as yet not been demonstrated in plant extracts. Work carried out using Ins-1,4,5P3 isolated from animal tissues nevertheless points towards a potential physiological role of this isomer in plants. Thus, Ins1,4,5-P3 has been demonstrated to release Ca2+ from microsomes isolated from zucchini hypocotyls (7), corn coleoptiles (21), and oat roots (26) and to affect Ca2 + fluxes across plasma membranes in isolated carrot protoplasts (23). Other intrinsic features of the animal phosphoinositide system find parallels in plants including low cytoplasmic Ca2+ activities, presence of Ca2 +-binding proteins with regulatory properties, and Ca2 + and Ca2 + -calmodulindependent protein kinases (1). Protein kinases bearing some resemblance to the animal protein kinase C have recently been reported to be present in various plant tissues (8, 19, 25). However, it remains to be shown that hydrolysis of phosphoinositides in plants occurs in response to external stimuli. Agonist-induced hydrolysis of PI-4,5-P2 in animal systems is mediated by a phospholipase(s) C (phosphoinositidase C). Although phospholipase C (PLC) has not received much attention in plant research, three recent reports (6, 11, 14) contain evidence for PLC activity in plant cells with defined activity towards phosphatidylinositol. The presence of PI as a major constituent of plant membranes is well established, and recently the presence of other inositol-containing phospholipids, tentatively identified as PIP and PIP2 has been reported (4, 10, 27). In this study we describe the incorporation of various radioactive tracers into inositol-containing phospholipids in suspension-cultured tomato leaf cells. The data are interpreted with regard to the turnover and chemical structure of these lipids. MATERIALS AND METHODS Materials. myo-[2-3H]Inositol, [2-3H]glycerol, and [32P]orthophosphate were obtained from Amersham, U.K.; Cellulose MN 300 HR was from Macherey Nagel, Duren, West Germany; and silica gel H was from Sigma. PI-4-P and PI-4,5-P2 were ex bovine

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brain and obtained from Sigma. All chemicals and solvents used were 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 acid and 5 AM 2-isopentenyl adenine. Cells were subcultured weekly and grown in 50 ml of medium in 250 ml conical flasks at 27°C under 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 added to each culture together with either myo-[2-3H]inositol (total activity 40 ,Ci) or [2-3H]glycerol (total activity 50 ,Ci). Cells were incubated as described above until extraction. Extraction of Lipids. After various incubation times, approximately 0.2 to 0.5 g fresh weight of cells were subsampled from cultures under sterile conditions. The culturing medium was removed by rapid filtration on glass fiber filters. The cells were transferred to a cooled Kondes glass grinder containing 10 ml of ice-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 twophase system. The upper acidified phase was removed and discarded, and the bottom phase was washed twice with aliquots of 4 ml chloroform/methanol/0.6 N HCl, 3:48:47 (v/v/v). The resulting extract was centrifuged at 2000g for 5 min to remove curd-like material. The lipid-containing phase was recovered and ,ultransferred to 2 ml glass vials after weighing and sampling of 20 aliquots for monitoring of radioactivity. The lipid-containing extract was evaporated under a stream of nitrogen. The dried lipid samples were reconstituted in chloroform/methanol/H20 (75:25:2, v/v/v) and either used immediately for analysis by TLC or stored at - 25°C until use, after flushing of the vials with N2 and sealing of the tubes. Thin Layer Chromatography. Thin layer chromatography was performed on TLC plates containing either silica gel H (SHplates) or a mixture of silica gel H and cellulose (MX-plates, silica gel H/cellulose: 5:2 [w/w]). SH-plates were used for identification purposes exclusively, whereas MX-plates were employed for both identification and assessment of incorporated radioactivity. Both layer types were spread on 200 x 200 x 4 mm glass plates to a thickness of 250 ,um after inclusion of 1% Koxalate (w/v) and 2 mM EDTA in the silica gel slurry. Plates were activated at 110°C for 45 min before use. After spotting of the labeled lipid samples, plates were developed in one dimension in chloroform/acetone/methanol/acetic acid/H20 (40: 15:13:12:8; v/v/v/v/v). Lipids were visualized by exposure toI2 vapor and radioactive compounds by autoradiography. After development of autoradiograms, TLC plates were treated with Stripmix as described by Redgwell et al. (22). Radioactive spots were identified, cut from the plates, and transferred to scintillation vials. Radioactivity was determined by liquid scintillation spectrophotometry (Beckmann LS 2800) using ASC II (Amersham) scintillation fluid. Presentation of Data. Incorporation data are presented relative to total incorporation of tracer into total extractable phospholipids (PL). We feel that this type of representation best illustrates the relative turnover rates within the lipid pool. RESULTS

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

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

with authentic standards, by use of phosphate- and amino groupspecific sprays (16) and by reference to published RF values for these compounds in the solvent system employed. Initial studies showed, in addition to PI, the presence of three major phosphate-containing compounds incorporating myo-[23H]inositol. These are indicated as compounds 3, 4, and 5, respectively. That no other major compounds incorporating myo[2-3H]inositol exist in these extracts has been shown by assay of radioactivity arising from this tracer in sequentially cut/scraped TLC-lanes and by fluorography of TLC-separations containing myo-[2-3H]inositol-labeled compounds exclusively (data not shown). Incorporation of [32P]Orthophosphate. Figures 2 to 5 show the

relative incorporation of 32Pi into PI and compounds 3 to 5, as expressed on the basis of total incorporation into phospholipids. As can be seen (Fig. 2), incorporation of label into PI amounts to 14 to 19% after short labeling times (0.5-2h) followed by a small drop to about 9% in the period from 24 to 144 h. Compound 5 incorporated 32Pi extremely rapidly, as can be seen from Figure 3. After 30 min incubation, it was the heaviest labeled of all phospholipids-in some experiments containing as much as 40% of total extracted activity. Similar results were obtained when tracer was added later in the cell cycle (data not shown). The relative level drops rapidly during the first 24 h of the labeling period to around 2% of total PL-32P. In contrast, 32Pi incorporation into compounds 3 and 4 is much slower, as evident from

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INOSITOL-CONTAINING LIPIDS IN CULTURED PLANT CELLS

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Figures 4 and 5. Compound 4 contains 1.2 to 1.5% of total PL32P after short labeling times, approaching a steady level of just under 1 % after 24 h. At the earliest sampling time (30 min), large variations were found in label incorporated into compound 3 (Fig. 5). The mean is 1.2% and thus comparable to the relative incorporation figures obtained after longer incubation times. Incorporation of [2-3H]Glycerol. The incorporation of [2-3H]glycerol into PI and compounds 4 and 5 relative to incorporation into total phospholipid is shown in Figure 6. Data for [2-3H]glycerol incorporation into compound 3 are not shown as very low levels of label were recovered (

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to 14% of total PL equalling approximately 0.4 nmol (mg cell

fresh weight) -1 32P-Labeling data after 48 h are consistent with chemical abundancies of PI of around 10 to 13% of total PL, which may suggest that the diesterphosphate has reached isotopic equilibrium by this time. A similar pattern was found for incorporation of [3H]glycerol into PI. Incorporation after 48 h suggests chemical amounts of 8 to 10% of total PL. After very long labeling times (i.e. 144 h), the percentage label of incorporated [2-3H]glycerol in PI was found to drop to around 5%. A smaller drop was registered in relative levels of 32P in PI at similar in-

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

cubation times. It is possible that a small decrease in chemical levels of PI occurs in the late stages of the growth cycle accompanied by an increase in triacylglycerols-a situation which would parallel findings from cultured algal cells (29). PI was, as expected, found to be the major inositol-containing phospholipid in the cells, containing 80% of the incorporated myo-[2-3H]inositol after incubation for 3 d. Chromatographic data suggest that compound 5 could be PIP. Several authors have presented evidence for the presence of inositol-containing phospholipids extracted from various plant tissues, which in both one- and two-dimensional TLC-systems are chromatographically indistinguishable from standard PI-4-P and PI-4,5-P2 (4, 10, 27). In this study, we found exact co-chromatography of compound 5 and standard PI-4-P employing both SH- and MX-plates. Compound 5 was found to incorporate myo[2-3H]inositol, [32P]orthophosphate, and [2-3H]glycerol in ratios which, when isotopic equilibrium had been reached, were consistent with a glycerophospholipid structure (Table I). Furthermore, fatty acids were identified as being present in compound 5, palmitate and linoleate dominating (BK Dr0bak, IB Ferguson, unpublished data). The ratios of 32P:3H-glycerol and 32P:3H-inositol, after long incubation times, were 1.8 when expressed on the basis of a 32P:3H-ratio in PI = 1. This suggests a chemical ratio of 2 mol of phosphate per mol of inositol and glycerol. The presented data, when seen in conjunction, establish beyond reasonable 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 either of these is identical with PI-4,5-P2. Both compounds incorporate [2-3H]inositol and [32P]orthophosphate. Incorporation of [2-3H]inositol suggests chemical levels of compound 3 comparable to PIP, with compound 4 being around half that quantity if equal numbers of inositol moieties are assumed. The results in Table I suggest 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 extent, being too low to determine accurately in compound 3 and around 0.04% of total phospholipid extractable 3H in compound 4. The ratio of 32P:3H-glycerol in these compounds is far too high to be compatible with either compounds 3 or 4 being a glycerolcontaining phospholipid. The possibility exists that both compounds 3 and 4 are inositol-containing phosphosphingolipids. Kaul and Lester (15) have described the presence of at least a dozen such compounds in tobacco leaves, many of which still

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Table I. Percentage of Incorporated [3H]Inositol and Ratios of [32p] to [3Hllnositol and [2'P] to [HI Glycerol in Compounds 3 to 5 and PI The percentage of incorporated [3H]inositol is calculated on basis of data from six independent experiments with three replicates using cells incubated for 3 d with the tracer prior to extraction. Ratios of [32P] to [3H]inositol are means of three independent experiments with three replicates from cells incubated with both tracers for 3 d prior to extraction. The ratio of [32P] to [3H]inositol is set to = 1 for PI, and other ratios are calculated relative to this. Ratios of [32P] to [3H]glycerol are calculated as for [32P] to [3H]inositol and represent four independent experiments with three replicates from cells incubated for 2 to 6 d with both tracers. (Data are ± SE.)

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remain to be further characterized. The small amount of label derived from [2-3H]glycerol found in the chromatographic region of compounds 3 and 4 is unlikely to originate from incorporation of secondary metabolites of the tracer. The tritium label of [23H]glycerol is lost in the conversion to acetate, so label is not incorporated into fatty acids. A more likely explanation is that a small quantity of PIP2 chromatographs in this region and is masked by the larger overlying quantities of compounds 3 and 4. Evidence supporting this view comes from two sources. Heim and Wagner (10) have presented results obtained from anion exchange chromatographic analysis of transacylated inositol-containing lipids which must be interpreted in favor of PIP2 being present in suspension-cultured plant cells, although in very small quantities. Recent work in our laboratories (BK Dr0bak et al., unpublished data) using anion exchange chromatography and HPLC analysis of inositolphosphate derivatives and transacylated [2-3H]inositol labeled phospholipids (glycerophosphorylinositides) isolated from suspension-cultured carrot cells has revealed that if PIP2 is present the amounts are likely not to exceed 0.05% of total phospholipids. Rapid turnover of polyphosphoinositides in animal tissues has been recognized for a number of years. The polyphosphoinositides incorporate 32P very rapidly into their 4 and 5 monoester phosphates (20) because of the very rapid addition and removal of these phosphates by kinases and phosphomonoesterases (for review, see Ref. 13). These enzymes make up two futile cycles (Fig. 7) whose function is believed to be to hold these lipids in a state of rapid turnover in readiness for receptor-stimulated phosphodiesteratic hydrolysis (28). Recently, evidence for the presence of PI and PIP kinases in plants has emerged. Sandelius and Sommarin (24) demonstrated that membranes isolated from dark-grown wheat contained PIand PIP-kinases with activity both toward endogenous and exogenous substrates. Our results showing a very rapid incorporation of 32P into PIP are in favor of a highly active PI kinase being present in plant cells. The very low levels of PIP2, however, suggest that the second futile cycle in Figure 7 (that responsible for formation/degradation of PIP2), if it exists, has its equilibrium heavily biased to the left. This can be due to either high phosphatase activity or low kinase activity. There is abundant evidence 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 animal membranes. PI-4-phosphokinase and PIP-5-phosphokinase are represented by a and b and PIP2-5-phosphomonoesterase and PIP-4-phosphomonoesterase by c and d.

PI 80% + 5 1 1

plants only after such an activation are appreciable levels of PIP, formed. The observations reported in this paper have several implications for further work on the putative phosphoinositide signalling system in plants. The presence in plant cells of several ill-characterized, lipid-extractable compounds containing both inositol and phosphate moieties strongly emphasizes the need for direct identification whenever conclusions with regard to plant phosphoinositides are drawn. The presented results suggest that PIP2, if indeed present in cultured plant cells, is a very minor component of the inositide pool. As yet, we have no information on its turnover rate. However, it is clear from 32P incorporation data that PIP is metabolized strikingly fast. It remains to be elucidated whether this is directly related to signal response coupling or perhaps reflects a truly futile interconversion between PI and PIP. LITERATURE CITED 1. ALLAN EF, AJ TREVAWAS 1987 The role of calcium in metabolic control. In PK Stumpf, EE Conn, eds, The Biochemistry of Plants, Vol 9. Academic Press, New York. In press 2. BERRIDGE MJ, RF IRVINE 1984 Inositol trisphosphate, a novel second messenger in cellular signal transduction. Nature 312: 315-321 3. BIELESKI RL, IB FERGUSON 1983 Physiology and metabolism of phosphate and its compounds. In A Lauchli, RL Bieleski, eds, Encyclopedia of Plant Physiology, New Series, Vol 15. Springer Verlag, Berlin, pp 422-449 4. Boss WF, M MASSEL 1985 Polyphosphoinositides are present in plant tissue culture cells. Biochem Biophys Res Commun 132: 1018-1023 5. BROEKMAN Ml 1984 Phosphatidylinositol-4,5-bisphosphate may represent the site of release of plasma membrane-bound calcium upon stimulation of human platelets. Biochem Biophys Res Commun 120: 226-23 6. CONNETT RJA, DE HANKE 1986 Breakdown of phosphatidylinositol in soybean callus. Planta 169: 216-221 7. DR0BAK BK, IB FERGUSON 1985 Release of Ca2+ from plant hypocotyl microsomes by inositol-1,4,5-trisphosphate. Biochem Biophys Res Commun 130: 1241-1246 8. ELLIOTT DC, JD SKINNER 1986 Calcium-dependent, phospholipid-activated protein kinase in plants. Phytochemistry 25: 39-44 9. GAMBORG OL, JP SHYLUK 1981 Nutrition, media & characteristics of plant cell & tissue cultures. In TA Thorpe, ed Plant Tissue Culture. Academic Press, New York, pp 21-44 10. HEIM S, KG WAGNER 1986 Evidence of phosphorylated phosphatidylinositols in the growth cycle of suspension cultured plant cells. Biochem Biophys Res Commun 134: 1175-1181 11. HELSPER JPFG, PFM DEGROOT, HF LINSKENS, JF JACKSON 1986 Phosphatidylinositol phospholipase C activity in pollen of Lilium longiflorum. Phytochemistry 25: 2053-2055 12. INHULSEN D, R NIEMEIER 1978 Inosit-phosphate aus Lemna minor L. Z Pflanzenphysiol 88: 103-116 13. IRVINE RF 1982 The enzymology of stimulated inositol lipid turnover. Cell Calcium 3: 295-309 14. IRVINE RF, AJ LETCHER, RMC DAWSON 1980 Phosphatidylinositol phosphodiesterase in higher plants. Biochem J 192: 279-283 15. KAUL K, RL LESTER 1975 Characterization of inositol-containing phosphosphingolipids from tobacco leaves. Plant Physiol 55: 120-129 16. KREBS KG, D HEUSSER, H WIMMER 1969 Spray reagents. In E Stahl, ed, Thin-layer Chromatography. Springer-Verlag, Berlin, pp 854-912 17. MANDAL NC, BB BISWAS 1970 Metabolism of inositol phosphates: Part II-

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Biosynthesis of inositol polyphosphates in germinating seeds of Phaseolus Ind J Biochem 7: 63-67 MORSE MJ, RC CRAIN, RL SATTER 1987 Phosphatidylinositol cycle metabolites in Samanea saman pulvini. Plant Physiol 83: 640-644 OLAH Z, Z Kiss 1986 Occurrence of lipid and phorbol ester activated protein kinase in wheat cells. FEBS Lett 195: 33-37 PALMER S, PT HAWKINS, RH MICHELL, CJ KIRK 1986 The labelling of polyphosphoinositides with [32P]Pi and the accumulation of inositol phosphates in vasopressin-stimulated hepatocytes. Biochem J 238: 491-499 REDDY ASN, BW POOVAIAH 1987 Inositol 1,4,5-trisphosphate induced calcium release from corn coleoptile microsomes. J Biochem 101: 569-573 REDGWELL RJ, NA TURNER, RL BIELESKI 1974 Stripping thin layers from chromatographic plates for radiotracer measurements. J Chromatogr 88: 2531 RINCON M, WF Boss 1987 Myo-inositol trisphosphate mobilizes calcium from aureus.

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fusogenic carrot (Daucus carota L.) protoplasts. Plant Physiol 83: 395-398 24. SANDELIUs A, M SOMMARIN 1986 Phosphorylation of Phosphatidylinositol in isolated plant membranes. FEBS Lett 201: 282-286 25. SCHAFER A, F BYGRAVE, S MATZENAUER, D MARM- 1985 Identification of a calcium- and phospholipid-dependent protein kinase in plant tissues. FEBS Lett 187: 25-28 26. SCHUMAKER KS, H SZE 1987 Inositol 1,4,5-trisphosphate releases Ca2+ from vacuolar membrane vesicles of oat roots. J Biol Chem 262: 3944-3946 27. STRASSER H, C HOFFMANN, H GRISEBACH, U MATERN 1986 Are polyphosphoinositides involved in signal transduction of elicitor-induced phytoalexin synthesis in cultured plant cells. Z Naturforschung 41c: 717-724 28. TURNER PR, MP SHEETZ, LA JAFFE 1984 Fertilization increases the polyphosphoinositide content of sea urchin eggs. Nature 310: 414-415 29. WETTERN M 1980 Lipid variation of the green alga Fritschiella tuberosa during growth in axenic batch culture. Phytochemistry 19: 513-517