Changes in Phosphatidylinositol Metabolism in Response to Hyperosmotic Stress in Daucus Carota 1. Cells Grown in Suspension Culture'

Plant Physiol. (1993) 103: 637-647

Changes in Phosphatidylinositol Metabolism in Response to Hyperosmotic Stress in Daucus carota 1. Cells Grown in Suspension Culture'

Myeon H. Cho, Stephen B. Shears, and Wendy F. BOSS* Department of Botany, North Carolina State University, Raleigh, North Carolina 27695-761 2 (M.H.C., W.F.B.); and Laboratory of Cellular and Molecular Pharmacology, National lnstitute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709 (S.B.S)

in the Samanea saman pulvini is there evidence for stimulus- Carrot (Daucus carota L.) cells plasmolyzed within 30 s after mediated turnover of polyphosphorylated inositol phospho- adding sorbitol to increase the osmotic strength of the medium lipids and inositol phosphates within the rapid time scale from 0.2 to 0.4 or 0.6 osmolal. However, there was no significant seen in animal cells (Morse et al., 1987). Although results change in the polyphosphorylated inositol phospholipids or inositol from in vitro studies and from IP3 microinjection in vivo have phosphates or in inositol phospholipid metabolism within 30 s of shown that Ir3 can release Ca2+from internal stores such as imposing the hyperosmotic stress. Maximum changes in phospha- vacuoles and tonoplast vesicles (Drcibak and Ferguson, 1985; tidylinositol 4-monophosphate (PIP) metabolism were detected at 5 min, at which time the cells appeared to adjust to the change in Schumaker and Sze, 1987; Ranjeva et al., 1988; Gilroy et al., osmoticum. There was a 30% decrease in [3H]inositol-labeledPIP. 1990), the complete pathway from a stimulus to Ca2+release The specific activity of enzymes involved in the metabolism of the has yet to be demonstrated in higher plants. inositol phospholipids also changed. The plasma membrane phos- When studying the functions of the inositol phospholipids, phatidylinositol (Pl) kinase decreased 50% and PIP-phospholipase one needs to realize the multifaceted effects of the inositol C (PIP-PLC) increased 60% compared with the control values after phospholipids on cell physiology. For example, in addition 5 min of hyperosmotic stress. The PIP-PLC activity recovered to to being the sources of second messengers, PIP and PIPz control levels by 10 min; however, the PI kinase activity remained also can directly affect cell physiology by regulating P-type below the control value, suggesting that the cells had reached a ATPases (Varsanyi et al., 1983; Schafer et al., 1987; Memon new steady state with regard to PIP biosynthesis. If cells were pretreated with okadaic acid, the protein phosphatase 1 and 2A et al., 1989a; Chen and Boss, 1991), protein kinase C (Chau- inhibitor, the differences in enzyme activity resulting from the han and Brockerhoff, 1988; Chauhan et al., 1989), and actin hyperosmotic stress were no longer evident, suggesting that an polymerization and nucleation sites (Lassing and Lindberg, okadaic acid-sensitive phosphatase was activated in response to 1985; Forscher, 1989). It has been shown that both PI and hyperosmotic stress. Our work suggests that, in this system, PIP is PIP kinase are present in the F-actin fractions from both plant not involved in the initial response to hyperosmotic stress but may (Tan and Boss, 1992) and animal cells (A431 mouse fibroblast be involved in the recovery phase. cells) (Payrastre et al., 1991) and that the specific activities of the lipid kinases in the F-actin fraction change in response to externa1 stimuli. These data suggest that there is a change in The signal transduction mechanisms involving inositol intracellular enzyme distribution or covalent modification of phospholipid-derived second messengers, IP3 and diacylglyc- the enzymes. In addition, we recently (Yang et al., 1993) erol, are well characterized in many animal cells (Majerus et have found a protein that not only regulates PI kinase activity al., 1986; Berridge and Irvine, 1989; Michell, 1992). In these but also binds and bundles actin and has translational elon- systems the response includes a rapid and transient decrease gation factor-la activity. Thus, changes in the metabolism of in PIPz and at least a 3-fold increase in Ir3.The increased IP3 these very negatively charged phospholipids could be a part releases calcium from internal stores, specifically the ER of a complex intracellular communication system among the (Streb et al., 1983), thus activating calcium-dependent en- plasma membrane, cytoskeleton, and protein synthesis zymes and altering cell physiology. machinery . Most components of the inositol phospholipid-mediated Changes in inositol phospholipids had been shown to occur signal transduction pathway have been shown to be present in Dunaliella salina as a result of hyperosmotic stress (Ein- in higher plants (for reviews, see Einspahr and Thompson, 1990; RincÓn and Boss, 1990; Chen et al., 1991; Gross and Boss, 1992; Hetherington and Drcibak, 1992). However, only Abbreviations: I, inositol; IP, inositol monophosphate; Ir2,inositol 1,4-bisphosphate; IP3, inositol 1,4,5-trisphosphate; I-1-P, inositol ' This research was supported by grant No. DCB-8812580 from 1-phosphate; I-4-P, inositol 4-phosphate; 1-1,3,4-P3, inositol 1,3,4- the National Science Foundation and in part by the North Carolina trisphosphate; PI, phosphatidylinositol; PIP, phosphatidylinositol Agricultura1 Research Service. 4-monophosphate; PIP2, phosphatidylinositol 4,5-bisphosphate; * Corresponding author; fax 1-919-515-3436. PLC, phospholipase C; MeOH, methanol; OSM, osmolal. 638 Cho et al. Plant Physiol. Vol. 103, 1993 spahr et al., 1988), and changes in inositol phosphates have transfer. The conditioned culture medium had an osmolality been reported for beet roots exposed to different osmoticum of 0.2 OSM, determined by freezing point depression using an (Srivastava et al., 1989). Because cells grown in suspension Osmette Precision Osmometer and a pH of 4.2. Hyperosinotic culture provide a relatively uniform system for biochemical conditions were produced by adding 2.5 mL of 2 or 4 OSM studies, we decided to study the effects of hyperosmotic stress sorbitol in conditioned medium containing 2 mM Mes baffer on inositol lipid metabolism in this system in more detail. For (pH 4.2) to conditioned medium (22.5 mL, pH 4.2). This gave these experiments we kept the cells in their normal medium a final osmoticum equivalent to 0.4 and 0.6 OSM, respectkely. and applied stress by adding sorbitol to the medium rather An equal volume of conditioned medium containing :! mM than placing the cells in sorbitol alone. Mes buffer (pH 4.2) was added for the control. At 30 s before Plasmolysis has been used over the years by cell biologists the termination of the treatments or at time zero for the 30-s to study the properties of the plasma membrane of plant cells time points, the cells were quickly poured into conical tubes (Pfeffer, 1877; Lee-Stadelmann and Stadelmann, 1976). Plas- and centrifuged at 2000g for 30 s. Supernatant was pciured molysis of cells in response to hyperosmotic conditions is off, and cells were immediately placed on ice and homoge- rapid (within seconds) and readily visible with the light nized or extracted for inositol phospholipid or inositol phos- microscope. Rapid membrane depolarization occurs as the phate analysis. cells plasmolyze (Slayman, 1982); however, the mechanism Cells were pretreated with okadaic acid for 1 h following of signal transduction has not been elucidated. Tolerance to the 30-min preequilibration. Okadaic acid was added iising osmotic stress is important agriculturally. Recent advances in a stock solution of 155 p~ okadaic acid in 10% (v/v) DlMSO this field show promising results in altering cell physiology to give a final concentration of 0.15 ~LMokadaic acid and to withstand hyperosmotic stress (Tarczynski et al., 1993) 0.01% DMSO. The equivalent volume of 10% DMSO was and emphasize the importance of understanding the mecha- added as a control. nisms by which cells respond and adjust to hyperosmotic conditions. Observations of Plasmolysis The question we have addressed in this work is what role do the inositol phospholipids play in cellular responses to For the microscopy studies, the carrot suspension culture hyperosmotic stress? Our data indicate that the inositol phos- cells (0.1 g in 4.5 mL of conditioned medium, pH 4.2) were pholipids are involved in the recovery phase as the cells placed in a small Erlenmeyer flask (25 mL) and treated adjust to hyperosmotic conditions and that this response may hyperosmotically by adding 0.5 mL of 2 or 4 OSM sorbitol in be mediated by an okadaic acid-sensitive phosphatase. conditioned medium containing 2 IT~MMes (pH 4.2), to give a final osmolality of 0.4 and 0.6 OSM, respectively. A drop of MATERIALS AND METHODS cells was placed on the glass slide, and plasmolysis was observed over time using a Zeiss IM 35 microscope with a Plant Material X63 neofluor lens. The first observations were made 30 to Wild carrot (Daucus carota L.) cells grown in suspension 40 s after the addition of osmoticum. culture were transferred weekly and used 4 d after transfer as previously described (Chen and Boss, 1990). [3Hllnositol Labeling Cells (approximately 0.25 g fresh wt 25 mL-' of med:ium) Chemicals were used 3 d after transfer. myo-[2-3H]Inositol(0.2pCi rnL-' my0-[2-~H]Inositol(24.40Ci mmol-') and PI-4-P (inositol- for lipid analysis and 0.4 pCi mL-' for inositol phosphate 2-3H[N]) (9.90 Ci mmol-') were purchased from New Eng- analysis) was added to the cells for 18 h. The cells were land Nuclear, and [y3'P]ATP was purchased from ICN harvested by brief centrifugation (2000g for 30 s). Radiochemicals. 3H-Labeled inositol phosphate standards (I- 1-P, I-4-P, Ir2,I- 1,3,4-P3, Ir3,I- 1,3,4,5 -tetrakisphosphate, Extraction and Separation of lnositol Phosphates in HPLC I-hexaphosphate) were obtained from New England Nuclear. The centrifuged cells were quenched with a final concen- [14C]I(1,3,4)P3 was prepared from ['4C]inositol-labeled pa- tration of 4% (v/v) perchloric acid (Kirk et al., 1990) for rotid acinar cells (Hughes et al., 1989). Dowex-1 (X8), chloride inositol phosphate analysis. Perchlorate-quenched cell ex- form mesh, 200-400), and okadaic acid were purchased (dry tracts were neutralized with freon/octylamine (Kirk et al., from Sigma, and okadaic acid stock (155 p~)was prepared 1990), and the inositol phosphates therein were resolvetl on in 10% DMSO according to manufacturer's guide. A11 other an Adsorbosphere 5 p-SAX HPLC column (Alltech Associ- chemicals were analytical grade. ates, Deerfield, IL) using a slight modification of the salt gradient previously described (Menniti et al., 1990) generated Hyperosmotic Treatment and Okadaic Acid Pretreatment from water and buffer (1 M ammonium dihydrogen phos- The cells from severa1 125-mL culture flasks were com- phate, pH 3.35 with phosphoric acid): O to 10 min, 0% buffer; bined into a 500-mL flask and redistributed by pipetting 22.5 10 to 85 min the buffer increased linearly from O to 35%; 85 mL of medium and cells into 125-mL flasks. The flasks were to 130 min, the buffer increased linearly from 35 to 80%. placed on a shaker for 30 min at 120 rpm and 25OC to Half-minute fractions were collected for 80 min, and 1-min preequilibrate the cells. The 30-min equilibration period is fractions were collected thereafter. Scintillant was added to essential to stabilize the cells and minimize the effects of each of the fractions, and the radioactivity was assessed. Phosphoinositide Metabolism and Hyperosmotic Stress 639

Lipid Extractio Separatiod nan n PLC Assay

For lipid analysis 1.5 mL of ice-cold CHCl3:MeOH (1:2, PLC activity was assayed according to the method of Melin v/v) was added to the centrifuged cells, and the lipids were . (1992)al t standare e Th . d reaction mixture containeM m 0 d5 extracted and separated by TLC as described by Cho et al. Tris-HC 6.0)H (p M0 l 2 ,M Ca a 2Ca( + 2+/EGTA mixturs ewa (1991). Briefly e LK5th , D (Whatman) silic l plateage s were used as described by Owen [1976]), 0.2 mM PIP, and 4 to 6 presoaked in 1% potassium oxalate for 80 s, dried in a Mg of membrane protein in a final volume of 50 ^L- The microwav 0 mind 1 develope an r , fo e n CHC^MeOHi d : reactio s startestoce additioP th nwa PI ky a db solutiof no n 3 1 NH4OH:H2O (86:76:6:16, v/v/v/v). The distribution of the containing H-labele (110P nmol"dm PI 0dp 0.1n i ) - %de 3H- and 32P-labeled lipids was quantitated with a Bioscan oxycholate, which was prepared by drying the lipid under a System 500 imaging scanner. The efficiency was 1% for 3H strea nitrogef mo n followe sonicatioy db 0.1n i % deoxycho- and 10% for 32P. 32P-Labeled phospholipids were visualized late for 10 min. The reaction was stopped after 5 min at 25°C by autoradiography. by addition of 1 mL of chloroform:methanol (2:1, v/v). After HC1N 1 ,f vortexingo L additiofi 0 d brie25 an ,f nfo centrifugation in a table-top centrifuge, the radioactive reaction prod- Plasma Membrane Isolation ucts were recovered in the upper phase, analyzed by ion- Plasma membranes were isolated by aqueous two-phase exchange chromatography on a Dowex AG 1-X8 column partitioning as described by Chen and Boss (1990). The final (Berridge et al., 1983), and quantitated by liquid scintillation plasma membrane pelle s resuspende TrisM wa t m / 0 3 n i d counting. Mes buffer (pH 6.5) containing 15 mM MgQ2 and kept on untie ic enzyme l th use r dfo e PIP-PLassayse th r Fo . C assay, RESULTS plasma membranes were resuspended in 50 mM Tris-HCl, pH 6.0. Protein was determined by the method of Lowry et Hyperosmotic Stress Induces Rapid Plasmolysis al. (1951) with BSA as a standard. The osmolality of the medium in which the carrot cells were growin s determinegwa e approximatelb o dt OSM2 y0. . Analysi I KinasP f so e Cells in conditioned medium were turgid and contained large starch granules (Fig. 1A). When sorbito e s addeth wa l o t d PI kinase activity was measured in the absence and pres- medium to give the equivalent of a 0.4 OSM solution, the outer ence of exogenous substrate. Equal amounts of plasma mem- cellclustea n i s r coul seee db plasmolyzo t n e slightly (Fig. brane protein wer e controe th use r d treatefo dan l d cells a finaIB) t A l. concentratio 6 OSM0. f , o nplasmolysi s wa s within each experiment. The protein amount ranged from 15 to 40 fig for the experiments reported. The plasma membranes Tris-MeM m wer 0 3 es f resuspendebuffeo H L n (p r 0 4 n i d

6.5) containin MgClM m 25 .g1 Phosphorylatio s startenwa d stocP y addinb AT k pL 0 f solution1 o g , which contained 10 nCi of [7-32P]ATP (7000 Ci mmol~'), 6 mM ATP, 0.05% (v/v) Triton X-100, and 5 mM Na2MoO4 in 30 mM Tris/Mes containin MgClM 6.5)H m (p 2giv5 o t g,1 efinaa l concentra- Na ATPM M 2m MoO m 1 , 2 d 0.011. tio4 an ,f no % Trito- nX 100. When exogenous substrate (PI, 25 ^g/tube) was added, the final concentration of Triton X-100 was 0.2% and the pH was 8.0. The PI was presolubilized in 2% Triton X-100. The tubes were shake hany nb d vigorousl t rooya n everm mi y1 temperature, and after 10 min, the reaction was stopped by adding 1.5 mL of ice-cold CHCl3:MeOH (1:2, v/v). The lipids were kept on ice until they were extracted.

ATPase Assay Vanadate-sensitive ATPas s assayeewa addiny L db n 0 g5 plasme oth f a membrane-rich fraction (30-40 ^f proteingo ) /u0 f reactioLo 35 o t n mixtur givo et efinaa l concentratiof no Figure 1. Plasmolysis induced by hyperosmotic treatments using OS6 0. M d sorbitoan 4 0. conditionen i l d medium. Carrot cells were 0.01% Triton X-100MgSOTris/MesM M M m m m 3 0 , 43 0 , 5 , treated hyperosmoticall OS6 0. M d addin y ysorbitob an e 4 th g 0. o t l KC1, 1 mM NaN3 in the presence or absence of 500 MM vanadate as described by Gallagher and Leonard (1982). The conditioned medium. Small aliquots of cells were placed on a glass slide, and plasmolysis was observed with a Zeiss IM 35 microscope stocP reactioAT k s starteM m nwa 5 addiny 1 db a ju 0 f Lo g10 at X63 describes 0a "Materialn di Methods.d san controe Th , A l" solutiot rooa n mcontinued mi ntemperaturean 0 3 r dfo e .Th cell conditionen si d medium OS4 cell, B ;0. Mt sa afte min1 r, cell C ; s reaction was stopped by the addition of 250 pL of 20% ice- OS6 a OS6 0. , trecovern cell M0. (i D M t ; sa aften s aftemi 5 4 yr5 r cold TCA. The Pi released was determined according to the phase). Arrowheads denote starch granules. Arrows denote th e method of Taussky and Shorr (1953). positio plasme th f no a membran celd elan wall. 640 Cho et al. Plant Physiol. Vol. 103, 1993 clearly evident and complete in a11 cells within 30 s. The of PIP by PLC. One potential mechanism by which such photograph in Figure 1C was taken after 45 s. The plasmo- putative regulation could be achieved is by covalent modifi- lyzed cells could be seen to regain volume slightly over a 5- cation of the enzymes or of regulatory proteins. Therefore, min period (Fig. 1D). After 5 to 10 min the volume stabilized. we isolated the plasma membranes of carrot cells and assayed The average decrease in diameter of the cells in 0.6 OSM PI kinase activity and PIP hydrolysis by PLC as well as the sorbitol was 15 & 4.8% (n = 6). plasma membrane vanadate-sensitive ATPase activity, specifically to search for any stable changes in the inherent The Distribution of [3H]lnositol-Labeled Lipids activities of these enzymes. Changes after Hyperosmotic Stress Hyperosmotic Stress Decreases the Plasma Membrane In the [3H]inositol-labeled carrot cells the predominant [3H]- Vanadate-Sensitive ATPase Activity inositol-labeled lipid was PI (82-85% of the total, Table I). [3H]PIP and [3H]PIP2were relatively minor constituents (ap- Initially as a means of characterizing the plasma membrane proximately 8 and 0.5%, respectively). When carrot cells were fractions, we studied the vanadate-sensitive ATPase activity treated hyperosmotically with sorbitol in conditioned me- in plasma membranes isolated from stressed and nonstressed dium to give an osmoticum of 0.4 or 0.6 OSM, the total amount cells. Greater than 85% of the ATPase activity in the plasma of 3H-labeled lipid did not change significantly at either 30 s membrane fraction from the carrot cells was inhibited by or 5 min (Table I). Furthermore, within the time frame that vanadate (data not shown). The specific activity of the van- plasmolysis was complete (30 s), there were no significant adate-sensitive ATPase decreased when cells were treated changes in any of the [3H]inositol-labeledlipids based either hyperosmotically at 0.4 OSM by adding sorbitol to the condi- on percentage of total 3H-lipid or on cpm (Table I). tioned medium (Fig. 2). A reduction in the ATPase-specific After the 5-min treatment at either 0.4 or 0.6 OSM sorbitol, activity was detected within 1 min. The maximum decrease the proportion of [3H]PIP in the 3H-labeled lipids decreased was observed after 5 min of hyperosmotic stress. by 25 and 32%, respectively, relative to the controls (Table I, P < 0.05). This effect on PIP, which appears to be a secondary Hyperosmotic Stress lncreases the Activity of PIP-PLC response to osmotic stress, was not associated with any significant change in the levels of [3H]PIP2.Because we only Plasma membranes prepared from the stressed cells exhib- detected changes in [3H]PIP, we focused our studies on PIP ited a transient increase in PIP hydrolysis by PLC. No ecfect metabolism. was observed after 2 min of stress. A 60 & 6.4% stimulation The changes in [3H]PIPin response to osmotic stress could in activity was observed after 5 min, and by 10 min the rate have resulted from stress-induced changes in the kinasel of hydrolysis of PIP had returned to the control leve1 (Fig. 3). phosphatase cycle that interconverts PI and PIP. Osmotic Analysis of the water-soluble products by Dowex-1 anion- stress also might have caused an increased rate of hydrolysis exchange chromatography showed that more than 90% were

Table 1. Changes in the distribution ~f[~H]inosito/lipids in response to 0.4 and 0.6 OSM hyperosmotic stress for 30 s and 5 min Carrot cells were labeled with [3H]inositol(1.25 pCi 0.1-' g fresh weight) for 18 h and treated hyperosmotically at 0.4 and 0.6 OSM in conditioned medium plus sorbitol for 30 s and 5 min. For the control, conditioned medium (0.2 OSM) was used. The experimental procedures are described in "Materials and Methods." [3Hllnositol-Labeled Lipids Treatment PI Lvso-PI PIP Lvso-PIP PIP2 30 s Control 83.0 f 2.7" 2.5 f 0.7 8.1 f 0.9 5.3 f 2.0 0.6 f 0.1 (7496 f 54)b (1 73 f 6) (673 f 45) (324 f 23) (43 f 4) 0.4 OSM 83.1 f 2.8 2.7 f 0.7 7.5 f 1.2 5.2 f 2.1 0.5 f 0.1 (7464 f 89) (204 f 40) (676 f 74) (302 f 18) (45 f 5) 0.6 OSM 82.3 f 2.7 3.2 f 1.1 7.6 1- 0.8 5.3 f 2.1 0.6 f 0.2 (7489 f 41) (201 f 24) (660 f 19) (316 +- 18) (51 f 2) 5 min Control 83.1 f 1.6 2.6 f 0.6 8.2 f 0.8 5.1 1.8 0.5 f 0.2 (7511 f 53) (214 f 44) (738 f 9) (345 f 17) (38 f 10) 0.4 OSM 84.2 f 1.5 2.3 f 0.6 6.3 f 0.7 4.6 f 1.8 0.6 2 0.2 (7360 f 27) (259 f 86) (549 f 15) (318 f 14) (38 f 10) 0.6 OSM 85.3 f 1.5 2.3 f 0.5 5.5 f 0.3 4.1 f 0.9 0.7 f 0.1 (7557 f 106) (179 f 24) (505 f 8) (327 f 52) (50 f 9)

a The numbers are the percentages of total [3H]inositol-labeledlipids and expressed as the means f SD of five or six values from three different experiments. The numbers in parentheses are the cpm of each inositol lipid and are the average of two values from one representative experiment. Phosphoinositide Metabolism and Hyperosmotic Stress 64 1

110 I these experiments the same fresh weight of cells was used I = for each treatment, and the total radioactivity recovered from O each treatment was almost identical. There were no signifi- L .I- c cant increases in [3H]IP, [3H]IP2,or [3H]IP3after subjecting O cells to osmotic stress (0.4 or 0.6 OSM) for either 5 min or 30 o s (data not shown). Thus, we conclude that in the carrot cells y. O stress-dependent increases in PLC-mediated hydrolysis of PIP are insufficient to alter the steady-state changes in levels vs > of inositol phosphates to a substantial extent. .-.I- .-> .I- Hyperosmotic Stress Decreases PI Kinase Activity in o 40 - a lsolated Plasma Membranes - al 30 To determine whether or not there was a difference in PI v) kinase activity, plasma membranes were isolated from control a 20 - a and hyperosmotically treated cells. In vitro phosphorylation I- - a 10 in the presence of [-p3’P]ATP indicated that plasma membranes from hyperosmotically stressed cells formed 40% less O [32P]PIPthan membranes from the control cells. This decrease 024681012 in PIP formation was not due to limited substrate availability Time (min) because a similar decrease in PIP formation was observed in the presence of exogenous PI (25 &each assay) (Table 11). When changes in PI kinase activity were monitored over Figure 2. Time course of changes in plasma membrane vanadate- time, slight but significant changes were observed consist- sensitive ATPase activity in response to 0.4 OSM hyperosmotic stress. Celis were treated hyperosmotically (0.4 OSM) as described in ”Ma- ently in plasma membranes from control cells (Fig. 5). The terials and Methods.” Plasma membranes were isolated by aqueous effect was transient and may be caused in part by a “touch”- two-phase partitioning, and the vanadate-sensitive ATPase was induced response upon addition of the conditioned medium. assayed. The results are means & SD of four values from two More importantly at a11 times, the effect of osmotic stress was experiments. observed over and above that of the conditioned medium. After 2 min of hyperosmotic stress, there was a slight decrease in the amount of [32P]PIPformed in the in vitro phosphoryl- IP2 (data not shown). Thus, the decrease in [3H]PIPin stressed cells could have been partly caused by increased hydrolysis by PLC. h .-c 20 E T

[3H]lnositol Phosphates in lntact Cells Did Not Change after Hyperosmotic Stress The analysis of [3H]inositol phosphates in [3H]inositol- labeled plant cells is complicated by the large array of addi- tional water-soluble metabolites (Loewus and Loewus, 1982; RincÓn et al., 1989; Coté et al., 1990). It is very difficult to specifically assay [3H]inositol phosphates against this high background of 3H-labeled compounds. In our experiments we have adapted an anion-exchange HPLC procedure that “Ido has been used successfully to resolve a number of inositol u-o 2 4 6 8 10 12 phosphates in extracts of animal cells (Balla et al., 1989; Menniti et al., 1990). The column was initially characterized Time (min) with 3H-labeled standards of I-I-P, I-4-P, Ir2, I-1,3,4-P3, Ir3, 1-1,3,4,5-tetrakisphosphate,and I-hexaphosphate. Each sam- Figure 3. Time-course study of the effects of hyperosmotic stress ple also contained an interna1 standard of [‘4C]I-1,3,4-P3to on PIP-PLC activity of plasma membranes isolated from hyperos- account for run-to-run variability. motically treated and nontreated carrot suspension-culture cells. The extract from control cells described in Figure 4 con- Cells were treated hyperosmotically at 0.4 OSM in conditioned tained small 3H-labeled peaks that coincided with the elution medium plus sorbitol for 2, 5, and 10 min. Conditioned medium was used for the control. Plasma membranes were isolated by positions of I-I-P and I-4-P (combined total = 8089 dpm) aqueous two-phase partitioning as described in “Materials and plus IP2 (1,464 dpm). Ir3was not consistently detected. Two Methods.” PLC activity was assayed using [3H]PIP as a substrate unidentified 3H peaks that migrated between IPz and IP3 and according to the method of Melin et al. (1992). Open circles, an unidentified 3H peak between IP and IP2 (a11 denoted with control; closed circles, hyperosmotic stress. The results are the ?) were observed to increase in the cells treated at 0.6 OSM. In means & SD of six values from three experiments. 642 Cho et al. Plant Physiol. Vol. 103, 1993

5000 of the large amount of diacylglycerol in the plasma m.em- branes. Lysophosphatidic acid, PIP, and an as yet unidenti- c 4000 fied phosphorylation product were also formed. PIP2 was .--O barely detectable, which is consistent with previous studies o of plasma membranes from higher plants (Sandelius and 2 3000 )I Sommarin, 1986; Sommarin and Sandelius, 1988; Chen and 2 Boss, 1990; Memon and Boss, 1990; Gross and Boss, 1992). na 2000 I- Pretreatment of Cells with Okadaic Acid Eliminates the ‘- 1000 Effect of Hyperosmotic Stress on PI Kinase and PIP-PLC Activity O The effect of hyperosmotic stress on PI kinase and I’IP- 5000 - PLC activity persisted in isolated plasma membranes. Because protein phosphorylation/dephosphorylation is a rapid and 4000 - .- effective means of altering enzyme activity and because there .Lo was evidence for phosphatases decreasing PI kinase activity 2 3000 - (Chen and Boss, 1990; Memon and Boss, 1990), we as,ked -\ whether inhibiting dephosphorylation would affect the re- I ,I(I)P + l(4)P sponse of the membrane enzymes to hyperosmotic stress. For a 2000 - a 1(1*4,5)P these experiments we used okadaic acid, a polyether fatty r acid, that inhibits protein phosphatases 1 and 2A (Bialojan “, “, 1000 and Takai, 1988). Okadaic acid is hydrophobic and readily enters cells. O The effect of okadaic acid on the change in the PIP-PLC 5000 and PI kinase activity was studied both in vivo and in viitro. For the in vivo experiments cells were pretreated with okadaic acid (0.15 PM, final concentration) in DMSO or DMSO alone .-2 4000 CI for 1 h. Okadaic acid pretreatment did not affect the plas- o molysis (data not shown). As with the non-okadaic acid- 2 3000 .c pretreated cells, plasmolysis was complete within 30 s. In 2 okadaic acid-pretreated cells, hyperosmotic stress no loriger na 2000 resulted in an increased PIP-PLC activity (Table 111). It is - interesting that okadaic acid pretreatment decreased the spe- 3 1000 cific activity of PIP-PLC in both the control and hyperosmotic Y stressed cells to the same basal value (approximately 6 nimol min-’ mg-’ of protein). O O 20 40 60 80 Okadaic acid pretreatment also eliminated the difference

Elution Time (min) Table II. The PI kinase activity of plasma membranes isolated from Figure 4. HPLC elution profile of inositol phosphates from control hyperosmotically treated and nontreated carrot suspension-culture and stressed cells. Cells were labeled with [3H]inositol(2.5 pCi 0.1 cells g-’ fresh weight) for 18 h and treated hyperosmotically at 0.4 and Carrot suspension-culture cells were treated hyperosmotically 0.6 OSM in conditioned medium plus sorbitol or in conditioned for 5 min at 0.4 OSM in conditioned medium plus sorbitol and medium alone for 5 min (A, control; B, 0.4 OSM; C, 0.6 OSM). Total conditioned medium alone was used for the control. The plasma [’Hlinositol phosphates were extracted and separated in HPLC as membranes were isolated by aqueous two-phase partitioning. The described in “Materials and Methods.” The inositol phosphates PI kinase activity was assayed in the absence and presence of were identified by comparison to the elution times of standards. exogenous substrate (Pl) as described in ”Materials and Methods.” The elution time for [’4C]1(1,3,4)P3,an interna1 standard, is indicated by the large arrow in each profile. PI Kinase Activity Percentage of [32P]PIPformed Control pmol mg-’ min-’ ation assay in the presence of exogenous substrate. As with Using endogenous substrate the PIP-PLC activity, the maximum decrease was found after Control 265.9 f 24.0” 1 O0 Hyperosmotic 150.4 +- 40.4 56.7 f 21.4 5 min of stress (Fig. 5). In contrast to the PIP-PLC activity Using exogenous substrate (Fig. 3), the activity of PI kinase did not recover by 10 min Control 1395.8 k 13.4 1O0 but remained at a low leve1 (Fig. 5). In the in vitro phos- Hyperosmotic 650.4 +- 236.8 46.6 +- 24.0 phorylation assay in which only endogenous substrate was a The data are the means 4 SD of at least six values from three used, i.e. isolated plasma membranes alone, the major phos- different experiments. phorylation product was phosphatidic acid. This is indicative Phosphoinositide Metabolism and Hyperosmotic Stress 64 3

h .-c To determine whether or not the stress-regulated okadaic E 2000 acid-sensitive phosphatase could be recovered with the plasma membrane, we isolated membranes from stressed and nonstressed cells and added okadaic acid (0.01-1 p~)to the phosphorylation reaction mixture. At a concentration of 0.01 p~,okadaic acid had no effect on the PI kinase activity of the membranes isolated from hyperosmotically stressed cells and decreased (by 15%) the [32P]PIPformed by membranes from control cells (Fig. 6). At higher concentrations (0.1 and 1 p~),okadaic acid decreased the [32P]PIPformed by membranes from hyperosmotically stressed cells. Importantly, in the in vitro experiments at all concentrations of okadaic acid aEzo added, the effect of osmotic stress on PI kinase activity was N 2 4 6 8 10 12 still evident (Fig. 6). *? The effect of okadaic acid on the plasma membrane PIP- Time (min) PLC also was studied. Adding okadaic acid to the isolated plasma membranes decreased the specific activity of PIP- Figure 5. Time course of changes in PI kinase activity in response PLC by 40 and 20% for the control and hyperosmotic cells, to 0.4 OSM hyperosmotic stress. Cells were treated hyperosmotically respectively. It is interesting that adding okadaic acid to the (0.4 OSM) for various times, and for the control the same amount of plasma membranes isolated from hyperosmotically stressed buffer in conditioned medium was added. Plasma membranes were cells decreased the specific activity of the PIP-PLC to ap- isolated from cells in each treatment by aqueous two-phase parti- proximately that of untreated membranes from control cells. tioning. PI kinase activity was assayed in the presence of exogenous Although the specific activity of the PIP-PLC decreased, the substrate. Open circles, control; closed circles, hyperosmotic stress. specific activity of PIP-PLC from hyperosmotically stressed The results are the means SD of four values from two different f cells remained higher than the okadaic acid-treated controls, experiments. i.e. adding okadaic acid to isolated plasma membranes did not overcome the effect of hyperosmotic stress. in PI kinase activity resulting from hyperosmotic stress. The DISCUSSION PI kinase activity of plasma membrane from the okadaic acid-pretreated cells did not decrease when the cells were We have shown that the metabolism of PIP changes as a hyperosmotically stressed (Table IV). DMSO alone (the minus result of hyperosmotic stress. After 5 min of stress there was okadaic acid control) had no effect on the response to osmotic a decrease in [3H]inositol-labeled PIP from whole cells. This stress. The data suggested that an okadaic acid-sensitive was associated with a concomitant increase in PIP-PLC and phosphatase(s) was activated in response to hyperosmotic a decrease in PI kinase-specific activity assayed in vitro. The stress and that the phosphatase(s) was involved in the stress- observed changes in PIP metabolism did not correlate with induced increase in PIP-PLC activity and decrease in PI the initial plasmolysis, which was complete within 30 s. kinase activity. The fact that the effects of hyperosmotic stress on the

Table 111. Effect of okadaic acid in vivo and in vitro on PIP-PLC activity Cells were pretreated with 0.15 PM okadaic acid for 1 h. An equivalent volume of DMSO was used for the control. After pretreatment,cells were treated hyperosmotically at 0.4 OSM in conditioned medium plus sorbitol. Plasma membranes were isolated by aqueous two-phase partitioning. For the in vitro treatment, okadaic acid (1 PM, final concentration)was added to reaction mixture. PIP-PLC was assaved as described in "Materiais and Methods." Assay Percentage of Pretreatment Treatment PLC Activity Condition Control nmol min-' mg-' Okadaic acid - Control 7.52 f 0.82a 1O0 + 5.87 f 0.08 78.0 f 1.1 - Hyperosmotic 10.66 f 0.48 141.7 f 6.4 + 5.63 f 0.51 74.9 f 6.8 Okadaic acid Control - 12.83 f 1.64 1 O0 + 7.96 f 0.86 62.0 f 6.7 Hyperosmotic - 17.01 f 1.16b 132.6 zk 9.1 + 13.10 f 0.92 102.1 f 7.2 "The numbers are the averages zk SD of four values from two different experiments. bThe numbers are averages f SD of three values from two experiments. 644 Cho et al. Plant Physiol. Vol. 103, 1993

Table IV. Effect of okadaic acid in vivo and in vitro on PI kina5e activity Cells were pretreated with 0.15 p~ okadaic acid for 1 h. An equivalent volume of DMSO was used for the control. After pretreatment, cells were treated hyperosmotically at 0.4 OSM in conditioned medium plus sorbitol. Plasma membranes were isolated by aqueous two-phase partitioning. For the in vitro treatment, okadaic acid (1 p~,final concentration) was added to reaction mixture. PI kinase was assayed as described in "Materials and Methods." Assay PI Kinase Percentage of Pretreatment Treatment Condition Activity Control pmol min-' mg-' Okadaic acid - Control 261 f 36.4" 1 O0 + 216 f 17.0 82.8 rt 9.2 - Hyperosmotic 172 2 23.8 65.8 f 12.9 + 233 f 23.2 89.3 f 12.6 Okadaic acid Control - 293 & 21.7 1o0 + 250& 6.6 85.3 & 3.2 Hyperosmotic - 210 & 11.7 71.7& 5.6 i- 166 f 36.7 56.7 f 12.5

a The numbers are the averages & SD of four values from two different experiments. plasma membrane enzymes persisted in the isolated mem- that the transient increase in PLC activity in vitro caused a branes indicated that the enzymes or some regulator of limited increase in the rate of release in [3H]IP2or that osmotic enzyme activity was altered. Although there is no evidence stress enhanced the hydrolysis of Ir2. Severa1 laboratories for covalent modification of PI kinase per se (Carpenter and have shown that the inositol phosphates are metabolized Cantley, 1990), our data suggest that the changes in PIP rapidly by plant homogenates and plant membranes anel that metabolism observed as a result of hyperosmotic stress are the plant inositol phosphate phosphatases are not very sen- mediated by an okadaic acid-sensitive phosphatase or by sitive to lithium (Loewus and Loewus, 1982; Drdbak et al., other okadaic acid-sensitive processes such as cytoskeleton 1988; Joseph et al., 1989; Memon et al., 1989b). Thus, newly reorganization (Fernandez et al., 1990) or intracellular trans- released IP2 may have been quickly degraded to inositol. port (Lucocq et al., 1991). In any event, within the limits of our measurements, ino- Although PIP-PLC activity recovered to control values by sitol phospholipids do not appear to be involved ir1 the 10 min, the PI kinase did not. These data suggest that, in primary signaling in response to hyperosmotic stress in carrot these cells, PI kinase may be a more critica1 and sustaining cells. It is possible that selective pools of PIP and PIP2 are factor for controlling steady-state PIP levels. Indeed, the PI involved in the early signaling event; however, the magnitude kinase activity may reflect the physiological state of the cell. of change certainly is not comparable to that of highly The decrease in the percentage of [3H]PIP and the tendency responsive animal cells such as the blowfly salivary gland for the percentage of [3H]PI to increase after 5 min of hyper- (Bemdge, 1983) or iris muscle (Akhtar and Abdel-Latif, osmotic stress would be consistent with activation of a PIP 1980). The change in PIP metabolism in the carrot cells phosphatase (monoesterase) as well as a decrease in PI kin- appears to be secondary to the initial stimulus, perhaps ase activity; however, we could not detect PIP phosphatase prolonging or facilitating in this instance a change in cell activity in isolated plasma membranes assayed with endog- shape and ultimately reflecting a change in the metabolic enous [32P]PIPor exogenous [3H]inositol PIP as substrates state of the cell. (data not shown). Einspahr et al. (1988) reported that increased [32P]PIPand We started each experiment with the same fresh weight of [32P]PIP2occurred as a result of short-tem treatment of the cells. For each treatment, the inositol lipids and inositol halotolerant algae, D. salina, with NaCl. Based on these phosphates were monitored as changes in cpm recovered as experiments we anticipated seeing an increase in [3H]inosit~l- well as relative changes in the distribution of each lipid or labeled PIP and PIP2 after hyperosmotic stress, but we found inositol phosphate as a percentage of the total 3H recovered. decreased recovery of [3H]PIP. The difference in the data [3H]PIPdecreased both in cpm and as percentage of total 3H- might be due to differences in systems, halotolerant algae lipid. Because the [3H]PIP was 10-fold higher than the [3H]- versus carrot cells, labeling positions, and, therefore, potential PIPl and because the [3H]IP3was so low, it seems unlikely pools of labeled lipids, [32P]Piversus [3H]inositol,and stress- that an increase in the flux of PIP to PIP2 and Ir3 makes a inducing substances, NaCl versus sorbitol, respectively. Also, significant contribution to the decrease in [3H]PIP. this discrepancy between algae and higher plants ma,y be The profile of water-soluble 3H compounds indicates the explained by the difference in regulation of PLC. For exam- complexity of inositol metabolism in plants, as others have ple, there is no evidence for effects of GTP or GTP-7-5 on shown (for review, see Coté et al., 1990; Loewus et al., 1990). the PLC-mediated breakdown of polyphosphoinositides in We did not detect a net increase in [3H]IP or [3H]IP2 after higher plants (Melin et al., 1987; McMurray and Irvine, 1988; osmotic stress. However, we could not exclude the possibility Tate et al., 1989; Pica1 et al., 1992), but the activity of PLC Phosphoinositide Metabolism and Hyperosmotic Stress 645

400 the osmotically stressed cells suggested that an initial decrease in ATPase activity may be unrelated to the level of PIP in ?IE plasma membranes; however, the later decrease in ATPase .-c after 5 min may reflect the lower levels of PIP as the cells E reach a new steady state. Negatively charged inositol lipids - can directly affect the activity of P-type ATPases (Varsanyi -o) O et al., 1983; Schafer et al., 1987; Memon et al., 1989a; Chen E and Boss, 1991). Q v Within the context of signal transduction mechanisms the W changes in inositol metabolism that we observed are slow o) and are probably down stream from the initial response. E L Slayman (1982) has shown that the plasma membrane of O Y- yeast cells depolarizes rapidly, within 30 s, in response to 1 hyperosmotic stress, followed by a slower recovery period that lasts severa1 minutes. Such a depolarization could indicate a change in ion flux or membrane structure. Stretch- activated ion channels have been shown to be present in

O‘ tobacco cells grown in suspension culture and guard cells O 0.01 0.1 1 I (Edwards and Pickard, 1987; Schroeder and Hedrich, 1989). These channels are activated by stretching the plasma mem- Okadaic Acid (1 0-6M) branes, and therefore, their open probability may be changed rapidly in response to mechanical stimuli or as a result of Figure 6. The effect of okadaic acid in vitro on PI kinase activity in volume or turgor changes. Physiological responses within the plasma membranes isolated from control and stressed cells. Cells cells could be mediated by a resultant increase in cytosolic were treated hyperosmotically (0.4 OSM) for 5 min as described in calcium or by some other factors, such as a change in H+, K+, “Material5 and Methods.” Okadaic acid (155 PM in 10% DMSO) was added to the lipid phosphorylation reaction mixture to give the or C1- ions. Calcium has been shown to activate PIP-PLC final concentrations indicated. The PI kinase activity was assayed in (Melin et al., 1987, 1992; Tate et al., 1989; Pical et al., 1992) the absence of exogenous substrate. Open circles, control; closed and inhibit PI kinase activity (Kamada and Muto, 1992) in circles, hyperosmotic stress. The results are the means & SD of four higher plants. values from two different experiments. One consequence of decreased PIP is a potential loss of gelsolin or profilin binding and a resultant increase in actin severing and a decrease in actin nucleation sites (Janmey and Stossel, 1987; Janmey et al., 1987; Lind et al., 1987). In vitro the PLC-mediated breakdown of polyphosphoinositides in studies indicate that profilin can bind to PIP and PIPl and higher plants (Melin et al., 1987; McMurray and Irvine, 1988; Tate et al., 1989; Pical et al., 1992), but the activity of PLC protect them from hydrolysis by PLC (Forscher, 1989; Gold- from D. salina has been reported to be stimulated by the schmidt-Clermont et al., 1990; Machesky et al., 1990). It is nonhydrolyzable GTP analog, GTP-7-S (Einspahr et al., not yet clear, however, whether the increase in cytosolic 1989). calcium and the profilin release precede or follow PIP hy- Srivastava et al. (1989) studied the effects of mannitol on drolysis in vivo. The data suggest that profilin could link cell signaling at the membrane level to reorganization of the IP3 production in red beet slices. IP3 was measured as [3H]IP3 displaced from the IP3-binding protein. The amount of pu- cytoskeleton. Because a considerable portion of the PI and tative IP3 decreased 10 min after beet root slices were placed PIP kinase activity is associated with the actin filament frac- in 0.4 M mannitol and increased after 10 min in 0.2 M tion in isolated plasma membranes (Tan and Boss, 1992), if mannitol. If the red beet slices were plasmolyzed in 0.4 M there is a change in the cytoskeletal structure (eg a loss of mannitol, the decrease in IP3 would be consistent with the actin filaments and actin-binding proteins associated with down regulation of the inositol lipid metabolism observed in the plasma membrane), this could contribute to the decrease the plasmolyzed carrot cells. in plasma membrane-associated PI kinase. Salt stress resulted in reduced plasma membrane ATPase Based on these results we propose the following working activity of tomato roots (Gronwald et al., 1990), suggesting model for carrot cells hyperosmotically stressed with sorbitol: that salt stress may impair the catalytic efficiency of the Hyperosmotic stress induces membrane depolarization and ATPase either by affecting the synthesis of positive or nega- plasmolysis within 30 s. An okadaic acid-sensitive phospha- tive effectors or by affecting the lipid composition of the tase that is not associated with the plasma membrane is membrane. The carrot cell plasma membrane vanadate-sen- activated. After about 5 min the cells are in a recovery phase sitive ATPase activity also was reduced by hyperosmotic in which the cells begin to adjust to the new osmoticum. stress. The change in plasma membrane vanadate-sensitive During the recovery phase, PIP-PLC is transiently activated ATPase activity previously has been shown to closely corre- while there is a sustained decrease in PI kinase activity. The late with the PIP kinase activity in response to the cell-wall- result is a decrease in PIP. The net decrease in PIP would degrading enzyme, Driselase (Chen and Boss, 1990). Results contribute to the decrease plasma membrane ATPase and of the time-course study of ATPase and PI kinase activity in affect cytoskeleton reorganization. As a result of these and 646 Cho et al. Plant Physiol. Vol. 103, 1993

other intracellular events, the physiological state of cells esterase in Dunaliella salina membranes. Plant Physiol 90 changes, and cells reach a new steady state. 1115-.1120 Einspahr KJ, Thompson GA Jr (1990) Transmembrane signalling via phosphatidylinositol 4,5-bisphosphate hydrolysis in plants. Received April 19, 1993; accepted June 19, 1993. \ Plant Physiol93: 361-366 Copyright Clearance Center: 0032-0889/93/103/0637/11. Fernandez A, Brautigan DL, Mumby M, Lamb NJC (1990) Protein phosphatase type-1, not type-2A, modulates actin microfilament integrity and myosin light chain phosphorylation in living non- LITERATURE ClTED muscle cells. J Cell Biol 111: 103-112 Akhtar AA, Abdel-Latif A (1980) Requirement for calcium ions in Forscher P (1989) Calcium and polyphosphoinositide control of acetylcholine-stimulated phosphodiesteratic cleavage of phospha- cytoskeletal dynamics. Trends Neurosci 12: 468-474 tidyl-myo-inositol4,5-bisphosphatein rabbit iris smooth muscle. J Gallagher SR, Leonard RT (1982) Effect of vanadate, molybdate, Biochem 192: 783-791 and azide on membrane-associated ATPase and soluble phospha- Balla T, Hunyady L, Baukal AJ, Catt KJ (1989) Structures and tase activities of corn roots. Plant Physiol 70 1335-1340 metabolism of inositol tetrakisphosphates and inositol pentakis- Gilroy S, Read ND, Trewavas AJ (1990) Elevation of cytoplasmic phosphate in bovine adrenal glomerula cells. J Biol Chem 264 calcium by caged calcium or caged inositol trisphosphate initiates 9386-9390 stomatal closure. Nature 346 769-771 Berridge MJ (1983) Rapid accumulation of inositol trisphosphate Goldschmidt-Clermont P, Machesky LM, Baldassare JJ, Padlard reveals that agonists hydrolyse polyphosphoinositides instead of TD (1990) The actin-binding protein profilin binds to PIP:! and phosphatidylinositol. Biochem J 212: 849-858 inhibits its hydrolysis by phospholipase C. Science 247: 1575-1578 Berridge MJ, Dawson RMC, Downes CP, Heslop JP, Irvine RF Gronwald JW, Suhayda CG, Tal M, Shannon MC (1990) Reduction (1983) Changes in the levels of inositol phosphates after agonist- in plasma membrane ATPase activity of tomato roots by salt stress. dependent hydrolysis of membrane phosphoinositides. Biochem J Plant Sci 66 145-153 212: 473-482 Gross W, Boss WF (1992) Inositol phospholipids and signal trans- Berridge MJ, Irvine RF (1989) Inositol phosphates and cell signall- duction. In DPS Verma, ed, Control of Plant Gene Expression. The ing. Nature 341: 197-205 Telford Press, Caldwell, NJ, pp 17-32 Bialojan C, Takai A (1988) Inhibitory effect of a marine-sponge Hetherington AM, Drdbak BK (1992) Inositol-containing lipids in toxin, okadaic acid, on protein phosphatases. Specificity and ki- higher plants. Prog Lipid Res 31: 53-63 netics. Biochem J 256 283-290 Hughes PJ, Hughes AR, Putney JW Jr, Shears SB (1989) The Carpenter CL, Cantley LC Jr (1990) Phosphoinositides kinases. regulation of the phosphorylation of inositol 1,3,4-trisphosphate Biochemistry 29 11147-11156 in cell-free preparations and its relevance to the formation of Chauhan A, Chauhan VPS, Deshmukh DS, Brockerhoff H (1989) inositol 1,3,4,6-tetrakisphosphatein agonist-stimulated rat parotid Phosphatidylinositol4,5-bisphosphatecompletely inhibits phorbol acinar cells. J Biol Chem 264 19871-19878 ester binding to protein kinase C. Biochemistry 28: 4952-4956 Janmey PA, Iida K, Yin HL, Stossel TP (1987) Polyphosphoinositide Chauhan VPS, Brockerhoff H (1988) Phosphatidylinositol-4,5-bis- micelles and polyphosphoinositide-containing vesicles dissociate phosphate may antecede diacylglycerol as activator of protein endogenous gelsolin-actin complexes and promote actin assembly kinase C. Biochem Biophys Res Commun 155: 18-23 from the fast-growing end of actin filaments blocked by ge1:jolin. Chen Q, Boss WF (1990) Short-term treatment with cell wall deg- J Biol Chem 262: 12228-12236 radation enzymes increases the activity of the inositol phospholipid Janmey PA, Stossel TP (1987) Modulation of gelsolin function by kinases and the vanadate-sensitive ATPase of carrot cells. Plant phosphatidylinositol4,5-bisphosphate.Nature 325 362-364 Physiol94 1820-1829 Joseph SK, Esch T, Bonner WD Jr (1989) Hydrolysis of inositol Chen Q, Boss WF (1991) Neomycin inhibits the PIP and PIPl phosphates by plant cell extracts. Biochem J 264 851-856 stimulation of plasma membrane ATPase activity. Plant Physiol Kamada Y, Muto S (1992) CaZf regulation of phosphatidylinositol 96 340-343 tumover in the plasma membrane of tobacco suspension culture Chen Q, Brglez I, Boss WF (1991) Inositol phospholipids as plant cells. Biochim Biophys Acta 1093: 72-79 second messengers. In G Jenkins, W Schulch, eds, Molecular Kirk CJ, Morris AJ, Shears SB (1990) Inositol phosphate second Biology of Plant Development. Company of Biologist, Cambridge, messengers. In K Siddle, JC Hutton, eds, Peptide Hormone Action, UK, pp 159-175 A Practical Approach. IRL Press, Oxford, UK, pp 151-184 Cho MH, Chen Q, Okpodu CM, Boss WF (1992) Separation and Lassing I, Lindberg U (1985) Specific interaction between phospha- quantitation of [3H]inositol phospholipids using thin-layer chro- tidylinositol 4,5-bisphosphate and profilactin. Nature 314 matography and a computerized 3H imaging scanner. LC/GC 10: 472-474 464-468 Lee-Stadelmann OY, Stadelmann EJ (1976) Cell permeability and Cote GG, Quarmby LM, Satter RL, Morse MJ, Crain RC (1990) water stress. In O Lange, L Kappen, ED Schulze, eds, Water and Extraction, separation, and characterization of the metabolites of Plant Life-Problems and Modern Approach, Ecological Studies the inositol phospholipid cycle. In DJ Morré, WF Boss, FA Loewus, Vol 19. Springer-Verlag,New York, pp 268-280 eds, Inositol Metabolism in Plants. Wiley-Liss, New York, Lind SE, Janmey PA, Chaponnier C, Herbert T-J, Stossel TP (lL987) pp 113-138 Reversible binding of actin to gelsolin and profilin in human Drdbak BK, Ferguson IB (1985) Release of Ca2+from plant hypo- platelets extracts. J Cell Biol 105: 833-842 cotyl microsomes by inositol-1,4,5-trisphosphate.Biochem Bio- Loewus FA, Everard JD, Young KA (1990) Inositol metabolism: phys Res Commun 130 1241-1246 precursor role and breakdown. In DJ Morré, WF Boss, FA Loewus, Drdbak BK, Ferguson IB, Dawson AP, Irvine RF (1988) Inositol- eds, Inositol Metabolism in Plants. Wiley-Liss, New York, pp containing lipids in suspension-cultured plant cells. An isotopic 21-46 study. Plant Physiol 87: 217-222 Loewus MW, Loewus FA (1982) myo-Inositol-I-phosphatasefrom Edwards KL, Pickard BG (1987) Detection and transduction of the pollen of Lilium longiflorum thunb. Plant Physiol 70: 765--770 physical stimuli in plants: the cell surface in signal transduction. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein NATO AS1 Ser H 12: 41-66 measurement with the Folin phenol reagent. J Biol Chem 193 Einspahr KJ, Peeler TC, Thompson GA Jr (1988) Rapid changes in 265-275 polyphosphoinositide metabolism associated with the response Lucocq J, Warren G, Pryde J (1991) Okadaic acid induces Golgi of Dunaliella salina to hypoosmotic shock. J Biol Chem 263 apparatus fragmentation and arrest of intracellular transport. :I Cell 5775-5779 Sci 100: 753-759 Einspahr KJ, Peeler TC, Thompson GA Jr (1989) Phosphatidyli- Machesky LM, Goldschmidt-Clermont PJ, Pollard TD (1990) The nositol 4,5-bisphosphate phospholipase C and phosphomono- affinities of human platelet and Acanthamoeba profilin isoforms Phosphoinositide Metabolism and Hyperosmotic Stress 64 7

for polyphosphoinositides account for their relative abilities to RincÓn M, Boss WF (1990) Phosphoinositides as second messengers inhibit phospholipase C. Cell Regul 1: 937-950 in plants. Zn DJ Morri., WF Boss, F Loewus, eds, Plant Inositide Majerus PW, Connolly TM, Deckmyn H, Ross TS, Bross TE, Ishii Metabolism. Wiley-Liss, New York, pp 173-200 H, Bansal VS, Wilson DB (1986) The metabolism of phosphoi- RincÓn M, Chen Q, Boss WF (1989) Characterization of inositol nositide-derived messenger molecules. Science 234 1519-1526 phosphates in carrot (Daucus carota L.) cells. Plant Physiol 89 McMurray WC, Irvine RF (1988) Phosphatidylinositol4,5-bisphos- 126-132 phate phosphodiesterase in higher plants. Biochem J 249 877-881 Sandelius AS, Sommarin M (1986) Phosphorylation of phosphati- Melin P-M, Pical C, Jergil B, Sommarin M (1992) Polyphosphoi- dylinositols in isolated membranes. FEBS Lett 201: 282-286 nositide phospholipase C in wheat root plasma membranes. Partia1 Schafer M, Behle G, Varsanyi M, Heilmeyer LMG Jr (1987) Ca2+ purification and . characterization. Biochim Biophys Acta 1123: regulation of 1-(3-sn-phosphatidyl)-lD-myo-inositol4-phosphate 163-169 formation and hydrolysis on sarcoplasmic reticular Ca2+-transport Melin P-M, Sommarin M, Sandelius AS, Jergil B (1987) Identifi- ATPase. Biochem J 247: 579-587 cation of Ca2+ stimulated phosphoinositide phospholipase C in Schroeder JI, Hedrich R (1989) Involvement of ion channels and isolated plant plasma membranes. FEBS Lett 223 87-91 active transport in osmoregulation and signalling of higher plant Memon AR, Boss WF (1990) Rapid light-induced changes in phos- cells. Trends Biochem 14: 187-192 phoinositide kinases and H+-ATPase in plasma membrane of Schumaker KS, Sze H (1987) Inositol 1,4,5-trisphosphate releases sunflower hypocotyls. J Biol Chem 265: 14817-14821 CaZ+from vacuolar membrane vesicles of oat roots. J Biol Cell262: Memon AR, Chen Q, Boss WF (1989a) Inositol phospholipids 3944-3946 activate plasma membrane ATPase in plants. Biochem Biophys Slayman CL (1982) Charge-transport characteristics of a plasma Res Commun 162 1295-1301 membrane proton pump. In AN Martonosi, ed, Membranes and Memon AR, RincÓn M, Boss WF (1989b) Inositol trisphosphate Transport. Plenum Press, New York, pp 485-490 metabolism in carrot (Daucus carota L.) cells. Plant Physiol 91: Sommarin M, Sandelius AS (1988) Phosphatidylinositol and phos- 477-480 phatidylinositol phosphate kinases in plant plasma membranes. Menniti Oliver KG, Nogimori K, Obie JF, Shears SB, Putney Biochim Biophys Acta 958: 268-278 FS, Srivastava A, Pines M, Jacoby (1989) Enhanced potassium uptake JW Jr (1990) Origins of myo-inositol tetrakisphosphates in agonist- B and tumover by hypertonic man- stimulated rat pancreatoma cells. Stimulation by bombesin of myo- phosphatidylinositol-phosphate nitol shock. Physiol Plant 77: 320-325 inositol breakdown to myo-inositol 1,3,4,5,6-pentakisphosphate Streb H, Irvine RF, Berridge MJ, Schultz I(1983) Release of Caz+ 3,4,5,6-tetrakisphosphate.J Biol Chem 265: 11167-11176 from nonmitochondrial intracellular store in pancreatic acinar cells Michell RH (1992) Inositol lipids in cellular signalling mechanisms. by inositol 1,4,5-trisphosphate. Nature 306: 67-68 Trends Biochem 17: 274-276 Tan Z, Boss WF (1992) Association of phosphatidylinositol kinase, Morse MJ, Crain RC, Satter RL (1987) Light-stimulated inositol phosphatidylinositol monophosphate kinase, and diacylglycerol phospholipid tumover in Samanea saman. Proc Natl Acad Sci USA kinase with the cytoskeleton and F-actin fractions of carrot (Daucus 84 7075-7078 carota L.) cells grown in suspension culture: response to cell wall- Owen JD (1976) The determination of the stability constant for degrading enzymes. Plant Physiol 100: 21 16-2120 calcium-EGTA. Biochim Biophys Acta 451: 321-325 Tarczynski MC, Jensen RG, Bohnert HJ (1993) Stress protection of Payrastre B, van Bergen PMP, Henegouwen EN, Breton M, den transgenic tobacco by production of the osmolyte mannitol. Sci- Hartigh JC, Plantvid M, Verkleij AJ, Boonstra J (1991) Phos- ence 259 508-510 phoinositide kinase, diacylglycerol kinase, and phospholipase A Tate BF, Schaller GE, Sussman MR, Crain RC (1989) Characteriza- activities associated to the cytoskeleton: effect of epidermal growth tion of polyphosphoinositide phospholipase C from the plasma factor. J Cell Biol 115 121-128 membrane of Avena saliva. Plant Physiol91: 1275-1279 Pfeffer W (1877) Osmotische untersuchungen: Studien zur zellme- Taussky HH, Shorr E (1953) A microcolorimetric method for the chanik, translated by GR Kepner and EJ Stadelmann, 1985. Van determination of inorganic phosphorus. J Biol Chem 202: 675-685 Nostrand Reinhold Co, New York Varsanyi M, Tolle HG, Heilmeyer LMG Jr, Dawson RMC, Irvine Pical C, Sandelius AS, Merlin P-M, Sommarin M (1992) Poly- RF (1983) Activation of sarcoplasmic reticular Ca2+-transportATP- phosphoinositide phospholipase C in plasma membranes of wheat ase by phosphorylation of an associated phosphatidylinositol. (Triticum sativum L.), Plant Physiol 100 1296-1303 EMBO J 2 1543-1548 Ranjeva R, Carrasco A, Boudet AM (1988) Inositol trisphosphate Yang W, Burkhart W, Cavallius J, Merrick WC, Boss WF (1993) stimulates the release of calcium from intact vacuoles isolated from Purification and characterization of a phosphatidylinositol 4-ki- Acer cells. FEBS Lett 230 137-141 nase activator in carrot cells. J Biol Chem 268: 392-398