Phosphatidylinositol and phosphatidylinositolphosphate kinases in plant plasma membranes

268

Blochimica et Biophysics Acta 958 (1988) 268-278

Elsevier BBA 52132

Phosphatidylinositol

and phosphatidylinositolphosphate

kinases

in plant plasma membranes Marianne

Sommarin

a and Anna Stina Sandelius

b

a Institute of Plant Physiology, University of Lund, Lund and b Department of Plant Physiology, University of G&eborg, Gijteborg (Sweden)

(Received 17 July 1987) (Revised manuscript received 19 October 1987)

Key words: Polyphosphoinositide;

Lipid kinase; Phosphatidylinositol Phospholipid; Phosphorylation

diphosphate; Phosphoinositide;

Both phosphatidylinositol (PI) and phosphatidylinositolphosphate (PIP) kinase activities were present in plasma membrane fractions isolated from shoots and roots of dark-grown wheat (Triticrrm aestiuum L.) by aqueous polymer two-phase partition. The enzymes phosphorylated their respective endogenous substrates as well as exogenously added substrates (PI and phosphatidylinositol 4-monophosphate, PI-4P), to form PIP and phosphatidylinositol diphosphate (PIP,). The reactions were dependent on ATP. Phosphorylation of added PI reached maximum activity around 0.75 mM ATP, while the ATP requirement for maximal activity was higher both for phosphorylation of added PI-4P (1.25 mM ATP) and of endogenous lipids (1.5 mM ATP). Optimal Mg2+ concentration varied between 5 mM (endogenous PI phosphorylation) and 15 mM (phosphorylation of exogenous PI). The Mg2+ requirement could be substituted only partially by Mn” and not at all by Ca”. Phosphorylation of endogenous lipid substrates was inhibited by Triton X-100 concentrations above 0.015%, while phosphorylation of exogenous substrates was stimulated several-fold by up to 0.5% Triton X-100. Triton X-100 also influenced the optimal pH range of the reactions. While phosphorylation of endogenous PI and PIP was optimal at pH 6.5-7 without Triton X-100 in the assay medium, addition of 0.010% Triton X-100 extended the optimal pH range up to pH 8.6. Phosphorylation of exogenous lipids were optimal at pH 7.8-8.2. At optimal conditions and with endogenous substrates, PIP formation was 125-225 and 40-90 pmol/mg protein per min in shoot and root plasma membranes, respectively, and PIP, formation lo-25 and 4-8 pmol/mg protein per min, respectively. With exogenous substrates, the corresponding rates increased 8-20-times. These results demonstrate the close resemblance between the characteristics of PI and PIP kinase activities in plant membranes with corresponding activities in animal plasma membranes. It is, however, not yet known if polyphosphoinositide metabolism in plant cells resembles the corresponding metabolism in animal cells also in function, that is, in acting as a signal-transducing system for internal Ca2+ mobilization.

Abbreviations: DG, diacylglycerol; IP-,, inositol trisphosphate; PA, phosphatidic acid; PI, phosphatidylinositol; PI-4P, phosphatidylinositol 4-monophosphate; PIP, phosphatidylinositol monophosphate; PI-4,5P,, phosphatidylinositol 4,5-diphosphate; PIP,, phosphatidylinositol diphosphate. Correspondence: M. Sommarin, Institute of Plant Physiology, University of Lund, P.O. Box 7@7, S-220 07 Lund, Sweden.

Introduction Plant growth and development is governed by environmental (e.g., light, temperature, gravity, inorganic and organic nutrients, water potential) and internal (e.g., hormones, water potential) signals. The

OOOS-2760/88/$03.50 0 1988 Elsevier Science Publishers B.V. (Biomedical Division)

nutritional correlative

status, manners

269

in which these signals affect a plant is not understood [l-3], but it has been suggested that hormones act as integrating agents [l]. Plant hormone research is presently making progress in several crucial areas, such as quantification of hormone concentrations in tissues [4], elucidation of hormone metabolism [5-81, identification of hormone transport carriers [9], and isolation and characterization of hormone receptors [lo-121. The signal-transducing system, that is, the reaction or chain of reactions that transmit the information of an activated receptor to a physiological response, has not yet been elucidated for any plant hormone. However, there are indications that several plant hormones affect the cytoplasmic concentration of Ca2+ [13]. The two major signal-transducing systems in animal cells utilize cyclic AMP and Ca2+, respectively, as second messengers. The system leading to an increase in the concentration of cytoplasmic Ca2+ involves hydrolysis of a plasma membrane localized lipid, phosphatidylinositol 4,5-diphosphate (PI-4,5P,) [14,15]. PI-4,5P, hydrolysis is catalyzed by a phospholipase C, and the coupling of an activated receptor to the phospholipase is stimulated by GTP [14,15]. The hydrolysis products are inositol trisphosphate (191) and diacylglycerol (DG). IP, is released to the cytoplasm, and acts to mobilize Ca2+ from internal stores in the cell, thus increasing the cytosolic Ca*+ concentration [14-161. DG remains a membrane component and can, together with Ca”, stimulate protein kinase C [14,17]. The first indication of the existence of a polyphosphoinositide metabolism in plants also was the demonstration by Boss and Masse1 [18] that a cell suspension culture of Caucus carota incorporated myo-[2-‘Hlinositol into PI, PIP and PIP,. Another approach to study the possible involvement of polyphosphoinositide metabolism in signal transduction in plants was used by Drobak and Ferguson [19], who showed that added IP, stimulated the release of Ca2+ from Ca2+-loaded microsomal membrane vesicles. Our work on polyphosphoinositide metabolism in isolated plant membranes represents yet another approach. We have demonstrated the capability of isolated plant membranes to form PIP and PIP,

when incubated with [y-j2P]ATP, and that the reactions involved were localized predominantly in plasma membrane fractions [20]. We present here a characterization of PI and PIP kinases in plasma membrane fractions isolated from shoots and roots of dark-grown wheat. The similarities between the properties found for the plant PI and PIP kinases with those reported for corresponding activities in animal membranes may be considered to support the emerging assumption that polyphosphoinositide metabolism could be involved in signal-transducing mechanisms also in plants. Materials and Methods Materials. All lipids were obtained from Sigma. [y-32P]ATP was synthesized according to the method of Chang et al. [21]. All other chemicals used were of analytical grade. Plant material and plasma membrane isolution. The shoots (leaves and coleoptiles) and roots of 7-day-old dark-grown wheat seedlings (Triticum aestiuum L. cv. Drabant) were harvested and the shoot and root microsomal membrane fractions (10000 X g (15 min) - 30000 X g (60 min) pellet) were isolated separately [22]. The plant growth conditions were as described [22]. Plasma membranes were isolated from the microsomal membrane fractions (containing not only plasma membrane-derived vesicles but also vesicles of intracellular membranes) by aqueous polymer two-phase partition [22]. Shoot and root plasma membranes were always isolated from the same plants and assayed in parallel. Isolated membrane fractions were kept on ice and assayed for PI and PIP kinase activities within 4 h of isolation. Membrane protein was determined according to the method of Markwell et al. [23] with bovine serum albumin as protein standard. Determination of lipid kinase activities. The standard reaction mixture comprised, unless stated otherwise in figure and table legends, in a final volume of 50 ~1: 2.5 pmol Hepes-KOH (pH 7.8) 0.75 (PI kinase) or 0.375 (PI-4P kinase) pmol 50 nmol dithioerythritol, 75 nmol [yMgCl,, ‘* P]ATP (specific activity, 250-450 cpm/pmol), and 20-25 pg membrane protein. Triton X-100 (0.005 mg) or 0.125 mg Triton X-100 plus 20 nmol

PI or 0.150 mg Triton X-100 plus 20 nmol PI-4P were included for determinations of endogenous PI and PIP kinase activities, exogenous PI phosphorylation, and exogenous PI-4P phosphorylation, respectively. External substrates were added as sonicated aqueous suspensions. Triton X100 was added to the membranes prior to addition of phosphoinositides. The reactions were run in duplicates and were started by the addition of ATP. The incubation time was 4 min, except for phosphorylation of added PI, where the incubation time was 2 min. The reactions were linear between 10 and 40 pg of protein. For time-course studies, the reaction mixtures were scaled-up and samples withdrawn at the indicated times. The reactions were stopped by the addition of 600 ~1 ice-cold chloroform/ methanol/ water (1 : 2: 0.6, by vol.). After addition of 150 ~1 chloroform (containing 0.05% (w/v) 2,6-di-tertbutyl-p-cresol and approx. 30 pg of a phosphoinositide mixture (Sigma)) and 120 ~1 2.4 M HCI, lipids were extracted by a modification of the method of Schacht [24] as described previously 1201. The lipid extracts were chromatographed on silica gel thin-layer chromatography plates, which had been impregnated with 1% (w/v) potassium oxalate and dried (lZO*C) for 2 h prior to use. The solvent system was chloroform/methanol/ 25% ammonia (Merck)/H~O (45 : 45 : 3.5 : 10, by vol.). PI-4P and PI-4,5P, (of bovine origin, Sigma) were used as standards. The radioactivity of the different lipids was determined by liquid scintillation counting as previously described [20]. The data presented are representative of 2-4 independent membrane isolations. The ratio of the rates of phosphorylation of endogenous substrates to exogenously added substrates was constant between different membrane isolations, whiIe the absolute rates varied up to 2-fold between different isolations. Duplicate analyses within an experiment deviated between 1 and 6% from the mean value. Identification of phosphotylated lipids. Plasma membrane fractions were incubated in a scaled-up reaction mixture for 30 min and the lipids were extracted. The lipid samples were chromatographed on potassium oxalate-impregnated thinlayer chromatography plates in either of the two systems: (1) chloroform/ methanol/ 25% am-

monia/H,O (45 : 45 : 3.5 : 10, by vol.) or (2) chloroform/ methanol/ acetic acid/ water (50 : 30: 8.4, by vol.) followed by development in the same dimension in chloroform/methanol/25% ammonia/H,0 (45 : 45 : 3.5 : 10, by vol.) for approx. 5 cm. This second development was necessary to move PIP, from the application point. The following authentic lipid standards were co-chromatographed with the samples: PI-4P, PL4,5P,, PI, lysoP1, PA, lysoPA, phosphatidylcholine, lysophosphatidylcholine, phosphatidylethanolamine, lysophosphatidylethanolamine, phosphatidylglycerol, lysophosphatidylglycerol, phosphatidylserine and lysophosphatidylserine. After development, the plates were exposed to iodine vapor to localize the lipids. After the iodine had evaporated, the silica gel area corresponding to respective sample lane was scraped off the plates in 3-5-mm increments perpendicular to the direction of the chromatographic separation. The ratioactivity of each increment was determined by liquid scintillation counting [20]. Results Lipids phosphorylated Plasma membrane fractions isolated from shoots and roots of dark-grown wheat phosphorylated exogenous (PI and PI-4P, Fig. 1) as well as endogenous substrates to form PIP and PIP,. The identities of the lipids were verified by thin-layer chromatography with standards in two different solvent systems. When no lipid substrates or when PI-4P was added to plasma membrane fractions, the major phosphorylated lipid was PA, which comprised 60-70s of the label after a 30 min incubation. Three other lipid spots contained “P, and two of these co-chromatographed with authentic lipid standards, namely PI4P and PI-4,5P,. When endogenous substrates were phosphorylated, the radioactivity of PIP was higher than that of PIP,, while the reverse was found for exogenous PI-4P phosphorylation. One 32P-containing spot on the chromatograms remains unidentified, This spot chromatographed between PI and PI-4P in both the acidic and the basic solvent systems, but did not co-chromatograph with any of the reference lipids in both solvent systems.

0

10

20 Segment

30

F

number

1. Thin-iayer chromatography of lipid extracts of plasma membranes incubate with [32PjATP and added PI or PI-4P. Lipid extracts were separated by thin-layer chromatography in chloroform/methanol/25% ammonia/H@ (d5: 45 : 3.5 : IO, by vol.). The plate was scraped in 3-S-mm segments starting from 0, the application point, to F1 the front. a-----0, plasma membrane incubated with PI, l------a, plasma membrane incubated with PIP. Fig.

When PI was added to an incubation, four phosphorylated lipids were detected after thinlayer chromatography of the lipid extract, three of which co-chromatographed with PIP, PIP, and PA, respectively, and an unknown compound which migrated below PIP and which may be 1ysoPIP (Wendy Boss, personal communication). When added PI was phospho~lated. the radioactitities of PIP, PIP, and PA formed comprised approx. 8O%, less than 5% and less than 5% respectively, of the recovered radioactivity. Effects of Trim X-100 Phosphorylation of exogenous substrates was markedly stimulated by inclusion of up to 0.5% Triton X-100 in the assay medium (Fig. 2a and b). PI phosphorylation was stimulated 7-S-fold in both shoot and root plasma membranes by inclusion of 0.25% Triton X-100 (Fig. Za), while PIP phosphorylation was stimulated 2-3-fold by 0.30% Triton X-100 (Fig. 2b). Phosphorylation of endogenous PI and PIP was, however, inhibited by concentrations of Triton X-1ocI above 0.015% (Figs. 2c and d). stimulation

Fig. 2. EfFects of Triton X-100 on PI and PIP kinase activities. Plasma membranes isolated from Shoots (0) and roots (A.) of dark-grown wheat were incubated with [y-‘* P]ATP and varying concentrations of T&on X-100. (A) Formation of PIP from added PI; (B) formation of PIP, from added PI4P: (C) formation of PIP from endogenous substrate; (D) formation of PIP, from endogenous substrate. Incubations with exogenous substrates (A, B) were at pH 7.8, while incubations with endogenous substrates (C, D) were at pH 7.0. All incubations contained 15 mM Mgs+.

of endogenous PI and PIP phosphorylatiun was obtained only with Triton X-100 concentrations between 0.0025 and 0.015%. PI and PIP kinases of shoot plasma n~embranes were sti~nulated approx. f&fold and those of root plasma membranes approx. l.Cfold by 0.010% Triton X-100 in the assay (Fig. 2c and d, insets). Triton X-100 concentrations above 0.015% may disturb membrane organization enough to render the endogenous lipid substrates inaccessible to the kinases. When exogenous lipid substrates were added to the incubation, a higher detergent concentration was needed to increase availability of the added substrate to the kinases. pN

optimum

When exogenous substrates were phosphorylated, the pH optimum for PI and PIP kinases were between 7.5 and 8.2 for shoot piasma membranes (Fig. 3a and b), and between 7.8 and 8.2

272

25

LipId substrate, nmol

Fig. 4. Effects of added lipid substrates on PI and PIP kinase activites. Plasma membranes isolated from shoots (0. 0) and roots (A., A) of dark-grown wheat were incubated with [y32P]ATP and varying amounts of PI or PI-4P. (A) Formation of PIP from added PI; (B) formation of PIP, from added PI (open symbols) or from added PI-4P (closed symbols). Phosphorylations of added PI were assayed in presence of 0.25% (w/v) Triton X-100, while phosphorylations of added PI-4P were assayed in presence of 0.30% (w/v) Triton X-100. All incubations contained 15 mM Mg*+.

0

’

I

1

6

7

8

1

j/-y-y-@ 6

7

8

PH

Fig. 3. Effects of pH on PI and PIP kinase activities. Plasma membranes isolated from shoots (0, 0) and roots (A, A) of dark-grown wheat were incubated with [y- 32P]ATP at different pH. (A) Formation of PIP from added PI; (B) formation of PIP, from added PI (open symbols) or from PI-4P (closed symbols); (C, E) formation of PIP from endogenous substrates in the presence (C) or absence (E) of 0.01% (w/v) Triton X-100; (D, F) formation of PIP, from endogenous substrates in the presence (D) or absence (F) of 0.01% (w/v) Triton X-100. The buffers used were 80 mM Mes-Tris (pH 5.5-6.0) 80 mM Hepes-KOH (pH 6.0-8.2) and 80 mM BTP-HCI (pH 8.2-8.6). All incubations contained 15 mM Mg2+.

for root plasma membranes (Fig. 3a and b). Phosphorylation of PIP (Fig. 3b) showed a much flatter pH profile around the optimum value than did PI phosphorylation (Fig. 3a). With only endogenous substrates present, the pH optimum for PI and PIP phosphorylation was in both shoot and root plasma membranes in the absence of Triton X-100 between 6.5 and 7 (Fig. 2e and f). The inclusion of 0.010% Triton X-100 in the assay extended the optimal pH range up to pH 8.6 (Fig. 2c and d), which suggests that Triton X-100 could activate the enzyme above pH 7.

Effects of externally added lipid substrates Phosphorylation of PI was linear with amount of substrate added up to 20 nmol PI in both shoot and root membranes (Fig. 4a). Further additions of PI stimulated the phosphorylation rates, but to a lesser extent. With only PI added as substrate, PIP, was also formed (Fig. 4b), although at much lower rates than when PI4P was added (Fig. 4b). Formation of PIP, from exogenously added PI4P showed a substrate optimum of 20 nmol added lipid (Fig. 4b). A TP dependability Phosphorylation of endogenous and exogenously added lipid substrates was dependent on ATP (Figs. 5 and 7). Phosphorylation of added PI reached maximum activity clearly around 0.75 mM ATP and retained the high activity up to 2 mM ATP in both shoot and root plasma membranes (Fig. 5a). ATP concentrations above 2 mM were inhibitory (data not shown). The maximum endogenous PI kinase activity was achieved at higher concentrations of ATP (1.5 mM), although 87% of maximal activity was reached at 0.75 mM ATP (Fig. 5~). The PIP kinase analyzed with exogenously ad-

213

Mg”,

ATP,

Fig. 6. Effects of Mg*+ on PI and PIP membranes isolated from shoots (0, dark-grown wheat were incubated with Mg*+ concentrations. (A) Formation (B) formation of PIP, from added PI added PI-4P (closed symbols); (C) PIP formation from endogenous

mM

Fig. 5. ATP dependency on PI and PIP kinase activities. Plasma membranes from shoots (0, 0) and roots (A, A) of dark-grown wheat were incubated in different concentrations of (y-‘*P]ATP. (A) Formation of PIP from added PI; (B) formation of PIP, from added PI (open symbols) or from added PI-4P (closed symbols); (C) PIP formation and (D) PIP, formation from endogenous substrates.

TABLE

mM

kinase activities. Plasma o) and roots (A, A) of [ Y-~*P]ATP at different of PIP from added PI; (open symbols) or from formation and (D) PIP, substrates.

I

THE EFFECTS OF DIVALENT IONS ON PHOSPHORYLATION (PI-4P) IN PLASMA MEMBRANES ISOLATED FROM SHOOTS

OF ENDOGENOUS AND EXOGENOUS AND ROOTS OF DARK-GROWN WHEAT

PI AND

PIP

Plasma membrane fractions were incubated in Hepes buffer (pH 7.8) without added lipids (-), with 20 nmol PI or with 20 nmol PI-4P. The phosphotylation rates obtained with 7.5 mM salt (added PI) or 15 mM salt (endogenous lipid substrate or added PI-4P) are presented minus the rate without salts in 0.5 mM EDTA. Substrate

Triton X-100

Lipid formed (pmol/pg

(% y/y)

shoot plasma

PIP formation _ 0.01% PI 0.25% PIP, formation _ 0.01 !%I PI 0.25% PI-4P 0.30%

per mm)

membranes

root plasma

membranes

Mg*+

Mn*+

Ca*+

Mg*++ 100 PM Ca*+

Mg’+

Mn* +

Ca*+

Mg*++ 100 PM Ca*+

126 1623

22 52

2.3 21

110 1115

37 1419

11 61

1.4 26

32 936

2 19 58

2.0 0.1 3.0

0 0.2 2.8

7.4 14 47

4.8 15 39

0.3 0 3.2

0 0 3.1

0.9 12 30

274

ded PI-4P was most active around 1.25 mM ATP (Fig. 5b), although 60-70% of maximal activity occurred already at 0.125 mM ATP. The endogenous PIP kinase activity (Fig. 5d) showed an ATP dependency rather similar to the endogenous PI kinase activity (Fig. 5b) with maximum activity at 1.5 mM ATP. For both PI and PIP kinase activities, the K, values for ATP were around 2. 1O-4 M. Ion requirement PI kinase activity measured with externally added PI was maximal at around 15 mM Mg*+ in both shoot and root plasma membranes (Fig. 6). However, when only endogenous PI was available, maximal activity was obtained at around 5 mM Mg*+ (Fig. 6). The Mg*+ requirement was somewhat different for PIP, formation. With exogenous PI-4P present, maximum activity was obtained already at 5 mM Mg*+ (Fig. 6). Maximum phosphorylation of endogenous PIP was in shoot plasma membranes at around 12.5 mM Mg*+, while for root membranes the Mg2+ concentrations that stimulated phosphorylation of endogenous PIP ranged between 5 and 40 mM (Fig. 6). for formation of PIP, from The Mg*+ requirement exogenously added PI more closely resembled phosphorylation of endogenous PIP than phosphorylation of exogenously added PI-4P. The dependencies on Mg *+ did not strictly follow Michaelis-Menten kinetics, presumably due to unspecific interactions of Mg*+ with membrane constituents, Triton X-100 and lipid substrates. The stimulatory effects of Mg2+ could only be partly substituted by Mn*+, and not at all by Ca*+ (Table I). Ca2+ added together with Mg*+ proved to be inhibitory compared to Mg*+ alone (Table I). We did not attempt to elucidate whether the Mg*+-stimulated Ca2+ acted by inhibiting kinases or by stimulating phospholipase action on the products formed by the kinase activities. Time-course studies and stability of PIP and PIP, formed Time-course experiments with exogenous substrates present showed that phosphorylation of PI to form PIP was linear with time for 4-6 min and approx. 2 min in shoot and root plasma membrane fractions, respectively (Figs. 7a and c). In

root plasma membranes, the reaction levelled off after 15 min, while the shoot plasma membranes continued to phosphorylate exogenous PI for over 30 min (Fig. 7). With exogenous PI as substrate, PIP, was also formed, although at less than half the rate compared with the phosphorylation of exogenously added PI-4P (Fig. 6b and d). Phosphorylation of added PI-4P to form PIP, was linear for 15 min in both shoot and root plasma membranes, after which the rate levelled off faster in the root than in the shoot membranes (Fig. 7b and d). To investigate if the decreased rates of PI and PIP phosphorylation at longer time incubations in root plasma membranes were due to a

I

I

I

I

A

I

I

B

L \1

f

,L

I

I I

I

I

I

I

IO

20

3c Time, mln

Fig. 7. Time-course studies of PI and PIP kinase activities in the presence of exogenous lipid substrates. Plasma membranes isolated from shoots and roots of dark-grown wheat were incubated with [Y-~~P]ATP and added lipid substrates for various lengths of time. Closed symbols, phosphorylation of PI or PI-4P; open symbols, phosphorylation of PI or PI-4P after addition (arrow) of 5 units of hexokinase and 0.5 pmol glucose to the incubation. (A) PIP formation from added PI in shoot plasma membranes; (B) PIP, formation from added PI (0, 0) or PI-4P (H, 0) in shoot plasma membranes; (C) PIP formation from added PI in root plasma membranes; (D) PIP, formation from added PI (A, A) or PI-4P (M, 0) in root plasma membranes.

215

B

1500

300

1000

200

lYii!id \1

500

100

0

0

;cn E 0 E a

750

250

0 0

10

20

30

the inclusion of 0.010% Triton X-100 in the assay (Fig. 8). Phosphorylation of endogenous PI to PIP showed a time dependency that was markedly similar whether Triton X-100 was present or not. The reactions were linear for much shorter times than when exogenous substrates were present, which may reflect the low content of PI in plant plasma membranes [25], but also that the ATP supply could be limiting. The time course of formation of PIP, from endogenous substrates, in both shoot and root plasma membranes, was markedly affected by Triton X-100. While the initial rates did not significantly differ with or without Triton, after 3 min of incubation, 0.010% Triton X-100 stimulated the reactions (Fig. 8b and d). The ATP dependency of phosphorylations of endogenous PI and PIP was demonstrated by the addition of hexokinase and glucose after 10 min incubation time. Approx. 40% of the PIP, formed prior to ATP depletion was degraded during the following 20 min in both shoot and root plasma membranes, irrespective of Triton X-100, while PIP proved more stable, with a 20% loss during the first 20 min after ATP depletion (Fig. 8).

0

10

20

30

Time, mln

Fig. 8. Time-course studies of PI and PIP kinase activities on endogenous lipid substrates in the absence or presence of a stimulatory concentration of Triton X-100. Plasma membranes isolated from shoots and roots of dark-grown wheat were incubated with [ Y-~*P]ATP for various lengths of time. Closed symbols, phosphorylation of endogenous substrates; open symbols, phosphorylation of endogenous substrates after addition (arrow) of 5 units of hexokinase and 0.5 pmol glucose to the incubation. (A) PIP formation and (B) PIP, formation in shoot plasma membranes; (C) PIP formation and (D) PIP, formation in root plasma membranes. In incubations were performed at pH 7.0 without Triton X-100 (A. A) or at pH 7.8 with 0.01% (w/v) Triton X-100 (0. 0).

limiting ATP supply, an additional 75 nmol of ATP was added to the assay after 10 min of incubation. The PI and PI-4P phosphorylation rates remained unaffected for the next 5 and 10 min, respectively, after which a small stimulatory effect was recorded (data not shown). To investigate the stability of the PIP and PIP, formed from exogenous substrates, hexokinase and glucose were added after 10 min of incubation to deplete the ATP supply. The results demonstrate the absolute ATP requirement of the kinases, and also that when exogenous substrates were phosphorylated the reaction products (PIP and PIP,) were not significantly degraded (Fig. 7). The time course for phosphorylation of endogenous PI and PIP was investigated with and without

Discussion Animal PI and PIP kinases are generally considered to be plasma membrane-localized [14]. However, although PI kinase has been found to be enriched predominantly in plasma membranes [ 141, in, e.g., rat liver, it is also present to a great extent in lysosomes [26], the Golgi apparatus [27], and the nuclear envelope [28]. PIP kinase has been found both as a soluble [29] and as a membranelocalized [30] enzyme. The membrane bound PIP kinase of, e.g., rat liver is localized mainly in plasma membranes, but is also present in the Golgi apparatus [30]. We have earlier shown that PI and PIP kinase activities of isolated plant microsomal membranes are enriched in plasma membrane fractions [20]. For, e.g., PI phosphorylation in shoots, 20-40% of the activity of a microsomal membrane fraction was recovered in the plasma membrane fraction, together with only 3-5% of the protein; the data for PI4P phosphorylation and for root membranes were similar [20]. It has been estimated

216

that plasma membranes comprise approx. 10% of the membrane area of cells in developing leaves [31]. In our case [20], we did not recover all plasma membrane-derived vesicles from the microsomal fraction and, consequently, not all plasma membrane-localized PI and PIP kinase activities. This means that we cannot exclude the possibility that PI and PIP kinase activities reside in other membranes besides plasma membranes. PIP and PIP, formation in animal cells has, in most cases, been studied in cell cultures in vivo [14], which only makes possible a few comparisons of our results with those obtained for animals. The rates of phosphorylation of endogenous lipid substrates presented here for plant plasma membranes are similar to those reported for plasma membranes isolated from human platelets and rat liver. In isolated shoot and root plasma membranes, PIP formation was around 125-225 and 40-90 pmol/mg protein per min, respectively, and PIP, formation around lo-25 and 4-8 pmol/ mg protein per min, respectively. For plasma membranes isolated from human platelets, the rates of PIP and PIP, formation were 40 and 73 pmol/mg protein per min, respectively (data from a 15 min incubation) [32]. For rat liver, the rate of PIP formation differed between plasma membrane fractions of different origins, being 250 and 2000 pmol PIP formed/mg protein per min for bile canalicular and blood sinusoidal regions of the plasma membrane, respectively, while formation of PIP, was around 100 pmol/mg protein per min in both regions [30]. For human placenta membranes, however, much higher rates, 10000 and 400 pmol/mg protein per min, were obtained for PIP and PIP, formation, respectively [33]. It should be noted that the investigations on animal PI and PIP kinases cited above [30,32,33] used plasma membrane fractions isolated from well-defined cell types, while in our case the plasma membrane fractions were isolated from whole organs (shoots and roots of wheat seedlings), and therefore may represent all cell types of the respective organ used. PI and PIP kinase activities in plant membranes probably vary with cell type and stage of development. Our results show that the properties of the activities, as studied on ‘average’ plasma membrane fractions, differ between shoots and roots.

The rates of PIP and PIP, formation [30,32,33] (present work) represent steady-state levels resulting from several reactions affecting the status of respective lipid in the membrane. While studies using [ y-‘* P]ATP to phosphorylate endogenous lipids in animal plasma membranes [30,32,33] reported that most of the lipid-associated label was recovered in PIP and PIP,, we recovered a high proportion of the 32P administrated in PA, PA formation is the result of DG kinase activity. In our case, we do not know the proportion of [32P]PAs which had originated from phospholipase C activity on PIP and PIP, followed by phosphorylation of the resulting DG. Therefore, we cannot exclude that the rates we report for PIP and PIP, formation in plant plasma membranes are underestimated. No quantifications of plant PIPS and PIP,s have yet been presented, but it can be assumed that, in analogy with the situation in animal membranes [14], the PIP and PIP, contents of plant membranes are considerably lower than the PI content. Indications that this might be the case come from studies of steady-state distributions of radioactivity in lipids isolated from cell suspension cultures incubated with either ~yo-[2-~H]inositol [18] or [y-32P]Pi [34]. The relative radioactivities of PI, PIP and PIP, were found to be 114: 2.4: 1 [18] and 100: 7: 1 [34] for the two cases, respectively. Plasma membrane vesicles isolated by aqueous polymer two-phase partition are considered to be orientated with the cytoplasmic side facing inwards and to be between 85 and 95% tightly sealed, based on ATPase latency studies [35]. The general assumption held for animal plasma membranes is that PI and PIP kinases are localized exclusively in the cytoplasmic exposed leaflet of the membrane [14]. If the same should hold true for plant PI and PIP kinases, plasma membrane vesicles isolated by aqueous polymer two-phase partition need to be made leaky in order to allow for substrate (ATP, exogenously added lipids) availability. In our case, plasma membrane vesicles isolated from wheat shoots and roots by two-phase partition phosphorylated both endogenous and exogenously added PI and PIP without the addition of Triton X-100. These phosphorylation activities showed pH optima, increased incorporations with

211

time, etc. It is possible that the assay conditions rendered the cytoplasmic side in vesicles leaky or that a minor portion of the population of membrane vesicles used exposed the cytoplasmic membrane leaflet to the assay medium. In order to determine in which leaflet of the plasma membrane the PI and PIP kinases are localized, methods to isolate plasma membrane vesicles of both orientations separately must be developed. A large proportion of the plasma membrane vesicles isolated were of an orientation where the substrate site initially was inaccessible, which was shown by the fact that added Triton X-100 stimulated the reactions. The present knowledge concerning polyphosphoinositide metabolism in plants stems from research on several different plant species and several levels of organization. In vivo capability of PIP and PIP, formation has been shown for hypocotyl segments of Glycine max [36], for pulvini isolated from Samanea saman [37], and for cell suspension cultures of D. carota [18], Catharanthus roseus [14], Petrosefinum crispum [38], G. max [38] and Acer pseudoplatanus [39]. The cell suspension cultures used [18,34,38] required the addition of a synthetic auxin, 2,4-dichlorophenoxyacetic acid, to growth and incubation media to hamper cell differentiation. Isolated membranes are capable of phosphorylating PI followed by phosphorylation of the product, PIP, to form PIP,. These reactions are localized to a great extent in the plasma membrane (T. aestiuum, [20], and present work), as is a polyphosphoinositide-specific phospholipase C of wheat shoots and roots [39]. It has also been shown that release of inositolphosphates from membranes isolated from cells incubated with [3H]inositol is stimulated by GTP or GTP analogues (A. pseudoplantanus, [40] and, further, that IP, can stimulate the release of Ca2+ from Ca2+-loaded membrane vesicles of unidentified subcellular origin (Curcurbita pepo, [19]) or of vacuolar membrane origin ( Auena satiua, [41]). Taken together, these results suggest that in plants a metabolism involving polyphosphoinositides occurs which is markedly similar to that connecting receptor activation with increased cyin animal cells. If toplasmic Ca2 + concentrations we expect polyphosphoinositide metabolism to

play a similar role in plant cells, the whole chain of events, from signal perception to IP,-stimulated increase of cytoplasmic Ca2+ concentration and physiological response, needs to be shown to occur in the sample plant cell. It also remains to be shown which signals, which cells, and at what stage of growth and development, polyphosphoinositide metabolism is utilized as a signaltransducing system in plants. Acknowledgements We wish to express our gratitude to Ms. Inger Rohdin for plasma membrane isolations, to Ms. Nancy Ilenius for drawing the figures, and to Ms. Nan Albertson for linguistic advice. This work was supported by grants from the Swedish Natural Science Research Council (M.S., A.S.S.), and The Carl Tesdorpf Foundation (M.S.). References 1 Trewavas, A. (1981) Plant Cell Environ. 4, 203-228. 2 Trewavas, A.J. (1982) Physiol. Plant. 55, 60-72. 3 Wareing, P.F. (1986) in Plant Growth Substances 1985 (Bopp, M., ed.), pp. l-9, Springer-Verlag, Berlin. 4 Crazier. A.. Sandbera. G.. Monteiro. A.M. and Sundbera. B. (1986) in Plant Growth Substances 1985 (Bopp, M., ed.), pp. 13-21, Springer-Verlag, Berlin. 5 McGaw, B.A., Scott, I.M. and Horgan, R. (1984) in The Biosynthesis and Metabolism of Plant Hormones (Crazier, A. and Hillman, J.R., eds.), pp. 105-133, Cambridge University Press, Cambridge. 6 Bandurski, R.S., Schultze, A. and Reinecke, D.M. (1986) in Plant Growth Substances 1985 (Bopp, M., ed.), pp. 83-91, Springer-Verlag, Berlin. 7 Beale, M.H., Hooley, R. and MacMillan, J. (1986) in Plant Growth Substances 1985 (Bopp, M., ed.), pp. 65-73, Springer-Verlag, Berlin. D.S., Spray, CR. 8 Phinney, B.O., Freeling, M., Robertson, and Silverthorne, J. (1986) in Plant Growth Substances 1985 (Bopp, M., ed.), pp. 56664, Springer-Verlag, Berlin. 9 Jacobs, M. and Short, T.W. (1986) in Plant Growth Substances 1985 (Bopp, M., ed.), pp. 2188226, Springer-Verlag, Berlin. 10 Lobler, M. and Kllmbt, D. (1985) J. Biol. Chem. 260, 9848-9853. 11 Lobler, M. and Kllmbt, D. (1985) J. Biol. Chem. 260, 9854-9859. S., Sotobayashi, T., Futai, M. and Fukui, T. 12 Shimomura, (1986) J. B&hem. 99, 1513-1524. 13 Hepler, P.K. and Wayne, R.O. (1985) Annu. Rev. Plant Physiol. 36, 397-439. 14 Abdel-Latif, A.A. (1986) Pharmacol. Rev. 38, 227-272.

278

15 16 17 18 19 20 21 22 23 24 25 26 27 28 29

Berridge, M.J. (1986) Cell Sci. Suppl. 4, 137-153. Berridge, M.J. and Irvine, R.F. (1984) Nature 312, 315-321. Nishizuka, Y. (1984) Nature 308, 693-698. Boss, W.F. and Masse], M.O. (1985) Biochem. Biophys. Res. Commun. 132, 1018-1023. Drabak, B.K. and Ferguson, I.B. (1985) Biochem. Biophys. Res. Commun. 130, 1241-1246. Sandelius, A.S. and Sommarin, M. (1986) FEBS Lett. 201, 282-286. Chang, K.J., Marcus, N.A. and Cuatrecasas, P. (1974) J. Biol. Chem. 249, 6854-6865. Sommarin, M., Lundborg, T. and Kylin, A. (1985) Physiol. Plant. 65, 27-32. Markwell, M.A.K., Hass, S.M., Beiber, L.L. and Tolbert, N.E. (1978) Anal. Biochem. 87, 206-210. Schacht, J. (1978) J. Lipid Res. 19, 1063-1067. Yoshida, S. and Uemura, M. (1986) Plant Physiol. 82, 807-812. Collins, CA. and Wells, W.W. (1983) J. Biol. Chem. 258, 2130-2134. Jergil, B. and Sundler, R. (1983) J. Biol. Chem. 258, 7968-7973. Smith, C.D. and Wells, W.W. (1983) J. Biol. Chem. 258, 9368-9373. Dongen, C.J.V., Zwiers, H. and Gispen, W.H. (1984) Biothem. J. 223, 197-203.

30 Lundberg, Biophys. 31 Forde, J. 32 Plantavid, L. (1986) 33 Urumow, 253-257. 34 Heim, S. Commun.

G.A., Jergil, B. and Sundler, R. (1985) Biochim. Acta 846, 379-387. and Steer, M.W. (1976) J. Exp. Bot. 27,1137-1141. M., Rossignol, L., Chap, H. and Douste-Blazy, Biochim. Biophys. Acta 875, 147-156. T. and Wieland, O.H. (1986) FEBS Lett. 207, and Wagner, K.G. (1986) Biochem. 134, 1175-1181.

Biophys.

Res.

35 Larsson, C., Kjellbom, P., Widell, S. and Lundborg, T. (1984) FEBS Lett. 171, 271-276. 36 Sandehus, AS. and Morn?, D.J. (1987) Plant Physiol. 84, 1022-1027. 37 Morse, M.J., Cram, R.C. and Satter, R.L. (1987) Plant Physiol. 83, 640-644. 38 Strasser, H., Hoffmann, C., Grisebach, H. and Matern, U. (1986) Z. Naturforsch. 41~. 717-724. 39 Melin, P.-M., Sommarin, M., Sandelius, AS. and Jergil, B. (1987) FEBS Lett. 223, 87-91. 40 Dillenschneider, M., Hetherington, A., Graziana, A., Alibert, G., Berta, P., Haiech, J. and Ranjeva, R. (1986) FEBS Lett. 208, 413-417. 41 Shumaker, K.S. and Sze, H. (1987) J. Biol. Chem. 262, 3944-3946.