Activation of phospholipase D by calmodulin antagonists and mastoparan in carnation petals - 21709y

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Activation of phospholipase D by calmodulin antagonists and mastoparan in

carnation petals

de Vrije, T.; Munnik, T.

DOI

10.1093/jexbot/48.314.1631

Publication date

1997

Published in

Journal of Experimental Botany

Link to publication

Citation for published version (APA):

de Vrije, T., & Munnik, T. (1997). Activation of phospholipase D by calmodulin antagonists

and mastoparan in carnation petals. Journal of Experimental Botany, 48(314), 1631-1637.

https://doi.org/10.1093/jexbot/48.314.1631

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Journal of Experimental Botany, Vol. 48, No. 314, pp. 1631-1637, September 1997

Journal of Experimental Botany

Activation of phospholipase D by calmodulin antagonists

and mastoparan in carnation petals

Truus de Vrije1'3 and Teun Munnik2

1 Agrotechnological Research Institute (ATO-DLO), PO Box 17, 6700 AA Wageningen, The Netherlands 2 Institute for Molecular Cell Biology, BioCenter Amsterdam, University of Amsterdam, Kruislaan 318,

1098 SM Amsterdam, The Netherlands Received 2 May 1997; Accepted 15 May 1997

Abstract

An in vivo assay for phospholipase D (PLD; EC 3.1.4.4) activity, based on its transphosphatidylation property, is described in detail and was used to study putative post-translational regulation mechanisms of PLD activ-ity in carnation [Dianthus caryophyllus L.) petals. A variety of agents was applied to petal discs. The cal-modulin (CaM) antagonists propranolol, /V-(6-amino-hexyl)-5-chloro-1 -naphthalenesulphonamide (W7) and N-(6-aminohexyl)-1 -naphthalenesulphonamide (W5), stimulated PLD activity in a dose-dependent manner. EGTA partially inhibited the stimulation by the CaM antagonists. Erythrosin B, an inhibitor of CaM-dependent P-type Ca2 + -ATPases, slightly stimulated

PLD activity. The results suggest that part of the stimu-lation of PLD activity by CaM antagonists is due to an increased intracellular Ca2 + -concentration. PLD

activ-ity was stimulated by mastoparan in a dose- and time-dependent manner. The signal-like activation kinetics suggests that mastoparan activates PLD (in)directly via a G protein.

Key words: Phospholipase D, CaM antagonists, mastopa-ran, Dianthus, calcium.

Introduction

Phospholipase D hydrolyses the phosphate ester between the phosphatidate moiety and the head group of glycero-phospholipids. The enzyme was first discovered in carrot roots and spinach leaves (Hanahan and Chaikov, 1947) and has since been shown to be widely distributed throughout the plant kingdom (Quarles and Dawson, 1969; Heller, 1978). Despite its high activity in plants,

the physiological function of PLD in growth and devel-opment is still not clear. PLD could be involved in phospholipid turnover that maintains cell viability and homeostasis (reviewed by Dawidowicz, 1987). Additionally, the observation that the phospholipid con-tent of petals, fruits and leaves decreased during senes-cence (McArthur et al., 1964; Ferguson and Simon, 1973; Beutelmann and Kende, 1977; Thompson et al., 1982) led to the hypothesis that PLD initiated this lipid break-down (Brown et al., 1990; Paliyath and Droillard, 1992). The idea was partly based on changes in PLD activity that were measured using in vitro assays (Herman and Chrispeels, 1980; Borochov et al., 1982; Salama and Pearce, 1993), but recently, it was shown that regulation of PLD activity is more complicated. During castor bean germination and leaf senescence, not only the total PLD content was changed, but also three isoforms were differ-entially expressed (Dyer et al., 1994; Ryu and Wang, 1995). Furthermore, the intracellular distribution of PLD changed, with increasing activity becoming associated with its substrate in membranes (Xu et al., 1996). This suggests that post-translational mechanisms contribute to the regulation of PLD activity.

Post-translational regulation mechanisms apply to PLD involved in signalling. A recent study of the green alga

Chlamydomonas illustrated that G-protein activation

trig-gered immediate increases in PLD activity (Munnik et al., 1995), generating PtdOH which is a second messenger in animal cells (Liscovitch and Cantley, 1994, 1995), and which had dramatic effects on Chlamydomonas, causing deflagellation (Munnik et al., 1995).

In order to investigate the different functions of PLD in higher plants, activity must be measured in vivo, and in a preliminary study it was shown that this is possible with live carnation petals by using the

transphosphatidyl-3 To wtiom correspondence should be addressed. Fax: +31 317 475347. E-mail: g.j.devrije@ato.dlo.nl

1632 Vrje and Munnik

ation activity of PLD (Munnik et al., 1995). Here, the assay conditions are described in full detail and data presented to show that PLD is activated by CaM antagon-ists and mastoparan in vivo. The results are discussed in relation to possible post-translational activation mechanisms.

Materials and methods

Plant material

Carnation (Dianthus caryophyllus L.) flowers cv. White Sim were obtained from a commercial grower and transported dry to the laboratory. Stems were re-cut to 20 cm and placed in deionized water under controlled environmental conditions (Wolteringera/., 1993a). Just before use, discs (0.8 cm diameter, approximately 15 mg fresh weight) were cut from the middle part of the petals. Only the petals of the outer whorl were used.

Materials

[9,10 (n)-3H]Palmitic acid (40-60 Cimmol"1) was obtained

from Amersham (Buckinghamshire, UK). The analytical grade organic solvents and TLC Silica 60 on plastic sheets were from Merck (Darmstadt, Germany). Phospholipid standards, phos-pholipase D (cabbage), erythrosin B, DL-propranolol, W5, W7, and mastoparan (Vespula lewisii) were purchased from Sigma (St Louis, MO, USA). Synthetic mastoparan and mastoparan

17 (Masl7) were obtained from Bachem (Bubendorf, Switserland). A standard of PtdBut was prepared by conversion of egg-yolk PtdCho into PtdBut by phospholipase D in the presence of 5% (v/v) w-butanol (Comfurius and Zwaal, 1977).

Lipid labelling, extraction and analyses

Petal discs (a maximum of 10) were labelled with [3H]palmitic

acid (lOfiCi) in 2 ml labelling buffer (25 mM MES, 0.05% Tween 20, pH 5.5) by vacuum-infiltration (Munnik et al.,

1994). After 5 min, the vacuum was released and the discs were incubated at room temperature for the times indicated. Incubations were stopped by immersing each petal disc in 600 /xl of CHC13:MeOH:HC1 (100:100:1, by vol.) and freezing the

samples in liquid nitrogen. Lipids were directly extracted after thawing and the petal discs were re-extracted with 450 /A of the same solution. Lipid fractions were pooled and a two-phase separation induced by the addition of 500 ^.1 CHC13 and 375 fi\

0.9% (w/v) NaCl, 1 mM EGTA. The organic lower phase was washed once with 375 ^1 MeOH : H2O: HC1 (50:50:1, by vol.).

Lipid extracts were dried under a stream of nitrogen at 30 °C and stored at - 2 0 ° C until further use.

For TLC analyses, lipids were dissolved in CHCI3: MeOH

(9:1, v/v) and samples applied to silica TLC plates which were then developed in the organic upper phase of ethyl acetate: iso-octane:HAc:H2O (13:2:3:10, by vol.). Lipid spots were

revealed with iodine vapour and identified by co-migration with unlabelled lipid standards. The Rf values for the lipids were:

PtdCho and Ptdlns, 0.01; PtdEtn and PtdGro, 0.06; PtdOH, 0.24; monogalactosyl diacylglycerol, 0.44. Spots of interest were excised and their radioactivity was determined by liquid scintillation counting.

In vivo Phospholipase D assay

Separate petal discs were labelled at room temperature in 250 /xl labelling buffer containing 3.5 jiCi [3H]palmitic acid by

vacuum-infiltration. After release of the vacuum, incubations were

started by the simultaneous or successive addition of rc-butanol (1 vol% in 250 /il of the labelling buffer) and the test reagents (stock solutions of DL-propranolol, W5, W7, mastoparan, and Masl7 in deionized water) as specified in the figure legends. Incubations were continued for the times indicated, the reactions quenched and the lipids extracted as described above.

A mixture of unlabelled PtdBut, PtdOH, and PtdCho in CHCl3:MeOH (9:1, v/v) was added to the dried samples. The

total samples were chromatographed using three solvents consecutively, with intermediate drying. First, CHC13 was used

to reduce 3H-counts in the background due to the presence of

unincorporated palmitic acid in the samples. Second, acetone was used to remove monogalactosyl diglyceride which otherwise co-migrated with PtdBut (RF value PtdBut, 0.43). Third, the

TLC plate was developed using the ethyl acetate-iso-octane solvent described above to separate PtdBut and PtdOH from the rest of the phospholipids.

PLD activity is presented as the formation of PtdBut which is expressed as the level of 3H-counts in PtdBut as a percentage

of the label recovered from the structural phospholipids (SPL), PtdCho, Ptdlns, PtdEtn, and PtdGro. The 3H-counts in the

PtdBut fractions were corrected for those migrating at the position of PtdBut in control samples incubated without n-butanol. Lipid fractions isolated from petal discs incubated for 1 h with [3H]palmitate in the presence or absence of w-butanol

(0.5%) contained on average (n = 6-12, + SE): SPL, 216 100±24 000 dpm; PtdBut (not corrected), 1180±120 dpm; background PtdBut, 470 ±45 dpm.

Measurement of ion leakage

Excised petals from the outer whorl were placed with their cut base in 3 ml of a 4% n-butanol solution for 24 h. Thereafter, ion leakage was measured as described by Woltering et al. (19936). Briefly, 10 petals were immersed in 30 ml deionized water and gently shaken. Conductivity of the diffusate was measured between I and 3 h. The total conductivity was determined after two cycles of freezing and thawing. Conductivity was expressed as the percentage ion leakage per hour relative to the total conductivity of the petals.

Results

?H\Palmitic acid labelling of lipids

Carnation petal discs were incubated with [3H] palmitic

acid, their lipids extracted at regular intervals, and the lipids separated by TLC. After 24 h, about half of the

3H-label in the lipid extract was present as phospholipids.

Most was incorporated into PtdCho plus Ptdlns (56%) and PtdEtn plus PtdGro (42%). Approximately 1.3% of the label was recovered as PtdOH. The rest of the 3

H-label was recovered as glycolipids (3%) or migrated nearer the solvent front, probably representing diacylglycerols, triacylglycerols and unincorporated palmitic acid.

Incorporation of 3H into phospholipids increased

lin-early during the first 8 h, usually reached a maximum within 24 h (Fig. 1 A) and then remained at the same level up to 48 h, suggesting that isotopic equilibrium had been reached. The 3H-label was incorporated into PtdOH and

the structural phospholipids in the same relative propor-tions during the whole labelling period (Fig. IB),

indicat-600 10 15 20 25 ~ 80 0. "5 o c O Q.

8

*f

-f_

1

-I

1

Fig. 1. Incorporation of [3H]palmitate into the phospholipids of

carnation petals. Petal discs were labelled with [3H]palmitate

(5^Ciml~') for increasing times up to 24 h. Lipids were extracted, separated by TLC and analysed as described in Materials and methods. (A) Data are expressed as the sum of the radioactivity in PtdCho, Ptdlns, PtdEtn, and PtdGro. The experiment was repeated once with essentially similar results. (B) Data are expressed as the relative 3H incorporation per phospholipid species, ( • ) PtdOH; (O) PtdCho plus Ptdlns; (A) PtdEtn plus PtdGro. The results are means±SE of 2-17 values from different experiments. PL, phospholipids.

ing that [3H] palmitic acid was incorporated into those

phospholipids at equal rates.

Phospholipase D activity in carnation petals and its stimulation by calmodulin antagonists

In order to measure PLD activity, the ability of [3H]palmitate-labelled petal discs to form [3H] PtdBut

was monitored in the presence of H-butanol, because a unique property of PLD is that it can transfer the phosphatidyl group of its substrate to a primary alcohol (Dawson, 1967; Yang et al., 1967). Thus when 0.5% n-butanol was added to prelabelled petal discs PtdBut was formed and, after 60 min, approximately 0.2% of the label in phospholipids was converted to [3H]PtdBut (Fig. 2A,

closed circles). 15 30 45 60 Time (mln) 10 0_ W "a o m •o a B / 0 1 2 3 4 Butanol (%)

Fig. 2. Formation of PtdBut in control ( • ) and propranolol-treated

(O) petal discs as a function of the incubation time (A) and concentration (B) of n-butanol. Petal discs were incubated with [3H]palmitate (3.5jtCi/25O Ml) and propranolol (5mM) for 2h (A) and 1 h (B). (A) n-Butanol (0.5%) was present during the last period of the incubation for the indicated times. (B) n-Butanol was present at the indicated concentrations for 1 h. The results are means±SE, from different experiments. SPL, structural phospholipids

The local anaesthetic propranolol, a CaM antagonist, (Volpi et al., 1981), was tested for its effect on in vivo PLD activity. When 0.5% n-butanol was added to petal discs pretreated with 5 raM propranolol, PtdBut forma-tion was rapidly elevated above the control and increased for at least 60 min (Fig. 2A, open circles). The increase was linear with increasing H-butanol concentrations up to 2% (Fig. 2B). Membrane permeability was not adversely affected in as much as treatment with 4% «-butanol for 24 h did not increase ion leakage (conductivity: control petals, 0.42% h"1, SE±0.02; n-butanol-treated petals,

0.48% h"1, SE±0.07).

PtdBut formation was measured after 1 or 17 h of prelabelling with [3H]palmitate (see Fig. 1A for relative

incorporation levels) using 0.5% n-butanol and 5 mM propranolol. After 1 h under both labelling conditions,

1634 Vrje and Munnik 4

2

Fig. 3. Time-course of PLD-dependent PtdBut formation upon stimula-tion with propranolol. Petal discs were stimulated with propranolol (5mM) for increasing times up to 2 h. Labelling with [3H]palmitate was earned out in the presence of n-butanol (0.5%) for 2 h. The results are means±SE of 3-10 values from different experiments. SPL, structural phospholipids.

approximately 2.4% of the phospholipid label was con-verted into PtdBut. Therefore, in subsequent experiments, PLD activity was routinely measured using petal discs incubated with 0.5% «-butanol and [3H]palmitate

during 1 h.

Addition of 5 mM propranolol to petal discs rapidly stimulated the formation of [3H] PtdBut and after 2 h of

treatment [3H]PtdBut accumulated up to 10-fold (Fig. 3).

The effect was dose-dependent and increased up to 10 mM (Fig. 4A). Higher concentrations of propranolol inhibited the incorporation of [3H]palmitate into the structural

phospholipids (data not shown).

The naphthalenesulphonamides W7 and W5, two other CaM antagonists, also stimulated PLD activity in a dose-dependent manner (Fig. 4B). W7 (closed circles) was found to be more active than W5 (open circles), which correlated well with their order of potency (Hidaka and Tanaka, 1983). Higher concentrations of these CaM antagonists again inhibited the incorporation of label into the structural phospholipids.

Mastoparan stimulates PLD activity

The G protein activator mastoparan was used to get insight into the possible involvement of G proteins in the regulation of PLD activity in carnation petals. Mastoparan stimulated PLD activity in a concentration-and time-dependent manner (Fig. 5A, B). At 25 ^M, activity was stimulated more than 5-fold. Mas 17, a masto-paran analogue which is a far less potent activator of G proteins (Higashijima et al., 1990), did not significantly stimulate PLD activity (Fig. 5A). The effect of mastopa-ran was very rapid, half-maximal activity being reached

c o E o (0 Q _J Q_ C JO 10 o CO 0 1 2 Compounds (mM)

Fig. 4. Stimulation of PLD activity by CaM antagonists. (A) Propranolol; (B) naphthalenesulphonamides W7 ( • ) and W5 (O) Petal discs were incubated with [3H]palmitate, ^-butanol (0.5%) and CaM antagonists at the indicated concentrations for 1 h. The data are means ±SE of 2—4 values from different experiments.

within 5 min, producing kinetics of stimulation that were very different to those found for propranolol (Fig. 3).

Besides an increase in [3H]PtdBut formation, treatment

of petal discs with either mastoparan or propranolol also resulted in a stimulation of the percentage of 3H-label in

PtdOH. Interestingly though, mastoparan and proprano-lol differed in the ratio of PtdBut/PtdOH formation. To understand this point one must appreciate that the prim-ary alcohol competes with water to accept the phosphati-dyl group. Thus the PtdBut to PtdOH ratio is determined by the affinity of PLD for the acceptor and its local concentration. Even when stimulated, the PtdBut to PtdOH ratio is expected to be constant. Indeed, that was found for both stimulation by mastoparan and proprano-lol (Fig. 6). However, the PtdBut: PtdOH ratio for masto-paran was 1:5.5 (SE + 0.1, n = 2, data were derived from two series of concentrations of mastoparan), while that for propranolol was 1:3.2 (SE±0.3, n = S, data were derived from a series of concentrations of propanolol and kinetic experiments).

10 15 20 25 Mastoparan (uM) Q_ W 75 o m Si 0 15 30 45 Time (mln)

Fig. 5. Mastoparan stimulates PLD activity dose- (A) and time-dependent (B). Petal discs were incubated with [3H]palmitate and

n-butanol (0.5%) for l h (A) Mastoparan ( • ) and Masl7 (O) were present at the indicated concentrations for 1 h. The results are means±SE, n ^ 2 from different experiments. (B) Mastoparan was added during the last period of the incubation for the indicated times Data are from one experiment except those from 0, 5 and 60 min which are expressed as means±SE (n = 9, 2 and 3, respectively). SPL, structural phospholipids.

Discussion

It has been shown that phosphatidylation of primary alcohols can be used as a measure of in vivo PLD in plant tissues. The advantages of this assay are first that phos-phatidylalcohols are stable products while PtdOH and, for example, choline from PtdCho, are not. Second, PLD activity can be determined in vivo because the formation of phosphatidylalcohols is specific for PLD activity while that of PtdOH is not. That transphosphatidylation is indeed a property of PLD was demonstrated by introdu-cing a PLD gene from Ricinus communis into E. coli and showing that only when the gene was expressed were extracts of the bacterium able to hydrolyse phosphatidyl-choline and transphosphatidylate ethanol (Wang et cil., 1994). Similar results have recently been obtained using

2 x O Si 0 1 2 3 PtdBut (% of total SPL)

Fig. 6. Linear relationship between PtdBut- and PtdOH-formation. PLD activity was stimulated by different concentrations of mastoparan (0 to 50 p.M) ( • ) for 1 h or by 5 mM propranolol ( O ) for different times (0, 0.5, 1, 2 h). PtdOH formation was expressed as the level of labelled PtdOH as a percentage of the sum of the radioactivity of the structural phospholipids (SPL).

a human PLD gene expressed in insect cells (Hammond

et ah, 1995).

In order to use the transphosphatidylation assay for PLD activity, the phosphatidyl moiety of the PLD sub-strate must be labelled. If the subsub-strate has not been determined, this means that all structural phospholip-ids must be effectively labelled. Incubating discs in [3H]palmitate achieves this goal, and in our system it was

incorporated into all major phospholipids at equal rates. When 0.5% n-butanol was then added, the formation of PtdBut increased linearly with time. While it is tempting to use higher concentrations of n-butanol to facilitate the detection of PtdBut, one should realize that alcohols can themselves activate signalling enzymes such as PLC and PLD (Hoek et al, 1992; Musgrave et al, 1992; Munnik

et al, 1995). Higher concentrations of n-butanol added

to the carnation petals certainly increased PtdBut forma-tion, but whether this reflects PLD activation or just an increase in substrate concentration was not tested.

The ability to measure PLD activity in vivo allowed the study of some regulatory aspects of PLD. The CaM antagonists W7, W5, and propranolol stimulated PLD activity to a varying extent. PLD itself is not a CaM-regulated enzyme, but CaM-dependent enzymes in plants have been described. CaM antagonists have been shown to raise cytosolic calcium levels in plant protoplasts (Gilroy et al, 1987), perhaps by inhibiting CaM-depend-ent P-type Ca2 + -ATPases in the plasma- and ER

mem-branes (Dieter and Marme, 1980, 1981; Briskin, 1990; Evans et al, 1991; Askerlund and Evans, 1992). Since in

vitro PLD activity has been shown to be

calcium-dependent (Heller, 1978) and since the recently cloned PLD genes from plants contain a potential

calcium-1636 Vrje and Munnik

binding domain (Wang et al., 1994; Ueki et al., 1995), calcium could be a general trigger that activiates PLD. Propranolol, W7 and W5 were effective at relatively high concentrations ranging from 0.5 to 5 mM, but their order of potency (W7> propranolol «W5) correlated with their /CJ0s for CaM-dependent phosphodiesterase activity

(Volpi et al., 1981; Hidaka and Tanaka, 1983), suggesting that all three worked via CaM. The fact that EGTA (5mM) partially inhibited propranolol-stimulated PLD activity (to 70% of the control) suggests that part of the effect of propranolol is due to an influx of extracellular Ca2 + . Additionally, the significant stimulation of PLD

by erythrosin B (50 pM, 1.6-fold increase), a specific inhibitor of P-type Ca2 + -ATPases (Rasi-Caldogno et al.,

1989; Askerlund and Evans, 1992), implicates calcium as the common denominator in all these responses. This is in agreement with a recent study where it is proposed that activation of PLD upon wounding of castor bean leaves is mediated by an increase in cytoplasmic Ca2+

(Ryu and Wang, 1996). Intracellular Ca2 +-measurements

will be needed to provide conclusive evidence about a possible role of Ca2+ in regulation of PLD in vivo. Besides

specific effects of the CaM antagonists, aspecific effects such as perturbation of the membranes by these lipophilic molecules might contribute to a stimulation of PLD activity.

Mastoparan also stimulated PLD activity in carnation petals. It is thought to mimic the peptide domain of agonist-bound receptors that activates G proteins (reviewed by Ross and Higashijima, 1994). As such it could increase the intracellular Ca2 +-concentration via a

G protein activated Ca2 +-channel or via PLC. There are

several reports that mastoparan stimulates PLC activity in plants (Quarmby et al., 1992; Legendre et al., 1993; Drabak and Watkins, 1994; Quarmby and Hartzell, 1994; Cho et al., 1995). By increasing the production of inositol-(l,4,5)trisphosphate, calcium can be released from intracellular stores and this could again stimulate PLD activity. Another possibility is that PLD is directly coupled to a G protein. This could account for the signal-like activation kinetics and is in agreement with the recent study of PLD activation in the alga Chlamydomonas (Munnik et al., 1995), where it was shown that treatments that are known to raise the calcium concentration and cause deflagellation, did not activate PLD, whereas mas-toparan and other G protein activators did activate PLD. Lastly, mastoparan could directly activate PLD as was indicated recently for animal cells (Mizuno et al., 1995). Both the kinetics of stimulation and the ratio of PtdBut:PtdOH formation for mastoparan and proprano-lol were different. The differences in kinetics could reflect different mechanisms of PLD stimulation or could be determined by different uptake rates or physical properties such as membrane affinity. The different ratios of PtdBut to PtdOH indicate that mastoparan and propranolol

could stimulate or inhibit enzymes that affect PtdOH levels such as PLC in combination with diacylglycerol kinase, PtdOH kinase, and PtdOH phosphatase (Munnik

et al., 1996). An alternative explanation is that

mastopa-ran and propmastopa-ranolol activate two different isoforms. The idea of different PLD isoforms in higher plants has already been established in castor bean based on immuno-blotting and activity assays (Dyer et al., 1994). In addi-tion, three cDNAs have recently been identified in

Arabidopsis (Dyer et al., 1995). The possibility that PLD

isoforms can be differentially activated by CaM antagon-ists and mastoparan is new, exciting and deserves more attention.

Acknowledgements

This work was supported by the Commodity Board for Ornamental Horticulture in The Netherlands. The authors are grateful to Alan Musgrave and Ernst Woltering for critically reading the manuscript and to Mariska Nijenhuis-de Vries for carrying out the ion leakage experiments.

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