Gibberellin-regulated phosphatidylcholine biosynthesis in suspensioncultures of Apium graveolens

Andrew Burgess and Carole Rolph*Department of Applied Biology, University of Central Lancashire, PR1 2HE, Preston, UK; *Author forcorrespondence

Received 15 March 2000

Key words: CDP-base pathway, Gibberellic acid, Methyltransferase pathway, Phosphatidylcholine, Signal trans-duction

Abstract

Recent studies with mammalian tissues have shown that phosphatidylcholine (PC) plays a key role in signaltransduction mechanisms involved in cell proliferation and differentiation. This investigation attempts to show ifPC has a role in gibberellin-stimulated plant signal transduction. With respect to cell differentiation, GA3-treat-ment caused an overall increase in the elongation ratio exhibited by the cells. The effects of GA3 on the biosyn-thetic route of PC were investigated. Carbon flux through the CDP-base pathway was significantly reduced uponGA3 treatment. Whereas, radiolabelling studies with tritiated methionine showed a GA3 concentration-dependentincrease in the flux of intermediates through the methyltransferase pathway indicating a possible link betweenmethyltransferase-derived PC and cell differentiation.

Abbreviations: PC – Phosphatidylcholine, MP – methyltransferase pathway, ST – signal transduction

Introduction

For many years, azole xenobiotics have been usedextensively used to regulate plant growth. They mayact by either preventing gibberellin biosynthesisthrough inhibition of the ent-kaurene oxidase (Dalz-iel and Lawrence 1984) or by modulation of sterolbiosynthesis via 14�-demethylase inhibition (Haugh-an et al. 1988). In some cases, both biosynthetic path-ways may be perturbed by azole treatment (Burden etal. 1989, 1990).

The exact mechanism(s) by which sterols may reg-ulate plant cell division in celery (Apium graveolens)cell suspension cultures has been under investigationfor many years in the laboratories of Rolph and Goad.Preliminary studies using these cultures indicated thatazole – induced inhibition of cell growth coincidedwith reduced CTP: cholinephosphate cytidylyltrans-ferase activity, the rate-limiting enzyme of the CDP-base pathway involved in the production of PC(Rolph and Goad 1991). Later, Parkin et al. (1995)

showed that in growth, arrested celery cultures, whichhad previously been treated with azole xenobiotics,restoration of cell division could be achieved throughthe non-sterol activation of CT. These studies werethe first to describe PC involvement in plant cell pro-liferation.

Initial studies, in which scientists attempted to linkGA3-elicited responses to phospholipid mediated sig-nal transduction were performed on the aleurone tis-sues of barley and wheat. Several investigationsshowed that GA3 treatment did not alter either thequantitative or qualitative phospholipid content (Firnand Kende 1974; Jusatis et al. 1981; Vakharia et al.1987). However, the work of Vakharia et al. (1987)extended the previous studies in that they were ableto show that GA3 treatment of wheat aleurone tissueresulted in increased turnover of the phosphatidylcho-line headgroup. In addition, in GA3-treated tissues theincorporation of exogenous choline into phosphati-dylcholine was also very much reduced.

113Plant Growth Regulation 34: 113–117, 2001.© 2001 Kluwer Academic Publishers. Printed in the Netherlands.

During the last decade, in studies on mammalianSystems, choline phospholipids have been shown tobe important in signal transduction mechanisms withrespect to the regulation of protein kinase C (PKC)isoforms involved in cell proliferation and differen-tiation. This phenomenon may be facilitated throughPC hydrolysis resulting in the generation of PKC iso-form-specific diacylglycerol (DAG) molecular spe-cies. The production of distinct molecular DAG spe-cies may be attributed to the fact that PC can besynthesised via two biosynthetic routes: the CDP-base pathway and the PE methyltransferase pathway.Many PKC isoforms involved in cell proliferation aremodulated by DAG molecular species derived fromPC formed through the CDP-base pathway (Zeisel1993). In contrast, PKC isoforms involved in cell dif-ferentiation have been shown to be sensitive to DAGsderived from PE methyltransferase synthesised PC(Sesca et al. 1996; Tessitore et al. 1997).

With these studies in mind, the investigation re-ported herein attempts to elucidate whether phos-phatidylcholine produced via the PE methyltrans-ferase pathway has a possible role in GA3-stimulatedsignal transduction in suspension cultures of Apiumgraveolens.

Materials and methods

Materials

[3H-methyl] choline (3.03 PBq mol−1) and [3H-meth-yl] methionine (0.6 GBq mol−1) were purchased fromAmersham International PLC, UK.

Culture growth

Suspension cultures of celery (Apium graveolens cv.New Dwarf White) were grown on Murashige andSkoog medium (pH 5.5) (Flow Laboratories), supple-mented with 2,4-dichlorophenoxyacetic acid (2.45�M), kinetin (2.97 �M) and sucrose (0.08 M, Sigma).Cells were cultured according to Haughan et al.(Haughan et al. 1988). Gibberellin A3 was dissolvedin ethanol and added to the growth medium under as-ceptic conditions. Control cultures were grown in thepresence of ethanol.

Growth measurement

Dry cell weights were determined according to themethod of Parkin et al. (1995). Cellular dimensionswere monitored using a microscope (Nikon) fittedwith a calibrated graticule.

Extraction and analysis of lipids

Lipid extracts were prepared according to Rolph andGoad (1991) and individual phospholipid classes sep-arated by thin layer chromatography on silica G 60plates in a solvent system comprising of chloroform/methanol/acetic acid/water (170:30:20:7, by volume).Lipid bands were visualised and identified by co-chromatography as previously described (Rolph et al.1989). Monomethyl- and dimethyl-phosphatidyletha-nolamine were identified by co-chromatography andby developing the plates with ninhydrir. Fatty acidmethyl esters (FAMEs) were prepared from total lipidextracts by transmethylation with 2.5% H2SO4 (v/v)in anhydrous methanol at 70°C for 2 h. The resultingFAMEs were then extracted into petroleum ether andanalysed via gas chromatography as outlined by Par-kin et al. (1995). Individual FAMEs were identifiedby comparison with the relative retention times ofauthentic standards.

Incorporation of [3H-methyl] choline into polarlipids

[3H-methyl] choline (0.6 mBq) was added to incuba-tion flasks containing 50 ml of log phase culturesgrown in the presence of GA3. In order to monitorcholine uptake, aliquots of cell free media (super-natant from 4 000 × g (5 mm) centrifuged samples)were subjected to scintillation counting using Euros-cint A (Mensura Technology Ltd, Wigan UK) on aBeckman LS 5801 scintillation counter.

After a 2 h incubation period, cells were harvestedand lipids extracted according to Rolph and Goad(Rolph and Goad 1991). Incorporation into cholinelipids was determined by scintillation counting of thetotal polar lipid extract.

Incorporation of [3H-methyl] methionine into polarlipids

Log-phase cells, with or without GA3 (50 ml cul-tures), were incubated with [3H-methyl] methionine(0.9 MBq) for a period of 4 h. Cultures were har-

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vested by centrifugation (4 000 × g for 5 min) and lip-ids extracted according to Rolph and Goad (1991).Individual phospholipid classes were separated, visu-alised and identified as outlined above. Phospholipidbands were scraped and then scintillation counted.

Results and discussion

Gibberellin treatment and cell elongation

The effects of GA3 on cell elongation were monitoredover an eight day growth period (Table 1). Results arepresented as cell elongation ratios calculated fromcell length divided by width. The presence of GA3 inthe growth media resulted in changes in the elonga-tion ratios which proved initially to be GA3 concen-tration dependent. This trend was consistent for ap-proximately 2 d, after which time, the cells lost somepolarity and moved towards a more spherical form,prior to further elongation.

Gibberellin treatment and fatty acid composition

Total lipid extracts were prepared from log-phase cel-ery cultures treated with either 10 �M or 50 �M GA3

for 6 d. On the whole, the fatty acid composition didnot change upon exposure to GA3 (Table 2). How-ever, a slight increase in the relative proportions oflinoleic acid was apparent. This observation is con-sistent with the studies of Grindstaff et al. (1996) onGA3-treated barley aleurone.

Figure 1. Effect of GA3 on choline uptake. GA3 treated cells wereincubated with tritiated choline. The uptake of the radio-label fromthe growth medium was monitored with time. Results are meansof duplicate assays.

Figure 2. CDP-base pathway flux in the presence of GA3. The ac-cumulation of radio-labelled phosphatidylcholine in the presence ofGA3 was determined after a 2 h incubation period with tritiatedcholine. Results are means of duplicate assays.

Table 1. Effect of GA3 treatment on cell elongation

Day Elongation Ratio

[GA3] = 0 �M [GA3] = 10 �M [GA3] = 50 �M

0 1.90 1.90 1.90

2 1.55 2.47 5.35

6 1.80 2.19 2.53

8 1.48 5.23 3.95

The effects of GA3 treatment on cell elongation were monitoredwith time. Results are the mean values of 20 cell measurementsgiven as an elongation ratio. (Elongation ratio = cell length: cellwidth)

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Phosphatidylcholine metabolism

The effects of GA3 treatment on PC biosynthesiswere assessed using the radiolabelled precursors [3H-methyl] choline and [3H-methyl] methionine.

CDP-base pathway fluxGibberellin treatment was shown to have little effecton the initial rate of the uptake of exogenous cholinefrom the growth medium (Figure 1). However, in con-trast to the work of Vakharia et al. (1987) on wheataleurone, after an incubation time of 10 min, theamount of [3H] choline taken up by the GA3 treatedcells reached maximal values (0.4nmoles × mg−1 d wt, 10 �M; 0.6 nmoles × mg−1 d wt,50 �M GA3). Meanwhile, control cells continued totake up the radio-label for approximately a further 30min.

With respect to the choline flux through the CDP-base pathway, GA3-treated cells accumulated loweramounts of radiolabelled PC than their non-treatedcounterparts (Figure 2). A similar situation has beendemonstrated in wheat aleurone (Vakharia et al. 1987)and in growth-arrested celery cell suspension cultures(Rolph and Goad 1991). Nevertheless, which enzy-mic step(s) is/are modulated by GA3 treatment re-mains to be elucidated. However, in an early study byTanaka and Tolbert, the first enzyme in the pathway,choline kinase was shown to display reduced activityin GA3-treated squash (Tanaka and Tolbert 1966).

Methyltransferase pathway fluxCelery cell suspension cultures were incubated for 4h with radio-labelled methionine in order to deter-mine the effects of GA3 treatment on the flux of in-termediates through the methyltransferase pathway(Figure 3). In control cells, the distribution betweenmonomethylphosphatidylethanolamine (MMPE),dimethylphosphatidylethanolamine (DMPE) and PC

was approximately equal. Upon 10 �M GA3 treat-ment, a greater proportion of the label accumulatedin PC, than in the two partially methylated PE phos-phoglycerides. This trend was further accentuatedwhen cells were exposed to 50 �M GA3. In this in-

Table 2. Effect of GA3 treatment on the acyl composition of total lipid fractions

Relative Proportions of Acyl Chains (%)

Fatty Acid [GA3] = 0 �M (n = 3) [GA3] = 10 �M (n = 5) [GA3] = 50�M (n = 5)

Palmitate 28 ± 2 31 ± 12 25 ± 6

Stearate 4 ± 1 4 ± 3 4 ± 2

Oleate 15 ± 3 10 ± 4 9 ± 2

Linoleate 47 ± 4 49 ± 7 54 ± 5

�-Linolenate 7 ± 1 6 ± 2 8 ± 1

Apium cultures were grown +/− GA3 for 6 d, after which lipids were extracted and analysed by gas chromatography. Results are means± SEM

Figure 3. Methyltransferase pathway flux in the presence of GA3.Cells +/− GA3 were incubated with tritiated methionine for a 4 hincubation period. Lipids were then extracted, individual phospho-lipids separated chromatographically and the distribution of radio-label determined by scintillation counting. PC, phosphatidylcho-line; DMPE, dimethylphosphatidylethanolamine; MMPE, monom-ethylphosphatidylethanolamine. Results are means of duplicate as-says.

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stance, up to 70% of the radiolabel was found to beassociated with PC. These results are consistent withthe findings of Mirbahar and Laidman, in which GA3-treated wheat aleurone exhibited increased turnoverof PS and PE, the phospholipid precursors of the MP-derived PC (Mirbahar and Laidman 1982).

In several studies, GA3 treatment has been shownto have little effect on the total cellular PC content(Jusatis et al. 1981; Grindstaff et al. 1996). It is pos-sible that the decrease in the CDP-base pathway fluxmay be compensated for by increased methyltrans-ferase pathway activity. A similar situation, in whichthe two pathways have been shown to be co-ordi-nately regulated in celery cells, has been demon-strated previously (Rolph and Goad 1991). The abovefindings support the original hypothesis that methyl-transferase-derived PC may play a role in plant celldifferentiation. Nevertheless, previous studies (Jusa-tis et al. 1981; Mirbahar and Laidman 1982; Evinsand Varner 1971) have shown that the extent of GA3

exposure to be critical, with respect to the perceivedpattern of phospholipid metabolism, a parameterwhich must be taken into account in future studies.

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