Institute of Molecular Cell Biology, BioCentrum, University of Amsterdam, 1098 SM Amsterdam, The Netherlands

Background and objectives
Plants recognize pathogen elicitors and activate their defences. Elicitors often bind to receptors exposed on the surface of the plasma membrane. They transmit the information into the cell via effector enzymes that produce second messengers. Receptors often communicate with the effector enzymes via trimeric G-proteins. Second messengers can be derived from phospholipids in the plasma membrane. For example, when phospholipase C (PLC) hydrolyses phosphatidylinositol 4,5-bisphosphate (PtdlnsP2) it produces the second messengers inositol 1,4,5-trisphosphate (InsP3) and diacylglycerol (DAG) [1]. DAG is rapidly phosphorylated to produce two more potential messengers, phosphatidic acid (PtdOH) and diacylglycerol pyrophosphate (DGPP) [2]. Lipid second messengers can also be produced by phospholipase D (PLD), which hydrolyses a structural phospholipid such as phosphatidylcholine, producing PtdOH directly. In its turn, PtdOH can again be phosphorylated to DGPP. We are determining whether elicitors activate PLC and PLD signalling cascades in activating defence responses.

Material and methods
Tomato cell suspension cultures were incubated with 32P-orthophosphate for 5 h to label all the phospholipids. They were then treated with mastoparan, a G-protein activator, or with xylanase, a widely used aspecific elicitor. At time intervals from 30 s to 10 min, samples were taken, the lipids extracted, separated by TLC and quantitated. Most treatments were carried out in the presence of 0.25% 1-butanol to assess PLD activity. When this enzyme hydrolyses a phospholipid it transfers the phosphatidyl group to water, but in the presence of 1-butanol, some phosphatidyl groups are transferred to the alcohol, forming phosphatidylbutanol (PtdBut). The amount formed is a relative measure of PLD activity.

Results and conclusions
The major structural lipids in tomato cells are phosphatidyl-choline, -ethanolamine, -inositol and -glycerol. They accounted for about 85% of the total radioactivity in the lipid extract after labelling. PtdOH and PtdInsP and PtdlnsP2 were identified as minor lipids accounting for about 1.6, 3 and 0.2% of the total lipid radioactivity, respectively. 32P-Incorporation studies showed that they incorporated label much faster that the structural lipids. This probably reflects their faster turnover, which is typical of signalling lipids [1]. Treatment of 32P-labelled cells with mastoparan resulted in a time- and dose-dependent increase in 32P-PtdOH and 32P-DGPP. There was a concomitant decrease in 32P-PtdlnsP and 32P-PtdInsP2, indicating that PLC was activated. However, in the presence of butanol, the production of 32P-PtdBut was also stimulated. The inactive mastoparan analogue masl7 was without effect. These results indicate that mastoparan activates both PLC and PLD. Incubation of 32P-labelled cells with xylanase resulted in a time- and dose-dependent decrease in radioactive PtdInsP and PtdlnsP2 correlated with a rise in PtdOH and DGPP. There was no increase in PtdBut, suggesting that xylanase specifically activates PLC.

1. Munnik T, Irvine RF, Musgrave A, 1998. Biochimica et Biophysica Acta 1389, 222-272.
2. Munnik T, de Vrije T, Irvine RF, Musgrave A, 1996. Journal of Biological Chemistry 271, 15708-15715.