Plant Physiol, September 2002, Vol. 130, pp. 90-101

Molecular Identification of Cytosolic, Patatin-Related Phospholipases A from Arabidopsis with Potential Functions in Plant Signal Transduction¹

Universität Hannover, Institut für Zierpflanzenbau, Baumschule und Pflanzenzüchtung, Herrenhäuser Strasse 2, D-30419 Hannover (A.H., S.R., M.Z., G.F.E.S.); and Universität Hamburg, Institut für Allgemeine Botanik, Ohnhorststrasse 18, D-22609 Hamburg (H.Q.)

Rapid activation of phospholipase A (PLA) by auxin or plant-pathogen interaction suggests a function in signal transduction for this enzyme, but the molecular identification of a cytosolic PLA carrying out this function remains open. We isolated four cDNA sequences from Arabidopsis (ecotype Columbia), AtPLA I, AtPLA IIA, AtPLA IVA, and AtPLA IVC, which are members of the patatin-related PLA gene family in plants and which are homologous to the animal Ca²⁺-independent PLA₂ gene family. Expression was measured by reverse transcriptase-polymerase chain reaction, and AtPLA I transcripts were found preferentially in shoots, AtPLA IIA and AtPLA IVA in roots, and AtPLA IVC in flowers. Transient expression of the four PLA-green fluorescent protein fusion proteins in tobacco (Nicotiana tabacum) leaves showed they were located in the cytosol and not in the vacuoles. Surprisingly, AtPLA::green fluorescent protein was also localized to chloroplasts. The enzymatic activity of the purified recombinant AtPLA IVA toward phosphatidylcholine was dependent on Ca²⁺, saturated at 0.5 mM, and had a pH optimum of about 7.0. It had both PLA₁ and PLA₂ specificity. The enzyme showed in vitro highest sensitivity toward the PLA₂ inhibitors palmitoyltrifluoromethyl ketone (PACOCF₃, K_i approximately 30 nM), arachidonyltrifluoromethyl ketone (AACOCF₃, K_i approximately 25 µM), and tetrahydro-3-(1-naphtalenyl)-2H-pyran-2-one (K_i approximately 200 nM) and was also sensitive to other previously used inhibitors 5,8,11,14-eicosatetraynoic acid (K_i approximately 3 µM) and nordihydroguajaretic acid (K_i approximately 15 µM). The influence of these PLA₂ inhibitors on elongation in etiolated Arabidopsis seedlings was tested, and tetrahydro-3-(1-naphtalenyl)-2H-pyran-2-one and 5,8,11,14-eicosatetraynoic acid inhibited hypocotyl elongation maximally at concentrations close to their K_i in vitro.