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

Abstract

Abstract 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, andAtPLA 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, andAtPLA I transcripts were found preferentially in shoots,AtPLA IIA and AtPLA IVA in roots, andAtPLA 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  iapproximately 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 theirK  i in vitro.