Molecular identification of cytosolic, patatin-related phospholipases A from Arabidopsis with potential functions in plant signal transduction.
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 Ca(2+)-independent PLA(2) 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 Ca(2+), saturated at 0.5 mM, and had a pH optimum of about 7.0. It had both PLA(1) and PLA(2) specificity. The enzyme showed in vitro highest sensitivity toward the PLA(2) inhibitors palmitoyltrifluoromethyl ketone (PACOCF(3), K(i) approximately 30 nM), arachidonyltrifluoromethyl ketone (AACOCF(3), K(i) approximately 25 microM), 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 microM) and nordihydroguajaretic acid (K(i) approximately 15 microM). The influence of these PLA(2) 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.
Holk
André
A
Universität Hannover, Institut für Zierpflanzenbau, Baumschule und Pflanzenzüchtung, Herrenhäuser Strasse 2, D-30419 Hannover. holk@zier.uni-hannover.de
Rietz
Steffen
S
Zahn
Marc
M
Quader
Hartmut
H
Scherer
Günther F E
GF
D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000201:enzymology / Q000235:genetics*; D029681:Arabidopsis Proteins / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism; D002118:Calcium / Q000494:pharmacology; D002265:Carboxylic Ester Hydrolases / Q000235:genetics / Q000378:metabolism; D003600:Cytosol / Q000201:enzymology; D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics; D004791:Enzyme Inhibitors / Q000494:pharmacology; D015971:Gene Expression Regulation, Enzymologic; D018506:Gene Expression Regulation, Plant; D049452:Green Fluorescent Proteins; D006863:Hydrogen-Ion Concentration; D018546:Hypocotyl / Q000187:drug effects / Q000254:growth & development; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D010740:Phospholipases / Q000235:genetics* / Q000378:metabolism; D010741:Phospholipases A / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism; D010802:Phylogeny; D018515:Plant Leaves / Q000235:genetics / Q000378:metabolism; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism; D018517:Plant Roots / Q000235:genetics / Q000378:metabolism; D018547:Plant Stems / Q000235:genetics / Q000378:metabolism; D011133:Polysaccharide-Lyases; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D015398:Signal Transduction / Q000235:genetics / Q000502:physiology*; D013379:Substrate Specificity; D014026:Nicotiana / Q000235:genetics
D029681:Arabidopsis Proteins; D018076:DNA, Complementary; D004791:Enzyme Inhibitors; D008164:Luminescent Proteins; D010940:Plant Proteins; D011993:Recombinant Fusion Proteins; C043222:patatin protein, Solanum tuberosum; D049452:Green Fluorescent Proteins; D010740:Phospholipases; C468915:phospholipase IIA, Arabidopsis; C468916:phospholipase IVA, Arabidopsis; C468917:phospholipase IVC, Arabidopsis; D002265:Carboxylic Ester Hydrolases; D010741:Phospholipases A; D011133:Polysaccharide-Lyases; C468914:AT1G67750 protein, Arabidopsis; D002118:Calcium