First published online August 29, 2002; 10.1104/pp.006288
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
André
Holk,*
Steffen
Rietz,
Marc
Zahn,
Hartmut
Quader, and
Günther F.E.
Scherer
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,
Ki approximately 30 nM),
arachidonyltrifluoromethyl ketone (AACOCF3,
Ki approximately 25 µM), and
tetrahydro-3-(1-naphtalenyl)-2H-pyran-2-one (Ki approximately 200 nM) and
was also sensitive to other previously used inhibitors
5,8,11,14-eicosatetraynoic acid (Ki
approximately 3 µM) and nordihydroguajaretic acid
(Ki 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
Ki in vitro.
1
This work was supported by the Deutsche
Forschungsgemeinschaft (grant no. Sche207/9-1), by the Bundesland
lower saxony (VW-Vorab), the European Union (grant no.
IC15-CT98-0118), and by the Bundesministerium für Forschung und
Technologie/Deutsches Zentrum für Luft- und Raumfahrt (project
no. 50WB0010).
*
Corresponding author; e-mail holk{at}zier.uni-hannover.de; fax
49-511-762-2654.
© 2002 American Society of Plant Physiologists
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