The existence of phospholipase A2 activity in plant mitochondria and its activation by hyperosmotic stress in durum wheat (Triticum durum Desf.)

Abstract

The activity of mitochondrial phospholipase A2 (PLA2) was shown for the first time in plants. It was observed in etiolated seedlings from durum wheat, barley, tomato, spelt and green seedlings of maize, but not in potato and topinambur tubers and lentil etiolated seedlings. This result was achieved by a novel spectrophotometric assay based on the coupled PLA2/lipoxygenase reactions using 1-palmitoyl-2-linoleoyl-sn-glycero-3-phosphatidylcholine as substrate; the mitochondrial localisation was assessed by checking recovery of marker enzymes. Durum wheat mitochondrial PLA2 (DWM-PLA2) showed maximal activity at pH 9.0 and 1mM Ca2+, hyperbolic kinetics (Km=90±6μM, Vmax=29±1nmolmin−1mg−1 of protein) and inhibition by methyl arachidonyl fluorophosphonate, 5-(4-benzyloxyphenyl)-4S-(7-phenylheptanoylamino)pentanoic acid and palmityl trifluoromethyl ketone. Reactive oxygen species had no effect on DWM-PLA2, that instead was activated by about 50% and 95%, respectively, under salt (0.21M NaCl) and osmotic (0.42M mannitol) stress imposed during germination. Contrarily, a secondary Ca2+-independent activity, having optimum at pH 7.0, was stress-insensitive. We propose that the activation of DWM-PLA2 is responsible for the strong increase of free fatty acids recently measured in mitochondria under the same stress conditions [Laus, et al., J. Exp. Bot. 62 (2011) 141–154] that, in turn, activate potassium channel and uncoupling protein, able to counteract hyperosmotic stress.