Copper excess triggers phospholipase D activity in wheat roots

Abstract

Wheat seedlings ( Triticum durum Desf.) were incubated in 100 μM Cu 2+ for different periods of time ranging from 1 min up to 16 h. Following metal addition a rapid intake of copper ions into the roots was observed. Cu 2+ induced an accumulation of both phosphatidic acid and phosphatidylbutanol within 1 min of incubation, the latter indicating a very rapid induction of phospholipase D (PLD) activity. The highest PLD stimulation was detected after 2 h from copper addition and decreased almost to the initial value at increasing times. Cycloheximide treatment of roots lowered phosphatidylbutanol accumulation because of a reduced PLD activity. The expression profile of a T. durum putative PLD-encoding gene showed a peak after 1 h of treatment as well, indicating that enhanced gene expression contributed to the increase in PLD activity. In the absence of copper ions, roots treated with the G protein activator mastoparan showed increases in phosphatidic acid and phosphatidylbutanol similar to those detected with the metal. PLD activity was also stimulated by cholera toxin. Two putatively G protein α subunit encoding sequences were isolated and no significant differences in transcription activity following Cu 2+ addition were observed. In copper-treated roots an early production of superoxide generated both by total and membrane-bound NADPH oxidase occurred. The G protein inhibitor suramin as well as the PLD antagonist 1-butanol abolished copper-induced superoxide production.