Nitric oxide triggers phosphatidic acid accumulation via phospholipase D during auxin-induced adventitious root formation in cucumber

Abstract

Auxin and nitric oxide (NO) play fundamental roles throughout plant life. NO is a second messenger in auxin signal transductionleading to root developmental processes. The mechanisms triggered by auxin and NO that direct adventitious root (AR)formation are beginning to be unraveled. The goal of this work was to study phospholipid (PL) signaling during the auxin- andNO-induced AR formation in cucumber (Cucumis sativus) explants. Explants were labeled with 32P-inorganic phosphate andtreated with the auxins indole-3-acetic acid or 1-naphthylacetic acid, or the NO donor S-nitroso N-acetyl penicillamine, in thepresence or absence of the specific NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide. PLs wereseparated by thin-layer chromatography and quantified. We report that the signaling PLs phosphatidic acid (PA), phosphatidylinositolphosphate, and phosphatidylinositol bisphosphate accumulated within 1 min after auxin or NO treatment. Bothauxin and NO evoked similar and transient time course responses, since signaling PLs returned to control levels after 20 or30 min of treatment. The results indicate that auxin relies on NO in inducing PA, phosphatidylinositol phosphate, andphosphatidylinositol bisphosphate accumulation. Furthermore, we demonstrate that auxin and NO trigger PA formation viaphospholipase D (PLD) activity. Explants treated for 10 min with auxin or NO displayed a 200% increase in AR numbercompared with control explants. In addition, PLD activity was required for the auxin- and NO-induced AR formation. Finally,exogenously applied PA increased up to 300% the number of ARs. Altogether, our data support the idea that PLD-derived PA isan early signaling event during AR formation induced by auxin and NO in cucumber explants.