Arabidopsis PLDζ2 regulates vesicle trafficking and is required for auxin response

Phospholipase D (PLD) and its product, phosphatidic acid (PA), play key roles in cellular processes, including stress and hormonal responses, vesicle trafficking, and cytoskeletal rearrangements. We isolated and functionally characterized Arabidopsis thaliana PLD zeta 2, which is expressed in various tissues and enhanced by auxin. A PLD zeta 2-defective mutant, pld zeta 2, and transgenic plants deficient in PLD zeta 2 were less sensitive to auxin, had reduced root gravitropism, and suppressed auxin-dependent hypocotyl elongation at 29 degrees C, whereas transgenic seedlings overexpressing PLDz2 showed opposite phenotypes, suggesting that PLD zeta 2 positively mediates auxin responses. Studies on the expression of auxin-responsive genes and observation of the beta-glucuronidase (GUS) expression in crosses between pld zeta 2 and lines containing DR5-GUS indicated that PLD zeta 2, or PA, stimulated auxin responses. Observations of the membrane-selective dye FM4-64 showed suppressed vesicle trafficking under PLD zeta 2 deficiency or by treatment with 1-butanol, a PLD-specific inhibitor. By contrast, vesicle trafficking was enhanced by PA or PLD zeta 2 overexpression. Analyses of crosses between pld zeta 2 and lines containing PIN-FORMED2 (PIN2) enhanced green fluorescent protein showed that PLD zeta 2 deficiency had no effect on the localization of PIN2 but blocked the inhibition of brefeldin A on PIN2 cycling. These results suggest that PLD zeta 2 and PA are required for the normal cycling of PIN2-containing vesicles as well as for function in auxin transport and distribution, and hence auxin responses.