Arabidopsis PLDζ2Regulates Vesicle Trafficking and Is Required for Auxin Response

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Abstract
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 PLDζ2, which is expressed in various tissues and enhanced by auxin. A PLDζ2-defective mutant, pldζ2, and transgenic plants deficient in PLDζ2 were less sensitive to auxin, had reduced root gravitropism, and suppressed auxin-dependent hypocotyl elongation at 29°C, whereas transgenic seedlings overexpressing PLDζ2 showed opposite phenotypes, suggesting that PLDζ2 positively mediates auxin responses. Studies on the expression of auxin-responsive genes and observation of the β-glucuronidase (GUS) expression in crosses between pldζ2 and lines containing DR5-GUS indicated that PLDζ2, or PA, stimulated auxin responses. Observations of the membrane-selective dye FM4-64 showed suppressed vesicle trafficking under PLDζ2 deficiency or by treatment with 1-butanol, a PLD-specific inhibitor. By contrast, vesicle trafficking was enhanced by PA or PLDζ2 overexpression. Analyses of crosses between pldζ2 and lines containing PIN-FORMED2 (PIN2)–enhanced green fluorescent protein showed that PLDζ2 deficiency had no effect on the localization of PIN2 but blocked the inhibition of brefeldin A on PIN2 cycling. These results suggest that PLDζ2 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.