Naphthylphthalamic acid is enzymatically hydrolyzed at the hypocotyl-root transition zone and other tissues of Arabidopsis thaliana seedlings

During prolonged storage, solid naphthylphthalamic acid (NPA) turns purple due to the formation of the dye, 1,1ʹ-azonaphthylene (ANA). ANA forms oxidatively from two molecules of the NPA degradation product, α-naphthylamine (αNA). At concentrations ≥ 30 μM, solutions of `purple NPAʹ stained Arabidopsis thaliana seedlings specifically at the hypocotyl-root transition zone and other regions. Staining was caused by the aggregation of ANA and could be reconstituted using undegraded NPA and either αNA or ANA. Studies with [3H]-NPA confirmed that NPA is localized at sites of staining. Genestein, curcumin and quercitin inhibited the staining reaction. Liquid chromatography-mass spectral (LC-MS) analysis of the ANA in the stained tissue indicated that ANA is synthesized at sites of staining. It was postulated that the amide bond of NPA is enzymatically cleaved, producing αNA. The αNA combines to form ANA, which aggregates to yield an insoluble precipitate. Consistent with this hypothesis, NPA amidase activity was detected in purified plasma membranes. The NPA amidase activity was activated by 500 μM MnCl2 and inhibited by phloretin, genestein, quercitin, bestatin and EDTA.