Localization and characterization of soluble and plasma membrane aminopeptidase activities in Arabidopsis seedlings

We previously demonstrated that N,1-naphthylphthalamic acid is hydrolyzed at the root-hypocotyl transition zone and other regions of Arabidopsis thaliana seedlings, and that this reaction, like NPA-induced growth inhibition, is strongly promoted by blue light. In addition, NPA amidase activity was detected in plasma membrane-enriched fractions obtained from Arabidopsis seedlings. To further investigate this phenomenon, we tested the hypothesis that the arylamidase(s) responsible for NPA hydrolysis may also have aminopeptidase activity. The responses of Arabidopsis seedlings to various aminopeptidase substrates were tested. The hydrolysis of Tyr-, Trp-, Pro- and Gly-Pro-β-naphthylamide aminopeptidase substrates was shown to be histochemically localized at the root-hypocotyl transition zone and other regions where NPA hydrolysis also occurs. Blue light stimulated the in vivo activity of Tyr- and Pro-aminopeptidase activities, and far-red light stimulated the activity of the Trp-aminopeptidase. These same substrates also induced NPA-like growth inhibitory effects. In parallel experiments, aminopeptidase activities were detected in the supernatant and plasma membrane fractions of seedling extracts. The soluble AP activities resemble previously described neutral aminopeptidases with specificity for aromatic residues. The plasma membrane fraction hydrolyzed Tyr-, Trp-, Ala-Pro- and Pro-AP substrates, and also exhibited activity against Phe- and Leu-substrates. Many of the properties of the aminopeptidases, such as pH optima, metal requirements and responses to inhibitors, overlap with those of the previously characterized NPA amidase, suggesting that the latter may represent the combined activities of multiple aminopeptidases.