Extracts of light-grown Cucumis sativus L. seedlings catalyzed the oxidation of indole-3-acetaldehyde to indole-3-acetic acid. No added cofactors were required. Inhibitor studies indicated that the enzyme is a metafloflavoprotein. While indole-3-aldehyde, benzaldehyde, and phenylacetaldehyde partially inhibited the oxidation of indole-3-acetaldehyde, suggesting that they may serve as alternative substrates, it is proposed that indoleacetaldehyde is the major substrate in vivo. 2,4-Dichlorophenoxyacetic acid strongly inhibited the indoleacetaldehyde oxidase activity, and it is proposed that this enzyme may be subject In vivo to feedback inhibition by indole-3-acetic acid. The enzyme was activated by brief heating or by treatment with mercaptoethanol.The young cucumber shoot is an excellent system for studies of auxin biosynthesis and its regulation. The growth of the intact seedling is strongly promoted by exogenously supplied IAA or synthetic auxins (5), suggesting a regulatory mechanism which disallows saturating auxin synthesis under normal conditions. We have isolated and characterized several of the enzymes presumed to be responsible for auxin synthesis in cucumber (1,2,6,12) and have shown that certain of them are subject to regulation (7; and H. M. Brown and W. K. Purves, manuscript in preparation). The present report extends these studies to include the characterization of the cucumber indole-3-acetaldehyde oxidase and its regulatory properties.The derivation of IAA from IAAld5 in vivo has been demonstrated by several workers, and it is now accepted that the enzymic oxidation of IAAld to IAA is the terminal step in auxin biogenesis (11). Rajagopal (9, 10) has described the properties of a partially purified aldehyde oxidase activity from Avena, and Wightman and Cohen (15) MATERIALS AND METHODS Preparation of Enzyme. Seeds of Cucumis sativus L. cv. National Pickling (Burpee Seed Co.) were soaked for 2 hr in tap water and sown in vermiculite saturated with tap water. Seedlings grew for 7 days under a 14-hr light, 10-hr dark cycle at 25 C. Shoots were homogenized in ice cold 50 mm tris buffer (pH 7.3) containing 1 mM MgCl2, for 90 sec in a Waring Blendor. The homogenate was filtered through cheesecloth (eight layers) and centrifuged for 20 min at 10,000g. The supernatant fluid was filtered through glass wool to remove the lipid pellicle. For some experiments, this filtrate served as the enzyme preparation, while others involved a heat-treated preparation obtained as follows.Aliquots (10 ml) of the filtrate were immersed in a 60 C circulating water bath and, at selected times, plunged into an ice bath. Except for the time study, the standard heating time was 2.5 min. The heated and chilled samples were centrifuged for 15 min at 10,000g. The supernatant fluid was decanted and used as "heat-activated" enzyme.Some untreated or heat-activated preparations were also subjected to a pH precipitation procedure. The pH was slowly lowered to 5 with 1 N HCl at 0 to 4 C with constant stirring. The preparation wa...

Subcellular fractionation of cucumber (Cucumis sativus L.) seedlings was achieved, and two of the enzymes in the auxin biosynthetic pathway were localized. NADH-specific indoleacetaldebyde reductase activity was observed only in the cytosol fractions obtained from separated hypocotyl and cotyledon tissue. In contrast, a portion of the NADPHspecific indoleacetaldehyde reductase activity was associated with a microsomal fraction derived from these tissues. The NADPH-specific indoleacetaldehyde reductase was consistently found to be more firmly associated with the microsomsd frction derived from hypocotyls than with that from the cotyledons. These results indicate a division of the terminal steps of auxin biogenesis into at least two subceflular compartments.Two distinct enzymes which catalyze the reduction of indole-3-acetaldehyde to indole-3-ethanol with the concomitant oxidation of NAD(P)H have been isolated and characterized from cucumber seedlings (2). An important role for IEt3 in indole-3-acetic acid biosynthesis now seems well established, for several experimental findings have suggested that lEt metabolism plays a regulatory role in this pathway (5,12,19). We have previously shown that cucumber seedlings contain multiple amine oxidases potentially responsible for the in vivo oxidation of tryptamine to IAAld (11) and have recently discovered that cucumber seedlings contain at least two separate IEt oxidases (unpublished results). The observation that several of the enzymes responsible for IAA synthesis exist in multiple forms makes it likely that intracellular and/or tissue compartmentation of the pathway plays a role in the regulation of auxin synthesis. We now report the subcellular and organ compartmentation of the NADPHand NADH-specific indoleacetaldehyde reductases in cucumber seedlings. MATERIALS AND METHODSPlants. Seeds of Cucumis sativus L. cv. National Pickling (Burpee) were sown in vermiculite after soaking 1 hr in tap H20.