Cucumber Seedling Indoleacetaldehyde Oxidase

Abstract

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...

Indoleacetaldehyde Reductase of Cucumis sativus L

“…The data are plotted by the LineweaverBurk method; nonlinear plots are indicative of nonhyperbolic kinetics, and concave plots ( Possible Roles for IAAld Reductase in the Regulation of Auxin Biosynthesis. We have reported that IAA is a potent inhibitor of highly purified IEt oxidase (10), and experiments with a crude preparation of IAAld oxidase activity from cucumber seedlings have shown that this activity is inhibited by 2,4-D, a chemical analog of IAA (2). These results suggest that IAA regulates its own synthesis through feedback inhibition of the terminal enzyme of the pathway.…”

“…The (100%) control rate was 39 nmol NADPH oxidized per min per ml enzyme. Controls have shown that the difference in ionic strength between, for example, 2 Figure 2 shows the reaction kinetics of the NADH-linked IAAld reductase activity. The Lineweaver-Burk plots indicate a sequential mechanism (both substrates add to the enzyme before any product dissociates) and are consistent with either random or ordered addition of the substrates.…”

Indoleacetaldehyde in Cucumber Seedlings

“…Some of these are directly involved in the metabolism of indole-3-acetaldehyde. They include three amine oxidases which can oxidize tryptamine to IAAld4 (7), an indoleethanol oxidase which oxidizes indole-3-ethanol to IAAld (8,13), IAAld reductases which reduce IAAld to IEt (1,3), and an IAAld oxidase which oxidizes IAAld to IAA (2). These demonstrations could be considered irrelevant to the actual biosynthesis of auxin unless it is shown that IAAld occurs naturally in the cucumber seedling.…”

Indole-3-Ethanol Oxidase in Phycomyces blakesleeanus Bgff

“…Under the assumption that at least under some physiological conditions IAAld' may be a major precursor of IAA (formed either from indole-3-acetaldoxime [12], tryptamine, or indole-3-pyruvic acid [27]), the reversible oxidation-reduction of the aldehyde to IEt by IAAld reductase may reduce the rate of IAA biosynthesis and thus IAA concentration (4,27). Upon changing physiological conditions lEt may be 'reactivated' by oxidation to IAAld and IAA via lEt oxidase (23) and IAAld oxidase (3).…”