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(2S)‐Flavanone 3‐hydroxylase from flowers of Petunia hybrida catalyses the conversion of (2S)‐naringenin to (2R, 3R)‐dihydrokaempferol. The enzyme could be partially stabilized under anaerobic conditions in the presence of ascorbate. For purification, 2‐oxoglutarate and Fe2+ had to be added to the buffers. The hydroxylase was purified about 200‐fold by a six‐step procedure with low recovery. The Mr of the enzyme was estimated by gel filtration to be about 74000. The hydroxylase reaction has a pH optimum at pH 8.5 and requires as cofactors oxygen, 2‐oxoglutarate, Fe2+ and ascorbate. With 2‐oxo[1‐14C]glutarate in the enzyme assay dihydrokaempferol and 14CO2 are formed in a molar ratio of 1:1. Catalase stimulates the reaction. The product was unequivocally identified as (+)‐(2R,3R)‐dihydrokaempferol. (2S)‐Naringenin, but not the (2R)‐enantiomer is a substrate of the hydroxylase. (2S)‐Eriodictyol is converted to (2R,3R)‐dihydroquercetin. In contrast, 5,7,3′, 4′, 5′‐pentahydroxy‐flavanone is not a substrate. Apparent Michaelis constants for (2S)‐naringenin and 2‐oxoglutarate were determined to be respectively 5.6 μmolx1−1 and 20 μmolx1−1 at pH 8.5. The Km for (2S)‐eriodictyol is 12 μmolx1−1 at pH 8.0. Pyridine 2,4‐dicarboxylate and 2,5‐dicarboxylate are strong competitive inhibitors with respect to 2‐oxoglutarate with Ki values of 1.2 μmolx1−1 and 40 μmolx1−1, respectively.

Two isoenzymes of an NADP' -dependent cinnamyl alcohol dehydrogenase and an NAD + -dependent aliphatic alcohol dehydrogenase were extracted from cell suspension cultures of soybean (Glycine max L., var. Mandarin) which form lignin during growth. These enzymes could be separated from each other by chromatography on DEAE-cellulose and hydroxyapatite. The cinnamyl alcohol dehydrogenase isoenzymes were partially purified by (NH&SO, fractionation, and column chromatography on DEAE-cellulose, SephadexG-100, and hydroxyapatite. The molecular weight of the enzymes were estimated by the elution volumes from a Sephadex G-100 column and were found to be about 43000 (isoenzyme 1) and 69000 (isoenzyme 2). Maximum rates of reaction were observed in the case of coniferyl alcohol oxidation at pH 9.2 (isoenzyme 1) and pH 8.8 (isoenzyme 2); in the reverse reaction pH 6.5 was optimal for isoenzyme 2.Whereas isoenzyme 1 is specific for coniferyl alcohol, isoenzyme 2 can also oxidize cinnamyl alcohol and a number of substituted cinnamyl alcohols. K , values for substituted cinnamaldehydes are 3 -11 times lower than for the corresponding alcohols. Neither isoenzyme reacted with benzyl alcohol, anisic alcohol or ethanol. Substrate inhibition for the forward and reverse reaction was found with isoenzyme 2 but not with isoenzyme 1. The equilibrium constant was determined to be about lo9 in favour of coniferaldehyde reduction. The possible role of the cinnamyl alcohol dehydrogenase in lignin biosynthesis is discussed.We have previously shown that an enzyme preparation from cell suspension cultures of soybean (Glycine max) catalyses the NADPH-dependent reduction We now describe the partial purification of two specific cinnamyl alcohol dehydrogenase isoenzymes from soybean cell suspension cultures. MATERIALS AND METHODS MatrriulsSubstituted cinnamic acids and coniferyl alcohol were purchased from C. Roth (Karlsruhe). Lithium aluminium-tris (tert-butyloxyhydride) was obtained from Fluka AG (Buchs, SG, Switzerland). All biochemicals were purchased from Boehringer Mannheim GmbH (Mannheim) or Serva (Heidelberg).Svnthesis of Substituted Cinnamyl Alcohols and Cinnamaldehydes p-Coumaryl alcohol [7] and sinapyl alcohol [8] were synthesized according to published methods. In analogy to these methods, 3,4-dimethoxycinnamyI alcohol (A, , , = 267 nm) was obtained from methyl 3,4-dimethoxycinnamate by reduction with LiAIH,. p-Coumaryl, coniferyl and sinapyl alcohol were purified by paper chromatography on Whatman 3 MM (prewashed with 10mM EDTA, 10% acetic acid, methanol, water) with the solvent system of butanol (water-saturated) containing 2 7,; NH3 [9]. For the purification of 3,4-dimethoxycinnamyl alcohol, 2 %, acetic acid was used as solvent system.

A radioimmunoassay specific for glyceollin I was used to quantitate this phytoalexin in roots of soybean (Glycine max [L.] Merr. cv Harosoy 63) after infection with zoospores of either race 1 (incompatible) or race 3 (compatible) of Phytophthora megasperma Drechs. f. sp. glycinea Kuan and Erwin. The sensitivity of the radioimmunoassay and an inmmunofluorescent stain for hyphae permitted quantitation of phytoalexin and localization of the fungus in alternate serial cryotome sections from the same root. The incompatible interaction was characterized by extensive fungal colonization of the root cortex which was limited to the immediate vicinity of the inoculation site. Glyceollin I was first detected in extracts of whole roots 2 hours after infection, and phytoalexin content rose rapidly thereafter. Significant concentrations of glyceollin I were present at the infection site in cross-sections (42 micrometers thick) of such roots by 5 hours, and exceeded 0.6 micromoles per milliliter (ECg, in vitro for glyceollin I) by 8 hours after infection. Longitudinal sectioning (14 micrometers thick) showed that glyceollin I accumulated particularly in the epidermal cell layers, but also was present in the root cortex at inhibitory concentrations. No hyphae were observed in advance of detectable levels of the phytoalexin and, in most roots, glyceollin I concentrations dropped sharply at the leading edge of the infection. In contrast, the compatible interaction was characterized by extensive unchecked fungal colonization of the root stele, with lesser growth in the rest of the root. Only small amounts of glyceollin I were detected in whole root extracts during the first 14 hours after infection. Measurable amounts of glyceollin I were detected only in occasional cross-sections of such roots 11 and 14 hours after infection. The phytoalexin was present at inhibitory concentrations in the epidermal cell layers, but the inhibitory zone did not extend appreciably into the cortex. Altogether, these data support the hypothesis that the accumulation of glyceollin I is an important early response of sojbean roots to infection by P. megasperma, but may not be solely responsible for inhibition of fungal growth in the resistant response.The production of phytoalexins is a well-documented response of plants to infection with a wide variety of microorganisms (7). The various lines of evidence supporting a role for these anti- ' Supported by the Deutsche Forschungsgemeinschaft (SFB 46) A disease that has received considerable attention as a model host-pathogen system is root and stem rot of soybean (Glycine max) cause by Phytophthora megasperma f. sp. glycinea. In the field, this pathogen most commonly infects soybean plants under conditions of water-logged soils (12). The natural infective propagules are zoospores, and the most frequent site of infection is the plant root. Young seedlings are most severely damaged. Most studies on the biochemistry of this plant-pathogen interaction have relied on infecting mechanically wounded h...

Cambial sap of spruce (Picea abies) proved to be a good source for isolation of cinnamoyl-CoA reductasc and cinnamyl alcohol :NADP+ dehydrogenase. Apparently homogeneous enzymes were obtained by a multistep procedure including dye-ligand chromatography and for the reductase also affinity chromatography on (coenzymc A)-agarose. An improved purification procedure for the reductase from soybean cell cultures is also reported.Molecular weights and subunit composition of reductase and dehydrogenase from spruce are very similar to those of the corresponding enzymes from soybean.Reduction of feruloyl-CoA to coniferaldehyde catalysed by the reductase is a freely reversible reaction with an equilibrium constant of 5.6 x M at pH 6.25. A strong dependence of the Michaelis constants on the type of buffer was found. For reductase the K,-value of feruloyl-CoA in phosphate buffer (5.2 pM) is about 14-times smaller than in citrate buffer (73 pM).Pronounced differences in substrate specificities between the enzymes from spruce and soybean were found, which reflect the different lignin composition of gymnosperms and dicotyledenous angiosperms. From the kinetic constants of the enzymes it can be concluded that under physiological conditions feruloyl-CoA is the preferred substrate for the reductase from both sources whereas sinapoyl-CoA is a substrate only for the soybean reductase and sinapyldehyde a substrate only for the soybean dehydrogenase.4-Coumaroyl-CoA is a poor substrate for the reductase from both spruce and soybean.This result is consistent with the low content of 4-coumaryl alcohol units in gymnosperm and angiosperm lignin.