Formation of Nitrite from Hydroxylamine in the presence of illuminated chloroplast lamellae is inhibited by superoxide dismutase but not by catalase, indicating that the superoxide free radical ion and not H2O2 is responsible for the oxidation of hydroxylamine. Decarboxylation of α-keto acids on the other hand is strongly inhibited by catalase but only slightly by superoxide dismutase. Light-dependent hydroxylamine oxidation and decarboxylation of α-keto acids can be used, therefor, as specific and sensitive probes for the determination of either the superoxide free radical ion or hydrogen peroxide, respectively. Photosynthetic oxygen reduction in the presence of ferredoxin, (monitored by the above method) yields both H2O2 and O2·-. The addition of an oxygen reducing factor (ORF, solubilized by heat - treatment of washed chloroplast lamellae) instead of ferredoxin, however, stimulates only the production of H2O2 , while O2·- - formation is not observed. The cooperation of ferredoxin and ORF during photosynthetic oxygen reduction by chloroplast lamellae apparently produces H2O2 not only by dismutation of O2·-, but also by a separate mechanism involving ORF.
Ethylene production by sugar beet (Beta vulgaris L.) leaf discs is inhibited by white (or red, >610 nm) light or by wounding. In contrast, in wounded leaf discs, ethylene production is stimulated by light. The effect of light on wounded leaf discs has been studied by using an in vitro system which mimics the loss of compartmentation in the wounded leaf.Chlorophyll-free extracts from sugar beet leaves stimulate the production of the superoxide free radical ion (as a prerequisite for ethylene formation) by Muminated chloroplast lamellae. The substance from the crude leaf extracts which is active in stimulating the production of the superoxide free radical ion has been identified as 3-hydroxytyramine (dopamine). Exogenous dopamine between 5 jLM and 100 FM stimulates ethylene formation by iluminated chloroplast lameflae from methional. It also stimulates the production of the superoxide free radical ion, the formation of which apparently involves both a lamellar phenoloxidase and photosynthetic electron transport as a 1-electron donor, and is cyanide-sensitive. Ethylene (1) seems to be responsible for the induction of the enzymes involved in the biosynthesis of several phenolic compounds, the reactions participating in the resistance to infections, fruit ripening, and the senescence of higher plants. Ethylene production, in turn, is initiated by physiological or artificially induced stress conditions. The biochemical mechanism of the induction of ethylene biosynthesis is unknown. However, the prerequisites for ethylene formation by higher plant tissue seem to be an oxygen-activating system together with an appropriate ethylene donor such as methionine, 2-keto-4-mercaptomethyl butyrate, methional (3-mercaptomethyl propanal), or linolenic acid (1,19,24,34). In the case of the model compounds 2-keto-4-mercaptomethyl butyrate or 3-mercaptomethyl propanal, the OH radical produced after monovalent oxygen reduction has been shown to be the oxidant (5, 10-12) yielding ethylene and other products (35).In the present communication, we wish to report on experiments concerning the induction of monovalent oxygen reduction (production of O2--) and ethylene formation in chloroplasts from sugar beet leaves, involving the function of endogenous 3-hydroxytyramine (dopamine) as the cofactor of monovalent oxygen reduction. (13). The activity units of superoxide dismutase were determined as described by McCord and Fridovich (23). Ethylene formation was monitored as previously described (10-12).3-Hydroxytyramine was isolated from sugar beet leaves according to the procedure of Gardner et al. (16). The aqueous phase from sugar beet leaves (see ref. 16) was evaporated to dryness and dissolved in methanol containing 0.25% concentrated HCI. The filtered solution was loaded onto a cellulose column (prepared by stirring 15 g cellulose [Avicel, Merck-Darmstadt] in a 150-ml butanone-2-formic acid-acetone-water mixture, 40:1:2:6, v/v). The column was washed with increasing amounts of the same solution. The fractions containing 3-hydroxyt...
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