Anaerobic Stress in Germinating Castor Bean, Ethanol Metabolism, and Effects on Subcellular Organelles

Abstract

Endosperms from castor beans (Ricinus communis) germinated for 0 to 6 days were exposed to anoxia for 0 to 15 hours. Ethanol, the only alcohol detected by gas chromatography in the tissue, accumulates to a concentration of 15 millimolar during the first 2 to 4 hours of anoxia and subsequently decreases. The absolute amount of ethanol varies from 10 micromoles per 5-day endosperm after 4 hours anoxia to less than 1 micromole in 2-day endosperm after 4 hours. Lactate content is 2 micromoles or less per endosperm. Alcohol dehydrogenase and pyruvate decarboxylase activities, which are localized in cytosolic fractions, are not greatly affected by anoxia. The recoveries of the marker enzymes and protein in endoplasmic reticulum (ER) and mitochondrial fractions decrease during anoxia. After 15 hours, the recovery of NADPH cytochrome c reductase is 15% of that in controls, fumarase is 50%, and catalase is 75%.Glyoxysomes and ER are capable ofconverting ethanol to acetaidehyde which was measured using the fluorogenic reagent, 5,5-dimethyl-1,3-cyclohexanedione. The glyoxysomal activity is dependent on a hydrogen peroxide-generating substrate and the ER is dependent on NADPH. However, these activities are less than 3% of the alcohol dehydrogenase activity.Hypoxic stress and alcoholic fermentation are common features of plant metabolism. Ethanol is produced by flooded seedlings (1, 18, 19), ice encased leaves (2), root nodules (20), and tissues exposed to S02 and 03 (10). The endosperm of germinating castor bean endosperm is an appropriate model to study anaerobic stress and alcohol metabolism. This tissue responds to anoxia by generating ethanol and smaller amounts of lactate (11). ER, mitochondria, and glyoxysomes, subcellular components which depend on 02 have been isolated and well characterized (3, 6, 21). The glyoxylate cycle has been shown to participate in ethanol metabolism (17). In the earlier investigation of anoxic castor bean endosperm, Kobr and Beevers (1 1) followed ethanol accumulation for 2 h in endosperms after 5 d germination. However, plant tissues are often capable of producing ethanol for longer periods of time (1,4).One purpose of this study was to determine the extent and regulation ofethanol production in castor bean during prolonged anoxia as a function of seedling age. Also, we examined the possibility that other alcohols might be produced and metabolized during anoxia. Because the endosperm initially contains very little carbohydrate (7), it was expected that little or no ethanol would be generated prior to the establishment of gluconeogenesis. We explored the possibility that the capacity for ethanol generation might be regulated by pyruvate decarboxylase and alcohol dehydrogenase, the enzymes responsible for the final steps of ethanol production.The second major aim of this study was to examine the subcellular locations of ethanol metabolism and the effects of anoxia on 02 requiring organelles. It has been reported that rat liver microsomal Cyt P450 and peroxisomal catalase are capable ...

Catalase Activity and Post-anoxic Injury in Monocotyledonous Species

“…The 'catalase' and 'peroxidase' modes of catalase activity were examined in an earlier study (Oshino et al, 1973) The second molecule of peroxide functions as a hydrogen donor in the catalase reaction, whereas ethanol takes this role in the peroxidatic reaction. In conditions of excess ethanol in plants, such as after a period of anaerobiosis, oxidation of this fermentation product to acetaldehyde could be accomplished not only by ADH but also by catalase provided there is a slow but continuous peroxidase generation source (Donaldson et al, 1985). The ability to oxidize ethanol aerobically has been demonstrated in a wide variety of plants and plant tissues (Cossins, 1978).…”

“…In a recent study on germinating castor bean, Donaldson, Soochan, and Zaras (1985) argued that ethanol accumulated during anaerobic stress could be oxidized by any of three separate systems, including one located in the endoplasmic reticulum and dependent on NADPH, and one which was confined to glyoxysome and dependent on a peroxideproducing substrate such as a fatty acid. The third alternative, alcohol dehydrogenase, requiring NAD for the oxidation of ethanol, was cited as the major reaction taking place.…”

Dynamics of Acetaldehyde Production during Anoxia and Post-Anoxia in Red Bell Pepper Studied by Photoacoustic Techniques

“…Prolonged periods of anoxia are expected to cause damage to mitochondria and, consequently, longer times of post-anoxic aeration will be required to obtain full or partia1 oxidative capacity. In support of this, a diminished recovery of marker enzymes and proteins in mitochondrial fractions was reported after 6 to 15 h of anoxia in excised castor bean endosperms (Donaldson, 1985).…”

Immunological Detection of Acetaldehyde-Protein Adducts in Ethanol-Treated Carrot Cells

“…Samples of culture media were obtained and immediately assayed for acetaldehyde using the dimedone reagent as described by Donaldson et al (6) with minor modifications (15).…”