Oxidation of Proline by Plant Mitochondria

Abstract

Mitochondria isolated from etiolated shoots of com ( Zen mays), wheat (Tddk aestum, baley ( HM "*are), soybean ( Glychie max L.Merr.), and mung bean ( Phasebhis awueus) exhibited a prol-pndent 02 uptake subject to respiratory controL ADP/O ratios with proline as substrate were intermediate between ratios obtained with exogenous NADH and malate + pyruvate as substrates. Isotope studies showed proline metabolism to be dependent on 02, but not NAD. The major ninhydrin-positive product formed via A1-pyrroline-5-carboxylic acid was glutamate. Mitochondria were capable of further metabolism of glutamate, as radioactive CO2, organic acids, and aspartate were recovered after 114CIprolne feeding expeiments. These results demonstrate the mitochondrial association and 02 dependence of plant proline metabolism.In mammals, insects, yeasts, and bacteria, the metabolism of proline is begun by conversion to P5C2 (5, 7, 8) or in some cases P2C (2, 8). This conversion is typically mitochondrial, and is catalyzed by proline oxidase, an 02-dependent flavoprotein (5,7,8,21). Activity of this enzyme apparently has not been demonstrated in higher plants. Instead, several workers have reported the presence of proline dehydrogenase, a nonparticulate, NADlinked enzyme which has been suggested, but not proved, to catalyze P5C formation in vivo (10)(11)(12)19). Doubt that proline dehydrogenase is the in vivo catalyst for proline oxidation has arisen from the necessity to assay it at high pH (above pH 10, refs. 10 and 12) and from the observation that it co-purifies with P5C reductase (10), the NADH-linked proline biosynthetic enzyme that is typically stable and present in relatively high activity in a variety of tissues (6,10,12,16,17). These considerations led us to look for proline oxidation by mitochondria isolated from higher plants. MATERIALS AND METHODSPlant Material. Mitochondria were isolated from 3-day etiolated shoots of corn (Zea mays cv. WF9[NJ x B37) and wheat ( Triticum aestivum cv. Abe), from 4-day etiolated shoots of barley (Hordeum vulgare cv. Prior) and from 4-day etiolated hypocotyls of soybean (Glycine max L. Merr. cv. Amsoy 71) and mung bean (Phaseolus aureus). Corn seedlings were grown at 30 C on moist paper towels saturated with 0.1 mM CaCl2. The other seedlings were grown in moist Vermiculite at room temperature (24-28 C).Mitochondrial Preparation. The procedure was the same as previously described for corn mitochondria (13 were clipped, ground in a mortar and pestle, and the homogenate filtered through cheesecloth. After a low speed centrifugation to remove debris, mitochondria were peileted (28,000g, 6 min), resuspended in 0.4 M sucrose, and the suspension clarified by centrifugation at low speed. Mitochondria were then centrifuged through a 0.6 M sucrose cushion (17,500g, 18 min) with final suspension in 0.4 M sucrose. Mitochondrial protein was measured by the Lowry procedure (9).02 Uptake Measurements. 02 concentration was monitored with a Clark 02 electrode (Yellow Springs Instrument Co.) in a stirred...

Submitochondrial Location and Electron Transport Characteristics of Enzymes Involved in Proline Oxidation

“…Proline oxidase is severely inhibited (2,10) while P5C dehydrogenase activity (as measured by NAD+ reduction) is dependent upon detergent activation in corn mitochondria as in those of barley (3). As shown in Figure 2A, proline oxidase exhibited a gradual inhibition with increasing detergent and had negligible activity at concentrations above 0.020%1o.…”

“…Since proline oxidation had been found to support 02 uptake (2), an experiment was performed to see if P5C oxidation would also result in 02 utilization. The addition of either proline or P5C was found to support 02 uptake.…”

“…Proline oxidation is a mitochondrial process (2,10) that can be inhibited by water stress (16). Although extensive research has been conducted on proline accumulation and its significance during water stress, little has been directed toward elucidating the inhibition of proline oxidation.…”

Effects of the Proline Analog l-Thiazolidine-4-carboxylic Acid on Proline Metabolism

“…The oxidation of proline to glutamate occurs within mitochondria (3,7,8), and is believed to provide energy and carbon skeletons for a number of processes. During water stress, inhibition of proline oxidation and stimulation of proline synthesis cause proline to accumulate (2,20).…”

BIOCHEMICAL EFFECTS OF SULPHUR DIOXIDE ON PROLINE METABOLISM IN THE ALGA TREBOUXIA SP.

“…The fall in proline might be due to inhibition of proline synthesis by sulphite or to an increase in rate of degradation. Two possible degradative pathways are known; one of them is an oxygen-dependent reaction catalysed by proline oxidase (Boggess, Koeppe, and Stewart, 1978;Boggess and Stewart, 1980); the other starts • with a dehydrogenase reaction catalysed by a NAD'*'-dependent proline dehydrogenase (Rena and Splitstoesser, 1974) which converts proline to pyrroline-5-carboxylate (cf. review by Adams and Frank, 1980).…”