Inhibition of Proline Oxidation by Water Stress

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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...

Section: Resultssupporting

confidence: 59%

Oxidation of Proline by Plant Mitochondria

Boggess¹,

Koeppe²,

Stewart³

1978

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“…Thus, if any other compounds were labeled, they represented a very small percentage of the total. Table I shows that the distribution of 'IC in oxidized products of proline was similar to that previously reported (17), except for a greater percentage of 14C recovered in y-aminobutyrate and correspondingly less in glutamate. The recovery of label in yaminobutyrate from proline is quite variable from experiment to experiment.…”

Section: Methodssupporting

confidence: 69%

“…Evidence has been presented previously that water stress inhibits proline oxidation (3,17). This effect of stress contributes to proline accumulation under stress conditions but cannot account for all of it (17).…”

mentioning

confidence: 99%

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Metabolism of [5-³H]Proline by Barley Leaves and Its Use in Measuring the Effects of Water Stress on Proline Oxidation

Stewart¹,

Boggess²

1978

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The objective of these expenments was to determine the fate of tritium from the 5 position of proline aad to assess the vaidity of its loss to H20 as a measure of proline oxidation. When 15-3H1prollne was fed to barley (Horueum vulgare) (6). Second leaves were excised and allowed to wilt under a bank of incandescent lamps at a height adjusted to cause 25% of the original leaf weight to be lost in about I hr. Turgid leaves were kept with the cut end in water during this time. Radioactive compounds were added in 5-,ul amounts to the cut end of the leaf.After incubation, leaves were frozen in liquid N2 then crushed in 1 ml of ice-cold water and 0. I-ml aliquots of the water were taken for 3H20 recovery (12). The leaves were ground in methanolchloroform so that the final methanol-chloroform-water was 12:5:3 (v/v/v) as prescribed by Bieleski and Turner (2). After twodimensional TLC of soluble compounds or acid hydrolysates of protein, radioactivity was measured by liquid scintillation with a Beckman LS-100 counter. Thin layer spots were placed in vials, 0.5 ml of 80% (v/v) ethanol was added followed by 5 ml of cocktail containing 7 g/l Butyl PBD,2 0.5 g/l PBBO, and 100 ml/l BBS-3 in toluene. Gain was adjusted to give the same external standard ratio that was obtained when there was <1% spillover of 3H counts into "C window in unquenched samples (i.e. [3Hjtoluene in the cocktail). Proline accumulation may represent a general response to stress since proline accumulates under salt and cold stresses (9, 10) as well as water stress (14,16). Evidence has been presented previously that water stress inhibits proline oxidation (3, 17). This effect of stress contributes to proline accumulation under stress conditions but cannot account for all of it (17). Proline oxidation rates have been determined by measured tritium in H20 after providing plants or plant parts with 13). The technique can be nondestructive and is convenient for assessing the effects of stress on proline oxidation, but its validity depends on the presumptions that tritium from proline is incorporated only into cellular water and that this water equilibrates readily with the external medium. Before applying this technique to study the effects of stress on proline metabolism, we wanted to determine the fate of tritium from [5-3HJproline and to assess the validity of proline oxidation rates measured by the 3H loss technique by comparing those rates with the rates obtained from oxidation of ['4Clproline in the same leaves. These were the objectives of the experiments reported in this paper. MATERIALS AND METHODSBarley (Hordeum vulgare cv. Prior) seedlings were grown in soil in the greenhouse for about 2 weeks until the second leaf was fully expanded. Each measurement of radioactivity was an average of three replicate leaves weighing 166 + 20 mg. Radioactive compounds were fed and samples analyzed as previously described 'Supported by the Graduate College and the Sciences and Humanities

“…During water stress in some plants, proline accumulates because of increased proline synthesis and an inhibition of proline oxidation (13). Since proline must pass through the mitochondrial inner membrane into the matrix for oxidation to occur, it is possible that proline oxidation in vivo may be regulated at either the transport, dehydrogenase, or electron transport chain level.…”

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confidence: 99%

Energetics of Proline Transport in Corn Mitochondria

Elthon¹,

Stewart²,

Bonner³

1984

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The mechanism of proline entry into the matrix region of isolated corn mitochondria (Zea may: L. Mol7 x B73) was investigated by measuring osmotically induced changes of mitochondrial size (changes in AS2.) in combination with oxygen uptake measurements. Using NADH oxidation to generate the electrochemical gradient, we have determined that proline transport is stereospecific and that it can be inhibited by the proline analog L-thiazolidine-4-carboxylic acid. Proline transport has been investigated in deenergized mung bean mitochondria (1) and was found to be partially stereospecific and reversibly inhibited by sulfhydryl reagents. These are properties consistent with a protein-mediated mechanism of transport. Proline transport has also been investigated in mitochondria isolated from spinach leaves (15).In this paper, we have followed oxidation and transport at substrate concentrations normally used to determine ADP:O ratios. By following these processes in energized mitochondria, we have been able to show that proline enters the mitochondrial matrix in a stereospecific and partially energy-dependent manner. In addition, we provide evidence that proline transport is dependent upon the pH potential across the membrane and not upon the electrical potential. MATERIALS AND METHODSCorn seedlings (Zea mays L. Mo 17 x B73) were grown in the dark at 30 ± 2°C in moist vermiculite. Mitochondria were isolated from 3-to 4-d-old seedlings as before (6), except that often Tes was used rather than KH2PO4 and EDTA rather than EGTA2 in the grinding medium. In addition, a Moulinex blender was sometimes used rather than a mortar and pestle to disrupt the tissue. Protein was estimated by the method of Lowry et al. ( 11) using BSA (fraction V) as the standard. FCCP (2.0 mM) and valinomycin (0.9 mM) were solubilized in 100% ethanol. Residual valinomycin was removed from the oxygen electrode, cuvette, and stiffer by rinsing with 100% ethanol.