The conversion of proline to glutamic acid and hence to other soluble compounds (prolne oxidation) proceeds readily in turgid barley (Hordeum vulgare) leaves and is stimulated by higher concentrations of proline. This suggests that proline oxidation could function as a control mechanism for maintain*ag low ceilular levels of proline in turgid tissue.In water-stressed tissue, however, proline oxidation is reduced to negligible rates. These results are consistent with the idea that proline accumulation results from inactivation by water stress of normal control mechanisms. It seems likely that inhibition of proline oxidation is necessary in maintaining the high levels of proline found in stressed barley leaves.Radiotracer experiments have implied that the synthesis of proline from glutamic acid is stimulated by water stress (1, 10). We have confirmed and strengthened these earlier results, and presented evidence that the stimulatory effect of stress is likely to be on P5C2 formation (6), consistent with the implication of other experiments that loss of feedback control of proline biosynthesis occurs in barley leaves during water stress (4). These studies also produced evidence that proline oxidation may occur rapidly in barley leaves (4) and that water stress may reduce its rate (3,6). In this paper, we present more detailed information concerning proline oxidation and its response to proline concentration and water stress. MATERIALS AND METHODSGrowth of barley (Hordeum vulgare cv. Prior) plants and methods of wilting, feeding of radioactive precursors, and sample analysis have been described (6). Two-week-old plants were wilted intact by flooding the rooting medium with polyethylene glycol-4000 (30 g/100 ml) 1 hr before beginning the experiment; alternatively, second leaves were excised and wilted to 75% of initial fresh weight in a lighted fume hood. The latter wilting procedure (which also took about 1 hr) was preferred as it gave more reproducible rates of water loss. [14C]proline, radioactivity was recovered about 60% as soluble products and 40% as protein. After acid hydrolysis, the proteinbound radioactivity was found to be 85 to 90% proline, with the rest as glutamic and aspartic acids. In the soluble fraction, measurable radioactivity was recovered in glutamate, glutamine, aspartate, y-amino-butyrate, alanine, and several ninhydrinnegative spots. Traces of 14C were detected in glycine, serine, asparagine, citrulline, and arginine. Although not measured in this experiment, 14CO2 has constituted less than 1.5% of the total radioactivity under comparable conditions. Table I (column 1) shows the distribution of radioactivity in these metabolites as a percentage of the total soluble radioactivity. Glutamate was always the most heavily labeled proline metabolite, and in short experiments (10-15 min), was the only labeled compound detected autoradiographically. This suggests that metabolism of proline consists of proline oxidation (conversion to glutamate) followed by further metabolism of glutamate. In ...

Barley (ffordeum vulgare L. var. Prior) leaves converted more '4C-glutamic acid to free proline when water-stressed than when turgid; neither decreased protein synthesis nor isotope trapping by the enlarged free proline pools found in wilted tissue seemed to account for the result. This apparent stimulation of proline biosynthesis in wilted leaves was not observed when radioactive ornithine or P5C (A1-pyrroline-5-carboxylate, an intermediate following glutamate in proline synthesis) were used as proline precursors unless proline levels were high as a result of previous water stress. We interpret this to mean that any stimulation of proline synthesis by water stress must act on P5C formation rather than its reduction to proline. Experiments showing greater apparent conversion of 14C-glutamate to proline do not unequivocally prove that proline synthesis is stimulated by water stress, as P5C feeding studies show that proline oxidation is inhibited under comparable conditions. This inhibition could account, at least in part, for increased proline labeling, and must be considered an alternate possibility.Although several plants have been reported to accumulate free proline during periods of water deficit (2,12,15,16,18) or when subjected to other stress (7, 8), the biochemical changes linking water stress and proline accumulation are not well understood. Barnett and Naylor (2) and Morris et al. (13) have observed increased incorporation of 14C-glutamic acid into proline in wilted leaves as compared to turgid leaves of Bermuda grass and turnip. This increase in radiotracer incorporation might reflect a stimulation of proline biosynthesis by water stress, but the data presented did not fully eliminate the possibility that inhibited protein synthesis might account for the results.The Bermuda grass experiment was done after a long period of drought, so that the large amount of free proline already present during "4C-glutamate feeding could have acted as a trapping pool for radioactive proline, accounting for the results in the absence of an actual increase in the rate of conversion of glutamate to proline. In this paper we describe 14C-glutamate feeding experiments designed to avoid these difficulties in interpretation, and to localize the point in the proline biosynthetic pathway at which a stress-induced stimulation would most likely occur.

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...

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

The characteristics of the enzyme A'-pyrroline-5-carboxylic acid dehydrogenase from etiolated barley (Hordeum distichum) shoots have been examined. The bulk of the enzyme activity was found in the l0,OOOg pellet fraction, this activity being displayed only after detergent treatment of the suspended pellet. The enzyme was most active at pH 8, and activity was NAD-dependent. Enzyme activity was unaffected by either mannitol or sucrose in the reaction mixture up to a concentration of 0.45 M but was strongly inhibited by Cl-and, to a lesser extent, S02-. The inhibition attributable to KCI was reversed by increasing the concentration of A1-pyrroline-5-carboxylic acid in the reaction mixture.The enzyme A'-pyrroline-5-carboxylic acid dehydrogenase catalyzes the second step of proline oxidation, the conversion of PSC3 to glutamic acid. Crude preparations of the enzyme have been reported from mammalian liver (18,19), bacteria (5, 6), and yeast (10,11). Little is known of the enzyme from higher plants, the report of Stewart and Lai (15) being the first on this enzyme in such tissue. These authors, however, used tissues that do not accumulate proline when water stressed, and properties pertinent to stress conditions were not determined. The possibility that proline accumulation might arise from an inhibition of proline oxidation (1)