Amino Acid and Protein Metabolism in Bermuda Grass During Water Stress

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: 60%

Oxidation of Proline by Plant Mitochondria

Boggess¹,

Koeppe²,

Stewart³

1978

“…While glutamate is clearly involved in proline synthesis during water stress (1,3,6), the contribution of arginine has not been evaluated. Although leaf tissues typically contain little free arginine, the inexplicably low protein-arginine content of wilted Bermuda grass leaves (1) suggests that in at least some cases, arginine might contribute significantly to the build up of free proline that occurs during water stress. Experiments reported here were designed to clarify the involvement of arginine metabolism in the accumulation of proline by water-stressed barley leaves.…”

mentioning

confidence: 99%

“…Glutamic acid and arginine are likely precursors for water stress-induced proline synthesis, as either may be converted to P5C (Al-pyrroline-5-carboxylic acid) which is the immediate precursor of proline. While glutamate is clearly involved in proline synthesis during water stress (1,3,6), the contribution of arginine has not been evaluated. Although leaf tissues typically contain little free arginine, the inexplicably low protein-arginine content of wilted Bermuda grass leaves (1) suggests that in at least some cases, arginine might contribute significantly to the build up of free proline that occurs during water stress.…”

mentioning

confidence: 99%

Contribution of Arginine to Proline Accumulation in Water-stressed Barley Leaves

Boggess

Stewart

1976

Barley (Hordeum vulgare cv Prior) leaves converted L-U_'4C-arginine to labeled proline. Accumulation of radioactivity in proline was greater in wilted leaves, but only after 9 hours of incubation. As the increase in free proline was detectable after only 3 to 6 hours, it is likely that the observed stimulation of proline labeling represents a result rather than a cause of proline accumulation. Furthermore, the loss of total arginine during water stress was only 10 to 15% of the increase in proline. We condude that arginine probably contributes less than 1% of the carbon in the expanding proline pool of wilted barley leaves.Wilted leaves of certain plants accumulate quantities of free proline considerably greater than could be released through net protein breakdown (5). Most of the accumulated proline must, therefore, be synthesized from other compounds during the period of water stress. Glutamic acid and arginine are likely precursors for water stress-induced proline synthesis, as either may be converted to P5C (Al-pyrroline-5-carboxylic acid) which is the immediate precursor of proline. While glutamate is clearly involved in proline synthesis during water stress (1,3,6), the contribution of arginine has not been evaluated. Although leaf tissues typically contain little free arginine, the inexplicably low protein-arginine content of wilted Bermuda grass leaves (1) suggests that in at least some cases, arginine might contribute significantly to the build up of free proline that occurs during water stress. Experiments reported here were designed to clarify the involvement of arginine metabolism in the accumulation of proline by water-stressed barley leaves. MATERIALS AND METHODSExperimental procedures were as previously described (3). Briefly, second leaves of 2-week-old barley plants (Hordeum vulgare cv Prior) were excised and allowed to take up radioactive precursor (L-U-_4C-arginine, 50 mCi/mol, obtained from Amersham/Searle and purified by elution from Dowex 50 in the ammonium form) in 4 ,1l aqueous solution. Stressed leaves were previously excised and wilted to 75 % of initial fresh weight, and not rewatered after uptake of radioactive material; turgid leaves were kept in vials of distilled H20 for the rest of the experiment.

“…It is well established that proline accumulates rapidly in excised and intact leaves under water stress (3,7,11,12,17). However, relatively little attention has been paid to proline accumulation in senescing excised leaves.…”

Section: Introductionmentioning

confidence: 99%

“…It seems that accumulation of proline may, at least in part, result from an increased rate of synthesis, possibly due to a disruption of the normal feedback inhibition of proline synthesis. Potassium cyanide and 2,4-dinitrophenol strongly inhibited proUne accumulation, indicating that some energy compound(s) may participate in proline accumulation during senescence of excised rice leaves.It is well established that proline accumulates rapidly in excised and intact leaves under water stress (3,7,11,12,17). However, relatively little attention has been paid to proline accumulation in senescing excised leaves.…”

mentioning

confidence: 99%

Senescence of Rice Leaves

Wang¹,

Cheng²,

Kao³

1982

Proline content increased greatly in detached rice (Oryza sativa cv.Taichung Native 1) leaves during senescence. There was a slight but significant increase in proline level after one day of incubation, and, subsequently, proline accumulated relatively rapidly. By 4 days after excision, the level of proUne had increased 30-to 50-fold, which is similar to the level seen in the water-stressed detached rice leaves. It is unlikely that the proline accumulation in detached leaves is to be derived solely from protein hydrolysis, since the addition of L-glutamic acid increased the proline level during senescence. The proline analog, 3,4-dehydroproline, did not affect the level of proline during senescence. It seems that accumulation of proline may, at least in part, result from an increased rate of synthesis, possibly due to a disruption of the normal feedback inhibition of proline synthesis. Potassium cyanide and 2,4-dinitrophenol strongly inhibited proUne accumulation, indicating that some energy compound(s) may participate in proline accumulation during senescence of excised rice leaves.It is well established that proline accumulates rapidly in excised and intact leaves under water stress (3,7,11,12,17). However, relatively little attention has been paid to proline accumulation in senescing excised leaves. ABA is known to increase rapidly in excised leaves under water stress (2, 7). Proline accumulation can be induced by ABA in both excised leaves and intact plants (1,13). It has been found that ABA accumulation precedes a detectable change in proline level (2). Therefore, proline accumulation in response to water stress may be a response to accumulated ABA. Inasmuch as ABA has also been reported to increase during senescence (5, 6), proline may also be accumulated during senescence of excised leaves. The present communication describes the accumulation of proline during senescence of excised rice leaves. MATERIALS AND METHODSRice (Oryza sativa cv. Taichung Native 1) seedlings were cultured as previously described (8). The apical 3 cm of the third leaves of 9-d-old seedlings was used for experiments. A group of 10 segments was floated on 10 ml of test solution in a 50-ml flask.Incubation was carried out in darkness at 27°C for the indicated period. Each treatment contained four replications.Chlorophyll and a-amino nitrogen were extracted and determined as described before (8). Chlorophyll and a-amino nitrogen were expressed as A665 and A570 per ten segments, respectively. ' Supported by research grant to C. H. Kao from the National Science Council of the Republic of China.2 To whom reprint requests should be addressed.Proline was extracted and its concentration determined following the method of Bates et al. (4). Leaf segments were homogenized with 3% sulfosalicylic acid and centrifuged. The supernatant was treated with acetic acid and acid-ninhydrin, boiled for 1 h and extracted with toluene. Then, its A at 520 nm was read. Proline content was expressed as,umol/10 segments. RESULTS AND DISCUSSIONThe senesc...