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

Abstract

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

Removal of Feedback Inhibition of Δ1-Pyrroline-5-Carboxylate Synthetase Results in Increased Proline Accumulation and Protection of Plants from Osmotic Stress

“…Pro catabolism is catalyzed by Pro dehydrogenase and P5C dehydrogenase (Hu et al, 1996;Peng et al, 1996). Suppression of Pro degradation has been demonstrated both in radiolabeling studies (Stewart and Boggess, 1978) and gene expression experiments (Kiyosue et al, 1996;Peng et al, 1996;Verbruggen et al, 1996). Recent studies have demonstrated that high Pro concentrations are present in the phloem sap of drought-stressed alfalfa (Girousse et al, 1996) and that the expression of a Pro-specific amino acid transporter is induced in response to water deficit and salt stress (Rentsch et al, 1996).…”

The Effects of Benzyladenine, Cycloheximide, and Cordycepin on Wilting-Induced Abscisic Acid and Proline Accumulations and Abscisic Acid- and Salt-Induced Proline Accumulation in Barley Leaves

“…Proline (12) determination methods and salt (4) and wilting (12) treatments have also been described. Leaves were fed by placing their cut ends in a vial containing various compounds and kept in room light (about 11 jsmol m-2 s-' [4]).…”

Accumulation and consumption of solutes in swards of Lolium perenne during drought and after rewatering

“…In an earlier experiment on these populations (Thomas, 1990), the greater accumulation of proline in leaf blades (an average of 101 /imol g^^,, compared with 31 m easured here) was probably a consequence of the artificial drought technique, which involved adding frequent small volumes of water and resulted in a series of short droughts. If, as was inferred from the difficulty of measuring i^^., drought damaged the leaf membranes in treatment D8 (Thomas, 1986), then proline may have accumulated because compartmentation between production (in the cytoplasm or cbloroplasts) and oxidation (in the mitochondria) was disrupted, as proposed by Stewart & Bogges (1978).…”