Vacuolar Deposition of Ascorbate-derived Oxalic Acid in Barley

Abstract

L-11-"4ClAscorbic acid was supplied to detached barley seedings to determine the subcellular location of oxalic acid, one of its metabolic products. Intact vacuoles isolated from protoplasts of labeled leaves contained I'4Cloxalic acid which accounted for about 70% of the intraprotoplast soluble oxalic acid. Tracer-labeled oxalate accounted for 36 and 72%of the "C associated with leaf vacuoles of seedlings labeled for 22 and 96 hours, respectively.OA2 is a widely occurring natural product of plants, yet little is known about its metabolism and function (3,10,15). The amount of OA formed and accumulated in different plant species can vary greatly (3,9,10,14). Moderate to high accumulators, such as spinach and rhubarb, may contain 0.2 to 1% OA (on a fresh weight basis), whereas low accumulators, such as barley and tomato, may contain one-tenth this amount. In the very high accumulator Halogeton glomeratus, Ca-OA can account for 30Yo ofthe dry weight ofthe plant (10). Glycolic, glyoxylic, and isocitric acids (8) and oxaloacetic (1) and ascorbic (7, 13) acids are known to donate carbon to OA in plants. The relative significance of these metabolites as precursors of OA has not been established.Ca-OA, in various crystalline forms, often is observed in plant vacuoles (2). It also has been reported to occur in cytoplasm, in cell walls, and in extracellular spaces. However, ultrastructural observations suggest that extracellular Ca-OA originates in vacuoles (see ref. 4 and references therein). Soluble OA salts are thought to be contained in vacuoles. In the study presented here, vacuolar deposition of OA formed from AA was tested in barley, a low level OA accumulator in which AA to OA conversion has been reported (9). MATERIALS AND METHODSSix 13-day-old seedlings of Hordeum vulgare cv. Moore were cut at soil level and recut under water in preparation for labeling. 70o ethanol. To prepare protoplasts, the leaves were sliced into 1-mm strips and the strips were incubated in a sterile solution containing 1% Cellulysin, 0.5% Macerase (both from Calbiochem), and 0.7 M mannitol. Leaves from 24 plants which had not been labeled were similarly treated to provide carrier protoplasts and vacuoles. Tissues were about 75% digested after incubation at 25 C and in room light for 4 h. After removal of undigested materials, labeled-tissue and carrier-tissue digests were combined and protoplasts were recovered by sedimentation at 500g. Protoplasts (0.4 ml) were washed three times with 40 ml 20 mm Mes-KOH (pH 5.5). One-fifth was removed to be used as a protoplast sample before protoplasts were sedimented from the third wash. The remaining four-fifths was used to prepare vacuoles, essentially as previously described for the preparation oftulip leafvacuoles (12). Vacuoles were released in 0.17 M KH2PO4/K2HPO4 (pH 8.0); Kphosphate), and most residual protoplasts and much of the resulting particulate material were sedimented at 500g. The supernatant was made 17% (w/v) with sucrose by addition of 60%o (w/v) sucrose and this solutio...

Relationships between Cadmium, Zinc, Cd-Peptide, and Organic Acid in Tobacco Suspension Cells

“…The remainder was centrifuged and the pellet gently suspended in lysis medium containing 0.08 M K2HPO4/KH2PO4 (pH 8), 10 mM DTT (28). The suspension was gently stirred at room temperature for 10 min to liberate vacuoles and then filtered through two layers of cheesecloth wet with lysis medium.…”

“…3) observed and noted earlier was not labeled. When [1-'4C] ascorbate (precursor of oxalate in plants) (28) …”

“…To monitor organic acids, cold Ca-acetate was added to 100,000g supernatants of 0.2% oxalic acid homogenates, the suspensions were centrifuged at 16,000g for 5 min at 4°C to sediment Ca-oxalate, and the pellets were washed with cold water and resedimented (28 Tris (pH 5.5) and one-fifth was centrifuged and the pellet was frozen and set aside for monitoring protoplasts. The remainder was centrifuged and the pellet gently suspended in lysis medium containing 0.08 M K2HPO4/KH2PO4 (pH 8), 10 mM DTT (28).…”

The Roles of Germin Gene Products in Plants Under Salt Stress

“…Moreover, glycolic and isocitric acids (Millerd et al, 1963), oxaloacetic acid (Chang & Beevers, 1968) are known to donate carbon to oxalic acid in plants. The relative significance of these metabolites as precursors of oxalic acid has not been established (Wagner, 1981). The enzymes involved in the synthesis and degradation of oxalic acid are of interest.…”

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