Regulation of H⁺ Excretion

Abstract

Osmotic shock, a 15-minute plasmolysis followed by a 15-minute rehydration in the cold, is a nondestructive technique which inhibits fusicoccinstimulated H+ excretion from oat mesophyll cells (Avena sativa L.). Osmotic shock also causes a loss of intracellular solutes and stimulates H+ uptake, but osmoregulation can still occur, and enhanced H+ uptake is observed only at low external pH. It Cl-CCP, carbonylcyanide, m-chlorophenyl-hydrazone.(Econowatt, Westinghouse, Corp.) and incandescent light at 145 M-2 One-quarter strength Hoagland solution was applied at time of sowing and on a daily basis after 4 d. The primary leaves were harvested 7 d after sowing, the lower (abaxial) epidermis was peeled away, and one or two 1-cm segments were cut approximately 1 cm below the tip.Segments to be osmotically shocked were first plasmolyzed on 0.6 M sorbitol, 29 mm sucrose, 5 mm Hepes (pH 7.0) for 15 min. This solution was then aspirated, the tissue moved to an ice bucket, and cold 5 mm Hepes (pH 7.0) was added. After rehydration for 15 min, the sections were transferred to an appropriate medium for estimates of H+ flux. Unshocked sections were treated identically except that the sorbitol plus sucrose was omitted from the Hepes buffer during the first 15-min treatment. Sucrose alone is without effect, but was included for future comparisons with protoplasts which are suspended in media containing sucrose.The presumptive net efflux of H+ was measured by floating five 1-cm peeled leaf segments on 2.5 ml of 1 mm Hepes (pH 6.5) plus 1 mm KCI in 20-ml beakers. To determine the alkalinization of the medium, segments were floated on Hepes-KCl adjusted to pH 4.8 with HCI. For both types of measurements, the beakers were shaken continuously at ambient light and temperature, and the pH of the medium was determined at intervals with a Model 26 Radiometer pH meter equipped with a Radiometer combination electrode. Data were converted to H+ equivalents with the aid of a titration curve for the solution used. All treatments were run in duplicate and experiments repeated on at least 2 separate d. Tissue osmolarity was determined by the methods of Van Volkenburgh and Cleland (personal communication). Ten peeled leaf segments were wrapped in aluminum foil after treatment and frozen on dry ice for I to 2 h. After thawing for 2 to 5 min, 5 to 10 pl of cytoplasm was expressed and placed on a filter paper disc in a Wescor Model 5100B vapor pressure osmometer. Osmolarities of incubation media were determined by freezing point depression with an Advanced Model 3L osmometer.For scanning electron microscopy, peeled or unpeeled leaf segments were fixed overnight in osmium vapor and then dehydrated in acetone and critical point exchanged. The tissue was mounted on copper pegs, coated with gold/palladium in a sputter coater, and examined with a JEOL Model JS35 SEM at an accelerating voltage of 25 kev. RESULTSPreliminary experiments indicated that leaf tissue must be peeled in order to measure H' flux or to perturb the membrane by osmotic shoc...

Osmotic Stress-Induced Polyamine Accumulation in Cereal Leaves

“…The lack of PEG effect is not due to obvious toxicity, since proline increases significantly, and leafviability as measured by the Evans Blue exclusion test (25) is not affected. PEG apparently does not penetrate into the cell wall space (17) while sorbitol diffuses freely, and interactions at the cell wall/membrane interface may be critical for the polyamine response. Overall, our results suggest that the increase in proline and putrescine during stress are mediated by different mechanisms.…”

Turgor Regulation in Osmotically Stressed Arabidopsis Epidermal Root Cells. Direct Support for the Role of Inorganic Ion Uptake as Revealed by Concurrent Flux and Cell Turgor Measurements

“…3) and literature data (Reid et al, 1984;Kinraide and Wyse, 1986;Reuveni et al, 1987;Kitamura et al, 1997;Teodoro et al, 1998;Zingarelli et al, 1999) suggest that osmotic stress causes rapid, significant, and prolonged hyperpolarization of plasma MP. Because the major source for generating the MP in higher plant cells is the activity of the electrogenic ATP-dependent H ϩ pump (Spanswick, 1981), it is not surprising that such a pump has long been considered as a potential target of osmotic stress (Rubinstein, 1982;Reinhold et al, 1984;Reuveni et al, 1987). Supporting evidence includes reports of significant osmotic-induced acidification of the bathing medium (Kinraide and Wyse, 1986;Reuveni et al, 1987;Zingarelli et al, 1999) and direct measurements of net H ϩ extrusion (Lew, 1998;Shabala et al, 2000).…”

H+ Fluxes in Excised Samanea Motor Tissue

“…H+ fluxes in some tissues are regulated in part by external pH (15,19). We therefore compared darkpromoted changes in the pH of the bathing solution when the initial pH was 5.5 to those obtained when the initial pH was 6.7 (Table II); changes in pH were then converted to H+ fluxes.…”

“…H+ fluxes through membranes of other plant cells have been monitored by measuring changes in the pH of a solution bathing excised groups of homogeneous cells (1,3,5,8,14,19). Inasmuch as the pulvinus is divided into two functionally different halves 'Supported by a grant from the National Science Foundation to R. L.…”

“…Previous investigators (9,21) have postulated that K+ fluxes into and out of pulvinar motor cells might also be coupled to oppositely directed H+ fluxes. H+ fluxes through membranes of other plant cells have been monitored by measuring changes in the pH of a solution bathing excised groups of homogeneous cells (1, 3,5,8,14,19). Inasmuch as the pulvinus is divided into two functionally different halves 'Supported by a grant from the National Science Foundation to R. L.…”