UV-Stimulated K⁺ Efflux from Rose Cells

Abstract

Irradiation of a washed suspension of cultured rose (Rosa damascena var. Gloire de Guilan) cells with about 1,680 joules per square meter of short wave ultraviolet (UV) light (254 nanometers) caused K+ to appear in the external medium. Short-term tracer (86Rb+) experiments confirmed the earlier suggestion (Wright, Murphy 1978 Plant Physiol 61: 434-436) that UV increases the efflux of K+; there was also a small decrease in influx of K+. There was a partial recovery of fluxes from the effects of UV radiation, but no net accumulation of K+ within 16 to 18 hours after the irradiation. The K+ appearing in the medium was matched by an equivalent amount of HC03-; it was suggested that HC03-was the principal counterion for the K+ flux induced by UV. The suspension was fine: few or no aggregates were larger than 0.5 mm in diameter. At this stage, packed cell volume was about 0.2 ml/ml of suspension, but the wet weight and displacement volumes of filtered cells indicated that the true protoplast volume was less than 0.06 ml/ml of suspension. Before each experiment, cells were filtered three times and resuspended in a volume of 0.01 M CaCl2 equal to the original volume of suspension. After the final wash, 10-ml volumes (3 x 107 cells in 10 mm CaCl2) were distributed into 100-ml beakers.Ion Concentrations. The concentration of K+ in the cell suspension was measured by placing a K+-sensitive glass electrode (Markson) and a separate calomel reference electrode directly in the suspension. The suspension was gently swirled and then allowed to settle. A stable reading was observed about 10 to 20 s after the swirling. The electrode combination and meter were calibrated each day with 10-4, 10-3, and 10-2 M KCI. The concentration of Cl-in the cell suspension was measured in a similar fashion using a solid-state Cl--sensitive electrode (Orion). Ca2+, NO3 , P043 , orffanic acids, and HCO3 were measured in cellfree filtrates. Ca + was measured by atomic absorption spectrometry (Perkin-Elmer). N03-was measured by HPLC (18). P043-was measured by the method of Fiske-SubbaRow (6). Organic acids were separated by HPLC (Bio-Rad HPX-87 ion exclusion column with 35 mm H2SO4 liquid phase) and detected by OD at 210 nm; malic, citric, succinic, and oxalic acids were used as standards. Titrations of acids and their conjugate bases were performed in filtrates that had been quickly overlaid with mineral oil to prevent exchange of CO2 with the atmosphere. Estimates of HCO3 amounts were calculated from titration curves by assuming that 94% of the anion became associated with H+ between pH 7.9 and pH 4.9.Influx and Efflux. Influx of 'Rb+ was measured by adding 0.5 ,uCi of [86Rb]Cl (Amersham-Searle, >86 mCi/mmol) to 10 ml of washed cells immediately after measuring K+ of the suspension. One-ml samples taken 1, 3, 6, and 10 min later were each filtered, washed with 30 ml of solution containing 20 mm KCI and 10 mM CaCl2, and dried. The washing took approximately 1 min. Dried filters were counted in 4 ml of dioxane-based scintillation cocktail (...

Ultraviolet-Stimulated KHCO3 Efflux from Rose Cells

“…UV radiation has been used as a tool to probe the regulation and integration of ion transport across the membranes of cells of several different organisms (2,7,13,14,19,21 …”

“…This change was not as great as was predicted from the observed HC03-loss. Analysis of nitrogenous compounds in the cell suggested that reduction of nitrate and synthesis of y-aminobutyric acid absorbed some of the protons formed by the synthesis and dissociation of bicarbonate.UV radiation has been used as a tool to probe the regulation and integration of ion transport across the membranes of cells of several different organisms (2,7,13,14,19,21 …”

“…When suspension-cultured rose cells are exposed to shortwave UV (254 nm), they respond within minutes with a rapid efflux of K+ ion ( 13). The irradiated cells retain viability, as measured by fluorescein diacetate, for 1 d, and they maintain their normal rate of respiration, showing that the loss of K+ does not result from the death of the cells.…”

“…The irradiated cells do not leak substantial amounts of nitrate, phosphate, chloride, or calcium ion, further indicating that the K+ efflux does not represent a general disintegration of the plasma membrane. Murphy and Wilson (13) reported that the appearance of K+ in the external medium was matched in part by an appearance of HCO3Q. The authors suggested that HCO3 , produced by hydration ofrespiratory CC2, served as a counterion to K+ in the efflux process.…”

“…Suspension cultured cells of Rosa damascena var Gloire de Guilan were grown as previously described (13). For experiments, we filtered 5-d cultures (50 ml) three times, resuspending the cells each time in 50 ml of solution containing 1 mM CaC12 and 0.1 mM KC1.…”