Acclimation to Low Temperature by Microsomal Membranes from Tomato Cell Cultures

Abstract

ABSTRACISealed vesicles were prepared from microsomal membranes from cell suspension cultures of tomato (Lycopersicon escidentum Mill cv VF36). ATP-dependent proton transport activity by the vesicles was measured as quenching of fluorescence of acridine orange. Measurements of proton transport were correlated with the activity of a nitrate-inhibitable ATPase. The initial rate of proton influx into the vesicles was strogly temperature dependent with a Q,, of 2 and a maximum rate near 35C. The data suggest that passive permeability did not increase at chilling temperatures but did increase rapidly with temperatures above 30°C. A comparison was made between membranes from cell cultures grown at 28C and 9C. The temperature optimum for proton transport broadened and shifted to a lower temperature range in membranes from cells maintained at 9C.determine the upper and lower limits for optimal functioning of the membrane.Suspension cultures of tomato cells have been used as model systems to study the effects of water stress (12) MATERIAIS AND METHODSCell Cultures. Callus of L. esculentum Mill cv VF36 was initiated from hypocotyls of aseptic seedlings. Suspension cultures were initiated from hypocotyl callus and were maintained as described previously (8). The suspension cultures were initiated 1 year prior to the experiments shown, and were maintained on a medium containing ammonium as the sole nitrogen source. The cultures were maintained by weekly subculture of 5 ml of cells (early stationary phase) into 175 ml of fresh medium in 500 ml Erlenmeyer flasks, and kept at 28°C on a reciprocal shaker at 60 cycles/min. ChilL:ng Treatment. Flasks, inoculated as described above, were maintained at 28°C for 4 d, then transferred to a temperature-controlled orbital shaker (New Brunswick Scientific, Edison, NJ) and maintained in a cold room at 9C and 180 rpm.Membrane Preparations. Cells were collected by vacuum filtration onto Whatman No. 4 filter paper on a Buchner funnel, and washed with an equal volume of distilled H20. Cells grown at 28°C were harvested and washed at room temperature; cells grown at 9°C were harvested and washed in a cold room at 4C.All other operations were carried out at 4C. Cells (10-20 g) were immediately weighed, placed in 250 ml ofhomogenization buffer with 0.5 mm diameter glass beads in a Bead Beater Cell Homogenizer (Biospec Products, Bartlesville, OK). The homogenate was filtered through two layers of Miracloth (Calbiochem), centrifuged at 3,000g for 5 min, the pellet discarded, centrifuged at l0,OOOg for 20 min to obtain the mitochondrial pellet, and at l00,OOOg for 35 min to obtain the microsomal pellet. The microsomal and mitochondrial pellets were resuspended in 20 to 40 ml of suspension buffer consisting of 0.25 M sucrose and either 5 mM Tris-HCI (pH 7.5) or 5 mM Pipes-Tris (pH 7.0) as indicated, placed in one or two centrifuge tubes of38 ml capacity, underlaid with 10 ml of 10% Dextran (70 kD) in suspension buffer, and centrifuged at 80,000g for 1 h. The membranes from the dextran interface we...

Effects of Temperature on the Coupled Activities of the Vanadate-Sensitive Proton Pump from Maize Root Microsomes

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Chilling-Induced Inactivation and Its Recovery of Tonoplast H+-ATPase in Mung Bean Cell Suspension Cultures

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Effect of Temperature on the Plasma Membrane and Tonoplast ATPases of Barley Roots

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