Proteins in Intercellular Washing Fluids from Leaves of Barley (Hordeum vulgareL.)

Abstract

Primary leaves of barley were detached, infiltrated with various buffers, and centrifuged to yield ‘intercellular washing fluid’ (IWF). Effective pH control of the IWF was obtained only with Tris, among all buffers tried. In these liquids, up to 30 proteins were detected by gradient gel electrophoresis. Intracellular protein from injured cells at the cut ends of leaves was present in IWF but did not contribute significantly to the total protein recovered in this liquid. The yield of protein in the IWF depended on the buffer used for infiltration and on the concentration of the buffer. Higher concentrations of buffer yielded more protein. In other experiments leaves were infiltrated with Tris, centrifuged, and then infiltrated a second time with this buffer containing various concentrations of the zwitterionic detergent CHAPS, a sulphobetaine derivative of cholate. Gel electrophoresis of the IWF obtained after the second centrifugation revealed protein ‘bands’ not detected when the detergent had been omitted from the infiltration buffer. The electrophoretic patterns of protein ‘bands’ in the gels differed depending on the CHAPS concentration used for infiltration. The effect of CHAPS on plasmalemma integrity was studied by observing infiltrated tissue with the electron microscope and by treating isolated protoplasts with the detergent. After infiltration with CHAPS at 0.6 mM or 2.0 mM no plasmalemma breaks were detected in leaves, and isolated protoplasts survived exposure to CHAPS at these concentrations for 2 h without bursting. Evidently, CHAPS at these low concentrations did not destroy the integrity of the plasmalemma; the additional protein recovered in the IWF under these conditions probably originated in the cell wall. Infiltration of leaves with 6.0 mM CHAPS resulted in breaks of the plasmalemma, in tissue collapse and leaf tip necrosis. Isolated protoplasts burst within minutes after being exposed to CHAPS at this concentration.