A novel method of measuring volume changes of mesophyll cell protoplasts and the effect of mercuric chloride on their osmotically-induced swelling

Abstract

A quick and accurate method of monitoring changes of volume of mesophyll cell protoplasts (MCP) of pea was devised using the difference in absorbance of the protoplasts at 440 nm and 750 nm (OD 440-750); when protoplasts expanded in response to changing the external medium from 0.4 M sorbitol to 0.3 M sorbitol OD 440-750 values increased and, conversely, when protoplasts were transferred from 0.4 to 0.5 M sorbitol, protoplasts contracted. The kinetics of expansion or contraction of the protoplasts could also be monitored using this method and the half-time for water exchange for expanding protoplasts (about 10 s) was slightly higher than that for contracting protoplasts. A study of the effects of the water channel blocker, mercuric chloride, on swelling protoplasts showed that 500 μM irreversibly damaged protoplasts, 5-10 μM had a negligible inhibitory effect on swelling while 100 μM had a large inhibitory effect often completely inhibiting swelling. A preliminary study indicated that mercapto-ethanol reversed the inhibitory effect of mercuric chloride on protoplast swelling.