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

Abstract

it is not always easy to prepare healthy, viable ones, and also it is not easy to measure the kinetics of their changing A quick and accurate method of monitoring changes dimensions in response to an osmotic gradient. Recent of volume of mesophyll cell protoplasts (MCP) of pea studies used vesicles prepared from root cells of wheat was devised using the difference in absorbance of the (Niemietz and Tyerman, 1997) or from suspension-cultured protoplasts at 440 nm and 750 nm (OD 440‐750); when tobacco cells (Maurel et al., 1997) and indirectly measured protoplasts expanded in response to changing the changes in volume by monitoring light scattering of a external medium from 0.4 M sorbitol to 0.3 M sorbitol suspension of vesicles with stopped-flow spectrofluorimetry. OD 440‐750 values increased and, conversely, when Such a system is diYcult to use with protoplasts since they protoplasts were transferred from 0.4 to 0.5 M sorbitol, sediment relatively quickly. In this study, however, a protoplasts contracted. The kinetics of expansion or method of indirectly measuring volume changes of protocontraction of the protoplasts could also be monitored plasts is described and the eVects of mercuric chloride and using this method and the half-time for water exchange mercaptoethanol on the volume changes induced by for expanding protoplasts (about 10 s) was slightly osmotic gradients are reported in order to investigate the higher than that for contracting protoplasts. A study involvement of water channels in such phenomena. of the effects of the water channel blocker, mercuric Mercury ions are reported to inhibit water movement chloride, on swelling protoplasts showed that 500 mM through water channels (aquaporins) while sulphydryl irreversibly damaged protoplasts, 5‐10 mM had a neg- reagents, such as mercaptoethanol, are reported to reverse ligible inhibitory effect on swelling while 100 mM had the inhibition (Steudle and Henzler, 1995). a large inhibitory effect often completely inhibiting swelling. A preliminary study indicated that mercaptoethanol reversed the inhibitory effect of mercuric Materials and methods chloride on protoplast swelling. Plant material and protoplast preparation