Fusion and fission of plasma-membrane material accommodates for osmotically induced changes in the surface area of guard-cell protoplasts

Abstract

Stomatal movement requires large and repetitive changes in cell volume and consequently changes in surface area. The patch-clamp technique was used to monitor changes in plasma-membrane surface area of individual guard-cell protoplasts (GCPs) by measuring membrane capacitance (C m ), a parameter proportional to the surface area. The membrane capacitance increased under hypoosmotic conditions and decreased after hypertonic treatment. As the speci®c capacitance remained constant, this demonstrates that osmotically induced changes in surface area are associated with incorporation and removal of membrane material. Osmotically induced fusion and ®ssion of plasma-membrane material was not aected by removal of extracellular Ca 2+ . Dialysing protoplasts with very low (<2 nM) or high (1 lM) Ca 2+ had no eect on changes in C m under hypo-and hyperosmotic conditions. However, the rate of change in surface area was dependent on the size of the dierence in osmotic potential applied. The larger the osmotic dierence and thus changes in membrane tension caused by water in¯ux or eux, the faster the change in C m . The results therefore demonstrate that osmotically induced fusion and ®ssion of plasma-membrane material in GCPs are Ca 2+ -independent and modulated by membrane tension.During stomatal movement, guard cells undergo large changes in cell volume and surface area over a period of minutes, as a result of changes in ion accumulation and consequently water in¯ux or eux. So far, little is known about the mechanism underlying osmotically induced changes in plasma-membrane area. Studies of the mechanical properties of the plant cell membrane have demonstrated that the maximum possible elastic stretching of membranes is only about 2% (Wolfe and Steponkus 1983). During stomatal movement, changes in surface area of about 40% occur (Raschke 1979). These large changes cannot be the result of stretching of the existing membrane, but require the insertion of new membrane material into the plasma membrane. Kell and Glaser (1993) postulated that water in¯ux causes an increase in membrane tension which results in an increase in exocytotic activity, a process which they proposed was regulated by membrane voltage and extracellular and intracellular Ca 2+ -activity.The formation of endocytotic vesicles in protoplasts from rye leaves after hyperosmotic treatment suggests that osmotic contraction of protoplasts is due to endocytosis and not membrane folding (Gordon-Kamm and Steponkus 1984). Similar results were found in guard-cell protoplasts (GCPs) after osmotically induced shrinkage (Diekmann et al. (1993). However, endocytotic vesicles were not observed in intact guard cells.To investigate the mechanisms involved in osmotically induced changes in the surface area in GCPs, I used the whole-cell patch-clamp technique. This technique allows the measurement of changes in surface area at a high time resolution by measuring the membrane capacitance (C m ), as well as controlling the cytosolic composition by dialysing the cell via the patch pip...

Changes in Surface Area of Intact Guard Cells Are Correlated with Membrane Internalization

“…Furthermore, it has been shown that the cross-sectional shape of guard cells is nearly circular at high turgor pressures, but becomes more flattened as they lose turgor (Raschke, 1979). This lends credibility to the first solution because flattening could produce changes in volume while maintaining a constant surface area.The problem of how guard cells maintain the integrity of the plasma membrane during volume changes has been addressed using osmotically induced changes in the volume of guard cell protoplasts (Homann, 1998;Kubitscheck et al, 2000). Those studies showed that vesicles were internalized 1 This work was supported by the National Science Foundation (grant no.…”

“…Thus, guard cells must either: (a) change shape such that plasma membrane surface area remains nearly constant while volume changes, or (b) move membrane between the plasma membrane and internal sources within the cell to accommodate changes in surface area. Although the literature on guard cells assumes the latter solution (Diekmann et al, 1993;Homann, 1998;Blatt, 2000), examples of both solutions can be found in animal cells. For example, mammalian erythrocytes change shape to maintain a constant surface area as volume is changed osmotically, but neurons continuously shuttle membrane between the endomembrane system and the plasma membrane to vary both surface area and volume (Morris and Homann, 2001).…”

Tansley Review No. 108

“…Very modest hydrostatic pressures gave rates of capacitance rise 50-fold higher than under elevated [Ca# + ] i without evidence of saturation and, in Vicia guard cell protoplasts, was insensitive to [Ca# + ] i (Homann, 1998). These results suggest a much larger pool of vesicles ready for fusion and\or their rapid supply to the plasma membrane, by contrast with secretion evoked by [Ca# + ] i .…”

“…Secretion activity independent of [Ca# + ] i has been intimated from work with barley aleurone protoplasts (Homann & Tester, 1997) and more recently in studies of guard cell protoplasts (Homann, 1998) and maize coleoptile protoplasts (Thiel et al, 1999). Homann & Tester (1997) found a small, but steady rise in capacitance that was independent of [Ca# + ] i level in the patch pipette.…”

“…Zorec & Tester (1993) observed that a small hydrostatic pressure applied via the patch pipette would evoke a steady increase in membrane capacitance in barley aleurone protoplasts. More recently, work with protoplasts of Vicia guard cells (Homann, 1998) has shown that both increases and decreases in plasma membrane surface area may be driven under an appropriate hydrostatic gradient. In both of these studies, release of the hydrostatic pressure was followed by recovery of the capacitance to its previous level.…”