A laser microsurgical method of cell wall removal allows detection of largeconductance ion channels in the guard cell plasma membrane

Abstract

Application of patch clamp techniques to higher-plant cells has been subject to the limitation that the requisite contact of the patch electrode with the cell membrane necessitates prior enzymatic removal of the plant cell wall. Because the wall is an integral component of plant cells, and because cell-wall-degrading enzymes can disrupt membrane properties, such enzymatic treatments may alter ion channel behavior. We compared ion channel activity in enzymatically isolated protoplasts of Vicia faba guard cells with that found in membranes exposed by a laser microsurgical technique in which only a tiny portion of the cell wall is removed while the rest of the cell remains intact within its tissue environment. "Laser-assisted" patch clamping reveals a new category of high-conductance (130 to 361 pS) ion channels not previously reported in patch clamp studies on plant plasma membranes. These data indicate that ion channels are present in plant membranes that are not detected by conventional patch clamp techniques involving the production of individual plant protoplasts isolated from their tissue environment by enzymatic digestion of the cell wall. Given the large conductances of the channels revealed by laser-assisted patch clamping, we hypothesize that these channels play a significant role in the regulation of ion content and electrical signalling in guard cells.

A type of voltage-dependent Ca2+ channel on Vicia faba guard cell plasma membrane outwardly permeates K+

“…In the study of Wu (1995), Vicia guard cell protoplasts were also used, but only inward K + channels at whole-cell level were discussed even if the outward currents were also bigger than normal ones (Wu 1995). Another work conducted by Miedema et al found inward and outward K + channels with high conductance of as much as 130-360 pS in intact Vicia guard cells (Miedema et al 1999). The channel conductance for high ones indicated in our present study is about 187 pS, which is close to that recorded in Miedema's study.…”

“…High-conductance cation channels have been observed on the plasma membrane of different plant species with different analytical methods including planar lipid bilayer and whole-cell and single-channel recordings from intact guard cells or protoplasts (White 1993;Wu 1995;Miedema et al 1999;Balleza et al 2005). Some of the high-conductance channels indicated in these reports showed K + permeation, and some of them showed Ca 2+ permeation as well.…”

A force of nature: molecular mechanisms of mechanoperception in plants

“…Since the discovery that cell-wall fragments produced during plant cell-wall degradation by pathogens serve as elicitors to trigger plant defence responses (Sequeira, 1983), it has become apparent that the cell wall is not only a target of cellular signalling but is also a vital source of information (Pennell, 1998;Wolf et al, 2012). Removal of the cell wall by enzymatic digestion yields protoplasts that retain at least some mechanosensitivity (Haley et al, 1995;Wymer et al, 1996) but a precise evaluation of the contribution of the cell wall to mechanoperception is difficult as these assays are known to alter membrane properties (Miedema et al, 1999). However, an elegant series of experiments by Hématy and co-workers showed that developmental defects in cell-wall assembly are actively monitored by plants and lead to adjustments in growth and development (Hematy et al, 2007).…”

In vivo femtosecond laser nanosurgery of cell wall enables patch-clamp on filamentous fungi

“…In this way, the polarity of the fungal cells is preserved, and a portion of the plasma membrane is accessible for patch-clamp measurements. Laser removal of the cell wall with patch-clamp recording has previously been used in some algae and plants [29][30][31][32]21 and only in two lamentous fungi 10,33 . However, these experiments were conducted more than twenty years ago with UV lasers and never evolved into a routine protocol.…”