Cytoplasm-enriched fragments ofChara: structure and electrophysiology

Abstract

Cytoplasm-enriched fragments prepared from internodal cells ofChara corallina by centrifugation contain membrane bound vesicles ranging in size from a few μm to hundreds of μm. If the fragments are incubated in artificial pond water (APW) of pH0 above ∼ 6.5, neutral red stains the inside of many vesicles bright crimson, suggesting the presence of inward proton-pumping. In APW of pH0 below 6 crimson vesicles are found less frequently. Under such conditions most vesicles remain unstained inside and some develop indistinct pink halos. After a few days most fragments form a central vacuole, which stains red, regardless of the pH0. The cytoplasmic layer still contains vesicles after vacuole formation. In order to identify the membrane bounding the vesicles various fluorescent probes were applied either by injection into the fragment or directly onto the vesicles released into artificial cytoplasm. Lucifer yellow or 6 COOH-F move readily across the tonoplast in intact cells, but did not enter any vesicles. On the other hand, the fluorescent cationic stain DIOC, which is used to highlight mitochondria and especially endoplasmic reticulum, stained the vesicle membrane. Numerous elliptical or kidney shaped nuclei in the flowing cytoplasm were highlighted with DAPI. In some fragments the nuclei formed large agreggates sometimes filling the width of the fragment. Patch-clamping the vesicles in artificial cytoplasm showed the presence of several kinds of channels, some displaying similar behaviour to the K+ channels observed in cytoplasmic droplets. Analogous to the plasmalemma of intact cells, the fragments without vacuoles displayed electrophysiological states dominated by either K+ conduction, H+ (or OH−) conduction or the proton pump. On the other hand, excitation transients in fragments were of low amplitude or absent altogether. Detailed comparisons of data from fragments and intact cells are shown. The effect of vacuole formation on fragment electrophysiology was also explored.