Vacuolar Membrane Lesions Induced by a Freeze-Thaw Cycle in Protoplasts Isolated from Deacclimated Tubers of Jerusalem Artichoke (Helianthus tuberosus L.)

Abstract

The processes of freezing injury in Jerusalem artichoke (Helianthus tuberosus L.) tubers were studied using protoplasts isolated from cold-acclimated and deacclimated tubers. Prior to freezing, protoplasts were preloaded with 10 microM fluorescein diacetate (FDA) in an isotonic sorbitol solution. After freeze-thawing at various temperatures, cell viability was evaluated under a fluorescence microscope. In cold-acclimated tubers, more than 80% of protoplasts survived freezing to -20 degrees C. By contrast, in deacclimated tubers, the cell survival abruptly declined after freezing to temperatures below -5 degrees C. Thus, freezing tolerance differed significantly between protoplasts isolated from cold-acclimated and deacclimated tubers. Two distinct types of cell injury, which were caused by either damage to plasma membrane (cell-lysis type) or by damage to the vacuolar membrane (abnormal-staining type), were observed, depending on the cold hardiness and freezing temperature. In the cells of the abnormal-staining type, shrinkage of the central vacuolar space and simultaneous acidification of the cytoplasmic space were characteristically observed immediately before complete cell-rehydration during thawing. The decrease in freezing tolerance of protoplasts after deacclimation was suggested to be due mainly to destabilization of the vacuolar membrane by freeze-induced dehydration stress.

Plant Freezing and Damage

“…In cereals, the plasma membrane is the membrane most vulnerable to this kind of damage; other membranes are also aected but often at a lower temperature. However , in other species and organs, damage to the tonoplast limits survival (Stout et al, 1980;Zhang and Willison, 1992b;Murai and Yoshida, 1998).…”

Alterations of Intracellular pH in Response to Low Temperature Stresses

“…For the measurement of the cytoplasmic pH by pH-ratiometry, it is critically important to load the fluorophore specifically into the cytoplasm (Yoshida 1994). For this purpose, protoplasts are more suitable than tissue sections because of the specific localization of the fluorophore in the cytoplasm after loading of the fluorophore under specified conditions (Murai and Yoshida 1998a).…”

“…These results indicate that the pH reduction lower than 6.5 in non-acclimated protoplasts is probably due to a release of H+ ions across the vacuolar membrane, apparently suggesting the role of the lesions associated with the vacuolar membrane in cell injury. In fact, we have recently suggested that the decrease in freezing tolerance of protoplasts isolated from Jerusalem artichoke tuber after deacclimation is mainly due to destabilization of the vacuolar membrane (Murai and Yoshida 1998a).…”

Dehydration Kinetics of Embryonic Axes from Desiccation‐sensitive Seeds: An Assessment of Descriptive Models

“…They include: water content, both absolute and relative (e.g. Berjak et al 1992; Sun et al 1994; Farrant 2000), water potential and related functions such as water activity (Roberts and Ellis 1989; Vertucci and Roos 1990; Tompsett and Pritchard 1993), cell volume (Merryman 1974; Steponkus 1979; Murai and Yoshida 1998), intracellular viscosity (Vertucci and Roos 1990; Koster 1991; Williams et al 1993; Leopold et al 1994; Buitink et al 1998; Leprince and Hoekstra 1998; Bryant et al 2001), intermolecular proximity (Lis et al 1982; Steponkus et al 1995) and structural water (Ladbrooke and Chapman 1969; Vertucci and Leopold 1984; Crowe et al 1990).…”