The freezing tolerance of cabbage petioles and asparagus shoot apexes was increased by preincubation with 0.8 M sugar solutions. In cabbage petioles with an initial freezing tolerance of -3 degrees C (temperature for 50% cell survival), as determined by both electrolyte leakage and fluorescein diacetate vital staining, the freezing tolerance was increased to -13 degrees C by incubation with sorbitol solutions for 3 h. In meristematic cells of asparagus shoot apexes with an initial freezing tolerance of -7.5 degrees C, as determined by fluorescein diacetate vital staining, the freezing tolerance was increased to -30 degrees C by incubation with 0.8 M sugar solutions for 3 h, although other cells in the shoot apexes were killed by higher freezing temperatures. During incubation of both cabbage petioles and asparagus shoot apexes with sugar solutions, sugars were intracellularly taken up by osmotically induced fluid-phase endocytotic vesicles, as indicated by comovement of Lucifer Yellows carbohydrazide (LYCH) observed with a confocal laser scanning microscope. The amounts of intracellularly taken up sugars increased concomitantly with the formation of endocytotic vesicles depending on the time of incubation in parallel with a gradual increase of freezing tolerance. However, the endocytotic vesicles and their contents were retained not only after prolonged incubation after maximum freezing tolerance had been achieved but also after recovery of these tissue cells to isotonic conditions or after freeze-thawing. These results suggest that although sugars are intracellularly taken up by endocytotic vesicles, they might be sequestered within vesicles, casting doubt on their protective role to the plasma membranes as a main site of freezing injury. The pretreatment with 1 mM p-chloromercuribenzenesulfonic acid (PCMBS), an inhibitor of sugar transport, reduced the amounts of intracellular sugar uptake without affecting the formation of endocytotic vesicles, suggesting that sugars were, at least partly, taken up by sugar transporters. In the pretreatment with PCMBS, the freezing tolerance of incubated tissues with sugar solutions was significantly reduced, although addition of PCMBS per se did not affect survival. These results suggest that sugars taken up by sugar transporters, rather than sugars taken up by endocytotic vesicles, are mainly responsible for the increased freezing tolerance of cabbage petioles and asparagus shoot apexes. Furthermore, we aimed to study the occurrence of fluid-phase endocytosis with LYCH in an isotonic condition. Our results indicated that uptake of LYCH by fluid-phase endocytotic vesicles was not detected microscopically in isotonic condition, although LYCH was spectrofluorimetrically taken up in isotonic condition. Spectrofluorimetric uptake of LYCH was inhibited by addition of probenecid, an anion transport inhibitor. These results suggest that in cabbage petioles and asparagus shoot apexes, LYCH is taken up by anion transport but not by fluid-phase endocytosis in isotonic condition, and up...
