Loading process of sugars into cabbage petiole and asparagus shoot apex cells by incubation with hypertonic sugar solutions

The freezing tolerance of cabbage petioles and asparagus shoot apexes was i ncreased by preincubation with 0.8 M sugar solutions. In cabbage petioles w ith an initial freezing tolerance of -3 degreesC (temperature far 50% cell survival), as determined by both electrolyte leakage and fluorescein diacet ate vital staining, the freezing tolerance was increased to -13 degreesC by incubation with sorbitol solutions for 3 h. In meristematic cells of aspar agus shoot apexes with an initial freezing tolerance of -7.5 degreesC, as d etermined by fluorescein diacetate vital staining, the freezing tolerance w as increased to -30 degreesC 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 sho ot apexes with sugar solutions, sugars were intracellularly taken up by osm otically induced fluid-phase endocytotic vesicles, as indicated by comoveme nt of Lucifer Yellows carbohydrazide (LYCH) observed with a confocal laser scanning microscope. The amounts of intracellularly taken up sugars increas ed concomitantly with the formation of endocytotic vesicles depending on th e time of incubation in parallel with a gradual increase of freezing tolera nce. 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 conditio ns 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 memb ranes as a main site of freezing injury. The pretreatment with 1 mM p-chlor omercuribenzenesulfonic acid (PCMBS), an inhibitor of sugar transport, redu ced the amounts of intracellular sugar uptake without affecting the formati on of endocytotic vesicles, suggesting that sugars were, at least partly, t aken up by sugar transporters. In the pretreatment with PCMBS, the freezing tolerance of incubated tissues with sugar solutions was significantly redu ced, although addition of PCMBS per se did not affect survival. These resul ts suggest that sugars taken up by sugar transporters, rather than sugars t aken up by endocytotic vesicles, are mainly responsible for the increased f reezing tolerance of cabbage petioles and asparagus shoot apexes. Furthermo re, we aimed to study the occurrence of fluid-phase endocytosis with LYCH i n an isotonic condition. Our results indicated that uptake of LYCH by fluid -phase endocytotic vesicles was not detected microscopically in isotonic co ndition, although LYCH was spectrofluorimetrically taken up in isotonic con dition. Spectrofluorimetric uptake of LYCH was inhibited by addition of pro benecid, an anion transport inhibitor. These results suggest that in cabbag e petioles and asparagus shoot apexes, LYCH is taken up by anion transport but not by fluid-phase endocytosis in isotonic condition, and uptake of LYC H by fluid-phase endocytosis is restricted to occur only in hypertonic cond ition.