Effects of cold-induced dysfunction of vacuoles upon homeostasis in intracellular ionic environment.

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Chilling sensitivity of mung bean cultured cells varied markedly depending on the growth phases. At the initial stages after subculture, cells were relatively insensitive to chilling, but chilling sensitivity increased abruptly thereafter and reached a maximum at the early stage of exponential growth. Upon further culturing, cell become chilling-tolerant, becoming most tolerant at the stage of stationary growth. We observed that cold sensitivity of the tonoplast H^+-ATPase in vivo changed markedly as a function of growth cycle. the enzyme was very sensitive to cold at the early stage of exponential growth and less sensitive at the late stage of exponential growth. Therefore, there exists an intimate correlation between sensitivities of cells to cold and the coldinsability of the H^+-ATPase. Biochemical studies on the cold-induced inactivation of the tonoplast H^+-ATPase indicated that a selective release of the peripheral subunits as a whole from the membrane sectors (16 kDa subunit).Changes in cytoplasmic pH in mung bean cultured cells were observed utilizing a cryomicroscope and a fluorescence ratio imaging system with special reference to the variations in chilling sensitivity during cell growth. In the extremely chilling-sensitive cells at the early stage of exponential growth, cytoplasmic pH was reduced abruptly upon cold incubation at 0ﾟC. However, in chilling- tolerant cells at the late stage of exponential growth, no change in the cytoplasmic pH was detected even after protracted chilling for up to 72 h.From the results, it is suggested that vacuolar proton translocating systems has an important role for maintaining of cell homeostasis, especially under such a cold environment.