In mung bean (Vigna radiata [L.] Wilczek) suspension-cultured cells, which are extremely chilling-sensitive at the early stage of exponential growth, cold-induced acidification of the cytoplasm has been shown to be closely correlated with the cold-induced inactivation of the vacuolar H+-ATPase [Yoshida (1994) Plant Physiol. 104: 1131]. To gain more insight into the mechanism of the cold-induced acidification of the cytoplasm in these suspension-cultured cells, we focused in the present study on changes in the vacuolar pH during cold incubation. The vacuolar pH was measured by fluorescence-ratio imaging cryomicroscopy, with CDCF-DA [5-(and 6-)carboxy-2'-7'-dichlorofluorescein diacetate] as a pH-sensitive probe. During cold incubation of the CDCF-loaded protoplasts from cells at the early stage of exponential growth, the vacuolar pH shifted toward alkaline values with time, increasing from 5.1 to 5.7 over the course of 8 hours. The increase in vacuolar pH was closely correlated with the acidification of cytoplasm. The cold-induced acidification of the cytoplasm appeared to be less dependent on the pH of the external medium. From the results, it appears that the cold-induced acidification of the cytoplasm probably reflects, to a considerable extent, the passive release of protons or proton equivalents from vacuoles as a consequence of the cold-induced inhibition of the ATP-driven transport of protons across vacuolar membranes.
