Low Temperature-Induced Alkalization of Vacuoles in Suspension-Cultured Cells of Mung Bean (Vigna radiata [L.] Wilczek)

In mung bean (Vigna radiata L.] Wilczek) suspension-culturedcells, which are extremely chilling-sensitive at the early stageof exponential growth, cold-induced acidification of the cytoplasmhas been shown to be closely correlated with the cold-inducedinactivation of the vacuolar H⁺-ATPase Yoshida (1994) PlantPhysiol. 104: 1131]. To gain more insight into the mechanismof the cold-induced acidification of the cytoplasm in thesesuspension-cultured cells, we focused in the present study onchanges in the vacuolar pH during cold incubation. The vacuolarpH 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-loadedprotoplasts from cells at the early stage of exponential growth,the vacuolar pH shifted toward alkaline values with time, increasingfrom 5.1 to 5.7 over the course of 8 hours. The increase invacuolar pH was closely correlated with the acidification ofcytoplasm. The cold-induced acidification of the cytoplasm appearedto be less dependent on the pH of the external medium. Fromthe results, it appears that the cold-induced acidificationof the cytoplasm probably reflects, to a considerable extent,the passive release of protons or proton equivalents from vacuolesas a consequence of the cold-induced inhibition of the ATP-driventransport of protons across vacuolar membranes. ¹Contribution no. 3814 from the Institute of Low TemperatureScience, Hokkaido University.