The externally derived portion of the hyperosmotic shock-activated cytosolic calcium pulse mediates adaptation to ionic stress in suspension-cultured tobacco cells

The influx of Ca2+ into the cytosol has long been suggested to serve as a signaling intermediate in the acquisition of tolerance to hyperosmotic and/or salinity stresses. Here we use aequorin-transformed suspension-cultured tobacco cells to directly assess the role of cytosolic calcium (Ca-cyt(2+)) signaling in salinity tolerance acquisition. Aequorin luminescence recordings and Ca-45 influx measurements using inhibitors of Ca2+ influx (Gd3+ and the Ca2+-selective chelator EGTA), and modulators of organellar Ca2+ release (phosphotipase C inhibitors U73122 or neomycin) demonstrate that hyperosmolarity, whether imposed by NaCl or by a non-ionic molecule sorbitol, induces a rapid (returning to baseline levels of Ca2+, within 10 min) and complex Ca-cyt(2+) pulse in tobacco cells, deriving both from Gd +-sensitive externally derived Ca influx and from U73122- and neomycin-sensitive Ca2+ release from an organelle. To determine whether each of the two components of this brief Ca2+ signal regulate adaptation to hyperosmotic shock, the Ca2+ pulse was modified by the addition of d, U73122, neomycin, or excess Ca2% and then cells were treated with salt or sorbitol. After 10 min the cell culture medias were diluted with additional hyperosmotic media to reduce the toxic affects of the modulators, and the growth of cells was measured after 1 week. Gd3+ treatment reduced growth in salt relative to control cells but not in sorbitol, and exposure to excess Ca2+ increased growth in salt but not in sorbitol. In contrast, exposure to inhibitors of IP3 formation had no effect on growth in salt or sorbitol. Therefore, although hyperosmotic treatment stimulates both Ca2+ influx and Ca2+ release from an internal Ca2+ depot, only Ca2+ influx has a measurable impact on ionic stress tolerance acquisition in tobacco cell suspensions. In contrast, osmoadaptation in these cells appears to occur independent of Ca2+ signaling. (c) 2006 Elsevier GmbH. All rights reserved.