An apoplastic Ca2+ sensor regulates internal Ca2+ release in aequorin-transformed tobacco cells

Removal of Ca2+ from tobacco suspension cell medium has two immediate effects on cytosolic Ca2+ fluxes: (i) externally derived Ca2+ influx (occurring in response to cold shock or hypo osmotic shock) is inhibited, and (ii) organellar Ca2+ release (induced by a fungally derived defense elicitor, caffeine, or hypo-osmotic shock) is elevated. We show here that the enhanced release of internal Ca2+ is Likely due to increased discharge from a caffeine-sensitive store in response to a signal transduced from an extracellular Ca2+ sensor. Thus, chelation of extracellular Ca2+ in the absence of any other stimulus directly activates release of intracellular Ca2+ into the cytosol, Evidence that this chelator-activated Ca2+ flux is dependent on a signaling pathway includes its abrogation by prior treatment with caffeine, and its inhibition by protein kinase inhibitors (K252a and staurosporine) and anion channel blockers (niflumate and anthracene-9-carboxylate). An unexpected characteristic of tobacco cell adaptation to low external Ca2+ was the emergence of a new Ca2+ compartment that was inaccessible to external EGTA, yet responsive to the usual stimulants of extracellular Ca2+ entry. Thus, cells that are exposed to EGTA for 20 min lose sensitivity to caffeine and defense elicitors, indicating that their intracellular Ca2+ pools have been depleted, Surprisingly, these same cells simultaneously regain their ability to respond to stimuli that usually activate extracellular Ca2+ influx even though all external Ca2+ is chelated, Because this gradual restoration of Ca2+ influx can be inhibited by the same kinase inhibitors that block EGTA-activated Ca2+ release, we propose that chelator-activated Ca2+ release from internal stores leads to deposition of this Ca2+ into a novel EGTA- and caffeine-insensitive compartment that can subsequently be activated by stimulants of extracellular Ca2+ entry.