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 Ca ²⁺ 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 Ca ²⁺ 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 Ca ²⁺ into a novel EGTA- and caffeine-insensitive compartment that can subsequently be activated by stimulants of extracellular Ca²⁺ entry.