Elevation of the Cytosolic Free [Ca(2+)] Is Indispensable for the Transduction of the Nod Factor Signal in Alfalfa

In root hairs of alfalfa (Medicago sativa), the requirement of Ca(2+) for Nod factor signaling has been investigated by means of ion-selective microelectrodes. Measured 50 to 100 μm behind the growing tip, 0.1 μm NodRm-IV(C16:2,S) increased the cytosolic free [Ca(2+)] by about 0.2 pCa, while the same concentration of chitotetraose, the nonactive glucosamine backbone, had no effect. We demonstrate that NodRm-IV(C16:2,S) still depolarized the plasma membrane at external Ca(2+) concentrations below cytosolic values if the free EGTA concentration remained low (≤0.01 mm). Externally added Sr(2+) was able to replace Ca(2+), and to some extent even enhanced the Nod-factor-induced depolarization, whereas with Mg(2+) it was decreased. This suggests that the Nod factor response is triggered by Ca(2+) from external stores. The addition of the endomembrane Ca(2+)-ATPase inhibitor 2,5-di(t-butyl)-1,4-benzohydroquinone, which presumably mobilizes Ca(2+) from Ins(1,4,5)P(3)-sensitive stores, mimicked the Nod factor response, i.e. increased the cytosolic free [Ca(2+)], triggered Cl(−)-efflux, depolarized the plasma membrane, and alkalized the root hair space. In all cases a refractory state toward Nod factor perception was produced, indicating a shortcut of Nod factor signal transduction by releasing Ca(2+) from internal stores. These latter results strongly support the idea that an elevation of cytosolic free [Ca(2+)] is indispensable for the transduction of the Nod factor signal, which is consistent with the role of Ca(2+) as a second messenger.