Elevation of the cytosolic free [Ca2+] is indispensable for the transduction of the nod factor signal in alfalfa

In root hairs of alfalfa (Medicago sativa), the requirement of Ca2+ for Nod factor signaling has been investigated by means of ion-selective microelectrodes. Measured 50 to 100 mu m behind the growing tip, 0.1 mu M NodRm-IV(C16:2,S) increased the cytosolic free [Ca2+] 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 Ca2+ concentrations below cytosolic values if the free EGTA concentration remained low (less than or equal to 0.01 mM). Externally added Sr2+ was able to replace Ca2+, and to some extent even enhanced the Nod-factor-induced depolarization, whereas with Mg2+ it was decreased. This suggests that the Nod factor response is triggered by Ca2+ from external stores. The addition of the endomembrane Ca2+-ATPase inhibitor 2,5-di(t-butyl)-1,4-benzohydroquinone, which presumably mobilizes Ca2+ from Ins(1,4,5) P-3-sensitive stores, mimicked the Nod factor response, i.e. increased the cytosolic free [Ca2+], 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 Ca2+ from internal stores. These latter results strongly support the idea that an elevation of cytosolic free [Ca2+] is indispensable for the transduction of the Nod factor signal, which is consistent with the role of Ca2+ as a second messenger.