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 microelec trodes. Measured 50 to 100 mu m behind the growing tip, 0.1 mu M NodRm-IV(C 16:2,S) increased the cytosolic free [Ca2+] by about 0.2 pCa, while the sam e concentration of chitotetraose, the nonactive glucosamine backbone, had n o effect. We demonstrate that NodRm-IV(C16:2,S) still depolarized the plasm a membrane at external Ca2+ concentrations below cytosolic values if the fr ee EGTA concentration remained low (less than or equal to 0.01 mM). Externa lly added Sr2+ was able to replace Ca2+, and to some extent even enhanced t he Nod-factor-induced depolarization, whereas with Mg2+ it was decreased. T his suggests that the Nod factor response is triggered by Ca2+ from externa l stores. The addition of the endomembrane Ca2+-ATPase inhibitor 2,5-di(t-b utyl)-1,4-benzohydroquinone, which presumably mobilizes Ca2+ from Ins(1,4,5 ) P-3-sensitive stores, mimicked the Nod factor response, i.e. increased th e cytosolic free [Ca2+], triggered Cl--efflux, depolarized the plasma membr ane, and alkalized the root hair space. In all cases a refractory state tow ard Nod factor perception was produced, indicating a shortcut of Nod factor signal transduction by releasing Ca2+ from internal stores. These latter r esults strongly support the idea that an elevation of cytosolic free [Ca2+] is indispensable for the transduction of the Nod factor signal, which is c onsistent with the role of Ca2+ as a second messenger.