Mobilization of Ca2+ by cyclic ADP-ribose from the endoplasmic reticulum of cauliflower florets

The NAD(+) metabolite cADP-Rib (cADPR) elevates cytosolic free Ca2+ in plan ts and thereby plays a central role in signal transduction pathways evoked by the drought and stress hormone abscisic acid. cADPR is known to mobilize Ca2+ from the large vacuole of mature cells. To determine whether addition al sites for cADPR-gated Ca2+ release reside in plant cells, microsomes fro m cauliflower (Brassica oleracea) inflorescences were subfractionated on su crose density gradients, and the distribution of cADPR-elicited Ca2+ releas e was monitored. cADPR-gated Ca2+ release was detected in the heavy-density fractions associated with rough endoplasmic reticulum (ER). cADPR-dependen t Ca2+ release co-migrated with two ER markers, calnexin and antimycin A-in sensitive NADH-cytochrome c reductase activity. To investigate the possibil ity that contaminating plasma membrane in the ER-rich fractions was respons ible for the observed release, plasma membrane vesicles were purified by aq ueous two-phase partitioning, everted with Brij-58, and loaded with Ca2+. T hese vesicles failed to respond to cADPR. Ca2+ release evoked by cADPR at t he ER was fully inhibited by ruthenium red and 8-NH2-cADPR, a specific anta gonist of cADPR-gated Ca2+ release in animal cells. The presence of a Ca2release pathway activated by cADPR at higher plant ER reinforces the notion that, alongside the vacuole, the ER participates in Ca2+ signaling.