Mobilization of Ca(2+) by Cyclic ADP-Ribose from the Endoplasmic Reticulum of Cauliflower Florets

The NAD(+) metabolite cADP-Rib (cADPR) elevates cytosolic free Ca(2+) in plants and thereby plays a central role in signal transduction pathways evoked by the drought and stress hormone abscisic acid. cADPR is known to mobilize Ca(2+) from the large vacuole of mature cells. To determine whether additional sites for cADPR-gated Ca(2+) release reside in plant cells, microsomes from cauliflower (Brassica oleracea) inflorescences were subfractionated on sucrose density gradients, and the distribution of cADPR-elicited Ca(2+) release was monitored. cADPR-gated Ca(2+) release was detected in the heavy-density fractions associated with rough endoplasmic reticulum (ER). cADPR-dependent Ca(2+) release co-migrated with two ER markers, calnexin and antimycin A-insensitive NADH-cytochrome c reductase activity. To investigate the possibility that contaminating plasma membrane in the ER-rich fractions was responsible for the observed release, plasma membrane vesicles were purified by aqueous two-phase partitioning, everted with Brij-58, and loaded with Ca(2+): These vesicles failed to respond to cADPR. Ca(2+) release evoked by cADPR at the ER was fully inhibited by ruthenium red and 8-NH(2)-cADPR, a specific antagonist of cADPR-gated Ca(2+) release in animal cells. The presence of a Ca(2+) release pathway activated by cADPR at higher plant ER reinforces the notion that, alongside the vacuole, the ER participates in Ca(2+) signaling.