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

The NAD1 metabolite cADP-Rib (cADPR) elevates cytosolic free Ca21 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 Ca21 from the large vacuole of mature cells. To determine whether additional sites for cADPR-gated Ca21 release reside in plant cells, microsomes from cauliflower (Brassica oleracea) inflorescences were subfractionated on sucrose density gradients, and the distribution of cADPR-elicited Ca21 release was monitored. cADPR-gated Ca21 release was detected in the heavy-density fractions associated with rough endoplasmic reticulum (ER). cADPR dependent Ca21 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 Ca21: These vesicles failed to respond to cADPR. Ca21 release evoked by cADPR at the ER was fully inhibited by ruthenium red and 8-NH2-cADPR, a specific antagonist of cADPR-gated Ca21 release in animal cells. The presence of a Ca21 release pathway activated by cADPR at higher plant ER reinforces the notion that, alongside the vacuole, the ER participates in Ca21 signaling.