NO-mediated [Ca2+]cyt increases depend on ADP-ribosyl cyclase activity in Arabidopsis

Abstract

cyclic ADP-ribose (cADPR) is a Ca2+-mobilising intracellular second messenger synthesised from nicotinamide adenine dinucleotide (NAD) by ADP-ribosyl cyclases (ADPR cyclases). In animals, cADPR targets the ryanodine receptor (RyR) present in the sarco/endoplasmic reticulum (SR/ER) to promote Ca2+ release from intracellular stores to increases the concentration of cytosolic free Ca2+ ([Ca2+]cyt) in Arabidopsis cADPR has been proposed to play a central role in signal transduction pathways evoked by the drought and stress hormone, abscisic acid (ABA) and the circadian clock. Despite evidence for the action of cADPR in Arabidopsis, no predicted proteins with significant similarity to the known ADPR cyclases have been reported in any plant genome database, suggesting either that there is a unique route for cADPR synthesis or a that homolog of ADPR cyclase with low similarity might exist in plants. We sought to determine whether the low-levels of ADPR cyclase activity reported in Arabidopsis are indicative of a bona fide activity that can be associated with the regulation of Ca2+ signalling. We adapted two different fluorescence-based assays to measure ADPR cyclase activity in Arabidopsis and found this activity has the characteristics of a nucleotide cyclase that is activated by nitric oxide to increase cADPR and mobilise Ca2+.