NO-Mediated [Ca2+]cyt Increases Depend on ADP-Ribosyl Cyclase Activity in Arabidopsis

S.M Abdul-Awal,Alison G Smith,Matthew P Davey,Alex A.R Webb,Carlos T Hotta,Antony N Dodd

doi:10.1104/pp.15.01965

Abstract

Cyclic ADP ribose (cADPR) is a Ca(2+)-mobilizing intracellular second messenger synthesized from NAD by ADP-ribosyl cyclases (ADPR cyclases). In animals, cADPR targets the ryanodine receptor present in the sarcoplasmic/endoplasmic reticulum to promote Ca(2+) release from intracellular stores to increase the concentration of cytosolic free Ca(2+) in Arabidopsis (Arabidopsis thaliana), and cADPR has been proposed to play a central role in signal transduction pathways evoked by the drought and stress hormone, abscisic acid, 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 that a 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 Ca(2+) signaling. We adapted two different fluorescence-based assays to measure ADPR cyclase activity in Arabidopsis and found that this activity has the characteristics of a nucleotide cyclase that is activated by nitric oxide to increase cADPR and mobilize Ca(2.)

Highlights

Cyclic ADP ribose is a Ca2+-mobilizing intracellular second messenger synthesized from NAD by ADP-ribosyl cyclases (ADPR cyclases)
Neither ADPR cyclase nor an equivalent of the ryanodine receptor has been identified in genomic databases, even though ADPR cyclase activity and Cyclic ADP ribose (cADPR)-evoked Ca2+ release from vacuoles and endoplasmic reticulum (ER) have been reported (Allen et al, 1995; Muir and Sanders, 1997; Leckie et al, 1998; Navazio et al, 2000; Sánchez et al, 2004). cADPR injected into guard cells causes stomatal closure (Leckie et al, 1998), and cADPR has been proposed to be involved in abscisic acid (ABA)-induced stomatal closure because 8-NH2-cADPR, a competitor of cADPR signaling, and nicotinamide, an inhibitor of ADPR cyclase activity, both reduced ABA-induced stomatal closure (Leckie et al, 1998)
We investigated the potential role of ADPR cyclase activity in nitric oxide (NO) signaling, because NO is a known regulator of the cADPR signaling pathway in animals (Galione et al, 1993; Willmott et al, 1996; Yu et al, 2000; Zhang and Li, 2006) and pharmacology suggests that NO-mediated increases in [Ca2+]cyt are cADPR dependent in Vicia faba (Garcia-Mata et al, 2003)

Summary

Introduction

Cyclic ADP ribose (cADPR) is a Ca2+-mobilizing intracellular second messenger synthesized from NAD by ADP-ribosyl cyclases (ADPR cyclases). We sought to establish whether the reported ADPR cyclase-like activity in Arabidopsis has functional characteristics of an enzyme involved in the generation of cADPR to mobilize Ca2+ in plant signaling networks; we investigated if the enzyme activity was correlated with stimulus-induced increases in cADPR and [Ca2+]cyt. The inhibition of SNAP-induced increases in [Ca2+]cyt by nicotinamide was suggestive of a role for ADPR cyclase in the elevation of [Ca2+]cyt by NO.

Results

Conclusion