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

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.)

Figure 1.

Nicotinamide abolished NO-induced [Ca²⁺]_cyt increases in Arabidopsis. A, Effect of cold water (4°C) on [Ca²⁺]_cyt in the absence or presence of nicotinamide (50 mm). B, Effect of H₂O₂ on [Ca²⁺]_cyt in the absence or presence of nicotinamide (50 mm). C, Effect of NaCl on [Ca²⁺]_cyt in the absence or presence of nicotinamide (50 mm). D, Effect of NO (SNAP) on [Ca²⁺]_cyt in the absence or presence of nicotinamide (50 mm). Twelve-day-old aequorin-expressing individual seedlings were incubated for 2 h with nicotinamide (50 mm). Cold water (4°C), 10 mm H₂O₂, 150 mm NaCl, and 300 μm SNAP (NO donor) were added at 15 s, and luminescence was measured for 896 s in a luminometer or multifunctional microplate reader. Data are presented as means of 12 biological replicates from three independent experiments (n = 12), and error bars represent se. Arrows indicate the time of stimulation. RT, Room temperature.

Figure 2.

NO evokes short-term [Ca²⁺]_cyt increases. A, SNP was added at 60 and 360 s (n = 5 for each treatment) after the start of the experiment, and [Ca²⁺]_cyt levels were measured for 600 s. B, SNAP was added to provide a final concentration of 150 μm or 0.5% ethanol control was added 60 s after the start of the experiment, and [Ca²⁺]_cyt levels were measured for 600 s (n = 19). C, SNAP to a final concentration of 150 μm was added at 60 s, and 300 μm cPTIO was added at 360 s (n = 21). D, Seedlings were incubated with 300 μm cPTIO for 300 s before the start of the experiment, when 150 μm SNAP was added at 60 s (n = 10). E, SNAP at a final concentration of 150 μm was added 60 s after the start of the experiment, and 50 mm nicotinamide was added 300 s later (n = 20). F, Nicotinamide (50 mm) was added 60 s after the start of the experiment, and 150 μm SNAP was added 300 s later (n = 8). G, SNAP at a final concentration of 150 μm was added 60 s after the start of the experiment, and 50 mm mannitol was added 300 s later (n = 5). H, Seedlings were incubated for 300 s in 1 mm GdCl₃ before the start of the experiment. SNAP at a final concentration of 150 μm was added after 60 s (360 s; n = 13). Arrows indicate the time of each drug addition. Error bars represent se.

Figure 3.

Identification of ADPR cyclase activity in Arabidopsis. A, Time course of ADPR cyclase activity in soluble protein extracts of leaves of Col-0 or plants transformed with 35S:Aplysia californica ADPR cyclase (35S:Ac ADPR cyclase). All components except substrate were added to the cuvette, then 200 μm NGD was added at 10 min, and fluorescence intensity was measured for another 10 min. B, ADPR cyclase activity of extracts of Col-0 leaves in the presence of NAD. NAD reduced the activity in a concentration-dependent manner. C, ADPR cyclase activity of Col-0 leaf extracts measured using NGD as a substrate (200 μm) in the presence of cADPR. D, Estimated ADPR cyclase activity in extracts of Col-0 based on the cyclization of NGD in the absence or presence of 200 μm NGD, 200 μm NAD as an alternative substrate, and when enzymatic activity had been inhibited by boiling for 10 min. E, Estimated ADPR cyclase activity in extracts of Col-0 based on the cyclization of NHD in the absence or presence of 200 μm NHD, 200 μm NAD as an alternative substrate, and when enzymatic activity had been inhibited by boiling for 10 min. F, Activity of ADPR cyclase in extracts of Col-0 and 35S:Ac ADPR cyclase plants calculated from a standard curve derived from A. californica ADPR cyclase (Supplemental Fig. S1). Fluorescence is in arbitrary units. Data are presented as means of three biological replicates of three independent experiments, and error bars represent se. TP, Total protein.

Figure 4.

ADPR cyclase activity in response to NO. A, ADPR cyclase activity in the soluble protein extract of SNAP (300 μm)-treated Col-0 plants. B, Effect of 300 μm SNAP on ADPR cyclase activity of the protein extracted from untreated Col-0 plants. C, ADPR cyclase activity in soluble protein extracts of cml24-4 and cml23-2 cml24-4 plants. D, ADPR cyclase activity in ABA (50 μm)-treated protein extracts of untreated Col-0 plants. Soluble protein extracts were prepared from 4- to 5-week-old plants, and equal amounts of protein (145 µg) were used to measure the ADPR cyclase activity by NGD or NHD assay. Data are presented as means of three biological replicates of three independent experiments, and error bars represent se. TP, Total protein.

Figure 5.

SNAP triggers [cADPR] accumulation in Arabidopsis. Three-week-old Arabidopsis seedlings grown in 12 h of light/12 h of dark were treated with 300 μm SNAP or methanol control (0.5% [v/v] MetOH) by flooding the plates for 1 min. Each plate contained an average of 20 seedlings, and all of them were harvested in each time point. Three independent replicates were harvested at the beginning of the time course and 5, 10, 30, and 60 min after drug treatment. [cADPR] was estimated by a coupled assay, and each sample was measured at least twice. Error bars represent se.