Extracellular nucleotides elicit cytosolic free calcium oscillations in Arabidopsis

Abstract

Extracellular ATP induces a rise in the level of cytosolic free calcium ([Ca(2+)](cyt)) in plant cells. To expand our knowledge about the function of extracellular nucleotides in plants, the effects of several nucleotide analogs and pharmacological agents on [Ca(2+)](cyt) changes were studied using transgenic Arabidopsis (Arabidopsis thaliana) expressing aequorin or the fluorescence resonance energy transfer-based Ca(2+) sensor Yellow Cameleon 3.6. Exogenously applied CTP caused elevations in [Ca(2+)](cyt) that displayed distinct time- and dose-dependent kinetics compared with the purine nucleotides ATP and GTP. The inhibitory effects of antagonists of mammalian P2 receptors and calcium influx inhibitors on nucleotide-induced [Ca(2+)](cyt) elevations were distinct between CTP and purine nucleotides. These results suggest that distinct recognition systems may exist for the respective types of nucleotides. Interestingly, a mutant lacking the heterotrimeric G protein Gβ-subunit exhibited a remarkably higher [Ca(2+)](cyt) elevation in response to all tested nucleotides in comparison with the wild type. These data suggest a role for Gβ in negatively regulating extracellular nucleotide signaling and point to an important role for heterotrimeric G proteins in modulating the cellular effects of extracellular nucleotides. The addition of extracellular nucleotides induced multiple temporal [Ca(2+)](cyt) oscillations, which could be localized to specific root cells. The oscillations were attenuated by a vesicle-trafficking inhibitor, indicating that the oscillations likely require ATP release via exocytotic secretion. The results reveal new molecular details concerning extracellular nucleotide signaling in plants and the importance of fine control of extracellular nucleotide levels to mediate specific plant cell responses.

Figure 1.

NTPs increase bioluminescence in aequorin-expressing transgenic Arabidopsis seedlings. A, Chemical structures of purine and pyrimidine derivatives. B, Individual 5-d-old aequorin seedlings were transferred to individual wells of a 96-well microplate and incubated overnight in reconstitution buffer containing coelenterazine. Each NTP was then applied at a final concentration of 100 μm. The line graph shows time-dependent changes in photon counts from representative wells of each treatment (bin size = 50 frames, 1 s, 20 bin smoothing). The inset shows a pseudocolored photon-counting image integrated over 400 s after nucleotide treatment calibrated to the inset scale.

Figure 2.

Effects of a PLC inhibitor and a Ca²⁺ channel inhibitor on ATP-induced [Ca²⁺]_cyt response in Arabidopsis seedlings. Arabidopsis seedlings harboring reconstituted aequorin were preincubated for 30 min with Gd³⁺ (a Ca²⁺ channel inhibitor; A and B) or U-73122 (a PLC inhibitor; C and D) at the indicated concentrations; 100 μm ATP was then applied. The remaining aequorin in the tissues was then discharged for [Ca²⁺]_cyt calculation (see “Materials and Methods”). A and C, Kinetic differences of ATP-induced bioluminescence in the presence or absence of Gd³⁺ (A) or U-73122 (C). B and D, Data were calculated as integrated [Ca²⁺]_cyt values over 10 to 60 s or 60 to 120 s and then converted into a relative value to mock treatment. Asterisks indicate statistically significant differences compared with the mock treatment control: * P < 0.05, ** 0.001 < P < 0.01, *** P < 0.001.

Figure 3.

Effects of NTPs on [Ca²⁺]_cyt response in Arabidopsis seedlings. Each integrated [Ca²⁺]_cyt was obtained from luminescence data recorded over 400 s after treatment and discharging of the remaining aequorin signal (see “Materials and Methods”). A and B, Histograms show means with se (n = 8) as integrated [Ca²⁺]_cyt values that were recorded after treatment with 100 μm (A) or 500 μm (B) NTPs. Different letters indicate statistically significant differences at P < 0.05. C, Dose-dependent curves of NTP-induced [Ca²⁺]_cyt responses.

Figure 4.

Effects of antagonists of mammalian P2 receptors on nucleotide-induced [Ca²⁺]_cyt response in Arabidopsis seedlings. Arabidopsis seedlings harboring reconstituted aequorin were preincubated for 30 min with antagonists for mammalian P2 receptors at the indicated concentrations (10–300 μm). The indicated nucleotide was then applied: ATP (A), GTP (B), ITP (C), and CTP (D). Ice-cold water was applied as a negative control (E). The integrated [Ca²⁺]_cyt values were calculated from normalized data of luminescence signal recorded over 400 s after treatment and discharging of the remaining aequorin signal (see “Materials and Methods”). Histograms show means with se as relative values to a single treatment of nucleotide. Nonselective P2 receptor antagonists are suramin, reactive blue-2 (RB2), and PPADS. Selective P2X antagonists are iso-PPADS, Evans blue (EB), and TNP-ATP. Mean [Ca²⁺]_cyt values of single nucleotide treatment controls were as follows: ATP, 1.9 ± 0.19 μm; GTP, 1.8 ± 0.15 μm; ITP, 1.3 ± 0.06 μm; CTP, 1.2 ± 0.04 μm; cold treatment, 2.6 ± 0.55 μm. Asterisks indicate statistically significant differences compared with a single application of nucleotides: * P < 0.05, ** 0.001 < P < 0.01, *** P < 0.001.

Figure 5.

Effects of nucleotides on [Ca²⁺]_cyt response in heterotrimeric G protein mutant backgrounds. NTPs (100 μm) were applied to three independent aequorin lines in gpa1-4, agb1-2, and gpa1-4;agb1-2 mutant backgrounds. Histograms show means with se as relative values (log₂ ratio) to the wild-type (WT) background control. Note that aequorin seedlings in agb1-2 and gpa1-4;agb1-2 backgrounds exhibited hyperresponsiveness to each of the nucleotides used. Mean [Ca²⁺]_cyt values of wild-type controls were as follows: ATP, 2.1 ± 0.36 μm; GTP, 2.0 ± 0.22 μm; ITP, 1.3 ± 0.06 μm; CTP, 1.2 ± 0.06 μm. Asterisks indicate statistically significant differences compared with the wild-type aequorin control: * P < 0.05, ** 0.001 < P < 0.01, *** P < 0.001.

Figure 6.

Effects of hydrolyzable and poorly hydrolyzable nucleotide analogs on [Ca²⁺]_cyt response in Arabidopsis seedlings. Native nucleotides or poorly hydrolyzable analogs were applied at a concentration of 100 μm. Experimental and analytical procedures were identical to those used in Figure 3. Different letters denote statistically significant differences (P < 0.05).

Figure 7.

NGXT191 elicited nucleotide-induced [Ca²⁺]_cyt oscillations. Arabidopsis seedlings harboring reconstituted aequorin were treated with 100 μm nucleotides after 30 min of preincubation with NGXT191. A, Histogram represents each integrated [Ca²⁺]_cyt response obtained from data recorded over 400 s upon nucleotide treatments. Arrows indicate the NGXT191 concentration that evoked nucleotide-induced [Ca²⁺]_cyt oscillations. B, ADP-induced [Ca²⁺]_cyt oscillations in the wild type. Arrows indicate amplitude peaks in the oscillations. C, Time-lapse movie of wild-type aequorin transgenic seedlings treated with ADP after preincubation with NGXT191 (see Supplemental Movie S1). Frames from the recording at the indicated times (s) are shown in a pseudocolored photon-counting image. D, ADP-induced [Ca²⁺]_cyt oscillations in the agb1-2 mutant background. E, Effect of the vesicle-trafficking inhibitor BFA on ADP-induced [Ca²⁺]_cyt oscillations. Ten micromolar BFA was applied together with NGXT191 for preincubation.

Figure 8.

Effects of NTPs on [Ca²⁺]_cyt response in Arabidopsis seedlings monitored using YC3.6. Arabidopsis seedlings expressing YC3.6 were treated with 10 μm ATP, CTP, or GTP as indicated. Changes in [Ca²⁺]_cyt were monitored at 5-s (A and B) or 3-s (C) intervals using confocal ratio imaging as described in “Materials and Methods.” [Ca²⁺]_cyt has been pseudocolor coded according to the inset scale. Representative of five or more roots per treatment are shown. A, ATP-induced changes in [Ca²⁺]_cyt imaged using YC3.6. Panels show time points in seconds following ATP treatment. Bar = 20 μm. B, ATP-induced changes in [Ca²⁺]_cyt monitored in individual cells shown in A. C, NTP-induced changes in [Ca²⁺]_cyt monitored in individual cell type a from A. CFP, Cyan fluorescent protein.