Ca2+-dependent phosphoregulation of the plasma membrane Ca2+-ATPase ACA8 modulates stimulus-induced calcium signatures

Abstract

Ca2+ signals are transient, hence, upon a stimulus-induced increase in cytosolic Ca2+ concentration, cells have to re-establish resting Ca2+ levels. Ca2+ extrusion is operated by a wealth of transporters, such as Ca2+ pumps and Ca2+/H+ antiporters, which often require a rise in Ca2+ concentration to be activated. Here, we report a regulatory fine-tuning mechanism of the Arabidopsis thaliana plasma membrane-localized Ca2+-ATPase isoform ACA8 that is mediated by calcineurin B-like protein (CBL) and CBL-interacting protein kinase (CIPK) complexes. We show that two CIPKs (CIPK9 and CIPK14) are able to interact with ACA8 in vivo and phosphorylate it in vitro. Transient co-overexpression of ACA8 with CIPK9 and the plasma membrane Ca2+ sensor CBL1 in tobacco leaf cells influences nuclear Ca2+ dynamics, specifically reducing the height of the second peak of the wound-induced Ca2+ transient. Stimulus-induced Ca2+ transients in mature leaves and seedlings of an aca8 T-DNA insertion line exhibit altered dynamics when compared with the wild type. Altogether our results identify ACA8 as a prominent in vivo regulator of cellular Ca2+ dynamics and reveal the existence of a Ca2+-dependent CBL-CIPK-mediated regulatory feedback mechanism, which crucially functions in the termination of Ca2+ signals.

Keywords: Arabidopsis thaliana; CBL-interacting protein kinases; Ca2+ signature; calcineurin B-like protein; phosphorylation; plasma membrane Ca2+-ATPase.

Fig. 1.

CIPK9 and CIPK14 interact with the ACA8 N-terminus in yeast two-hybrid assay. Serial dilutions of yeast PJ69-4A transformed with the ACA8 N-terminus (N-te) in the AD vector and all 26 CIPKs in BD vectors. Positive control: transformation with AD CBL1/BD CIPK1 vectors (white arrow; D’Angelo et al., 2006). Negative controls: transformation with AD ACA8 N-te/BD EV, BD CIPK9/AD EV, or BD CIPK14/AD EV (black arrows). Growth on selective SD-W-L-H medium supplemented with 2.5 mM 3AT indicates the interaction of ACA8 N-te with CIPK9 and CIPK14 (grey arrows). EV, empty vector; AD, activation domain; BD, binding domain.

Fig. 2.

CIPK9 and CIPK14 interact with CBL1 and ACA8 in BiFC assay. (A and B) GFP fluorescence associated with ACA8 in N. benthamiana leaves marks the cell periphery. The focus image (B) is a 5-fold magnification of the image in (A). The white arrow in the magnified image indicates the pattern of signal distribution in two neighbouring cells. (C–H) Biomolecular fluorescence complementation (BiFC) analyses of YN::CIPK9, YN::CIPK14, and YN::CIPK7 with, respectively, CBL1::YC (C, E, G) and ACA8::YC (D, F, H) in transiently transformed N. benthamiana leaves. CIPK9, CIPK14, and, to a lesser extent, CIPK7 show interaction with ACA8 as indicated by the reconstitution of the YFP fluorescence (reported as green colour) at the cell periphery that represents the plasma membrane. Results, collected 4 d after infiltration with A. tumefaciens, are from one experiment representative of at least five independent experiments. Scale bars=40 μm.

Fig. 3.

CIPK9 and CIPK14 specifically phosphorylate the ACA8 N-terminus in vitro. In vitro phosphorylation of the N-terminus (N-te) of ACA8 using StrepII-tagged recombinant CIPK9 or StrepII-tagged recombinant CIPK14. As negative controls, phosphorylation samples using purified GST as substrate or with N-te or GST alone were used. Assays were performed as described in the Materials and methods. Samples were solubilized and aliquots corresponding to 700 ng of N-te or GST were subjected to SDS–PAGE and autoradiography. Results are from one experiment representative of three. Numbers on the right refer to the mass of molecular weight markers.

Fig. 4.

Ca²⁺ signatures induced by leaf mechanical wounding in tobacco leaves expressing each of the tested proteins alone. (A–D) Nuclear Ca²⁺ concentration monitoring in N. benthamiana leaf cells transiently expressing NUP::YC3.6 alone (CNT, A) or co-expressing NUP::YC3.6 and ACA8::GFP (B), NUP::YC3.6 and CIPK9::GFP (C), or NUP::YC3.6 and CBL1::OFP (D). Leaves were challenged with wounding (arrow), and FRET variations (normalized FRET cpVenus/CFP ratio reported as ΔR/R₀) in single cells surrounding the wounded site were observed for ~400 s at 2 s intervals. Traces are the averages from the analysis of at least 25 independent cells. Insets: single plane confocal images of N. benthamiana epidermal cells from leaves used for wounding experiments, showing the simultaneous expression of the different expressed fluorescent proteins. (E and F) Comparison and statistical analysis of the height of the first and second peaks of the Ca²⁺ transients (as determined by single-cell analysis) reported as the normalized ΔR/R₀ (±SEM), measured in the different tested conditions. (G) Mean height of the second peak expressed as a percentage of the height of the first peak (±SEM). Asterisks indicate statistically significant differences (**P<0.05, ns=not significant) calculated using Student’s t-test. (H) Comparison of the number of epidermal cell nuclei in which the height of the second peak is <5% or >30% of the height of the first one in N. benthamiana leaves infiltrated with the different combinations of A. tumefaciens harbouring the different plasmids.

Fig. 5.

ACA8 interaction with CIPK9 and CBL1 influences Ca²⁺ signatures induced by leaf mechanical wounding. (A–D) Nuclear Ca²⁺ concentration monitoring in N. benthamiana leaf cells expressing NUP::YC3.6 alone (CNT, A) or co-expressing NUP::YC3.6, ACA8::YC and YN::CIPK9 (B), NUP::YC3.6, ACA8::YC, YN::CIPK9, and CBL1::OFP (C) and NUP::YC3.6, ACA8::YC, YN::CIPK9, and mutant CBL1-G2A::OFP (D). Leaves were challenged with wounding (arrow), and FRET variations (normalized FRET cpVenus/CFP ratio reported as ΔR/R₀) in single cells surrounding the wounded site were observed for ~400 s at 2 s intervals. Traces are the averages from the analysis of at least 30 independent cells. Insets: single plane confocal images of N. benthamiana epidermal cells from co-infiltrated leaves used for wounding, showing the simultaneous expression of the different expressed fluorescent proteins. (E and F) Comparison and statistical analysis of the height of the first and second peaks of the Ca²⁺ transients (as determined by single-cell analysis) reported as the normalized ΔR/R₀ (±SEM), measured in the different tested conditions. (G) Mean height of the second peak expressed as a percentage of the height of the first peak (±SEM). Asterisks indicate statistically significant differences (**P<0.05, ***P<0.01, ns=not significant) calculated using Student’s t-test. (H) Comparison of the number of epidermal cell nuclei in which the height of the second peak is <5% or >30% of the height of the first one in N. benthamiana leaves infiltrated with the different combinations of A. tumefaciens harbouring the different plasmids.

Fig. 6.

Cytosolic Ca²⁺ transients in response to wounding are altered in the aca8 single knock-out mutant. Leaves of wt or aca8 plants that constitutively express cytosolic YC3.6 were wounded (arrow). FRET changes were measured in a small area proximal to the wound site (~1 mm²). Traces (A) represent the raw FRET cp Venus/CFP ratio variations observed during the entire experiment and are the averages from the analysis of 23 independent wounding events performed on 23 independent leaves of 10 different plants for each genotype. (B) Statistical analysis of raw cpVenus/CFP ratios measured before challenging wt or aca8 leaves with wounding. (C) Statistical analysis of the normalized cpVenus/CFP ratio of the first and second peaks of the Ca²⁺ transients measured in wt or aca8 seedlings and reported as ΔR/R₀. Values are means ±SEM. Asterisks indicate statistically significant differences (**P<0.05) calculated using Student’s t-test.

Fig. 7.

The cytosolic Ca²⁺ signature in response to extracellular ATP (eATP) is altered in the aca8 single knock-out mutant. Root tips of 7-day-old wt or aca8 seedlings expressing NES::YC3.6 were treated for the indicated time with the specified eATP concentrations. Traces (A–D) represent the raw FRET cp Venus/CFP ratio variations in root tips observed during the entire experiment. The analyses were performed by considering the entire imaged root. (E) Statistical analysis of resting raw ratios measured before challenging wt or aca8 seedlings with eATP. (F) Statistical analysis of the normalized maximum FRET cpVenus/CFP ratio (°) reached after the administration of various eATP concentrations to wt or aca8 seedlings and reported as ΔR_max/R₀. (G) Statistical analysis of the normalized cpVenus/CFP ratio measured in wt or aca8 seedlings 250 s after challenging (Δ) and reported as ΔR/R₀. Values are means ±SEM. Asterisks indicate statistically significant differences (*P<0.1, **P<0.05, ***P<0.01, ns=not significant) calculated using Student’s t-test.