DORN1/P2K1 and purino-calcium signalling in plants: making waves with extracellular ATP

Abstract

Background and aims: Extracellular ATP governs a range of plant functions, including cell viability, adaptation and cross-kingdom interactions. Key functions of extracellular ATP in leaves and roots may involve an increase in cytosolic free calcium as a second messenger ('calcium signature'). The main aim here was to determine to what extent leaf and root calcium responses require the DORN1/P2K1 extracellular ATP receptor in Arabidopsis thaliana. The second aim was to test whether extracellular ATP can generate a calcium wave in the root.

Methods: Leaf and root responses to extracellular ATP were reviewed for their possible links to calcium signalling and DORN1/P2K1. Leaves and roots of wild type and dorn1 plants were tested for cytosolic calcium increase in response to ATP, using aequorin. The spatial abundance of DORN1/P2K1 in the root was estimated using green fluorescent protein. Wild type roots expressing GCaMP3 were used to determine the spatial variation of cytosolic calcium increase in response to extracellular ATP.

Key results: Leaf and root ATP-induced calcium signatures differed markedly. The leaf signature was only partially dependent on DORN1/P2K1, while the root signature was fully dependent. The distribution of DORN1/P2K1 in the root supports a key role in the generation of the apical calcium signature. Root apical and sub-apical calcium signatures may operate independently of each other but an apical calcium increase can drive a sub-apical increase, consistent with a calcium wave.

Conclusion: DORN1 could underpin several calcium-related responses but it may not be the only receptor for extracellular ATP in Arabidopsis. The root has the capacity for a calcium wave, triggered by extracellular ATP at the apex.

Keywords: Arabidopsis; ATP; DORN1; P2K1; calcium; reactive oxygen species; root; wave.

Fig. 1.

DORN1 governs the leaf eATP-induced [Ca²⁺]_cyt increase. (A) Mean ± s.e.m. [Ca²⁺]_cyt time course of individual excised leaves from 11-d-old Arabidopsis thaliana Col-0 constitutively expressing cytosolic aequorin. Plants were grown on solidified half-strength MS medium then leaves were assayed in liquid medium as described by Matthus et al. (2019). Control medium or with 1 mm ATP (pH 5.6) was added at 35 s (black triangle); n = 15–19 leaves in three independent trials. (B) As (A) but with 14-d-old Col-0 leaves, at pH 5.8. (C) Response of 14-d-old dorn1-1 leaves. (D) Total [Ca²⁺]_cyt mobilized (estimated as the area under the curve, after subtraction of mean pre-stimulus baseline) in response to control medium (grey points) or 1 mm ATP (green points) by 14-d-old Col-0, dorn1-1 and dorn1-3 leaves; Col-0 n = 14–17, dorn1-1 n = 16–17, dorn1-3 n = 18 in three independent trials. Each dot represents an individual data point, and the thick black line denotes the median. Different lower-case letters describe groups with significant statistical difference (P ≤ 0.05), while data points with the same letter indicate no statistical significance (P ≥ 0.05; Welch’s two sample t-test).

Fig. 2.

DORN1 governs the root eATP-induced and eADP-induced [Ca²⁺]_cyt increase. (A) Mean ± s.e.m. [Ca²⁺]_cyt time course of individual excised roots from 7-d-old Arabidopsis thaliana Col-0 and dorn1-1 constitutively expressing cytosolic aequorin. Control medium (10 mm CaCl₂, 0.1 mm KCl, 2 mm Tris/MES, pH 5.8) was added at 35 s (black triangle). No significant difference between genotypes was found. (B) Response of Col-0 and dorn1-1 to 0.1 mm ATP. (C) Response to 1 mm ATP. (D) Response to 1 mm ADP. (E) Total [Ca²⁺]_cyt mobilized (estimated as the area under the curve, after subtraction of mean pre-stimulus baseline) in response to control medium, 0.1 or 1 mm ATP, 1 mm ADP. n = 9–15 roots per genotype and treatment in three independent trials. Analysis of variance (ANOVA) with post-hoc Tukey test was used to assess statistical differences. Significance levels (P-values) in B–D: *** (<0.001); D: different lower-case letters indicate P < 0.05.

Fig. 3.

DORN1 abundance declines as primary root cells mature. (A–F) Confocal microscopy images of the different zones of 14-d-old Arabidopsis root expressing a DORN1-GFP fusion from the native promoter. Epidermis in the transition zone (TZ) is marked by brown rectangle, elongation zone (EZ) by pink, and mature zone (MZ) by yellow. Scale bar = 100 μm for all. (G) Background-subtracted DORN1-GFP fluorescence of epidermis in the transition zone (TZ, brown), elongation zone (EZ, pink) and mature zone (MZ, yellow) of 14-d-old Arabidopsis roots. GFP-signal was measured from the plasma membrane of epidermal cells and normalized to cells not expressing GFP; n = 24 (TZ/MZ) and n = 44 (EZ) from six individual plants. Analysis of variance (ANOVA) with post-hoc Tukey test was used to assess statistical differences, and different letters indicate significant difference (P < 0.001).

Fig. 4.

Localized ATP application causes spatially distinct [Ca²⁺]_cyt elevation in the root. (A) A 10-d-old Arabidopsis Col-0 seedling (expressing cytosolic GCaMP3; Vincent et al., 2017) was placed across a gap in the growth medium agar; scale bar: 1 mm. Twenty seconds after the start of image acquisition, 3 µL of control or 1 mm ATP solution was applied to the root tip (indicated by purple ‘1’), and at 295 s to the mature zone (indicated by purple ‘2’), and then imaged for in total 495 s. Regions of interest used for analysis (‘Roi’) are annotated with white boxes. (B–D) Mean ± s.e.m. GFP fluorescence intensity, background-subtracted. (E–G) Normalized mean ± s.e.m. GFP fluorescence (ΔF/F₀; Vincent et al., 2017). (H–J) Extracted normalized fluorescence maxima (ΔF_max/F₀) for ‘Phase 1’ (20–295 s) and ‘Phase 2’ (300–495 s). Data shown in (B, E, H) represent Roi A; (C, F, I) represent Roi B; and (D, G, J) represent Roi C. Data are from three independent trials, with n = 3 individual roots for control treatments and n = 6–9 individual roots per ATP treatment. Significance levels (P-values, Welch’s two sample t-test) in H–J: *** (<0.001), n.s. (>0.05). Assay medium as in Fig. 1A.

Fig. 5.

Localized ATP application to the mature zone causes spatially distinct [Ca²⁺]_cyt elevation in the root. (A) A 10-d-old Arabidopsis Col-0 seedling (expressing cytosolic GCaMP3; Vincent et al., 2017) was placed across a gap in the growth medium agar; scale bar: 1 mm. Twenty seconds after the start of image acquisition, 3 µL of control or 1 mm ATP solution was applied to the mature zone (indicated by orange ‘1’), and at 295 s to the root tip (indicated by orange ‘2’), and then imaged for in total 495 s. Regions of interest used for analysis (‘Roi’) are annotated with white boxes. (B–D) Mean ± s.e.m. GFP fluorescence intensity, background-subtracted. (E–G) Normalized mean ± s.e.m. GFP fluorescence (ΔF/F₀; Vincent et al., 2017). (H–J) Extracted normalized fluorescence maxima (ΔF_max/F₀) for ‘Phase 1’ (20–295 s) and ‘Phase 2’ (300–495 s). Data shown in (B, E, H) represent Roi A; (C, F, I) represent Roi B; and (D, G, J) represent Roi C. Data are from three independent trials, with n = 3 individual roots for control treatments and n = 6–9 individual roots per ATP treatment. Significance levels (P-values, Welch’s two-sample t-test) in H–J: *** (<0.001), n.s. (>0.05). Assay medium as in Fig. 1A.