Heterotrimeric G Protein-Regulated Ca2+ Influx and PIN2 Asymmetric Distribution Are Involved in Arabidopsis thaliana Roots' Avoidance Response to Extracellular ATP

Abstract

Extracellular ATP (eATP) has been reported to be involved in plant growth as a primary messenger in the apoplast. Here, roots of Arabidopsis thaliana seedlings growing in jointed medium bent upon contact with ATP-containing medium to keep away from eATP, showing a marked avoidance response. Roots responded similarly to ADP and bz-ATP but did not respond to AMP and GTP. The eATP avoidance response was reduced in loss-of-function mutants of heterotrimeric G protein α subunit (Gα) (gpa1-1 and gpa1-2) and enhanced in Gα-over-expression (OE) lines (wGα and cGα). Ethylenebis(oxyethylenenitrilo) tetraacetic acid (EGTA) and Gd³⁺ remarkably suppressed eATP-induced root bending. ATP-stimulated Ca²⁺ influx was impaired in Gα null mutants and increased in its OE lines. DR5-GFP and PIN2 were asymmetrically distributed in ATP-stimulated root tips, this effect was strongly suppressed by EGTA and diminished in Gα null mutants. In addition, some eATP-induced genes' expression was also impaired in Gα null mutants. Based on these results, we propose that heterotrimeric Gα-regulated Ca²⁺ influx and PIN2 distribution may be key signaling events in eATP sensing and avoidance response in Arabidopsis thaliana roots.

Keywords: Arabidopsis thaliana L.; auxin; calcium; extracellular ATP; heterotrimeric G protein.

Figure 1

Extracellular ATP avoidance response of Arabidopsis roots. (A) Seedlings of Arabidopsis thaliana (WS and Col-0 ecotype) grown in jointed medium. Note the bending roots. The concentration of ATP in the lower part is marked on top of the photos. (B) Time-lapse images of seedlings grown in jointed medium without (control) or with 0.5 mM ATP (ATP) in the lower part. The photograph time is marked on top of the photos. (C) Seedlings grown in U-shaped (left) or diagonal (right) jointed medium containing 0.5 mM ATP. Note the ATP avoidance response of roots. The small white arrows in the figure mark the different bending styles (bent to left or right) of seedlings at different position. (D) Seedlings grown in jointed medium containing 0.5 mM of a non-hydrolyzable ATP analog (bz-ATP) or some nucleotide phosphates (ADP, AMP or GTP). In (B–D) WS was used as material. Seedlings in (A,C,D) were photographed 5 days after transplantation. Triangles in (A,B,D) mark the joint line of the two media. In (C) the joint line of the two media is marked with a dotted line. The scale bar is showed on right side of (A).

Figure 2

Heterotrimeric Gα is involved in eATP avoidance response. (A) Seedlings of WS, Gα null mutants (gpa1-1, gpa1-2) and OE lines (wGα and cGα) grown in jointed medium containing 0, 0.3, and 0.5 mM ATP in the lower part, respectively. Note the different response of various genotypes to ATP. Seedlings were photographed 5 days after transplantation. In each photo series, the triangle marks the joint line of the two media. The scale bar is showed beside. (B,C) note the effect of ATP on root growth rate (B) and root curvature (C), respectively. In each experiment, root growth rate and curvature of at least 30 seedlings were measured, and data from 3 replicates were calculated to get the mean ± SD. Student's t-test p-values: ^*p < 0.05, ^**p < 0.01.

Figure 3

Ca²⁺ influx is necessary for eATP avoidance responses of Arabidopsis roots. (A) Seedlings of WS grown in jointed medium. EGTA (0.5 mM) or Gd³⁺ (50 μM) were added into 1/2 MS medium, then 0.3 mM ATP was added into the lower part of the jointed medium. The triangle marks the joint line of the two media. The scale bar is showed below. (B,C) note the root growth rate (B) and root curvature (C) before and after ATP treatments. Seedlings were photographed 5 days after transplantation. Primary root growth rate and curvature were measured in at least 30 seedlings, and data from 3 replicates were calculated to get the mean ± SD.

Figure 4

Heterotrimeric Gα is involved in eATP-stimulated Ca²⁺ influx in root cells. Ca²⁺ influx across the PM of root cell protoplasts was detected by whole-cell voltage clamping. Results from step-(1st to 3rd column) and ramp-(4th column) voltage clamping recordings in wild type (WS), Gα null mutants (gpa1-1 and gpa1-2) and OE lines (cGα and wGα). In each line, current traces before (control), and after 0.1 mM ATP treatment are shown in the 1st and 2nd column, respectively. The 3rd column shows the I–V relationship curve of step-voltage clamping (n = 6). The 4th column notes the current traces before and after 0.1 mM ATP treatment which were recorded by slow ramp-voltage clamping. The genotype is marked below each line on the left. The time/current intensity scale bar is showed on the lower left of the first line.

Figure 5

ATP-induced asymmetric distribution of DR5-GFP and PIN2-GFP in root tip cells. DR5-GFP or PIN2-GFP transgenic WS was used as material. Seedlings were grown in 1/2 MS medium for 4 days and transplanted onto 0.5 mM ATP-containing jointed medium. After 12, 24, 36, and 48 h, the fluorescence of DR5-GFP or PIN2-GFP in root tip cells was detected using confocal laser scanning microscopy (CLSM). (A) Fluorescence in representative root tips. In each series, the upper and lower line note the images of fluorescence and merged fluorescence/transmission, respectively. The scale bar is showed below. (B,C) note time-lapse analysis result of relative fluorescence intensity (the ratio of total fluorescence intensity after/before ATP treatment) of DR5-GFP (B) or PIN2-GFP (C) in root tip cells, respectively. (D,E) Note time-lapse analysis results of the ratio of fluorescence intensity (fluorescence intensity in right-side cells relative to that in left-side cells) of DR5-GFP (D) and PIN2-GFP (E), respectively. In each experiment, fluorescence intensity in up to 10 roots was measured. Data from 3 replicates were calculated to get the mean ± SD. Student's t-test p-values: ^*p < 0.05.

Figure 6

PIN2 null mutant did not respond to eATP. (A) Seedlings of Col-0 and PIN2 null mutant (pin2) grown in U-shape jointed medium containing 0 (control) or 0.5 mM ATP (ATP) in the lower part and two sides, respectively. Seedlings were photographed 5 days after transplantation. The dotted line marks the joint line of the two media. The scale bar is showed below. (B) Effect of ATP on root curvature. Root curvature of at least 30 seedlings was measured, and data from 3 replicates were calculated to get the mean ± SD. Student's t-test p-values: ^**p < 0.01.

Figure 7

Ca²⁺ is involved in eATP-induced asymmetric distribution of DR5-GFP and PIN2-GFP. DR5-GFP or PIN2-GFP transgenic WS seedlings grown in 1/2 MS medium for 4 days were transplanted onto jointed medium containing 0.5 mM EGTA (in both the upper and the lower part) and 0.5 mM ATP (in the lower part only). After 24 h, fluorescence was detected by using CLSM. (A) Fluorescence in representative root tip cells. The scale bar is showed below. (B,C) note the fluorescence intensity ratio (fluorescence intensity in right-side cells relative to that in left-side cells) of DR5-GFP (B) and PIN2-GFP (C), respectively. In each experiment, fluorescence intensity in up to 10 roots was measured. Data from 3 replicates were calculated to get the mean ± SD. Student's t-test p-values: ^*p < 0.05.

Figure 8

Heterotrimeric Gα is involved in eATP-induced asymmetric distribution of DR5-GFP and PIN2-GFP. DR5-GFP or PIN2-GFP transgenic WS, gpa1-1 and gpa1-2 seedlings were grown in 1/2 MS medium for 4 days and then transplanted onto 0.5 mM ATP-containing jointed medium. After 24 h, fluorescence was detected by using CLSM. (A) Fluorescence in representative root tip cells. The scale bar is showed below. (B,C) note fluorescence intensity ratio (fluorescence intensity in right-side cells relative to that in left-side cells) of DR5-GFP (B) and PIN2-GFP (C), respectively. In each experiment, fluorescence intensity in up to 10 roots was measured. Data from 3 replicates were calculated to get the mean ± SD. Student's t-test p-values: ^*p < 0.05.

Figure 9

Heterotrimeric Gα is involved in eATP-regulated functional gene expression. Seedlings grown in 1/2 MS medium for 4 days were transplanted onto a 0.5 mM ATP-containing jointed medium. After 30 and 60 min, roots were cut to extract total RNA, and the expression levels of several functional genes were measured by real-time qPCR. The relative level (fold of change) of gene expression in WS and Gα null mutants (gpa1-1 and gpa1-2) is showed. Gene names and the corresponding gene loci are noted in each figure. Data from 3 replicates were calculated to get mean ± SD. Student's t-test p-values: ^*p < 0.05, ^**p < 0.01.