Extracellular ATP in plants. Visualization, localization, and analysis of physiological significance in growth and signaling

Abstract

Extracellular ATP (eATP) in animals is well documented and known to play an important role in cellular signaling (e.g. at the nerve synapse). The existence of eATP has been postulated in plants; however, there is no definitive experimental evidence for its presence or an explanation as to how such a polar molecule could exit the plant cell and what physiological role it may play in plant growth and development. The presence of eATP in plants (Medicago truncatula) was detected by constructing a novel reporter; i.e. fusing a cellulose-binding domain peptide to the ATP-requiring enzyme luciferase. Application of this reporter to plant roots allowed visualization of eATP in the presence of the substrate luciferin. Luciferase activity could be detected in the interstitial spaces between plant epidermal cells and predominantly at the regions of actively growing cells. The levels of eATP were closely correlated with regions of active growth and cell expansion. Pharmacological compounds known to alter cytoplasmic calcium levels revealed that ATP release is a calcium-dependent process and may occur through vesicular fusion, an important step in the polar growth of actively growing root hairs. Reactive oxygen species (ROS) activity at the root hair tip is not only essential for root hair growth, but also dependent on the cytoplasmic calcium levels. Whereas application of exogenous ATP and a chitin mixture increased ROS activity in root hairs, no changes were observed in response to adenosine, AMP, ADP, and nonhydrolyzable ATP (betagammameATP). However, application of exogenous potato (Solanum tuberosum) apyrase (ATPase) decreased ROS activity, suggesting that cytoplasmic calcium gradients and ROS activity are closely associated with eATP release.

Figure 1.

Use of the CBD-luciferase reporter for live imaging of eATP distribution on the root surface of M. truncatula. A, Dose-response curve for ATP showing light intensity was strictly dependent on ATP concentration. B, Light production was dependent on the presence of ATP and substrate (luciferin). C, Projection of 36 confocal optical sections (700 nm each) showing an overview of a live root segment demonstrating eATP on several root hairs as revealed by CBD-luciferase assay; raw data pseudocolored green. D, Intensity-coded (inset range in D, where white areas show the strongest ATP concentration and black is the background) image of C showing ATP concentration gradients. All other images are single optical sections (700 nm thick). E, Section of a root surface showing increased eATP secretion in the interstitial spaces of epidermal cells. F, Medicago truncatula (A17) root hair showing light production primarily at the root hair tip. CBD-luciferase light production was greatly stimulated by the addition of exogenous ATP (100 μm; G) and by chitin elicitation (100 μg/mL chitin mixtures; H). Light production was decreased by the addition of potato apyrase (ATPase; 25 units/mL; 1 unit will liberate 1 μm of inorganic phosphate from ATP or ADP per min at pH 6.5 at 30°C; I) or 100 μm nonhydrolyzable ATP (βγmeATP; J). A negative control (i.e. without added luciferin) showing that there was no autofluorescence in root hairs in the absence (K) or presence (L) of CBD-luciferase treatment. Light production upon CBD-luciferase treatment was seen in the root hairs of a variety of plants. M, Arabidopsis (Columbia-0). N, T. aestivum (ET8). O, L. japonicus (Gifu). Light production in Figure 1, A and B, was measured using a luminometer (Veritas microplate luminometer; Turner BioSystem). All root hairs presented are captured in and around region B in Figure 5I. Scale bar = 10 μm.

Figure 2.

ATP distribution profile in M. truncatula root and enhanced eATP secretion is restricted to actively growing regions of the plant. I, All images are single confocal optical sections (700 nm). M. truncatula root (left, bright-field image) was divided into three sections designated as A, B, and C. The root apex is marked with an asterisk (*). Representative images showing light production from CBD-luciferase are shown (right) and correspond to the three regions. White areas show the strongest eATP concentration and black is the background (see scale; Fig. 1). II, CBD-luciferase activity was detected at interstitial spaces at different growth regions of the root (i.e. meristematic region [A and B], apical elongation region [C], basal elongation region [D], mature region [E], and epidermal cells of the etiolated hypocotyl [F]). White boxes were drawn on the images to highlight the areas of interstitial spaces between epidermal cells. Scale bar = 25 μm.

Figure 3.

Quantification of eATP and ROS intensity of M. truncatula. A, Quantification of ATP based on light intensity for the images presented in Figures 1 and 5I. Images were analyzed for intensity average in the Metamorph program, version 6.5 (Molecular Devices). Measurements were made at the root apices and if any contrast adjustments were needed, the adjustments were applied to all root hairs prior to analysis. Values (n > 8) are mean ± se in an experiment and representative of at least two independent experiments. B, Quantification of ROS production based on intensity for the images presented in Figure 6I. Images were analyzed for intensity (average) in the Metamorph program, version 6.5. Measurements were made at the root apices. Whenever contrast adjustments were needed, the adjustments were applied to all root hairs prior to analysis. Values (n > 8) are mean ± se in an experiment and representative of at least two independent experiments. C, Quantification of ATP based on intensity for the images presented in Figure 2I. A (proximal), B (middle), and C (distal) refer to the three root regions shown in Figure 2I. Images were analyzed for intensity average/sum in the Metamorph program, version 6.5. Measurements were made at the root hair apices. Whenever contrast adjustments were needed, the adjustments were applied to all root hairs identically prior to analysis. Values (n > 8) are mean ± se in an experiment and representative of at least two independent experiments.

Figure 4.

Immunofluorescence and immunogold labeling of CBD-luciferase protein showing uniform localization around the root hair and interstitial spaces of the root surface. A to F, Images are after fluorescent antibody (Ab) labeling. CBD-luciferase protein was localized using a mouse monoclonal antibody followed by Alexa fluor 568 conjugated secondary antibody on M. truncatula roots pretreated with CBD-luciferase protein. Root hair incubated with CBD preimmune serum and secondary antibody (A), control root hair incubated with both primary (anti-CBD) and secondary antibody (B), root hair incubated with luciferase preimmune serum (C and F), and root hair incubated with both primary (anti-luciferase) and secondary antibody (D and E). E and F, From the elongation region of the root apex (projection of 17 confocal optical sections, 700 nm each). All fluorescent images are single confocal optical sections (700 nm), except E and F. G to L, Images are after immunogold (10-nm gold particles) labeling with gold enhancement after binding of luciferase preimmune antibody or primary (anti-luciferase) antibody and obtained in a confocal microscope under reflection mode. G, I, and K are transmitted images of H, J, and K. Root hair incubated with luciferase preimmune serum and secondary antibody (H) and primary (anti-luciferase) antibody and secondary antibody (J and L). All root hairs presented are captured in and around region B in Figure 2I. Scale bar in A to D and G to L = 500 μm; E and F = 10 μm.

Figure 5.

ATP secretion is dependent on cytoplasmic calcium and ATP distribution is extracellular and localized at the plant cell wall. I, All images are single confocal optical sections (700 nm). A, Optical section of a root hair treated with CBD-luciferase showing eATP distribution. Similar root hairs treated with pharmacological agents for 1 h prior to CBD-luciferase application are shown in B (GdCl₃), C (LaCl₃), D (BAPTA), E (CaCl₂), and F (brefeldin A). See Fig. 3A for quantification of these treatments. Intensity-coding strip shown in A, where white areas show the strongest eATP concentration and black is the background. Scale bar = 10 μm. II, Optical section showing eATP (CBD-luciferase light activity) distribution on the root hair surface after plasmolysis with 5 m NaCl (30 min; A); transmission image of the same root hair showing the plasma membrane is retracted from the cell wall (B); and overlay of A and B, showing that light production was associated with the cell wall (C). All root hairs presented are captured in and around region B in Figure 3I. Scale bar = 500 μm.

Figure 6.

ROS profile and a time course analysis of ROS production in root hairs. I, All images are single confocal optical sections (700 nm). Optical section of control root hair treated with CM-H₂DCFDA showing ROS fluorescence (A); similar root hair applied with 1 mm ATP (B); 1 mm nonhydrolyzable ATP (βγmeATP; C); 1 mm adenosine (D); 1 mm AMP (E); 1 mm ADP (F); and 100 μg/mL chitin mixture (G). H, Exogenous potato apyrase (ATPase; 25 units/mL; 1 unit will liberate 1 μm of inorganic phosphate from ATP or ADP per min at pH 6.5 at 30°C) decreased ROS levels. I, Potato apyrase was applied after chitin treatment (30 min). There was also no fluorescence in the ROS negative control roots (J; i.e. in the absence of the CM-H₂DCFMA indicator dye), compared to control (A). Scale bar = 500 μm. II, There was no change in control (top, without added ATP) ROS levels and basal levels were maintained for at least 15 min. Addition of exogenous ATP increased ROS activity that gradually decreased to a basal level within 15 min (bottom). All root hairs presented are captured in and around region B in Figure 3I.