Extracellular ATP functions as an endogenous external metabolite regulating plant cell viability

Abstract

ATP is a vital molecule used by living organisms as a universal source of energy required to drive the cogwheels of intracellular biochemical reactions necessary for growth and development. Animal cells release ATP to the extracellular milieu, where it functions as the primary signaling cue at the epicenter of a diverse range of physiological processes. Although recent findings revealed that intact plant tissues release ATP as well, there is no clearly defined physiological function of extracellular ATP in plants. Here, we show that extracellular ATP is essential for maintaining plant cell viability. Its removal by the cell-impermeant traps glucose-hexokinase and apyrase triggered death in both cell cultures and whole plants. Competitive exclusion of extracellular ATP from its binding sites by treatment with beta,gamma-methyleneadenosine 5'-triphosphate, a nonhydrolyzable analog of ATP, also resulted in death. The death response was observed in Arabidopsis thaliana, maize (Zea mays), bean (Phaseolus vulgaris), and tobacco (Nicotiana tabacum). Significantly, we discovered that fumonisin B1 (FB1) treatment of Arabidopsis triggered the depletion of extracellular ATP that preceded cell death and that exogenous ATP rescues Arabidopsis from FB1-induced death. These observations suggest that extracellular ATP suppresses a default death pathway in plants and that some forms of pathogen-induced cell death are mediated by the depletion of extracellular ATP.

Figure 1.

Extracellular ATP and Cell Viability of Arabidopsis Cell Cultures. (A) ATP and phosphate (PO₄) standards (lane 1) and [³²P]H₃PO₄ fed to three independent cell cultures and medium aliquots taken at time 0 (lane 2) and 1 h later (lanes 3 to 5) showing the de novo synthesis and secretion of ATP. At 1 h, solutions of ATP traps were added, and 6 h later, medium samples were taken from the control culture (lane 6) and from cultures treated with 100 units/mL apyrase (lane 7) and glucose–hexokinase (lane 8). (B) Line graph of a pixel intensity profile showing relative levels of ATP in the 6-h samples (lanes 6 to 8) in (A). (C) Plasma membrane integrity, measured by Evans blue staining, of cell cultures in (A) over the first 1 h. (D) Dose response of cell viability to treatment with apyrase. (E) ATP and phosphate standards (lane 1), [³²P]H₃PO₄ (lane 2), and medium aliquots 1 h after adding radioactive phosphate (lanes 3 and 4) and just before adding 20 units/mL apyrase. Medium from control cells (lane 5) and cells treated with 20 units/mL apyrase (lane 6) 6 h after enzyme treatment. (F) Line graph of a pixel intensity profile across the ATP bands in lanes 5 and 6 of (E). Data and error bars represent means ± sd (n = 3). AU, arbitrary units.

Figure 2.

Effects of Various Treatments on the Viability of Arabidopsis Cell Cultures. (A) Effect of dialyzing or denaturing apyrase (Aps; 100 units/mL) and hexokinase (Hk; 200 units/mL) on cell viability. (B) Effect of various solutions on cell viability. Glc-6-P, glucose-6-phosphate. (C) Dose response of cell viability to glucose–hexokinase treatment. (D) Dose response of cell viability to AMP-PCP treatment. Data and error bars represent means ± sd (n = 3).

Figure 3.

Death of Intact Arabidopsis Tissues after Treatment with Extracellular ATP Traps. Top row, leaves treated on localized areas that developed localized lesions. Bottom row, entire leaves treated and beginning to die. These leaves eventually died except for the controls. Similar results were obtained when the solutions were infiltrated with a needle or needleless syringe. Glc-Hk, glucose–hexokinase. Bar = 6 mm.

Figure 4.

Death of Hydroponic Arabidopsis Plants Caused by Glucose–Hexokinase. (A) Healthy leaves of plants treated with glucose. (B) Dying leaves of plants treated with glucose–hexokinase. (C) Close-up photograph of leaves from plants treated with glucose–hexokinase showing both chlorosis and brown lesions of localized tissue death. (D) and (E) Roots of glucose-treated plants showing normal growth (D) and retarded roots of glucose–hexokinase-treated plants (E) 7 d after treatment. Fluorescein diacetate staining and microscopy confirmed that the roots in (E) had reduced viability.

Figure 5.

Glucose–Hexokinase Causes the Death of Arabidopsis Roots. Top row, root tissue from plants treated with glucose. Bottom row, root tissue from plants treated with glucose–hexokinase (Glc-Hk). Red channel, confocal images showing autofluorescence of the root tissues. Green channel, confocal images of fluorescein diacetate signals emitted by the same samples. White channel, images of the same samples captured using transmitted light under phase contrast. The extreme right column shows the merged images of the preceding three panels. It is clear that the glucose–hexokinase treatment caused the death of the roots, as there was no fluorescein diacetate signal in these samples. Bars = 200 μm.

Figure 6.

Death Triggered by Noninvasive Application of an ATP Analog. Arabidopsis plants were germinated on nutrient agar and transferred to plates with agar supplemented with 1 mM ATP (A) or 1 mM AMP-PCP (B). Photographs were taken after 4 weeks. (C) and (D) show close-up photographs of representative plants from (A) and (B), respectively. It is apparent that AMP-PCP caused the death of the treated plants.

Figure 7.

Reaction of Bean and Tobacco to Extracellular ATP Removal. (A) Control treatments with water (1), BSA (2), AMP/ADP (3), and glucose/glucose-6-phosphate/ADP (4) of tobacco. (B) Local lesions in response to apyrase (5) or glucose–hexokinase (6) treatment of tobacco. (C) Localized areas of a tobacco leaf treated with ATP (7) or AMP-PCP (8). (D) Tobacco treated with ATP. The arrow indicates the treated leaf. (E) Tobacco treated with AMP-PCP on the indicated leaf. (F) Untreated bean plant. (G) Water-treated (black arrow) and apyrase-treated (white arrow) bean leaves. (H) Bean leaves treated with glucose (black arrow) or glucose–hexokinase (white arrow). Bars = 2 cm for (A) to (C), 4 cm for (D) and (E), and 2 cm for (F) to (H).

Figure 8.

Extracellular ATP Is Required for the Viability of Maize Cell Cultures. Viability of maize cell suspension cultures after treatment with water (control), 1 mM ATP, 1 mM AMP-PCP, 100 units/mL apyrase, or 50 mM glucose + 200 units/mL hexokinase (Glc-Hk). Data and error bars represent means ± sd (n = 3).

Figure 9.

FB1 Treatment Induces the Depletion of Extracellular ATP That Precedes the Loss of Membrane Integrity. Light-grown Arabidopsis cell cultures were treated with methanol (control) or FB1 and spiked with [γ-³²P]ATP. (A) Growth medium aliquots from triplicate control (lanes 2 and 3) or FB1-treated (lanes 4 to 6) cultures 24 h after treatment and separated by TLC. Lane 1 contains free phosphate (PO₄) and ATP standards. (B) Level of radioactive ATP in growth medium (as a percentage of radioactivity initially added to the cell cultures) between 16 and 24 h. Gray bars, control cultures; white bars, FB1-treated cultures. (C) Changes in the integrity of the plasma membrane over 96 h in control (black bars) and FB1-treated (white bars) cell cultures assayed by the Evans blue method. Data and error bars represent means ± sd (n = 3).

Figure 10.

Extracellular ATP Attenuates FB1-Induced Cell Death in Arabidopsis Cell Cultures. (A) Light-grown cell cultures at a density of 5% (w/v) were treated with 1 μM FB1 at time 0 and spiked with 1 mM ATP at the given times. Cells were harvested at 120 h and incubated with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoium bromide (MTT) solution. (B) Cell cultures were treated with 1 μM FB1 at time 0, and increasing concentrations of ATP were added 40 h later. Cells were harvested at 120 h and processed as in (A). The formation of the purple formazan is an indication of viability; unstained cells are dead; + or – indicates addition or omission of the indicated compound, respectively.

Figure 11.

Nucleoside Triphosphates Rescue Arabidopsis Cells from FB1-Induced Death. (A) Light-grown cell cultures adjusted to a density of 5% (w/v) were treated with either 0.014% methanol (control) or 1 μM FB1 mixed with 1 mM of the indicated compounds. Cells were harvested 120 h later, and viability was determined via the MTT assay. (B) Cell cultures were treated with water (control), 1 mM AMP, or 1 mM UTP and ATP in the growth medium assayed by the luciferin–luciferase method. Data and error bars represent means ± sd (n = 3).

Figure 12.

ATP, but Not Phosphate, Rescues Arabidopsis Plants from FB1-Induced Death. (A) Hydroponic plants were treated with methanol (control) or 1 μM FB1 mixed with the indicated concentrations of ATP. Roots were stained 72 h later with Evans blue to determine viability. Bar = 1 μm; + or – implies addition or omission of the indicated compound, respectively. (B) Arabidopsis plants were germinated on nutrient agar and transferred after 5 d to plates with agar supplemented with 0.014% methanol (control), 1 μM FB1, or 1 μM FB1 mixed with either 1 mM ATP or 1 mM KH₂PO₄. Top row, retardation of growth in all treatments containing FB1. Middle row, close-up photographs of representative plants from corresponding plates in the top row. Bottom row, root tips from hydroponic plants subjected to the same treatments for 72 h and stained with Evans blue to reveal cell death. It is clear from the middle and bottom rows that only ATP was able to rescue the plants from FB1-induced death. Bar = 1 μm for the bottom row.