Atp na2

The phenotypic identification in this study can be divided into three parts. Part 1: To confirm the effect of eATP on hypocotyl change belonging to ethylene triple response, Col-0 seeds were sown on 1/2 MS medium with dark treatment; after germination, the seedlings were screened by a stereoscope (Stemi 2000-C, Zeiss, Oberkochen, Germany) and the hypocotyl length was calculated. Part 2: Col-0, etr1-1, and ein3-1eil1-1 were sown on a 1/2 MS agar plates without or with 100 mM NaCl for phenotypic analysis. Briefly, no fewer than 50 seeds of Col-0 and ethylene-insensitive mutants were sown onto each plate containing 1/2 MS medium subjected to 50 or 100 mM NaCl or not. Part 3: To determine the effect of eATP on the phenotype of ethylene-insensitive mutants, we added ATP-Na₂ (a donor of extracellular ATP, 300 μM, Sigma-Aldrich, St. Louis, MO, USA) on the salt plates (50 or 100 mM) based on a 1/2 MS medium. Each physiological experiment on a phenotype needed to be replicated at least three times. Root length and relative electrolyte leakage were computed after a week since the transfer to the light. The relative electrolyte leakage of the seedlings was evaluated with the method reported in Peever and Higgins [58 (link)].

Cells were placed into buffers containing 140 mM NaCl, 5 mM KCl, 1 mM MgCl₂, 10 mM HEPES and 10 mM glucose, adjusted to pH 7.4; supplemented with 1 mM EGTA for Ca²⁺-free buffer or with 2 mM CaCl₂ for Ca²⁺-containing buffer. For electro-stimulation, cells were placed into buffer containing 150 mM NaCl, 5.4 mM KCl, 2 mM MgCl₂, 10 mM HEPES, 1 mM glucose, 2.5 mM pyruvate, 5 mM creatine, 5 mM taurine and 2 mM CaCl₂. Drugs were added to buffers for stimulations [ATP-Na² (Sigma, A7699); ATP-Mg²⁺ (Sigma, A9187); 1 µM ionomycin (Abcam, ab-120370); 100 µM cyclopiazonic acid (CPA, Sigma, C1530); 1 µM thapsigargin (TG, Sigma, T9033); 1 µM SB216763 (Sigma, S3442); 10 µM TDZD8 (Sigma, T8325); 5 mM caffeine (Sigma, C0750)].

Enzyme activities were assayed in homogenized jejunum tissue. Samples were thawed at 4°C and homogenized in 10 times the volume of cold normal saline. The homogenates were then centrifuged at 20,000 ×g for 20 min at 4°C and the supernatant was collected for enzyme assays. Sucrase [Enzyme Commission (EC) 3.2.1.48] and maltase (EC 3.2.1.20) activity were assayed colorimetrically using sucrose and maltose as substrates, respectively. The activity was expressed as micromoles of glucose released per minute per gram of jejunum wet tissue. Alkaline phosphatase (EC 3.1.3.1) activity was determined by measuring the hydrolysis of p-nitrophenol at 37°C, and the unit of activity was expressed as per min per gram of jejunum wet tissue. Na⁺/K⁺ATPase (EC 3.6.1.3) activity was determined by measuring the liberation of phosphate from ATP-Na2 (No. A7699, Sigma-Aldrich) in two media: medium I (all ATPases system) and medium II (Na⁺/K⁺ATPase restrained system); and the activity of Na⁺/K⁺ATPase was calculated as the difference between phosphates liberated by each homogenate in the two media and was expressed as micromoles of phosphates per milligram homogenate protein or per milliliter of serum per h.