Total phosphatidic acid assay kit

Total PA content was measured by a coupled enzymatic reaction system using the Total Phosphatidic Acid Assay Kit (Cell Biolabs, Inc., San Diego, CA.). Experimental procedures were performed according to the manufacturer’s protocol with minor modifications. In brief, vegetative gametophytes from blades approximately 20 mm in length (0.05 g FW) were cultured in a 100 mL culture medium containing 50 μM ACC for 0, 3, and 7d. Samples were then ground in liquid nitrogen with a pestle and mortar. For each sample, the resulting homogenate was added to 0.75 mL methanol. Next, we added 1.15 mL 1 M NaCl and 1.25 mL chloroform to each sample and mixed the constituents thoroughly. After centrifugation at 4 °C for 10 min at 1500×g, the upper aqueous phase was discarded, and the lower chloroform phase was washed twice with a PEU solution, which was prepared by mixing 50 mL chloroform, 50 mL methanol, and 45 mL 1 M NaCl. Finally, the lower organic phase was dried under a gentle stream of nitrogen and dissolved in the provided Assay Buffer. Fluorescent signals were detected using a spectrofluorometer (FB-750, Jasco, Tokyo, Japan) at an excitation wavelength of 540 nm and an emission wavelength of 590 nm. Relative PLD production was quantified as a ratio of that PLD production at 0 d after ACC treatment. All data are presented as mean ± SD of five biological replicates.

PLD1 activity was estimated using the Amplex Red phospholipase D assay kit (Thermo Fisher) according to the manufacturer’s protocol using a FilterMax F5 multimode microplate reader (Molecular Devices). Phosphatidic acid production was determined using the Total Phosphatidic Acid assay kit (Cell Biolabs) according to the manufacturer’s protocol using a FilterMax F5 multimode microplate reader. VU0359595 and VU0155069 were purchased from Santa Cruz Biotechnology and were used at 1 μM for 1 h before treatment.