Fb 750

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.

Phospholipase D (PLD) activity was determined using an Amplex™ Red Phospholipase D Assay Kit (Thermo Fisher Scientific K.K., Tokyo, Japan), according to the manufacturer’s protocol with minor modifications. For the PLD activity assay, vegetative gametophytes from blades approximately 20 mm in length (0.05 g fresh weight; 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 0.5 mL of 50 mM Tris-HCl buffer at pH 8.0 and centrifuged at 4 °C for 5 min at 15,000×g. Enzyme activity was detected by adding 100 μL of the supernatant to the working solution of the Amplex Red reagent. The fluorescence was detected using a spectrofluorometer (FB-750, Jasco, Tokyo, Japan) at an excitation wavelength of 545 nm and an emission wavelength of 610 nm to decrease interference from autofluorescence caused by photosynthetic pigments. The resulting fluorescence was subtracted from the background fluorescence derived from samples that did not contain the Amplex Red reagent working solution. Relative PLD activity was calculated as a ratio of the measurement taken at 0 d to that of another taken after ACC treatment. All data are presented as mean ± standard deviation (SD) of five biological replicates.

The vegetative gametophytes (0.03-g fresh weight; FW) cultured in 100-ml medium containing 500-μM ACC were incubated at 15°C in a Petri dish with 10-ml PES medium containing 5-μM 2',7'-dichlorofluorescein diacetate (DCFH-DA, FUJIFILM Wako Pure Chemical Corporation) for 1 h. After incubation, algal thalli were rinsed in seawater, blotted dry, and ground in a mortar with a pestle under liquid nitrogen and extracted in 1 ml of 40-mM Tris-HCl buffer at pH 7.0. The homogenate was centrifuged at 10,000 ×g for 10 min and 500 μl of the supernatant was diluted to 2.5 ml with Tris-HCl buffer and used to measure fluorescence at 488 nm (excitation wavelength) and 525 nm (emission wavelength) with a spectrofluorometer (FB-750, Jasco, Tokyo, Japan). The data are presented as means ± SD of three independent experiments.