Aminoethoxyvinylglycine avg

The seeds were surface-sterilized as described previously^{63 (link)}, wiped dry with paper towels, and then stored in gas-tight containers with silica gel beads for three days before the experiments. For the germination assays of the 14 inbred lines under normal conditions or DE3 and BY815 under chemical treatment, the seeds were immersed in distilled water, 100 µM 1-aminocyclopropane-1-carboxylic acid (ACC) (Sigma–Aldrich, St. Louis, MO), 100 μM silver nitrate (Ag⁺) (Sigma–Aldrich), or 10 μM aminoethoxyvinylglycine (AVG) (Sigma–Aldrich) in Petri dishes containing two sheets of moist filter papers on the bottom and two layers of wet gauze on top of the seeds. The seeds were germinated in a growth chamber (28 °C, constant light) for seed imbibition, and germination was assessed based on the visual identification of radicle protrusion (approximately 1 mm) at the indicated time points. The number of germinated seeds was recorded as a percentage (%) of the total number of seeds tested.

A. rhizogenes-mediated root transformation of M. truncatula was performed as previously described [72 (link)]. For treatments with rhizobium LCOs, compound plants were transferred to agar-solidified buffered nodulation medium (BNM; 0.9 % Daishin agar (Duchefa, Haarlem, The Netherlands)) [73 (link)] containing 1 μM aminoethoxyvinylglycine (AVG) (Sigma, St. Louis, USA). After 3 days, ~100 μl of rhizobium LCOs (~10^-9 M) was pipetted on top of the root susceptible zone. After 3 h, roots were fixed in 90 % acetone and subsequently stained for GUS activity. For nodulation assays, compound plants were transferred to perlite and watered with Fåhraeus [74 (link)] medium without nitrate. One week after transfer, plants were inoculated with S. meliloti strain 2011 (OD₆₀₀ = 0.05-0.1). For the phosphate starvation experiment, compound plants were transferred into perlite and watered with half-strength Hoagland medium [75 ]. After one week, plants were removed from perlite and washed three times with demineralized water to get rid of the nutrient salts. Plants were re-planted in fresh perlite and watered with half-strength Hoagland medium with (200 μM PO₄^3-) or without (0 μM PO₄^3-) phosphate, respectively. After 5 days, plants were removed from perlite and stained for GUS activity.

Ethylene gas (99.5%) was purchased from GL Sciences, Inc. Japan. 1-Aminocyclopropane-1-carboxylic acid (ACC) and aminoethoxyvinylglycine (AVG) were from Sigma–Aldrich through Hirose kagaku, Ltd, Kobe, Japan; cobalt chloride (CoCl₂) and N-(1-naphthyl)phthalamic acid (NPA), from Wako Pure Chemical Industries, Osaka, Japan and Tokyo Chemical Industry, Tokyo, Japan, respectively. Ethylene was diluted with air, and the other reagents were dissolved in distilled water.