1 naphthylacetic acid naa

Stock solutions of Epibrassinolide (BL, Sigma-Aldrich, St. Louis, MO, USA), methyl jasmonate (MeJA, TCI, Tokyo, Japen), 1-naphthylacetic acid (NAA, Sigma-Aldrich, St. Louis, MO, USA), indole-3-acetic acid (IAA, Sigma-Aldrich, St. Louis, MO, USA), and salicylic acid (SA, Sigma-Aldrich, St. Louis, MO, USA) were prepared in 100% ethanol and diluted to the appropriate concentration with sterile distilled water containing 0.1% Triton X-100 for experiments. The same volume of ethanol with 0.1% Triton X-100 was used as a mock control. To evaluate the effects of BL, NAA, IAA, MeJA, and SA on RAVs, rice seedlings were sprayed with 10 μmol BL, 5 μmol NAA, 5 μmol IAA, 100 μmol MeJA, and 500 μmol SA, respectively. Eighty seedlings per hormonal treatment were sprayed, and samples were collected for testing at 3, 6, and 12 h.

Ten‐day‐old Huaidao No. 5 seedlings were grown in glass beakers (35–40 seedlings per beaker) and sprayed with 25 mL 1 μM 2,4‐dichlorophenoxyacetic acid (2,4‐D, Sigma‐Aldrich), 1 μM 1‐naphthylacetic acid (NAA, Sigma‐Aldrich, St Louis, MO, USA) or 100 μM DPI (MedChemExpress, Monmouth Junction, NJ, USA). Solutions also contained 0.1% Triton X‐100 and control plants were sprayed with 25 ml 0.1% Triton X‐100. For RT‐qPCR, samples of eight to ten plants per beaker were collected and pooled 0, 3, 6 or 12 h after spraying. Each experiment used not less than three biological repeats.

Cellulase R10 and Macerozyme R10 for enzymatic digest of leaf tissue were purchased from Melford Biolaboratories Ltd. Buffer W5 was 154 mM NaCl, 125 mM CaCl₂, 5 mM KCl, 2 mM MES pH 5.7. MMG solution was 0.4 M mannitol, 15 mM MgCl₂, 4 mM MES pH 5.7. Buffer W1 was 0.5 M mannitol, 20 mM KCl and 4 mM MES pH 5.7. Substances for hormonal treatments were abscisic acid (ABA), 1-naphthylacetic acid (NAA), trans-zeatin (t-zeatin), salicylic acid (SA) and methyl jasmonate (MeJA) and were purchased from Sigma Aldrich. Mock-treated wells received the amount of solvent present in the medium concentration of the three hormonal treatments or water. For analysis of marker specificity the treatment concentrations were 10 μM ABA, 500 nM NAA, 20 μM t-zeatin, 30 μM SA and 50 μM MeJA.
Lysis buffer was prepared as 5-fold stock solution using 125 mM Tris / H₃PO₄ (pH 7.8), 10 mM DTT, 10 mM DACTAA (Sigma D1383), 50% (v/v) glycerol, 5% (v/v) Triton X-100. LUC substrate was prepared using beetle luciferin (Promega E1602) as 1 mM luciferin, 30 mM HEPES (pH 7.8), 3 mM ATP (Sigma 797189) and 15 mM MgSO₄. GUS substrate was prepared using MUG (4-Methylumbelliferyl-β-D-glucuronide, Melford Biolaboratories Ltd. M65900) as 1 mM MUG, 10 mM Tris / HCl (pH 8.0) and 2 mM MgCl₂.