Free IAA and SA quantification
To quantify free IAA and SA in the rice, three genotypic lines of OE,
knockout, and NIP seedlings (30 days) were used. First, to prepare the
extract solution, 0.1 g cross-linked polyvinylpyrrolidone was added to
10 mL mod Bielesk solution with a methanol: water: formic acid volume
ratio of 75:20:5. Next, 100 mg fresh seedlings were ground in liquid
nitrogen, collected in a 1.5 mL tube containing 0.5 mL extract solution,
and ultrasonicated for 30 min in ice water, mixing and standing for 16
h. Subsequently, 1 mL dichloromethane was added to the tube and
ultrasonicated for 1 h. The mixture was then centrifuged for 15 min at 4
°C. The subnatant was transferred into a new 1.5 mL tube and dried with
blown nitrogen. The dried extract was then re-dissolved by adding 500 μL
chromatographic grade methanol aqueous solution (0.1% formic acid), and
filtered with a 0.2 μM NYLON66 organic filter membrane to obtain the
extraction solution containing plant hormones. Furthermore, the
extraction solution was purified via solid-phase extraction (SPE) using
a Waters C18-SPE column (1cc/100 mg; WAT023590), and eluted with 0.8 mL
methanol and 20 µL 50% acetic acid, which was analyzed using an Acquity
UPLC system (Waters e2695, USA) on a BEH C18 column (2.1 × 150 mm, 1.7
μm, Waters PN:186002353). The concentrations of IAA and SA (ng/g of
fresh seedlings) were determined by comparing them with standard curves
of IAA and SA peak areas.