S30 t7 high yield protein expression system

In order to analyze the activity of MjTTL5 and AtFTRc, the proteins were expressed by the S30 T7 High‐Yield Protein Expression System (Promega) and purified (Fig. S5); the proteins were diluted using EP buffer (0.01 M phosphate‐buffered saline (PBS), pH = 5.8, 500 mM imidazole).
The MjTTL5 activity analysis was performed as follows: 1 μl (100 ng) MjTTL5 or AtFTRc protein and PET28 protein was added to 10 μl Arabidopsis root total protein (1 μg), and then incubated at 4°C for 10 min. These mixtures were then added to 189 μl substrate solution (the substrate solution contained 100 μl 0.1% H₂O₂ and 89 μl distilled water), and the 1 μl EP buffer was added to 10 μl EX buffer as a control. The mixtures were then incubated at 22°C. At each time point indicated, the H₂O₂ content was determined as described earlier. The Arabidopsis total protein extracts were prepared as follows: 100 mg root tissues were collected and fully grounded in liquid nitrogen and then homogenized with 1 ml EX buffer (0.01 M PBS, pH = 5.8, 10 μl protease inhibitor). The homogenized materials were shaken intensely for 5–10 min before centrifugation at 20 817 g for 10 min at 4°C. The supernatants were collected and the total proteins were quantified using the Bradford method.

For protein expression, a S30 T7 High-Yield Protein Expression System (Promega) was used according to the manufacturer’s instructions. This kit contains all components used for expressing proteins in prokaryotic expression system and plasmid DNA encoding Renilla Luciferase as control protein.
To test the enzyme activities about 2 μg protein (YUCCA1, CKRC2 and Renilla Luciferase) (the amount was estimated from SDS-PAGE by comparison with the protein marker), NADPH (50 mM) 20 μL, FAD (2 mM) 2 μL, IPyA (50 mM) 0.4 μL and add nuclease-free water to a final volume of 100 μL. The mixture was incubated at 30 °C for 2 hours with vigorous shaking. For quantitative analysis, the reaction mixture was diluted 100 times before the measurement. The fluorescence intensities were measured at λex/λem = 363 nm/277 nm in a 1 cm quartz cell and with a slit at 2 nm for the excitation and 5 nm for the emission. Scan speed were 350 nm/min. The fluorescence values were quantified using a standard curve (y = 380.8x + 36.31).