Plant materials and cold treatment

Pyruvate treatment

Pyruvate treatment was performed as follows: Wheat seedlings of Fielder, EMS mutants, and CRISPR-Cas9-mediated TaPGK lines at the three-leaf stage were irrigated with fresh pyruvate solution (Solarbio, lot number P9810) to a final concentration of 10 mM, or an equal volume of distilled water (control) for three consecutive days. To prepare the pyruvate working solution, 2M NaOH solution was slowly added dropwise under continuous stirring to adjust the pH to approximately 7.0. TaPGK-edited plants and controls with Fielder background were exposed to cold stress at −4°C from 18 to 36 hours. All plants with Kronos background were exposed to cold stress at −3°C for 8 hours. All leaf tissue after cold treatment was collected and stored at −80°C.