RNA extraction and RT–qPCR

Total RNA was extracted from 50 to 100 mg seedlings using the Quick-RNA Miniprep Kit with on-column DNase I digestion (Zymo Research, Irvine, CA, USA). cDNA was synthesized with 2–2.5 µg total RNA using the Invitrogen SuperScript III Reverse Transcriptase and the Oligo(dT)₂₀ primer (Thermo Fisher Scientific, Waltham, MA, USA). For RT–qPCR, cDNA diluted in nuclease-free water was mixed with FastStart Universal SYBR Green Master (MilliporeSigma, Burlington, MA, USA) and gene-specific primers (Supplemental Table S3). RT–qPCR reactions were performed in triplicate with a Qiagen Rotor-Gene Q 5Plex real-time cycler (Qiagen, Germantown, MD, USA). Transcript levels of each gene were calculated relative to that of PP2A. A standard curve was performed for each gene to determine the linear range, efficiency, and reproducibility of the qPCR assay. The final RT–qPCR results were calculated by the 2^−ΔΔCt method using at least two biological replicates. Bar charts were generated using Prism 9 (GraphPad Software, San Diego, CA, USA).