Quantitative Gene Expression Analysis

Total RNA was extracted from all the samples using TRIzol reagent (Invitrogen, Germany), according to the manufacturer’s instructions. The RNA extraction procedure and cDNA synthesis followed the same procedures as detailed in our recently published papers (Ali et al., 2018 (link), 2020a (link)). For qRT-PCR, specific primer sets were designed and checked for primer specificity in Arabidopsis genome (Supplementary Table 6). qRT-PCR was performed in CFX96 Real-Time System machine (Bio-RAD, Hercules, CA, United States) using SYBR Premix Ex Taq^TM II (TaKaRa). Relative expression levels were normalized by Arabidopsis ACTIN7 gene and calculated using the 2^–ΔΔCt method (Schmittgen and Livak, 2008 (link)). Data were presented as means and +SD of three replications obtained from three independent biological experiments.