Quantification of Plant Gene Expression

RNA was isolated from control, drought-, and heat-treated plants as described by [96 (link),97 (link)]. To remove the genomic DNA contamination from RNA, samples were treated with DNase-I (Takara Bio. Inc., Shiga, Japan) at 37 °C for 30 min. cDNA was prepared using the iScriptTM cDNA synthesis kit (Bio-Rad, Hercules, CA, USA) at 46 °C for 20 min. cDNA was quantified using a NanoDrop (ND-1000, NanoDrop Technologies, Wilmington, DE, United States). Quantitative real-time PCR (qRT-PCR) was performed using the Applied Biosystems 7500 Fast Real-Time PCR (Applied Biosystems, Massachusetts, United States). Each qRT-PCR reaction was carried out with three technical replicates and repeated three times. The fold change was calculated based on mean 2^−ΔΔCT values [98 ]. Finally, this value was used for plotting graphs. Wheat actin (AB181991) was used as the internal control to normalize the data. Primer pairs were designed using PrimerQuest Tool (https://sg.idtdna.com/PrimerQuest/Home/Index accessed on 17 May 2021), and primers used in this work are listed in Table S9.