Plant Total RNA Isolation and qPCR Analysis

A Plant Total RNA Isolation Kit (FOREGENE, China) was used for total RNA extraction from differently treated samples (i.e., drought, salinity, or heat). A RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific, (Waltham, Massachusetts, USA) was used to synthesize purified cDNA with 1 μg of total RNA and oligo primers [53 (link)]. Light Cycler^® 480 II (Mannheim, Roche, Germany) and SYBR Green I Master Mix (Roche, Germany) were used to perform qPCR. The gene-specific primers were designed using the NCBI online software primer designing tool (https://www.ncbi.nlm.nih.gov/tools/primer-blast/) (accessed on 15 January 2023) (Table S2). Each qPCR reaction was conducted as follows: 0.2 μL of cDNA, 5 μL of 0.5 μM gene-specific primer pre-mixture, 10 μL of 2 × SYBR Green Master Mix, and 4.8 μL of water. Actin7 was used as the internal standard to normalize the expression levels for target genes. A melting curve was used to evaluate the specificity of amplification. All experiments had three biological replicates and technical replicates. The 2−ΔCT method was used for data calculation. Graph Pad Prism 9 was used to analyze and graph the expression data.