Measuring Stress-Related Flavonoid Gene Expression in Plants

The expression of stress-related flavonoid-related genes was measured by qRT-PCR in WT and transgenic lines of A. thaliana or apple calli. Total RNA was extracted from A. thaliana leaves or apple calli using the RNAprep Pure Plant Kit (Tiangen, Beijing, China). First-strand cDNA was synthesized using the PrimeScript™II 1st Strand cDNA Synthesis Kit (Clontech TaKaRa, Beijing, China). Quantitative RT-PCR was conducted using the ChamQ SYBR Color qPCR Master Mix Kit (Vazyme, Shanghai, China) with a QuantStudio™ 5 Real-Time PCR System. Each reaction was performed in triplicate, and data were analyzed as previously described [55 (link)]. Atactin1 (At2g37620) or MdActin (XM_029088423.1) was used as an internal control. All the quantitative PCR primers are listed in Table S1.
In addition, the expression level of mature miR156 was confirmed by stem-loop RT-PCR as described [56 (link)]. The cDNA synthesis was performed using the prime script first strand cDNA Synthesis Kit (Takara, China), according to the manufacturer’s instructions, using a stem-loop RT primer instead of an oligo (dT) primer (Table S1). Subsequently, PCR was performed to detect the expression level of miR156 using the miR156-specific forward primer and the stem-loop-specific reverse primer (Table S1). The apple 5.8S rRNA (GenBank accession no. AF186480) was used as a reference gene.