Extraction and Quantification of RNA from Grapevine and Nicotiana

Total RNA was extracted from the leaves of grapevine rootstock ‘A35’ and N. benthamiana using the improved CTAB method and TRIzol reagent (Invitrogen, Carlsbad, CA, USA), respectively, as previously described [3 (link), 22 (link), 23 (link)]. Then, RNA was used for cDNA synthesis with the PrimeScript^™ RT reagent kit with gDNA Eraser (Vazyme, Nanjing, China). Quantitative reverse transcription PCR (qRT-PCR) was performed according to the instructions of the SYBR^® PrimeScript^™ RT-PCR Kit (TaKaRa, Dalian, China) in the CFX96TM Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA). The actin1 (AY680701) and actin7 (XM_034827164) were used as internal references in the VvASMT1 expression pattern in grapevine under NaCl and mannitol stresses, and actin (XM_033660572.1) and Tubulin (N181029A17) genes were used as internal references to determine ROS scavenging-related target genes and VvASMT1 expression in Nicotiana benthamiana qRT-PCR analyses. All experiments were repeated at least three times, and all the primers used in this study are listed in S1 Table in S1 File. The data were automatically analyzed using the CFX Manager software program (version 1.1).