Ssofast sybr green supermix

Total RNA was extracted from inflorescences using PureZol RNA Isolation Reagent (Bio-Rad) following the manufacturer’s instructions. DNA was removed by DNase (Thermo Scientific) treatment. Isolated RNA samples were reverse transcribed using RevertAid First Strand cDNA Synthesis kit (Thermo Scientific) and oligo(dT)18 primers to initiate the reactions. Complementary DNA (cDNA) was amplified using the SSoFAST SYBR Green Supermix (Bio-Rad) in an iQ5 Real-Time RT-PCR (Bio-Rad) detection system using specific primers listed in Supplementary Table 1. Primers for the reference genes ACTINA8 ACT8 (At1g49240) and ARABIDOPSIS THALIANA RELATED TO UBIQUITIN 1 RUB1 (At1g31340) were used. Three technical replicates were performed for each situation. After 3 min at 95°C, a 10s denaturation step at 95°C was followed, and 45 cycles of 95°C at 10s and 60°C at 30s were performed. After amplification, the dissociation curve was acquired to verify the specificity of the amplification by heating the samples from 60 to 95°C. At the end of the PCR cycles, data were analyzed using the CFX Maestro™ Software (Bio-Rad) program.

RNA was isolated using RNeasy mini kit (Qiagen). Digested with DNaseI (Sigma-Aldrich) for 15 min at RT to remove DNA contamination, and subsequently, DNaseI was inactivated by heating at 70 °C for 10 min. cDNA was synthesized using Qscript supermix (Quanta Biosciences). Real-time PCR was performed using SSO fast SYBR green supermix (Bio-Rad) in a 7500 real-time PCR system (Applied Biosystems) using 7500 software v2.0.1. Melting curve analysis was performed to ensure the amplification of a single product. Primers used were the following: TCF-1-F-5′-AGGCCAAGAAGCCAACCATCAAGA and TCF-1-R-5′-ACTCTGCAATGACCTTGGCTCTCA; TCF-3-F-5′-TGCAGTGAGCGTGAAATCACCAGT and TCF-3-R-5′-AATGGCTGCACTTTCCTTCAGGGT; TCF-4-F-5′-TCGGCAGAGAGGGATTTAGCTGATGT and TCF-4-R-5′-CTTTCCCGGGATTTGTCTCGGAAACT; LEF1-F-5′-AAGCATCCAGATGGAGGCCTCTACAA and LEF1-R-5′-TGATGTTCTCGGGATGGGTGGAGAAA; β-catenin-F-5′-TCTTGCCCTTTGTCCCGCAAATCA and β-catenin-R-5′-TCCACAAATTGCTGCGTCCCA; EAAT2-F-5′-CCAAGCTTGGATCACTGCCCTGG and EAAT2-R-5′-CCAGCCCCAAAAGAGTCACCCACAA; GS-F-5′-TTGAGAAACTAAGCAAGCGGCACC and GS-R-5′-ATCCAGTTAGACGTCGGGCATTGT; and GAPDH-F-5′-CTTCAACGACCACTTTGT and GAPDH-R-5′-TGGTCCAGGGGTCTTACT. Fold change in messenger RNA (mRNA) expression was calculated by relative quantification using the comparative C_T method with GAPDH as the endogenous control.