Mj opticon system

For real-time quantitative PCR, the reaction was performed on an MJ Opticon system (MJ Research, Waltham, MA) using SYBR Green Real-Time PCR Master Mix (Toyobo Co., Ltd.). The amplification conditions were 95 °C for 10 minutes followed by 40 cycles of amplification (95 °C for 15 seconds, 58 °C for 15 seconds, and 72 °C for 30 seconds, followed by plate reading) and melting (50–95 °C, with plate readings every 1 °C). The sequences of the primers that were used for the real-time quantitative RT-PCR for ADR, NST1 and NST2 are listed in Supplementary Table S1. The Arabidopsis housekeeping gene UBQ10 was used as a normalization control with the primers RT-UBQ10-1 and RT-UBQ10-2. All of the experiments were repeated at least twice for reproducibility. The data were analyzed using Gene Expression Macro software (version 1.1, Bio-Rad) according to the manufacturer’s instructions. The ‘delta-delta method’ formula 2^{−[ΔCP sample − ΔCP control]}, where 2 represents perfect PCR efficiency, was used to calculate the relative expression of the genes. To calculate the statistical significance, unpaired T-tests were used.

For real-time quantitative RT-PCR, the reaction was performed on a MJ Opticon system (MJ Research, Waltham, MA) using SYBR^® Green Real-time PCR Master Mix (TOYOBO Co., LTD.). The amplification condition was 95 °C for 10 min, followed by 40 cycles of amplification (95 °C for 15 s, 58 °C for 15 s, 72 °C for 30 s and then plate reading) and melted (50–95 °C with plate readings every 1 °C) as described previously^{1 (link),2 (link)}. Sequences for the primers used for real-time quantitative RT-PCR for FYF, FYL1, FYL2, AGL6, AGL15, AGL19, AGL14, SOC1, EDF1, EDF2, EDF3, EDF4, ERF1, SAG12, BOP1, BOP2, IDA, and HAESA, were listed in Supplementary Table 1. The housekeeping gene UBQ10 was used as normalization control with the following primers: RT-UBQ10-1 and RT-UBQ10-2^{51 (link)}. Data were analyzed using Gene Expression Macro software (version 1.1, Bio-Rad).

For real-time quantitative RT-PCR, total RNA was isolated from wild-type (WT) and transgenic plants and used as templates. The transcript levels for each genes were determined using three biological replicates and normalized against UBQ10. The RT-PCR reaction was performed on an MJ Opticon system (MJ Research, Waltham, MA) using a SYBER Green Real-time PCR Master Mix (TOYOBO Co., LTD.). The amplification condition was 95 °C for 10 minutes, followed by 40 cycles of amplification (95 °C for 15 seconds, 58 °C for 15 seconds, 72 °C for 30 seconds followed by plate reading) and melting (50–95 °C with plate readings every 1 °C). Sequences for the primers used for real-time quantitative RT-PCR for GSF, GA20ox1, GA20ox2, GA20ox3, GA20ox4, GA2ox1, GA2ox2, GA2ox3, GA2ox4, GA2ox6, GA2ox7 and GA2ox8 are listed in Supplementary Table S1. The housekeeping gene UBQ10 was used as a normalization control with the following primers: RT-UBQ10-F and RT-UBQ10-4-2. All the experiments were repeated at least twice. The data were analyzed using the Gene Expression Macro software (Version 1.1, Bio-Rad).