Real-time quantitative PCR was run on an ABI 7500 real-time PCR system (Applied Biosystems) according to the manufacturer's recommendations. Real-time quantitative PCR reaction contained 25 μL 2× SYBR Premix Ex Taq (TaKaRa), 2 μL primer mix, 1 μL 50× ROX Reference Dye II, 4 μL cDNA, and 18 μL deionized water to make a total volume of 50 μL. After setting the amplification conditions, experiments were repeated twice. The primers used were as follows: ABA2 (At1g52340), 5′-ctcgctttggctcatttgc-3′ and 5′-ccgtcagttccaccccttt-3′; NCED3 (At3g14440), 5′-ccggtggtttacgacaagaa-3′, and 5′-cccaagcgttccagagatg-3′; and Actin2 (At3g18780), 5′-gctgagagattcagactgccca-3′ and 5′-cacagttttcgcgatccagac-3′.

For relative quantification the method of Pfaffl (2001) was used to determine the relative expression ratio. This determines the expression of the target gene relative to reference gene (ACTIN2) in a test sample compared with an untreated Col sample.