2.5 RNA isolation and quantitative real-time PCR
The young leaves and shoot buds were harvested after 24 h treatment at
4°C, then frozen in liquid nitrogen for RNA extraction. Nonstressed
seedlings were harvested as controls (0 h). More than 6 plants were
harvested and pooled in each treatment. Total RNA was extracted using
the Plant RNA Kit (Omega Bio-Tek). First-strand cDNA was synthesized
using the PrimeScript™ RT Reagent Kit (Takara, Otsu, Japan). A
quantitative real-time PCR (qRT-PCR) was conducted using SYBR ® Premix
Ex Taq™ (Takara) and the StepOnePlus Real-Time PCR Detection System
(Bio-Rad, CA, USA). MeACTIN was used as a reference gene for
normalizing transcript levels. Details regarding the primers used in
this study are provided in Table S3.