The apoplastic pH is a key determinant in the hypocotyl growth response to auxin dosage and light

For the dark vs. light auxin response set, light-grown seedlings were initially grown under dim light (1 μmol·m-2·s-1) for 4 days, then transferred to 80 μmol·m-2·s-1 white light for another 4 days. For dark-grown samples, cold stratified seeds were first exposed to 80 μmol·m-2·s-1 light for 12 h and then transferred to darkness for 3.5 days (D). These seedlings were collected and submerged in MS liquid medium supplied with DMSO (Mock) or the indicated concentrations of picloram. At the beginning of the treatment, the seedlings were vacuumed for 10 min and then returned to normal air pressure for another 35 min. Subsequently, hypocotyls of these treated seedlings were dissected and collected.
For RNA sequencing of 3- and 12-hour picloram or IAA treatments, after 12 hours of 80 μmol·m-2·s-1 light exposure, wild-type (Col-0) seeds were transferred to darkness and grown for 3 days. The seedlings were then submerged in liquid MS medium containing specified concentrations of picloram, equimolar amounts of DMSO (Mock), or IAA, as well as equimolar amounts of ethanol (Mock), for 3 and 12 hours, respectively. Hypocotyls were dissected and collected using microsurgical scissors and tweezers. Total RNA was extracted from the hypocotyls using a TaKaRa MiniBEST Plant RNA Extraction Kit (Takara, Cat# 9769). Three or four biological replicates of each treatment group were prepared.
RNA-Seq was performed on the Illumina HiSeq 4000 platform. A total of 125-bp paired-end sequencing reads were mapped to the Arabidopsis reference genome (TAIR10) using HISAT2, and reads mapped within exons were counted by HTSeq.