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.

关于黑暗 vs 光照条件下的生长素（auxin）响应数据集，光培养幼苗首先在弱光（1 μmol·m⁻²·s⁻¹）下生长4天，随后转移至80 μmol·m⁻²·s⁻¹的白光环境中继续培养4天。对于暗培养样本，经冷层积处理的种子先暴露于80 μmol·m⁻²·s⁻¹的光照下12小时，再转移至黑暗环境中培养3.5天（D）。收集这些幼苗并浸没于添加了DMSO（模拟处理，Mock）或指定浓度毒莠定（picloram）的MS液体培养基中。处理开始时，将幼苗真空处理10分钟，随后恢复至正常气压下35分钟。之后，解剖并收集这些处理后幼苗的下胚轴（hypocotyl）。

对于毒莠定或吲哚乙酸（IAA）处理3小时和12小时的RNA测序（RNA-Seq）样本，野生型（Col-0）种子在80 μmol·m⁻²·s⁻¹光照下暴露12小时后，转移至黑暗环境中生长3天。随后将幼苗浸没于含有指定浓度毒莠定、等摩尔浓度DMSO（模拟处理）或IAA、以及等摩尔浓度乙醇（模拟处理）的MS液体培养基中，分别处理3小时和12小时。使用显微手术剪和镊子解剖并收集下胚轴。采用TaKaRa MiniBEST植物RNA提取试剂盒（Takara，货号9769）从下胚轴中提取总RNA。每个处理组制备3或4个生物学重复。

RNA测序（RNA-Seq）在Illumina HiSeq 4000平台上进行。采用HISAT2将125 bp的双端测序 reads 比对至拟南芥（Arabidopsis）参考基因组（TAIR10），并通过HTSeq统计外显子区域内的比对 reads 数量。