ZAT14 OE and SMB-GR RNA sequencing

TRANSPLANTA ZAT14 OE line and Col-0 whole seedlings were treated with either DMSO or estradiol for 8 h. Then, they were harvested as 3 biological replicates for each condition. The whole seedlings of SMB-GR line were treated with DMSO or DEX for 6 h before they were collected as 3 biological replicates. Each replicate contained 25 seedlings. The RNA was extracted as described above. The quantity and quality of the RNA were checked using a NanoDrop 2000 (Nanodrop Technologies, Wilmington), and RNA integrity was confirmed on an Agilent 2100 Bioanalyzer (Agilent Technologies). The RNA sequencing was performed by VIB Nucleomics core as Illumina NEXT-seq with 75 bp paired-end sequencing. Sequencing quality as well as read mapping and summarization were performed with a software pipeline on an in-house Galaxy server. Briefly, the quality of raw data was verified with FastQC (https://www.bioinformatics.babraham.ac.uk/projects/fastqc/). Next, quality filtering was performed using Trimmomatic as described (Bolger et al. 2014). Reads were subsequently mapped to Arabidopsis genome (Araport11) (Cheng et al. 2017).

Differentially regulated genes (DEGs) were identified with the Edge-R software package in R (Robinson et al. 2010). Genes were considered as differentially expressed if their expression levels had an absolute Log₂(FC) > 1, P < 0.05, and false discovery rate (FDR) < 0.05. Upregulated genes upon inducible OE of ZAT14 (Supplemental Data Set S3) were determined by comparing gene expression levels in the ZAT14 overexpressor line treated with estradiol (ZAT14_OE_Estr) with the same line after mock treatment (ZAT14_OE_mock); this resulted in 1,126 upregulated genes. Secondly, we compared ZAT14_OE plants treated with estradiol with estradiol-treated WT plants; this resulted in 1,055 upregulated genes. To remove the genes regulated by estradiol treatment or transgenic insertion, these 2 upregulated gene lists were combined and 795 genes were obtained and regarded as upregulated upon the OE of ZAT14. The same method was performed to get the downregulated genes, by comparing ZAT14_OE_Estr with ZAT14_OE_mock or ZAT14_OE_Estr with WT_Estr, 663 genes and 815 genes were obtained, respectively. In total, 483 downregulated genes were obtained after combing these 2 downregulated gene lists.

GO enrichment analyses were carried out with the PLAZA 5.0 website (https://bioinformatics.psb.ugent.be/plaza/versions/plaza_v5_dicots). GO pathway enrichment bubble plot was plotted by https://www.bioinformatics.com.cn/en a free online platform for data analysis and visualization.

For representation factor calculation, the online software: http://nemates.org/MA/progs/overlap_stats.html was used. “The number of genes expressed in the RNAseq” was considered as the “Number of genes in the genome.”