ABSTRACT: Purpose: In plants, chloroplasts not only fix energy through photosynthesis, but they synthesize numerous metabolites which can act as important growth regulators by impacting nuclear gene expression. Upon developmental and external stimuli, signaling between the nucleus and chloroplasts is essential for maintaining plant cellular homeostasis. In this study, we applied an Illumina HiSeq 2500 platform to investigate the transcriptome changes of 2 Arabidopsis thaliana mutant lines, a T-DNA knock out muant allele of DIFFERENTIAL AND GREENING-LIKE (DAL), termed dal-2, and a dexmathasone (Dex)-induced overexpression line of DAL-INTERACTING F-BOX (termed DIF-OX2). Methods: Total RNA was isolated from 12-d-old Col-0 (wild type control) and DIF-OX2 seedlings grown on Gamborg’s B-5 (GM) medium containing 1% sucrose (GM-Suc), with or without 10 μM Dex, and 20-d-old dal-2 plants also grown on GM-Suc medium, and deep sequenced as 100-mers using the Illumina HiSeq 2500 platform (DNA Sequencing Facility, University of Wisconsin Biotechnology Center). Raw image files for each library were converted to FASTQ files by the standard Illumina pipeline and processed for quality control by Trimmomatic (http://www.usadellab.org/cms/?page=trimmomatic) to remove adapter and low quality sequences (phred=33, minimum length=36). Processed sequences were aligned to the A. thaliana Col-0 genomic sequence (from The Arabidopsis Information Resource (TAIR) version 10 at www.arabidopsis.org) via TOPHAT2 to identify accepted hits, which were then used to generate an absolute expression level (counts) of each locus by HTSeq (http://www-huber.embl.de/users/anders/HTSeq/). The resulting counts per locus were normalized to counts per million reads (CPM) among all 10 libraries and used to generate the list of differentially expressed (DE) genes between treatments or genotypes by edgeR (FDR < 5% or ABS[log2FoldChange (FC)] ≥ 1). Results: In total, 6,743 differentially expressed (DE) RNAs were identified using edgeR (FDR < 5%) in either dal-2 or DIF-OX2 (+Dex) seedlings, as compared to Dex-untreated or Dex-treated wild-type seedlings, respectively. Among these DE genes, the expression levels of 2,606 loci in dal-2 changed at a level greater than or equal to 2 fold compared to those in the Dex-untreated wild type control, but showed a strong positive correlation with their expression levels in DIF-OX2 (+Dex) (Spearman’s rank correlation rho = 0.72, p < 2.2e-16; Figure 5B-C). Conclusions: The similar transcriptome changes in both DIF-OX2 (+Dex) and dal-2 plants suggests that the DIF and DAL proteins are involved in the same pathway, which controls a retrograde signaling from chloroplasts to the nucleus, based on further biochemical analysis, cellular localization, and transcriptome comparisons with other public available microarray data from mutants impacting chloroplast retrograde signaling.
