Genome-wide expression and network analyses of mutants in key brassinosteroid signaling genes

BACKGROUND: Brassinosteroid (BR) signaling regulates plant growth and development in concert with other signaling pathways. Although many genes have been identified that play a role in BR signaling, the biological and functional consequences of disrupting those key BR genes still require detailed investigation. RESULTS: Here we performed phenotypic and transcriptomic comparisons of A. thaliana lines carrying a loss-of-function mutation in BRI1 gene, bri1–5, that exhibits a dwarf phenotype and its three activation-tag suppressor lines that were able to partially revert the bri1–5 mutant phenotype to a WS2 phenotype, namely bri1–5/bri1–1D, bri1–5/brs1–1D, and bri1–5/bak1–1D. From the three investigated bri1–5 suppressors, bri1–5/bak1–1D was the most effective suppressor at the transcriptional level. All three bri1–5 suppressors showed altered expression of the genes in the abscisic acid (ABA signaling) pathway, indicating that ABA likely contributes to the partial recovery of the wild-type phenotype in these bri1–5 suppressors. Network analysis revealed crosstalk between BR and other phytohormone signaling pathways, suggesting that interference with one hormone signaling pathway affects other hormone signaling pathways. In addition, differential expression analysis suggested the existence of a strong negative feedback from BR signaling on BR biosynthesis and also predicted that BRS1, rather than being directly involved in signaling, might be responsible for providing an optimal environment for the interaction between BRI1 and its ligand. CONCLUSIONS: Our study provides insights into the molecular mechanisms and functions of key brassinosteroid (BR) signaling genes, especially BRS1.ADDITIONAL FILE 1. List of differentially expressed genes as compared to WS2 in each line and group membership.ADDITIONAL FILE 2. List of marker genes being differentially expressed upon addition of external BRs or in line with a gain of mutation in BR signaling genes at least in 5 studies.ADDITIONAL FILE 3. GO enrichment for genes exclusively differentially expressed in each suppressor.ADDITIONAL FILE: Fig. S1. The T-DNA insertion site for bri1–1D (A), and microscopic images of 7-day old hypocotyl cells for WS2 (B), bri1–5 (C), bri1–5/bak1–1D (D), bri1–5/bri1–1D (E), bri1–5/brs1–1D (F). Fig. S2. PCA plot for assessing the reproducibility of the gene expression dataset. Samples taken from the same genotype are represented in the same color. The plot indicates high consistency between replicate samples as they are located close to each other when plotted on the first and second principal components. Fig. S3. RT-qPCR results for relative expression of selected genes and their corresponding values from microarray analysis. The values represent the log2 of relative expression (sample1/sample2). Rows indicate gene names and columns show the comparison between the indicated lines. Columns with pink header represent the RT-qPCR values, and columns with yellow header are microarray measurements. The red color on the heatmap indicates that the gene has been up-regulated in sample1 as compared to sample2, while blue indicates down-regulation. Fig. S4. Comparing genome-wide expression impact between bri1–5 suppressor lines. Fig. S5. Heatmap of expression of the marker genes that up/down regulation of their expression was confirmed by at least 5 independent references and also affected in the bri1–5 line of our study. For each line, the row-scaled normalized expression data of the 3 biological replicates are shown as adjacent columns. In each row the gradient red color indicates the higher expression for the gene compared to other samples while blue indicates the lower expression. Fig. S6. Pathway analysis (MapMan metabolism) showing for each mutant line the expression changes compared to WS2. Panel A: bri1–5, Panel B: bri1–5/bri1–1D, Panel C: bri1–5/brs1–1D, Panel D: bri1–5/bak1–1D. Fig. S7. Pathway analysis (MapMan: large enzyme families) showing for each mutant line the expression changes compared to WS2. Panel A: bri1–5, Panel B: bri1–5/bri1–1D, Panel C: bri1–5/brs1–1D, Panel D: bri1–5/bak1–1D. Fig. S8. Pathway analysis (MapMan: gene regulation) showing for each mutant line the expression changes compared to WS2. Panel A: bri1–5, Panel B: bri1–5/bri1–1D, Panel C: bri1–5/brs1–1D, Panel D: bri1–5/bak1–1D. Fig. S9. Expression pattern in each mutant line of genes related to ABA signaling, Glutathione metabolism, and ion related hemostasis as discussed in the main text. Mutant lines are represented in the x-axis. The y-axis indicates the log2 normalized expression value of the gene. Table S1. RT-qPCR test of log-fold change (log-FC) of the genes that are overexpressed by activation-tagging in the suppressors at the 7 days seedling stage. Table S2. Summary of the most significant results obtained by MapMan pathway analysis (metabolism, regulation and, large-enzyme families overview). Left column: enriched pathways; entries provide for each line the degree to which the pathway is enriched. P-values are FDR corrected using Benjamini-Hochberg). Table S3. Designed primers for RT-qPCR.The National Basic Research Program of China, Youth Innovation Promotion Association of the Chinese Academy of Sciences, the Ministry of Science, Research and Technology, Iran, the Fonds Wetenschappelijk Onderzoek-Vlaanderen (FWO) and UGent Bijzonder onderzoeksfonds.http://www.biomedcentral.com/bmcgenomicspm2021BiochemistryGeneticsMicrobiology and Plant Patholog