A stele-enriched gene regulatory network in the Arabidopsis root

Abstract

Tightly controlled gene expression is a hallmark of multicellular development and is accomplished by transcription factors (TFs) and microRNAs (miRNAs). Although many studies have focused on identifying downstream targets of these molecules, less is known about the factors that regulate their differential expression. We used data from high spatial resolution gene expression experiments and yeast one-hybrid (Y1H) and two-hybrid (Y2H) assays to delineate a subset of interactions occurring within a gene regulatory network (GRN) that determines tissue-specific TF and miRNA expression in plants. We find that upstream TFs are expressed in more diverse cell types than their targets and that promoters that are bound by a relatively large number of TFs correspond to key developmental regulators. The regulatory consequence of many TFs for their target was experimentally determined using genetic analysis. Remarkably, molecular phenotypes were identified for 65% of the TFs, but morphological phenotypes were associated with only 16%. This indicates that the GRN is robust, and that gene expression changes may be canalized or buffered.

Figure 1

A stele-enriched Arabidopsis GRN. Black edge, PDI; red edge, miRNA–mRNA interaction; green edge, PPI; circle, TF; square, miRNA. Interactions are represented from the top down, with targets located below their interacting TF.

Figure 2

In planta validation of protein–DNA interactions (PDIs) using chromatin immunoprecipitation (ChIP)-quantitative PCR (qPCR) (A–C), conditional induction (D), or genetic analyses (E). Results are representative of three (A–C) two (D, E (protein-coding genes)), and four (E, miRNAs) biological replicates. (A–C) Black bar, mock precipitation; gray bar, anti-GFP immunoprecipitated fraction. (A) The −1952 to −1851 base pair region of At3g43430 containing a NAC domain consensus sequence is enriched in the VND7pro:VND7∷GFP immunoprecipitated fraction. (B) The −422 to −253 base pair region of PHB is enriched in the OBP2pro:OBP2∷GFP immunoprecipitated fraction. (C) The −2437 to −2246 base pair region of PHV is enriched in the OBP2pro:OBP2∷GFP anti-GFP immunoprecipitated fraction. (D) Transcriptional activity of the 35S:OBP2∷glucocorticoid receptor line was induced with 10 μM dexamethasone (Dex) and gene expression monitored at 1 and 3 h after Dex exposure. At 1 h after Dex exposure, PHB expression was downregulated, while PHV expression was upregulated. (E) Interactions that are repressive, activating, or exerting no effect (circle) were determined using qPCR of the TF and its target in a mutant TF background. Edges are as described in Figure 1.

Figure 3

A predictive framework identifying regulatory relationships likely to have important roles in regulating expression of At5g60690 (REV), At2g34710 (PHB), miR399b and miR168a. Regulatory interaction strength is represented by edge and arrow width with thicker lines or arrowheads representing steeper slopes. P-value strength is represented by edge opacity with darker edges representing more significant interactions. Slopes were determined by plotting quantitative PCR (qPCR) expression values of the transcription factor (TF) and its target and fitting a line using weighted least squares regression (WLSR).