A Gene Regulatory Network for Root Epidermis Cell Differentiation in Arabidopsis

Abstract

The root epidermis of Arabidopsis provides an exceptional model for studying the molecular basis of cell fate and differentiation. To obtain a systems-level view of root epidermal cell differentiation, we used a genome-wide transcriptome approach to define and organize a large set of genes into a transcriptional regulatory network. Using cell fate mutants that produce only one of the two epidermal cell types, together with fluorescence-activated cell-sorting to preferentially analyze the root epidermis transcriptome, we identified 1,582 genes differentially expressed in the root-hair or non-hair cell types, including a set of 208 “core” root epidermal genes. The organization of the core genes into a network was accomplished by using 17 distinct root epidermis mutants and 2 hormone treatments to perturb the system and assess the effects on each gene's transcript accumulation. In addition, temporal gene expression information from a developmental time series dataset and predicted gene associations derived from a Bayesian modeling approach were used to aid the positioning of genes within the network. Further, a detailed functional analysis of likely bHLH regulatory genes within the network, including MYC1, bHLH54, bHLH66, and bHLH82, showed that three distinct subfamilies of bHLH proteins participate in root epidermis development in a stage-specific manner. The integration of genetic, genomic, and computational analyses provides a new view of the composition, architecture, and logic of the root epidermal transcriptional network, and it demonstrates the utility of a comprehensive systems approach for dissecting a complex regulatory network.

Tissue-Specific Profiling Reveals Transcriptome Alterations inArabidopsisMutants Lacking Morphological Phenotypes

“…For each of these paralogous gene pairs, homozygous recessive presumed loss-of-function mutations in one member (try, egl3, or myb23) cause no visible alteration in root epidermis (A) Simplified depiction of the gene regulatory network for Arabidopsis root epidermal cell differentiation. The TRY/CPC, EGL3/GL3, and MYB23/WER transcriptional regulators influence the expression of downstream genes in five distinct branches (Branches 1 to 5), based on their GL2, RHD6, and/or hormone dependence (for details, see Bruex et al, 2012). (B) Root phenotype of single and double mutants affecting the try/cpc, egl3/gl3, or myb23/wer loci.…”

“…To determine whether the single mutants lacking a visible phenotype (try, egl3, and myb23) exhibit transcriptional changes associated with root epidermis cell differentiation, an epidermal green fluorescent protein (GFP) marker, WER:GFP (Lee and Schiefelbein, 1999), was introduced into each mutant by crossing to enable fluorescence-activated cell sorting (FACS)-based enrichment for cells from the developing root epidermis tissue followed by gene expression assays using ATH1 microarray chips (Birnbaum et al, 2005;Bruex et al, 2012 Tusher et al, 2001]; t-statistic > 2.5, q-value < 0.05) exhibited significant overrepresentation for genes known to be preferentially expressed in differentiating root hair cells (a 487 COBL9:GFP-sorted gene set [Brady et al, 2007]; P values: 7.1E-16 [try], 9.4E-7 [egl3], and 0.04 [myb23]) and genes known to be differentially expressed in the root epidermis of hairy versus hairless mutants (a 208-member gene set, designated the "core root epidermal gene set" [Bruex et al, 2012] . A significant number of these differentially expressed genes was affected in more than one of the mutants ( Figure 2D; P value < 0.002 for each two-way and three-way comparison), and the overlapping set of genes are overrepresented for members of the core root epidermal gene set (P value < 0.001; see Supplemental Data Set 1 online).…”

“…The specification of the two cell types in the root epidermis, root hair cells, and non-hair cells is controlled by a suite of positive and negative transcriptional regulators Tominaga-Wada et al, 2011;Bruex et al, 2012;Grebe, 2012). Among these are three sets of transcription factors encoded by presumed paralogous genes, including one-repeat Myb type proteins TRIPTYCHON (TRY) and CAPRICE (CPC) that promote the hair cell fate, basic helix-loop-helix proteins ENHANCER OF GLABRA3 (EGL3) and GLABRA3 (GL3) that promote the nonhair fate, and R2R3-Myb-type proteins MYB23 and WEREWOLF (WER) that promote the non-hair fate (Figure 1).…”

Paired-End Analysis of Transcription Start Sites in Arabidopsis Reveals Plant-Specific Promoter Signatures

“…A large data set containing millions of tags that map stringently to a well-sequenced and well-annotated genome is likely to be sufficient for an accurate model, at least in the case of NP promoters. We expected that a tissue sample such as the root, with its complex regulatory network (Bruex et al, 2012;Lan et al, 2013), would be enriched for rapidly dividing cells where tissue-specific patterning is taking place, a setting expected to give rise to many sharp NP TSSs, in addition to more broadly distributed gene promoters typically associated with ubiquitously expressed genes.…”

Network Dynamics as an Inverse Problem

“…For instance, understanding interactions at cellular levels may help in identifying genes and pathways involved in the progression of specific human diseases [33]. Also, understanding how genes regulate themselves in gene regulatory networks may explain how cell differentiation is carried out in living organisms [80,[91][92][93]]. …”