Coexpression-based clustering of Arabidopsis root genes predicts functional modules in early phosphate deficiency signaling

Abstract

Phosphate (Pi) deficiency triggers the differential expression of a large set of genes, which communally adapt the plant to low Pi bioavailability. To infer functional modules in early transcriptional responses to Pi deficiency, we conducted time-course microarray experiments and subsequent coexpression-based clustering of Pi-responsive genes by pairwise comparison of genes against a customized database. Three major clusters, enriched in genes putatively functioning in transcriptional regulation, root hair formation, and developmental adaptations, were predicted from this analysis. Validation of gene expression by quantitative reverse transcription-PCR revealed that transcripts from randomly selected genes were robustly induced within the first hour after transfer to Pi-deplete medium. Pectin-related processes were among the earliest and most robust responses to Pi deficiency, indicating that cell wall alterations are critical in the early transcriptional response to Pi deficiency. Phenotypical analysis of homozygous mutants defective in the expression of genes from the root hair cluster revealed eight novel genes involved in Pi deficiency-induced root hair elongation. The plants responded rapidly to Pi deficiency by the induction of a subset of transcription factors, followed by a repression of genes involved in cell wall alterations. The combined results provide a novel, integrated view at a systems level of the root responses that acclimate Arabidopsis (Arabidopsis thaliana) to suboptimal Pi levels.

Figure 1.

Outline of the MACCU toolkit. A, Input data: a gene list (white nodes) and normalized microarray data from public databases. B, Coexpression relationships are identified by pairwise comparison of genes. Nodes without any relationships are removed. C, Generation of gene networks. D, Biologically meaningful information, such as gene symbols and colors indicating fold changes, can be added to the image. [See online article for color version of this figure.]

Figure 2.

Coexpression relationships of genes in RM1. The red nodes indicate up-regulated genes, and the white nodes represent genes that are not differentially expressed at this time point. The figure shows the expression of genes 1 h after transfer to Pi-deplete medium. [See online article for color version of this figure.]

Figure 3.

Short-term induction of genes with potential functions in Pi deficiency signaling derived from RM1. Relative transcript abundance was determined 10, 30, and 60 min after transfer to Pi-free medium by qRT-PCR and compared with control samples using the comparative C_T method. The expression of AT4 was determined as a control.

Figure 4.

Coexpression relationships of genes in RM2. The red nodes indicate up-regulated genes, and white nodes represent genes not differentially expressed at this time point. The figure shows the expression of genes 1 h after transfer to Pi-deplete medium. [See online article for color version of this figure.]

Figure 5.

Coexpression relationships of genes in RM3. The red nodes indicate up-regulated genes, and white nodes represent genes not differentially expressed at this time point. The figure shows the expression of genes 1 h after transfer to Pi-deplete medium. [See online article for color version of this figure.]

Figure 6.

Changes in root hair number after transfer to Pi-deplete medium. dat, Days after transfer.

Figure 7.

Root hair length of homozygous T-DNA insertion lines harboring mutations in genes of RM2. The red nodes indicate genes that cause the formation of longer root hairs under Pi-deficient conditions, when the transcript level is reduced or absent and supported by two different T-DNA insertion lines. Pink nodes also indicate mutants with longer root hairs, but supported only by one line. Blue nodes indicate shorter root hairs (validated by two lines), light blue nodes represent mutants with shorter root hairs supported by one line. Gray nodes indicate mutant lines that did not deviate from the wild type (validated by one line), and white nodes represent genes that were not tested. Green nodes denote genes in which two mutant lines showed opposite effects. P < 0.001 (Student’s t test) indicates a significant difference from the wild type.

Figure 8.

Phenotypes and root hair lengths of mutants harboring defects in genes from RM2. Black lines in the box plots indicate the median, and red lines indicate the mean. One hundred root hairs from five roots were measured for each genotype and growth type. P < 0.001 (Student’s t test) indicates a significant difference from the wild type (Wt). [See online article for color version of this figure.]

Figure 9.

Coexpression gene network around At1g74500. The gene network was determined with ATTED-II version 5.5 (http://atted.jp/). [See online article for color version of this figure.]