Functional genomic analysis of the AUXIN RESPONSE FACTOR gene family members in Arabidopsis thaliana: unique and overlapping functions of ARF7 and ARF19

Abstract

The AUXIN RESPONSE FACTOR (ARF) gene family products, together with the AUXIN/INDOLE-3-ACETIC ACID proteins, regulate auxin-mediated transcriptional activation/repression. The biological function(s) of most ARFs is poorly understood. Here, we report the identification and characterization of T-DNA insertion lines for 18 of the 23 ARF gene family members in Arabidopsis thaliana. Most of the lines fail to show an obvious growth phenotype except of the previously identified arf2/hss, arf3/ett, arf5/mp, and arf7/nph4 mutants, suggesting that there are functional redundancies among the ARF proteins. Subsequently, we generated double mutants. arf7 arf19 has a strong auxin-related phenotype not observed in the arf7 and arf19 single mutants, including severely impaired lateral root formation and abnormal gravitropism in both hypocotyl and root. Global gene expression analysis revealed that auxin-induced gene expression is severely impaired in the arf7 single and arf7 arf19 double mutants. For example, the expression of several genes, such as those encoding members of LATERAL ORGAN BOUNDARIES domain proteins and AUXIN-REGULATED GENE INVOLVED IN ORGAN SIZE, are disrupted in the double mutant. The data suggest that the ARF7 and ARF19 proteins play essential roles in auxin-mediated plant development by regulating both unique and partially overlapping sets of target genes. These observations provide molecular insight into the unique and overlapping functions of ARF gene family members in Arabidopsis.

Figure 1.

The ARF Gene Family of Arabidopsis. (A) Chromosomal location of ARF genes. The locations of 23 putative ARF genes on the Arabidopsis chromosomes (I to V) are shown according to version 5.0 of the Arabidopsis Genome annotation submitted to GenBank. Mutants that have been isolated in the ARF gene are shown on the left side of the chromosomes. The ARF genes clustered on chromosome I are boxed. (B) Phylogenetic analysis. An unrooted dendogram was generated using ClustalW (Thompson et al., 1994). TreeView was used to generate the graphical output (Page, 1996). The numbers at the branching points indicate the percentage of times that each branch topology was found during bootstrap analysis (n = 1000). The gene names, accession numbers, protein identifier, and the accession numbers of the full-length open reading frames (ORFs) used for this analysis are also shown. Predicted ORFs from the genomic annotation were used for ARF14, ARF15, ARF21, and ARF23 (pseudogene) genes. The full-length ORFs of ARF2, ARF6, ARF7, ARF8, ARF11, ARF12, ARF13, ARF19, ARF20, and ARF22 were constructed during this study. A differential spliced form of ARF13 has been cloned recently (accession number AY680406).

Figure 2.

Location of T-DNA Insertions in the ARF Gene Family Members. Boxes represent exons. T-DNA insertions with gray triangles denote lines whose characterization has been completed. T-DNA insertions with white triangles denote lines not yet characterized.

Figure 3.

Phenotype of Mature Mutant Plants. Three wild-type (left) and three mutant plants (right) are shown. The plants were grown at the same time. White dots indicate the boundaries between the wild-type and the mutant plants.

Figure 4.

Phenotypes of the arf7 arf19 Double Mutant. (A) Four-week-old soil-grown plants of the wild type, arf19-1, nph4-1, msg1-2, and arf7-1 (top) and the wild type, nph4-1 arf19-1, msg1-2 arf19-1, arf7-1 arf19-1, and slr-1 (bottom). (B) Seventeen-day-old seedlings of wild type, arf19-1, nph4-1, msg1-2, arf7-1, nph4-1 arf19-1, msg1-2 arf19-1, arf7-1 arf19-1, and slr-1. (C) Twenty-two-day-old seedlings of the wild type, nph4-1 arf19-1, and slr-1 grown on agar plates vertically. (D) Gravitropic response of 3-d-old dark-grown seedlings of the wild type, arf19-1, nph4-1, msg1-2, arf7-1, nph4-1 arf19-1, msg1-2 arf19-1, arf7-1 arf19-1, and slr-1. (E) Root hair formations of the wild type, arf19-1, nph4-1, and nph4-1 arf19-1. (F) Phototropism of 3-d-old dark-grown seedlings of the wild type, arf19-1, nph4-1, msg1-2, arf7-1, nph4-1 arf19-1, and arf7-1 arf19-1. Seedlings were exposed to unilateral blue light from the right for 8 h.

Figure 5.

Auxin Sensitivity of the Wild Type, arf7, arf19, arf7 arf19, and slr Mutants. (A) Inhibition of root growth by exogenous auxin. Each value represents the average of more than 10 seedlings. Bars represent se of the average. (B) and (C) Inhibition of hypocotyl elongation by exogenous auxin. Data represent the mean of hypocotyl length as a percent of controls (B) or of actual measurements (C). Bars represent se of the average. See Methods for experimental details.

Figure 6.

Expression of GUS in Pro_ARF7:GUS and Pro_ARF19:GUS Transgenics. (A) GUS expression in a 6-d-old light-grown Pro_ARF7:GUS seedling. (B) GUS expression in a 6-d-old light-grown Pro_ARF19:GUS seedling. (C) Root apex of a Pro_ARF7:GUS seedling primary root. (D) Root apex of a Pro_ARF19:GUS seedling primary root. (E) to (I) Pro_ARF7:GUS expression in the vascular tissue of mature primary root, lateral root primordia ([E] and [F], arrowhead), and developing lateral roots ([G] to [I]). (J) to (L) Pro_ARF19:GUS expression in entire tissue of primary root and developing lateral roots.

Figure 7.

Developmental Defects by ARF19 Overexpression. (A) to (C) Growth inhibition in 5-week-old plants (A), first true leaves (B), and 12-d-old light-grown seedlings (C). (D) Alteration of root architecture in 10-d-old seedlings. (E) Expression of ARF19 in overexpressing lines from 7-d-old light-grown seedlings. ARF gene expression was assessed by RT-PCR as described in Methods. The lanes are as follows: 1, the wild type; 2, arf19-1; 3, Pro_35s:ARF19 line 1; 4, Pro_35s:ARF19 line 2; 5, genomic DNA. Accumulation of the ACT8 transcript was used as an internal control. White and black arrowheads indicate the size of genomic and cDNA fragments, respectively.

Figure 8.

Global Gene Expression Profiling. MA plots (Dudiot et al., 2002) showing changes of auxin-regulated gene expression levels in the wild type, arf19-1, nph4-1, and nph4-1 arf19-1. Each plot represents the log ratio of the average of the auxin-treated samples (I) to the control samples (C) [M = log₂ (I/C)] versus overall average intensity [A = log₂√(I*C)]. The genes induced by auxin treatment (M > 1) are highlighted in red, and the genes repressed by auxin treatment (M < −1) are highlighted in green. The data were further analyzed for variance to extract statistically valid auxin-regulated genes (see Methods).

Figure 9.

Comparative Analysis of Genes Differentially Regulated by Auxin in nph4-1, arf19-1, and nph4-1 arf19-1. Differentially regulated genes in mutants among auxin-induced (A) and repressed (B) genes are shown. Each circle within the Venn diagram indicates numbers and percentages (in parentheses) of genes with repressed induction or repression levels. Only those genes with greater than twofold fold change ratio (FCR) in nph4-1, arf19-1, and nph4-1 arf19-1 were analyzed (see Methods). We defined each area of the Venn diagram from A to H, and each class was further divided into two subgroups based on their auxin-induced expression profiles in the wild type. The genes classified into class D are considered to be preferentially regulated by ARF7, and those classified into class F are considered to be preferentially regulated by ARF19. The genes classified into classes E and G are considered to be redundantly regulated by ARF7 and ARF19. The class A genes have similar expression profiles to class D genes. Likewise, class C genes have similar expression profiles to class F genes. The expression profiles of the representative genes from each class are shown in Supplemental Figure 5 online.

Figure 10.

The Expression Profiles of Representative Auxin-Regulated Genes in the Wild Type, nph4-1, arf19-1, and nph4-1 arf19-1. The data represent the average relative intensity expression level of control (open bar) or auxin-treated (blue bar) samples from triplicate experiments. Bars represent sd of the average. Boxes next to gene names indicate classification color codes according to Figure 9.

Figure 11.

Effect of the nph4-1, arf19-1, and nph4-1 arf19-1 Mutations on Global Gene Expression in Untreated Control Samples. (A) Induced genes in the mutants under control conditions. (B) Repressed genes in the mutants under control conditions. Each circle within the Venn diagram indicates the number of genes with greater than twofold induction or repression. (C) Expression profiles of induced classes of genes. (D) Expression profiles of repressed classes of genes. Data represent the average relative intensity expression levels of control (open bar) or auxin-treated (blue bar) samples from triplicate experiments. Bars represent sd of the average. Boxes next to gene names indicate classification color codes according to (A) and (B).