Functional genomic analysis of the AUXIN/INDOLE-3-ACETIC ACID gene family members in Arabidopsis thaliana

Abstract

Auxin regulates various aspects of plant growth and development. The AUXIN/INDOLE-3-ACETIC ACID (Aux/IAA) genes encode short-lived transcriptional repressors that are targeted by the TRANSPORT INHIBITOR RESPONSE1/AUXIN RECEPTOR F-BOX proteins. The Aux/IAA proteins regulate auxin-mediated gene expression by interacting with members of the AUXIN RESPONSE FACTOR protein family. Aux/IAA function is poorly understood; herein, we report the identification and characterization of insertion mutants in 12 of the 29 Aux/IAA family members. The mutants show no visible developmental defects compared with the wild type. Double or triple mutants of closely related Aux/IAA genes, such as iaa8-1 iaa9-1 or iaa5-1 iaa6-1 iaa19-1, also exhibit wild-type phenotypes. Global gene expression analysis reveals that the molecular phenotypes of auxin-treated and untreated light-grown seedlings are unaffected in the iaa17-6 and iaa5-1 iaa6-1 iaa19-1 mutants. By contrast, similar analysis with the gain-of-function axr3-1/iaa17-1 mutant seedlings reveals dramatic changes in basal and auxin-induced gene expression compared with the wild type. Expression of several type-A ARABIDOPSIS RESPONSE REGULATOR genes and a number of genes involved in cell wall biosynthesis and degradation is repressed in axr3-1/iaa17-1. The data suggest extensive functional redundancy among Aux/IAA gene family members and that enhanced stability of the AXR3/IAA17 protein severely alters the molecular phenotype, resulting in developmental defects.

Figure 1.

The Aux/IAA Gene Family of Arabidopsis. (A) Chromosomal location of Aux/IAA genes. The locations of 29 putative Aux/IAA 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 Aux/IAA gene are shown on the left side of the chromosomes. (B) Phylogenetic analysis. The gene names, accession numbers, protein IDs, and the accession numbers of the full-length ORFs used for this analysis are also shown. The full-length ORFs of IAA18, IAA19, IAA28, IAA29, IAA30, IAA31, IAA32, and IAA33 were constructed during this study.

Figure 2.

Alignment of the Aux/IAA Proteins Reveals Several Highly Conserved Domains. The deduced amino acid sequences of the Aux/IAA proteins were aligned using ClustalW (Thompson et al., 1994). The conserved domains I, II, III, and IV present in the Aux/IAA proteins are indicated at the top of the alignment. The sequences used in this analysis are the same as those used for constructing the phylogenetic tree shown in Figure 1B.

Figure 3.

Location of T-DNA or dSpm Insertions in the Aux/IAA Gene Family Members. Boxes represent exons. Insertions with gray triangles denote lines whose characterization has been completed. Insertions with white triangles denote lines not characterized.

Figure 4.

Growth Phenotype of Insertion Mutants of Various Aux/IAA Gene Family Members. Three wild-type (left) and three mutant plants (right) were grown simultaneously and are shown for each line. White dots indicate the boundaries between the wild-type and the mutant plants.

Figure 5.

Phenotypes of iaa8 iaa9 Double and iaa5 iaa6 iaa19 Triple Mutants during Developmental Stages. (A) Four-week-old soil-grown plants of the wild type and the iaa8-1 iaa9-1 double mutant. (B) Four-week-old soil-grown plants of the wild type or the iaa5-1 iaa6-1, iaa5-1 iaa19-1, and iaa6-1 iaa19-1 double mutants. (C) Four-week-old soil-grown plants of the wild type and the iaa5-1 iaa6-1 iaa19-1 triple mutant. (D) Root growth inhibition assay of wild-type and iaa5-1 iaa6-1 iaa19-1 triple mutant seedlings. Four-day-old seedlings were transferred to medium containing the indicated concentrations of IAA, and the increase in root length was measured after 5 d. Root length is shown as a percentage of the root length in the absence of auxin. Each point represents the average of at least 10 seedlings. Bars represent se. The double and triple mutants have been deposited with the ABRC (http://www.biosci.ohio-state.edu/∼plantbio/Facilities/abrc/abrchome.htm) and the Nottingham Arabidopsis Stock Centre (http://arabidopsis.info/).

Figure 6.

Global Gene Expression Profiling. M-A plots (Dudiot et al., 2002) showing changes of auxin-mediated gene expression in the wild type, iaa17-6, axr3-1/iaa17-1, and iaa5-1 iaa6-1 iaa19-1. Each plot represents the log₂ 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)] (Dudiot et al., 2002). The genes induced by auxin treatment (M > 1) are shown in red, and the genes repressed by auxin treatment (M < −1) are shown in green. The data were further analyzed for variance to extract the statistically valid auxin-regulated genes discussed in Tables 1 and 2 and Figure 8.

Figure 7.

Expression Characteristics of Cell Wall–Related Genes in Wild-Type, iaa17-6, axr3-1/iaa17-1, and iaa5-1 iaa6-1 iaa19-1 Seedlings. Data represent the average relative expression intensity of control (open bars) or auxin-treated (gray bars) samples from triplicate experiments. See Methods for further details. Bars represent sd.

Figure 8.

The Expression of Several Type A ARR Genes Is Suppressed in axr3-1/iaa17-1 Seedlings. Histograms show expression patterns of seven type A ARR genes in the wild type, iaa17-6, axr3-1/iaa17-1, and iaa5-1 iaa6-1 iaa19-1. Data represent the average relative expression intensity of control (open bars) or auxin-treated (gray bars) samples from triplicate experiments. The asterisks indicate genes whose expression is statistically different in wild-type (Col) and axr3-1/iaa17-1 seedlings. Bars represent sd.

Figure 9.

Comparision of the Auxin-Regulated Genes in the Wild Type with the Genes Whose Expression Is Altered in axr3-1/iaa17-1 Seedlings. (A) Venn diagram showing the number of overlapping and unique genes that are auxin induced in wild-type seedlings and whose expression is enhanced in control-treated axr3-1/iaa17-1 seedlings. (B) Venn diagram showing the number of overlapping and unique genes that are downregulated by auxin treatment of wild-type seedlings and downregulated in control-treated axr3-1/iaa17-1 seedlings. (C) Expression profiles of the seven overlapping genes whose expression is auxin induced in wild-type seedlings and enhanced in control axr3-1/iaa17-1 seedlings shown in (A). Data represent the average relative expression intensity of control (open bars) or auxin-treated (gray bars) seedlings from triplicate experiments. Bars represent sd. (D) Expression profiles of the six overlapping genes whose expression is downregulated in wild-type seedlings and downregulated in control-treated axr3-1/iaa17-1 seedlings shown in (B). (E) Expression characteristics of the SAUR-AC1 gene.