Genetic characterization of mutants resistant to the antiauxin p-chlorophenoxyisobutyric acid reveals that AAR3, a gene encoding a DCN1-like protein, regulates responses to the synthetic auxin 2,4-dichlorophenoxyacetic acid in Arabidopsis roots

Abstract

To isolate novel auxin-responsive mutants in Arabidopsis (Arabidopsis thaliana), we screened mutants for root growth resistance to a putative antiauxin, p-chlorophenoxyisobutyric acid (PCIB), which inhibits auxin action by interfering the upstream auxin-signaling events. Eleven PCIB-resistant mutants were obtained. Genetic mapping indicates that the mutations are located in at least five independent loci, including two known auxin-related loci, TRANSPORT INHIBITOR RESPONSE1 and Arabidopsis CULLIN1. antiauxin-resistant mutants (aars) aar3-1, aar4, and aar5 were also resistant to 2,4-dichlorophenoxyacetic acid as shown by a root growth assay. Positional cloning of aar3-1 revealed that the AAR3 gene encodes a protein with a domain of unknown function (DUF298), which has not previously been implicated in auxin signaling. The protein has a putative nuclear localization signal and shares homology with the DEFECTIVE IN CULLIN NEDDYLATION-1 protein through the DUF298 domain. The results also indicate that PCIB can facilitate the identification of factors involved in auxin or auxin-related signaling.

Figure 1.

Effects of PCIB on root growth and root tip morphology of Arabidopsis. A, Wild-type (Columbia-0) seedlings (n = 14) were germinated on GM with or without 20 μm PCIB and grown vertically under continuous light. Error bars indicate sd. Absence of bars indicates deviation is less than size of symbol. B, Roots from 5-d-old wild-type seedlings germinated and horizontally grown on GM with (right) or without (left) 20 μm PCIB were stained with 10 μg/mL propidium iodide. Bar = 100 μm. C and D, Roots from 5-d-old DR5∷GUS seedlings germinated and grown vertically on GM without (C) or with (D) 20 μm PCIB were stained with X-gluc for 15 h. Bar = 50 μm. E, A root from a 10-d-old QC46 seedling germinated and grown vertically on GM containing 20 μm PCIB was stained with X-gluc for 15 h. Arrow indicates primary root tip. Arrowhead indicates QC cells in secondary meristem. Bar = 100 μm. F, A root from a 14-d-old wild-type seedling germinated and grown vertically on GM containing 20 μm PCIB was stained with Lugol's solution (Merck KGaA) to visualize starch granules and was cleared with chloral hydrate (Willemsen et al., 1998). Arrow indicates primary root tip. Arrowhead indicates starch granules in the newly formed columella cells in secondary meristem. Bar = 100 μm. G, A root from a 10-d-old DR5∷GUS seedling germinated and vertically grown on GM with 20 μm PCIB was stained with X-gluc for 15 h. Arrow indicates original distal root tip. Arrowhead indicates secondary root meristem. Bar = 100 μm.

Figure 2.

Mapping of the aar mutants and identification of mutation in TIR1 and AtCUL1 loci. A, Map position of PCIB-resistant mutations. Approximate Arabidopsis Genome Initiative map positions (Mb) of mapping markers are shown in parentheses to the right of each chromosome. The interval to which each PCIB mutant maps is shown to the left of the chromosomes. For aar3-1, no recombinants were identified from 826 chromosomes for markers K5K13-8, K5K13-9, and K5K13-2. m31, m34, m85, and m100 have 1/396, 1/108, 1/20, and 3/104 recombinants at NGA112 and 0/792, 0/110, 0/20, and 0/150 recombinants at T20O10-1, respectively. aar5, m35, and m36 have 6/78, 1/40, and 3/60 recombinants at CIW5-1 and 4/120, 0/86, and 0/60 recombinants at T10P11-1, respectively. aar4 has 1/72 recombinants at both RCI 1B and T24H18 markers. B, Location of the m31 and m34 mutation sites in the TIR1 gene. Black and white boxes indicate exons in translated and untranslated regions, respectively. Lines indicate introns. C, Positions of T-DNA insertions in aar2 mutants in the AtCUL1 gene. D, Distribution of root length of wild-type and aar2-2 heterozygous seedlings germinated and grown vertically on 20 μm PCIB for 14 d.

Figure 3.

Phenotype of the aar mutants. A and B, Photographs of 3-d-old seedlings of aar mutants grown vertically for 8 d under white light on GM-MES medium without (A) or with (B) 20 μm PCIB. Two seedlings are shown for each mutant. C to E, Response to PCIB (C), 2,4-D (D), and IAA (E) in aar mutants. Seeds were germinated on GM-MES medium and grown under white light for 3 d. Germinated seedlings were transferred to media containing chemicals at indicated concentrations. Five days later, new root growth was measured and plotted as a percentage of root growth on medium without chemicals. Error bars represent sds of the means of 12 to 14 seedlings. F, Seedlings grown horizontally on GM-MES for 4 d under white light at 23°C. Two seedlings are shown for each. G and H, Hypocotyl length (G) and number of lateral roots (H) of 11-d-old seedlings. For lateral roots, only visible primordia that emerged from the main root were counted. Error bars represent sds of the means of 12 to 14 seedlings. I, Comparison of morphology of rosette leaves of the wild type and aar4. Plants were grown for 35 d on a 1:1 mixture of vermiculite and Metromix 350 (Scotts-Sierra Horticultural Products) without any supplementation at 23°C in a growth chamber under a 14-h-light and 10-h-dark regime.

Figure 4.

Responses of roots of the wild type and aar3-1 to IBA, other classes of phytohormones, and gravity. A to E, Germinated seedlings (3 d old) were transferred to media containing IBA (A), abscisic acid (ABA; B), 6-benzyladenine (BA; C), 1-aminocyclopropane-1-carboxylic acid (ACC; D), and methyl jasmonate (Me-JA; E) and grown vertically under white light. Elongated root length was measured after 5 d of incubation. Values are expressed as a percentage of root growth without hormones for each genotype. Each data point represents the mean ± sd of 12 to 14 seedlings. F, Distribution of root growth direction of the seedlings after 3 d of stimulation at 135° to the vertical (n = 25 for the wild type and 26 for aar3-1). Three-day-old seedlings were transferred to fresh GM-MES medium, given 135° gravity stimulus, and incubated in the dark for 3 d. The arrows indicate the vector of gravity before (1) and after (2) the commencement of gravity stimulus. The angles were grouped into 12 classes and expressed as percentage in a wheel diagram. [See online article for color version of this figure.]

Figure 5.

Molecular cloning of the AAR3 gene. A, Fine mapping with the PCR-based markers MMG15-2, MZN14-1, MLD15-1, MYI13-1, K5K13-2, MRI12-5, MXE2-3, and MUO22-1, which are located at the MYI13 and K5K13 regions on chromosome 3. The position of the 6.6-kb DNA fragment used for the complementation test is indicated with a hatched box. Open reading frames in this region were shown with black (for AAR3) and white arrows (for other open reading frames). Black and white boxes of AAR3 indicate exons in translated and untranslated regions, respectively. Lines indicate introns. The position of the mutation in aar3-1 is indicated with an asterisk, and the positions of the T-DNA insertion in aar3-2 and aar3-3 are indicated with open triangles. aar3-1 has a G-to-A mutation at the first base of the third intron, which may prevent normal splicing and result in generation of a truncated product. Nucleotides in the third intron in the wild type and the predicted stop codon in aar3-1 are shown in italics. B and C, The steady-state levels of the AAR3 transcript in various mutants analyzed by RT-PCR. Same volume (1 μL) of control DNA with different concentration (1 ng μL⁻¹, 0.1 ng μL⁻¹, 0.01 ng μL⁻¹, or 0.001 ng μL⁻¹) was used in the PCR reaction. D, Relative root length of aar mutants grown on PCIB or 2,4-D. Seeds were germinated on GM-MES medium containing 20 μm PCIB or 40 nm 2,4-D, and grown vertically under white light for 10 d. Root growth was measured and plotted as a percentage of root growth on control medium without the growth regulators. Error bars represent sds of the means of at least 16 seedlings. E, Segregation of PCIB-sensitive seedlings in the T₂ population of a transgenic aar3-2 line (line 12) transformed with the 6.6-kb fragment indicated as a hatched box in A. The seedlings were germinated and grown for 9 d on 20 μm PCIB. [See online article for color version of this figure.]

Figure 6.

Amino acid sequence of the AAR3 protein and alignment of the DUF298 domain region with other AAR3-like proteins. A, The AAR3 proteins are composed of 295 amino acid residues. The DUF298 domain is indicated with a red underline. NLSs bipartite (RRPHTGLRNIPGLKRKT) and pattern 7 (PGLKRKT) are shown by black and blue underlines, respectively. B, Nuclear localization of YFP-AAR3 in Arabidopsis protoplasts. Shown are protoplasts of Arabidopsis T87 suspension-cultured cells coexpressing 35S∷YFP and 35S∷NLS-tdTomato (top panels), and 35S∷YFP-AAR3 and 35S∷NLS-tdTomato (bottom panels). YFP-AAR3-associated fluorescence is localized in the nucleus, but nonfused YFP fluorescence is distributed in both the nucleus and the cytosol. NLS-tdTomato was used as the nuclear marker and its fluorescent signal was captured in the WIY channel. Bars = 10 nm. C, ClustalW alignment of the DUF298 domains of AAR3 and AAR3-like proteins. The amino acid sequence (amino acid nos. 72–177, shown in red underlines in A) of the DUF298 domain of the AAR3 protein (bold letters in the top line) was used for alignment with a Leu zipper-like protein of Oryza sativa (NP_001060359), DCN1-like protein 2 of human (Q6PH85) and mouse (Q8BZJ7), DCN1-like protein 1 of human (Q96GG9) and Drosophila (Q9VUQ8), At3g12760 protein from Arabidopsis (AAK93603), Leu zipper protein of O. sativa (BAD38167), DCN-1 of Schizosaccharomyces pombe (Q8WZK4), and Dcn1p of Saccharomyces cerevisiae (NP_013229). Amino acid residues identical and similar to the AAR3 sequence are colored with red and orange, respectively. Asterisks show Leu residues in the putative Leu zipper-like domain.

Figure 7.

Proposed model for PCIB resistance of the tir1 and atcul1 mutants and PCIB-dependent root growth inhibition. A, PCIB inhibits SCF^TIR1 activity and reduces auxin response. Depletion of auxin response directly results in root growth inhibition. B, PCIB indirectly promotes ectopic auxin accumulation in the root, which causes root growth inhibition through SCF^TIR1-dependent auxin signal transduction.