Overexpression of the Arabidopsis Gene <em>UPRIGHT ROSETTE</em> Reveals a Homeostatic Control for Indole-3-Acetic Acid^1,^[C]^[W]

RESULTS

The URO Gene Encodes a C2H2 Zinc-Finger Protein and Belongs to a Plant-Specific Gene Family

The uro mutant has multiple abnormal plant phenotypes that have been observed in several other auxin-defective mutants, such as lacking apical dominance with retarded reproductive development and having a very soft stem with the reduced xylem size and underdeveloped interfascicular fibers (Guo et al., 2004). To identify the URO gene, we first obtained a DNA fragment containing a part of the left border of a T-DNA sequence and a flanking Arabidopsis sequence by thermal asymmetric interlaced PCR (Liu et al., 1995). Sequence analysis revealed that the T-DNA left border was linked to the upstream sequence of the gene At3g23140, 164 bp from the start codon (Fig. 1A), and the gene At3g23140 was up-regulated in the uro mutant (see below). To prove whether At3g23140 is the URO gene and that its up-regulation could cause the uro phenotypes, we transformed wild-type and uro plants with several constructs (Fig. 1A).

Figure 1.

A T-DNA insertion in the URO locus causes the uro phenotypes. A, Diagram of the predicted structure in the uro locus and constructs used in plant transformation. Note that the T-DNA left-border (LB) sequence is linked to the 164-bp upstream sequence from the ATG of the URO gene. chr3, Chromosome 3; RB, right border. B to D, Mature wild-type (B), uro/+ (C), and uro (D) plants. E and F, RNA interference of the URO gene using the double-stranded URO sequence (p35S:dsAt3g23140) resulted in partial (E) or much better rescue (F) of the apical dominance defect in the transgenic uro plants. G and H, p35S:At3g23140/Ler (G) and pNOS:At3g23140/Ler (H) transgenic plants exhibited severe phenotypes with upright cotyledons or early appearing rosette leaves, and all these plants were arrested at the seedling stages. I, Transgenic plants, carrying the IAAH-At3g23140 construct (the upstream T-DNA sequence including IAAH, the entire URO coding region, and a part of downstream gene At3g23145), showed the uro-like phenotypes. Shown is a 4-week-old IAAH-At3g23140/Ler transgenic plant (I). Bars = 5 mm (B–F, I), 1 mm (G), and 2 mm (H).

First, we transformed uro with p35S:dsAt3g23140, in which an At3g23140 double-stranded RNA interference fusion gene driven by the 35S promoter (Fig. 1A). Compared with wild-type (Fig. 1B), uro/+ (Fig. 1C), and uro (Fig. 1D) plants, 30 out of a total of 51 p35S:dsAt3g23140/uro transgenic plants appeared nearly rescued, showing elongated inflorescence stems and normally shaped plant organs, although some plants in this group exhibited a slightly increased number of branches (Fig. 1F). Thirteen plants were weakly rescued, showing a partially restored apical dominance but a dwarf stature (Fig. 1E), while the other eight transgenic plants were with uro phenotypes (data not shown). Strikingly, the At3g21340 transcript level in the transgenic plants was correlated to the uro phenotype severity (Supplemental Fig. S1A). The p35S:dsAt3g23140 construct was also used to transform wild-type plants; however, all 12 transgenic plants that we obtained appeared normal (data not show). Next, 27 p35S:At3g23140/Landsberg erecta (Ler) and 76 pNOS:At3g23140/Ler transgenic plants were obtained, showing upright cotyledons (Fig. 1G) or rosette leaves (Fig. 1H), and all these plants were arrested at the cotyledon (for p35S:At3g23140/Ler plants) or seedling (for pNOS:At3g23140/Ler plants) stages. To determine the At3g23140 expression-driven sequence in the uro mutant, we transformed wild-type plants with the At3g23140 coding region, either fused to its 164-bp upstream sequence (pAt3g23140:At3g23140) or to the 2,986-bp upstream sequence containing the entire IAAH gene (IAAH-At3g23140; Fig. 1A). While transgenic plants carrying the first fusion did not yield plant phenotypic changes (data not shown), 82 out of 116 transgenic plants harboring IAAH-At3g23140 showed uro-like phenotypes in varying degrees of severity, and 27 out of 82 showed the strong bushy phenotype (Fig. 1I). Finally, wild-type plants were transformed with the 2,986-bp upstream sequence only (IAAH) or with a direct downstream gene (p35S:At3g23145; Fig. 1A), and the resulting eight IAAH/Ler and 10 p35S:At3g23145/Ler transgenic lines did not show altered plant phenotypes (data not shown). Based on the above results, we reasoned that the At3g23140 gene is URO.

The URO gene encodes a protein of 173 amino acids in length and has no predicted intron. A single C2H2 type of zinc-finger-like motif is located in the N terminus (Fig. 2A). A Leu-rich inhibition domain, which is present near the C terminus, contains a conserved motif (L/FDLNL/FXP) known as an ETHYLENE-RESPONSIVE ELEMENT-BINDING FACTOR-associated amphiphilic repression (EAR) motif (Fig. 2B; Fujimoto et al., 2000; Ohta et al., 2001; Hiratsu et al., 2003). An Arabidopsis genome-scale search revealed that URO belongs to a gene family containing 28 members (Englbrecht et al., 2004). Genes in this family are also called SUPERMAN (SUP)-like genes, as SUP was the first member to be cloned (Schultz et al., 1991). This gene family, which is plant specific (Englbrecht et al., 2004), includes members from other plant species such as rice (Oryza sativa), petunia (Petunia hybrida), and even the moss plant Physcomitrella (Fig. 2, A and B). The URO transcript levels in wild-type and mutant seedlings were analyzed by quantitative real-time reverse transcription (RT)-PCR. Compared with the wild type, the URO level was dramatically elevated in the uro/+ and uro seedlings (Fig. 2C). In different tissues analyzed in wild-type Arabidopsis, URO transcripts were only weakly detected in inflorescence (Fig. 2D), and these results were reproducible even with increased PCR cycles (Supplemental Fig. S2). The URO expression was strongly up-regulated in different plant tissues in the uro mutant, especially in stems and inflorescences. These results suggest that the uro phenotypes are likely to be caused by the abnormal URO expression.

Figure 2.

Sequence and expression pattern analyses of the URO gene. A and B, Amino acid sequence alignments show that the zinc-finger domain (A) and L/FDLNL/FXP, called EAR motif (B), of the URO protein share a high similarity to those of other plant homologs. The absolutely conserved residues are highlighted in yellow, highly conserved residues in blue, and conserved residues in green. C, Different expression levels of 6-d-old uro/+ and uro seedlings were shown by quantitative real-time RT-PCR. All values were normalized against the expression level of the ACTIN genes. Three biological replicates, each with three technical repeats, were performed, and the data are shown as the averages ± se. D, In different plant tissues, RT-PCR was used to detect URO transcript. In 5-week-old wild-type plants, URO transcripts were only weakly detected in inflorescence, whereas the transcription levels were dramatically elevated in several tissues of the uro mutants.

The uro Mutant Has Alterations in IAA Response and Homeostasis

To address the auxin-related phenotypes in the uro mutant, we first investigated the auxin response by performing a root elongation inhibition test. Wild-type and uro seedlings were first grown on a hormone-free medium for 3 d. Seedlings with similar root lengths were then transferred to new media containing a range of IAA concentrations. The length of the primary root was measured after 3 d growth on the new media. At lower concentrations (0.001–0.01 nm), IAA promoted root elongation in both wild-type and uro mutant seedlings, although the promotion in wild-type seedlings was more evident (Fig. 3, A and B). At a concentration of 0.1 nm, IAA promoted the root elongation of wild-type seedlings even more strongly, whereas elongation of uro roots was inhibited (Fig. 3, A and B). Treatment with 1 nm of IAA still promoted root elongation in wild-type seedlings, but severely affected uro root growth (Fig. 3, A and B). With a further increase in IAA concentration, root elongation was inhibited in both wild-type and uro seedlings (Fig. 3, A and B). These results suggest that root elongation in uro plants is hypersensitive to IAA. We also analyzed lateral root, and found that in the uro mutant the number of lateral roots was reduced as compared with wild-type plants (Supplemental Fig. S3).

Figure 3.

The increased URO expression resulted in the altered IAA response and homeostasis in the uro mutant. A and B, Roots elongation of uro showed a higher sensitivity to the IAA concentration. Data in A represent average ± se. Asterisks (* and **) indicate significant statistical differences by t test (P < 0.05 and P < 0.01, respectively) between wild type and uro. Ten to 15 seedlings were used in each measurement of the different IAA concentrations, with three biological replicates. C to F, Measurement of IAA contents in 12-d-old whole seedlings (C and D) and 4-week-old rosette leaves of wild-type and uro plants (E and F). The free, IAA-ester, and IAA-amino conjugates were respectively measured using three biological replicates (C and E), while the total IAA contents were calculated using the average of the three biological replicates from C and E, and the calculated results are shown in D and F, respectively. [See online article for color version of this figure.]

We next measured IAA levels in the uro mutant. Total IAA levels, including free, ester-, and amide-linked IAA, were similar in 12-d-old uro and wild-type aboveground seedlings (Fig. 3D) and 4-week-old rosette leaves (Fig. 3F). However, levels of each of these three IAA forms differed markedly in uro plants compared to wild-type plants (Fig. 3, C and E). In particular, free and ester-linked IAA levels were elevated, whereas amide-linked IAA levels decreased (Fig. 3, C and E). These results suggest that overexpression of the URO gene affects IAA homeostasis in Arabidopsis.

To provide additional evidence that the uro mutant contains increased levels of free IAA, we introduced the pDR5:GUS construct (Ulmasov et al., 1997) into uro mutant plants by crosses. Because the original pDR5:GUS plant is in the Columbia-0 background and the uro mutant is in Ler, we examined GUS reporter activity in progeny siblings with wild-type-like (pDR5:GUS/W), uro/+ (pDR5:GUS/uro/+), or uro (pDR5:GUS/uro) phenotypes. In young pDR5:GUS/W seedlings, GUS signals were weakly present in the root tip and distal parts of leaves (Fig. 4A). In comparison, GUS staining was observed throughout all aboveground tissues of pDR5:GUS/uro seedlings, and was highly accumulated in the vascular tissue (Fig. 4B). We then transformed pDR5:GUS/W plants with the pNOS:URO construct (Fig. 1A). In the resulting pNOS:URO/pDR5:GUS/W plants, the increased URO expression resulted in an even stronger accumulation of GUS staining throughout the entire seedling, with the plant apex and root tip both being highly stained (Fig. 4C). GUS signals in 5-week-old pDR5:GUS/uro/+ (Fig. 4, E and G) or pDR5:GUS/uro (Fig. 4H) plants were also strong, as compared to the pDR5:GUS/W plants (Fig. 4, D and F), especially in the stem.

Figure 4.

Auxin reporter expression and adventitious root formation in uro mutants. A and B, GUS staining of a 7-d-old wild-type (A) or uro (B) seedling carrying the pDR5:GUS construct. C, A 7-d-old pNOS:URO/pDR5:GUS/W transgenic plant. D to H, GUS staining of a 5-week-old wild-type (D and F), uro/+ (E and G), or uro/uro (H) plant carrying the pDR5:GUS construct. The strongest DR5::GUS expression is in stem in both uro/uro and uro/+ plants. I and J, When cotyledon explants were grown on IAA-free medium containing one-half Murashige and Skoog for 7 d, the adventitious roots were seen only in the uro cotyledon (J), not in the wild-type cotyledon (I). Bars = 2 mm (A–G), 1 mm (H), and 0.5 mm (I–J).

It was previously reported that explants from sur1, sur2, and yucca-D mutants, which contain higher concentrations of free IAA, are able to generate roots on auxin-free medium (Delarue et al., 1998; Zhao et al., 2001). To determine whether uro tissue could generate roots on hormone-free medium, uro cotyledons were used as explants. As expected, after 7 d growth on hormone-free media, root initiation was observed from uro cotyledons (Fig. 4J), but not from wild-type cotyledons (Fig. 4I). The pDR5:GUS staining and the root initiation experiments further support the idea that certain uro tissues contain a high level of free IAA.

Overexpression of the URO Gene Affects Auxin Homeostasis in Physcomitrella

The moss Physcomitrella belongs to the bryophytes, the evolutionary oldest group of land plants. A search of a genome sequencing database (www.cosmoss.org) revealed that Physcomitrella contains at least four putative URO homologs. To investigate whether the effect of URO overexpression on IAA homeostasis is evolutionarily conserved in other plant species, we analyzed p35S:URO and pNOS:URO transgenic Physcomitrella lines, whose motherline originally carried the pGH3:GUS construct (Bierfreund et al., 2003). As in the pDR5:GUS/Ler transgenic plants, free auxin is able to induce GUS expression in the pGH3:GUS line, and strength of GUS activity was correlated to auxin concentrations in the plants (Bierfreund et al., 2003). The pGH3:GUS motherline usually produced very weak GUS signals in the absence of IAA treatment (Fig. 5, A and D). Six independent p35S:URO/pGH3:GUS and five pNOS:URO/pGH3:GUS transgenic lines were obtained from a moss transformation experiment. The gametophytes of all transgenic lines had much stronger GUS signals than those of the motherline on IAA-free media (Fig. 5, B, C, E, and F). This indicates that the effect of URO overexpression on auxin homeostasis is evolutionarily conserved.

Figure 5.

Overexpression of the URO gene in moss Physcomitrella promoted the pGH3:GUS expression. A and D, Two-month-old Physcomitrella gametophores were analyzed. The motherline carrying pGH3:GUS showed weak GUS signals (A and D). B, C, E, and F, The motherline carrying either p35S:URO (B and E) or pNOS:GUS (C and F) resulted in strong GUS accumulations in gametophores. D to F are the magnification of the plants that were pointed by a black arrow in A, B, and C, respectively. Bar = 2 mm (A–C) and 0.5 mm (D–F).

The URO Gene Encodes a C2H2 Zinc-Finger Protein and Belongs to a Plant-Specific Gene Family

The uro mutant has multiple abnormal plant phenotypes that have been observed in several other auxin-defective mutants, such as lacking apical dominance with retarded reproductive development and having a very soft stem with the reduced xylem size and underdeveloped interfascicular fibers (Guo et al., 2004). To identify the URO gene, we first obtained a DNA fragment containing a part of the left border of a T-DNA sequence and a flanking Arabidopsis sequence by thermal asymmetric interlaced PCR (Liu et al., 1995). Sequence analysis revealed that the T-DNA left border was linked to the upstream sequence of the gene At3g23140, 164 bp from the start codon (Fig. 1A), and the gene At3g23140 was up-regulated in the uro mutant (see below). To prove whether At3g23140 is the URO gene and that its up-regulation could cause the uro phenotypes, we transformed wild-type and uro plants with several constructs (Fig. 1A).

Figure 1.

A T-DNA insertion in the URO locus causes the uro phenotypes. A, Diagram of the predicted structure in the uro locus and constructs used in plant transformation. Note that the T-DNA left-border (LB) sequence is linked to the 164-bp upstream sequence from the ATG of the URO gene. chr3, Chromosome 3; RB, right border. B to D, Mature wild-type (B), uro/+ (C), and uro (D) plants. E and F, RNA interference of the URO gene using the double-stranded URO sequence (p35S:dsAt3g23140) resulted in partial (E) or much better rescue (F) of the apical dominance defect in the transgenic uro plants. G and H, p35S:At3g23140/Ler (G) and pNOS:At3g23140/Ler (H) transgenic plants exhibited severe phenotypes with upright cotyledons or early appearing rosette leaves, and all these plants were arrested at the seedling stages. I, Transgenic plants, carrying the IAAH-At3g23140 construct (the upstream T-DNA sequence including IAAH, the entire URO coding region, and a part of downstream gene At3g23145), showed the uro-like phenotypes. Shown is a 4-week-old IAAH-At3g23140/Ler transgenic plant (I). Bars = 5 mm (B–F, I), 1 mm (G), and 2 mm (H).

First, we transformed uro with p35S:dsAt3g23140, in which an At3g23140 double-stranded RNA interference fusion gene driven by the 35S promoter (Fig. 1A). Compared with wild-type (Fig. 1B), uro/+ (Fig. 1C), and uro (Fig. 1D) plants, 30 out of a total of 51 p35S:dsAt3g23140/uro transgenic plants appeared nearly rescued, showing elongated inflorescence stems and normally shaped plant organs, although some plants in this group exhibited a slightly increased number of branches (Fig. 1F). Thirteen plants were weakly rescued, showing a partially restored apical dominance but a dwarf stature (Fig. 1E), while the other eight transgenic plants were with uro phenotypes (data not shown). Strikingly, the At3g21340 transcript level in the transgenic plants was correlated to the uro phenotype severity (Supplemental Fig. S1A). The p35S:dsAt3g23140 construct was also used to transform wild-type plants; however, all 12 transgenic plants that we obtained appeared normal (data not show). Next, 27 p35S:At3g23140/Landsberg erecta (Ler) and 76 pNOS:At3g23140/Ler transgenic plants were obtained, showing upright cotyledons (Fig. 1G) or rosette leaves (Fig. 1H), and all these plants were arrested at the cotyledon (for p35S:At3g23140/Ler plants) or seedling (for pNOS:At3g23140/Ler plants) stages. To determine the At3g23140 expression-driven sequence in the uro mutant, we transformed wild-type plants with the At3g23140 coding region, either fused to its 164-bp upstream sequence (pAt3g23140:At3g23140) or to the 2,986-bp upstream sequence containing the entire IAAH gene (IAAH-At3g23140; Fig. 1A). While transgenic plants carrying the first fusion did not yield plant phenotypic changes (data not shown), 82 out of 116 transgenic plants harboring IAAH-At3g23140 showed uro-like phenotypes in varying degrees of severity, and 27 out of 82 showed the strong bushy phenotype (Fig. 1I). Finally, wild-type plants were transformed with the 2,986-bp upstream sequence only (IAAH) or with a direct downstream gene (p35S:At3g23145; Fig. 1A), and the resulting eight IAAH/Ler and 10 p35S:At3g23145/Ler transgenic lines did not show altered plant phenotypes (data not shown). Based on the above results, we reasoned that the At3g23140 gene is URO.

The URO gene encodes a protein of 173 amino acids in length and has no predicted intron. A single C2H2 type of zinc-finger-like motif is located in the N terminus (Fig. 2A). A Leu-rich inhibition domain, which is present near the C terminus, contains a conserved motif (L/FDLNL/FXP) known as an ETHYLENE-RESPONSIVE ELEMENT-BINDING FACTOR-associated amphiphilic repression (EAR) motif (Fig. 2B; Fujimoto et al., 2000; Ohta et al., 2001; Hiratsu et al., 2003). An Arabidopsis genome-scale search revealed that URO belongs to a gene family containing 28 members (Englbrecht et al., 2004). Genes in this family are also called SUPERMAN (SUP)-like genes, as SUP was the first member to be cloned (Schultz et al., 1991). This gene family, which is plant specific (Englbrecht et al., 2004), includes members from other plant species such as rice (Oryza sativa), petunia (Petunia hybrida), and even the moss plant Physcomitrella (Fig. 2, A and B). The URO transcript levels in wild-type and mutant seedlings were analyzed by quantitative real-time reverse transcription (RT)-PCR. Compared with the wild type, the URO level was dramatically elevated in the uro/+ and uro seedlings (Fig. 2C). In different tissues analyzed in wild-type Arabidopsis, URO transcripts were only weakly detected in inflorescence (Fig. 2D), and these results were reproducible even with increased PCR cycles (Supplemental Fig. S2). The URO expression was strongly up-regulated in different plant tissues in the uro mutant, especially in stems and inflorescences. These results suggest that the uro phenotypes are likely to be caused by the abnormal URO expression.

Figure 2.

Sequence and expression pattern analyses of the URO gene. A and B, Amino acid sequence alignments show that the zinc-finger domain (A) and L/FDLNL/FXP, called EAR motif (B), of the URO protein share a high similarity to those of other plant homologs. The absolutely conserved residues are highlighted in yellow, highly conserved residues in blue, and conserved residues in green. C, Different expression levels of 6-d-old uro/+ and uro seedlings were shown by quantitative real-time RT-PCR. All values were normalized against the expression level of the ACTIN genes. Three biological replicates, each with three technical repeats, were performed, and the data are shown as the averages ± se. D, In different plant tissues, RT-PCR was used to detect URO transcript. In 5-week-old wild-type plants, URO transcripts were only weakly detected in inflorescence, whereas the transcription levels were dramatically elevated in several tissues of the uro mutants.

The uro Mutant Has Alterations in IAA Response and Homeostasis

To address the auxin-related phenotypes in the uro mutant, we first investigated the auxin response by performing a root elongation inhibition test. Wild-type and uro seedlings were first grown on a hormone-free medium for 3 d. Seedlings with similar root lengths were then transferred to new media containing a range of IAA concentrations. The length of the primary root was measured after 3 d growth on the new media. At lower concentrations (0.001–0.01 nm), IAA promoted root elongation in both wild-type and uro mutant seedlings, although the promotion in wild-type seedlings was more evident (Fig. 3, A and B). At a concentration of 0.1 nm, IAA promoted the root elongation of wild-type seedlings even more strongly, whereas elongation of uro roots was inhibited (Fig. 3, A and B). Treatment with 1 nm of IAA still promoted root elongation in wild-type seedlings, but severely affected uro root growth (Fig. 3, A and B). With a further increase in IAA concentration, root elongation was inhibited in both wild-type and uro seedlings (Fig. 3, A and B). These results suggest that root elongation in uro plants is hypersensitive to IAA. We also analyzed lateral root, and found that in the uro mutant the number of lateral roots was reduced as compared with wild-type plants (Supplemental Fig. S3).

Figure 3.

The increased URO expression resulted in the altered IAA response and homeostasis in the uro mutant. A and B, Roots elongation of uro showed a higher sensitivity to the IAA concentration. Data in A represent average ± se. Asterisks (* and **) indicate significant statistical differences by t test (P < 0.05 and P < 0.01, respectively) between wild type and uro. Ten to 15 seedlings were used in each measurement of the different IAA concentrations, with three biological replicates. C to F, Measurement of IAA contents in 12-d-old whole seedlings (C and D) and 4-week-old rosette leaves of wild-type and uro plants (E and F). The free, IAA-ester, and IAA-amino conjugates were respectively measured using three biological replicates (C and E), while the total IAA contents were calculated using the average of the three biological replicates from C and E, and the calculated results are shown in D and F, respectively. [See online article for color version of this figure.]

We next measured IAA levels in the uro mutant. Total IAA levels, including free, ester-, and amide-linked IAA, were similar in 12-d-old uro and wild-type aboveground seedlings (Fig. 3D) and 4-week-old rosette leaves (Fig. 3F). However, levels of each of these three IAA forms differed markedly in uro plants compared to wild-type plants (Fig. 3, C and E). In particular, free and ester-linked IAA levels were elevated, whereas amide-linked IAA levels decreased (Fig. 3, C and E). These results suggest that overexpression of the URO gene affects IAA homeostasis in Arabidopsis.

To provide additional evidence that the uro mutant contains increased levels of free IAA, we introduced the pDR5:GUS construct (Ulmasov et al., 1997) into uro mutant plants by crosses. Because the original pDR5:GUS plant is in the Columbia-0 background and the uro mutant is in Ler, we examined GUS reporter activity in progeny siblings with wild-type-like (pDR5:GUS/W), uro/+ (pDR5:GUS/uro/+), or uro (pDR5:GUS/uro) phenotypes. In young pDR5:GUS/W seedlings, GUS signals were weakly present in the root tip and distal parts of leaves (Fig. 4A). In comparison, GUS staining was observed throughout all aboveground tissues of pDR5:GUS/uro seedlings, and was highly accumulated in the vascular tissue (Fig. 4B). We then transformed pDR5:GUS/W plants with the pNOS:URO construct (Fig. 1A). In the resulting pNOS:URO/pDR5:GUS/W plants, the increased URO expression resulted in an even stronger accumulation of GUS staining throughout the entire seedling, with the plant apex and root tip both being highly stained (Fig. 4C). GUS signals in 5-week-old pDR5:GUS/uro/+ (Fig. 4, E and G) or pDR5:GUS/uro (Fig. 4H) plants were also strong, as compared to the pDR5:GUS/W plants (Fig. 4, D and F), especially in the stem.

Figure 4.

Auxin reporter expression and adventitious root formation in uro mutants. A and B, GUS staining of a 7-d-old wild-type (A) or uro (B) seedling carrying the pDR5:GUS construct. C, A 7-d-old pNOS:URO/pDR5:GUS/W transgenic plant. D to H, GUS staining of a 5-week-old wild-type (D and F), uro/+ (E and G), or uro/uro (H) plant carrying the pDR5:GUS construct. The strongest DR5::GUS expression is in stem in both uro/uro and uro/+ plants. I and J, When cotyledon explants were grown on IAA-free medium containing one-half Murashige and Skoog for 7 d, the adventitious roots were seen only in the uro cotyledon (J), not in the wild-type cotyledon (I). Bars = 2 mm (A–G), 1 mm (H), and 0.5 mm (I–J).

It was previously reported that explants from sur1, sur2, and yucca-D mutants, which contain higher concentrations of free IAA, are able to generate roots on auxin-free medium (Delarue et al., 1998; Zhao et al., 2001). To determine whether uro tissue could generate roots on hormone-free medium, uro cotyledons were used as explants. As expected, after 7 d growth on hormone-free media, root initiation was observed from uro cotyledons (Fig. 4J), but not from wild-type cotyledons (Fig. 4I). The pDR5:GUS staining and the root initiation experiments further support the idea that certain uro tissues contain a high level of free IAA.

Overexpression of the URO Gene Affects Auxin Homeostasis in Physcomitrella

The moss Physcomitrella belongs to the bryophytes, the evolutionary oldest group of land plants. A search of a genome sequencing database (www.cosmoss.org) revealed that Physcomitrella contains at least four putative URO homologs. To investigate whether the effect of URO overexpression on IAA homeostasis is evolutionarily conserved in other plant species, we analyzed p35S:URO and pNOS:URO transgenic Physcomitrella lines, whose motherline originally carried the pGH3:GUS construct (Bierfreund et al., 2003). As in the pDR5:GUS/Ler transgenic plants, free auxin is able to induce GUS expression in the pGH3:GUS line, and strength of GUS activity was correlated to auxin concentrations in the plants (Bierfreund et al., 2003). The pGH3:GUS motherline usually produced very weak GUS signals in the absence of IAA treatment (Fig. 5, A and D). Six independent p35S:URO/pGH3:GUS and five pNOS:URO/pGH3:GUS transgenic lines were obtained from a moss transformation experiment. The gametophytes of all transgenic lines had much stronger GUS signals than those of the motherline on IAA-free media (Fig. 5, B, C, E, and F). This indicates that the effect of URO overexpression on auxin homeostasis is evolutionarily conserved.

Figure 5.

Overexpression of the URO gene in moss Physcomitrella promoted the pGH3:GUS expression. A and D, Two-month-old Physcomitrella gametophores were analyzed. The motherline carrying pGH3:GUS showed weak GUS signals (A and D). B, C, E, and F, The motherline carrying either p35S:URO (B and E) or pNOS:GUS (C and F) resulted in strong GUS accumulations in gametophores. D to F are the magnification of the plants that were pointed by a black arrow in A, B, and C, respectively. Bar = 2 mm (A–C) and 0.5 mm (D–F).

DISCUSSION

The Cause of the uro Phenotypes

We previously reported isolation and characterization of the uro mutant, and proposed that the mutant phenotypes may be caused by defective auxin biology (Sun et al., 2000; Guo et al., 2004; Yuan et al., 2007). However, in previous studies of the URO gene, two important questions were unanswered. First, which gene corresponds to the uro abnormalities? And second, what is the molecular basis by which uro mutation affects plant development? In this work, we report the identification of the URO gene, which encodes a SUP-like zinc-finger protein. Overexpression of the URO gene results in the abnormal uro phenotypes. We also show that the uro auxin-related phenotypes are likely to be due to the disruption of auxin homeostasis. In answer to our first question, we have provided several lines of evidence to prove that the gene At3g23140 is URO. Transgenic plants harboring an At3g23140-containing genomic fragment phenocopied the uro mutant, and RNA interference of the At3g23140 gene in the uro mutant resulted in the rescue of the uro phenotypes. In addition, transgenic plants containing p35S:At3g23140 and pNOS:At3g23140 both partially mimicked uro. In answer to our second question, we demonstrated altered IAA homeostasis in uro by measuring free, ester-, and amino-linked IAA levels. We also showed indications for elevated IAA levels by a root regeneration experiment using uro cotyledon explants and increased GUS staining in uro lines containing the auxin-responsive pDR5::GUS reporter.

To understand the endogenous function of URO in plant development, we have searched several Arabidopsis databases for a loss-of-function uro mutant, but have not yet found one. The p35S:dsURO/uro construct restored the uro phenotypes, but p35S:dsURO/Ler transgenic plants did not have visible phenotypic changes. In addition, the uro mutant has multiple abnormal phenotypes in different plant tissues and organs, whereas the URO transcripts in wild-type plants could be detected only in the inflorescence. These suggest that the URO endogenous function may not correspond directly to the phenotype of the uro mutant. However, our data suggest that a regulatory system(s) for the IAA homeostasis control exists in plant, which respond to expression changes of URO or URO-like genes. The Arabidopsis genome has 28 members in this URO/SUP gene family, and it is possible that URO function is highly redundant. Arabidopsis and Physcomitrella both respond to URO overexpression by increasing IAA response, strongly suggesting the existence of an evolutionarily conserved molecular mechanism in which IAA homeostasis is affected by changes in the expression of certain regulatory genes. We noted that although both Arabidopsis and Physcomitrella respond to URO overexpression, phenotypic changes in the p35S:URO/pGH3:GUS and pNOS:URO/pGH3:GUS transgenic Physcomitrella lines were not observed. It is possible that different plant species may differ in sensitivity to the free IAA concentrations, which would cause changes in plant phenotypes.

Different Possibilities for Auxin Homeostatic Control

It has been proposed that one of the major functions of IAA conjugates is to act as a storage form of free IAA (Woodward and Bartel, 2005; Bajguz and Piotrowska, 2009). Plants can keep free IAA concentrations at a certain level, to meet their developmental requirements or to respond to their environment. This is accomplished, at least in one respect, as one important pathway by interconversion of free IAA and IAA conjugates. Levels of free IAA and its conjugates are controlled precisely in plants, with different species having distinct IAA conjugate profiles (Woodward and Bartel, 2005; Bajguz and Piotrowska, 2009). The balance between free and bound IAA could also be a useful, highly efficient way to regulate local free IAA concentrations. When needed, overaccumulated free IAA could result in an increased level of IAA conjugates. For example, the sur1 and sur2 mutants contain increased levels of both free and conjugated IAA (Boerjan et al., 1995). In both sur1 and sur2 cases, although the concentrations of free IAA and IAA conjugates were increased, the balance between free IAA and IAA conjugates was not obviously changed. The situation in sur1 and sur2 plants is quite different from that in uro plants. Although the level of free IAA is increased in the uro mutant, which is similar to sur1 and sur2, the level of the total IAA pool in uro does not seem to be markedly changed. In addition, remarkable changes occurred between different types of IAA: Increased levels of free and ester-conjugated IAA were apparently accompanied by a decrease in amide-conjugated IAA.

Enzymes that regulate homeostasis between free IAA and IAA conjugates have recently been identified. These include the IAA amidohydrolases, ILR1, ILL1, ILL2, and IAR3 (Bartel and Fink, 1995; Davies et al., 1999; LeClere et al., 2002; Staswick et al., 2005); several GH3 family gene-encoded IAA amino acid conjugate synthases (Tanaka et al., 2002; Staswick et al., 2005; Jain et al., 2006; Hakkinen et al., 2007; Park et al., 2007; Ludwig-Müller et al., 2009; Zhang et al., 2009); and an Arabidopsis enzyme that catalyzes a reaction to form IAA-Glc (Rosamond et al., 2002). In contrast to these enzyme-encoding genes, URO encodes a putative transcription factor. To understand whether URO overexpression could cause changes in the expression of these enzyme-encoding genes, we previously performed a genome-wide analysis of altered gene expression in uro seedlings using the Affymetrix GeneChip, which contains approximately 24,000 Arabidopsis genes (Yuan et al., 2007). Among the many genes with changes in expression, the YUCCA1 (At4g32540) transcript level was elevated by a Log₂ ratio of 2.2. Although it is not clear whether these genes are direct or indirect targets of URO, it is possible that URO overexpression could affect transcript levels of genes encoding important enzymes for IAA control, resulting in the alteration of auxin homeostasis.

Phenotypic Differences between uro and Other Mutants with Increased Free IAA Levels

Although uro and other mutants such as sur1, sur2, and yucca-D all contain increased free IAA levels than wild-type plants, their phenotypes vary. For example, sur1, sur2, and yucca-D have epinastic cotyledons, whereas cotyledons of uro are normal. However, the rosette leaves of uro seedling and cotyledon of p35S:URO transgenic plants grow vertically similar to the cotyledon petiole of YUCCA6-OX8 (YUCCA6 overexpression transgenic plant; Kim et al., 2007). Cotyledons of both p35S:URO/pDR5:GUS/W (Supplemental Fig. S4) and dark-grown yucca-D/pDR5:GUS (Zhao et al., 2001) seedlings exhibit extremely strong GUS staining, suggesting that the upright cotyledon phenotype may be closely related to the highly accumulated free IAA levels in the cotyledons.

Interestingly, although uro and yucca-D plants contain high free IAA levels, they have opposing apical dominance phenotypes. In yucca-D plants, apical dominance is increased, with an apparently reduced number of lateral branches (Zhao et al., 2001; Kim et al., 2007). However, the uro mutant displays a bushy plant stature, indicative of a loss of apical dominance (Guo et al., 2004). The stems of uro, but not yucca-D, show strong GUS staining in pDR5:GUS plants, reflecting a high level of free IAA. This may indicate a reduced efficiency in the basipetal IAA transport, which is generally thought to be necessary for the establishment of apical dominance (Prusinkiewicz et al., 2009; Shimizu-Sato et al., 2009).

MATERIALS AND METHODS

Plant Materials and Growth Conditions

The Arabidopsis (Arabidopsis thaliana) mutant uro, which is in the Ler genetic background, was generated previously from a T-DNA mutagenesis experiment (Sun et al., 2000). The medium for plant growth contained one-half Murashige and Skoog salt and 0.8% agar. Seeds on media were kept in 4°C for 3 d and then moved to a growth chamber at 22°C. For growing plants in soil, seeds were sowed in a 6:1 mixture of vermiculite and dark soil containing plant nutrition solution (Estelle and Somerville, 1987). The seeds were incubated in 4°C for 24 h and then moved to greenhouse at 20°C to 24°C. All plants were grown under continuous fluorescent illumination.

RT-PCR

PCR after RT of RNA and real-time RT-PCR analyses were performed according to our previous method (Xu et al., 2003; Fu et al., 2007). The gene-specific primers were used in the RT-PCR analysis: 5′-GGTACCATGAACCACCGGGACAAAC-3′ and 5′-GAATTCTTAATGATGACGATGACCGC-3′ for URO; and 5′-TGGCATCA(T/C)ACTTTCTACAA-3′ and 5′-CCACCACT(G/A/T)AGCACAATGTT-3′ for ACTIN.

Constructs for Plant Transformation

For each construct used in plant transformation, DNA fragments were first PCR amplified and verified by sequencing. The primers are listed as follows: 5′-GTTTATTTCGGCGTGTAGGACATG-3′ and 5′-GCATATTTTGTGAAGCTAGTTCGG-3′ for IAAH-At3g23140, 5′-ATTGATAAAACAATTTAGCCC-3′ and 5′-GCATATTTTGTGAAGCTAGTTCGG-3′ for pAt3g23140-At3g23140, 5′-GTTTATTTCGGCGTGTAGGACATG-3′ and 5′-GTGTGTGAACGGACACTAATTAG-3′ for IAAH, 5′-GTGTGTGAACGGACACTAATTAG-3′ and 5′-CCGCTGCAAGCTTAATG-3′ for p35S:At3g23140 and pNOS:At3g23140, and 5′-GTTCTTGTTGTCTGAAGTTGGGT-3′ and 5′-CTTGGAAGAATCCATGGAGTAG-3′ for p35S:At3g23145. Vectors pCAMBIA1301 (for IAAH-At3g23140 or IAAH, pAt3g23140-At3g23140, and pNOS:At3g23140) and pMON530 (for p35S:At3g23140 or p35S:At3g23145) were used in plant transformation. For 35S:dsAt3g23140, the amplified At3g23140 fragment was first subcloned into pFGC5941. The resulting construct was then subcloned into pFGC1008 for plant transformation.

IAA Determination

Extraction and measurement of endogenous IAA through gas chromatography-mass spectrometry (GC-MS) were performed according to a recently reported method (Ludwig-Müller et al., 2009) with slight modification, using aboveground parts of 12-d-old seedlings (grown on medium), or the second pair of rosette leaves from 4-week-old plants (grown in vermiculite). Briefly, approximately 100 mg fresh weight per sample was extracted with iso-propanol:acetic acid (95:5, v/v) and 100 ng ^C₆-IAA (Cambridge Isotope Laboratories) per sample was added as internal standard. For each sample three independent extractions were performed. The samples were incubated under continuous shaking (500 rpm) for 2 h at 4°C. The samples were then centrifuged for 10 min at 10,000g, the supernatant removed and the isopropanol evaporated under a stream of N₂. The samples were diluted 1:5 with water and further evaporated with N₂ until no acetic acid was present. For the determination of free IAA the aqueous residue was brought to pH 3.5 with 1 n HCl and extracted twice with equal volumes of ethyl acetate. The ethyl acetate phases were combined, centrifuged again for 10 min at 10,000g, the supernatant removed and placed in a glass vial. The ethyl acetate was evaporated under a stream of N₂ and the samples were suspended in 50 μL ethyl acetate. Methylation of samples was carried out by adding 950 μL freshly prepared diazomethane (Cohen, 1984). For GC-MS analysis the methylated samples were suspended in 30 μL ethyl acetate. Ester conjugates were hydrolyzed with 1 n NaOH for 1 h at room temperature and amide conjugates with 7 n NaOH for 3 h at 100°C under N₂. The latter samples were cooled on ice. The hydrolysate was filtered, the pH brought to 2.5, and the auxins were extracted twice with equal volumes of ethyl acetate and methylated as described for free IAA. GC-MS analysis was carried out on a Varian Saturn 2100 ion-trap mass spectrometer using electron impact ionization at 70 eV, connected to a Varian CP-3900 gas chromatograph equipped with a CP-8400 autosampler (Varian). For the analysis 1 μL of the methylated sample was injected in the splitless mode (splitter opening 1:100 after 1 min) onto a Phenomenex ZB-5 column, 30 m × 0.25 mm × 0.25 μm (Phenomenex), using helium carrier gas at 1 mL min^{. Injector temperature was 250°C and the temperature program was 70°C for 1 min, followed by an increase of 20°C min to 280°C, then 5 min isothermically at 280°C. For higher sensitivity, the} μSIS mode (Wells and Huston, 1995) was used. The endogenous concentrations of IAA were calculated according to the principles of isotope dilution (Cohen et al., 1986) monitoring the quinolinium ions at mass-to-charge ratio 130/136 (ions deriving from endogenous and ^C₆-IAA, respectively). Conjugated IAA was calculated by subtraction of the amount of free IAA from the amount obtained after hydrolysis with 7 m NaOH. Ester conjugates were calculated likewise by subtracting free IAA levels and amide conjugates were obtained after subtracting the ester-bound fraction from total conjugates.

Physcomitrella Culture and Transformation

Physcomitrella patens plants were cultured in liquid Knop medium or on solid Knop plates under a 16-h-light/8-h-dark regime (Bierfreund et al., 2003). Liquid cultures were mechanically disrupted every week to maintain the plants in the protonema stage. Gametophore development was induced by transferring protonema to the solid Knop medium. The Arabidopsis URO gene under the control of the NOS or 35S promoter was subcloned into a vector containing the hygromycin B phosphotransferase (hpt) selective marker. The p35S:URO-HPT or pNOS:URO-HPT fragment was then introduced into Physcomitrella carrying pGH3::GUS (Bierfreund et al., 2003) by a polyethylene glycol-mediated transformation method (Strepp et al., 1998). The transgenic Physcomitrella moss plants were verified by RT-PCR using URO-specific sequences as primers.

Sequence data from this article can be found in the GenBank/EMBL data libraries under accession numbers {"type":"entrez-protein","attrs":{"text":"ACB88838","term_id":"182623798","term_text":"ACB88838"}}ACB88838 (URO), {"type":"entrez-protein","attrs":{"text":"BAD07404","term_id":"41016079","term_text":"BAD07404"}}BAD07404 (LIF), {"type":"entrez-protein","attrs":{"text":"NP_187540","term_id":"15232631","term_text":"NP_187540"}}NP_187540 (TAC1), {"type":"entrez-protein","attrs":{"text":"AAY17042","term_id":"62865696","term_text":"AAY17042"}}AAY17042 (RAMOSA1), {"type":"entrez-protein","attrs":{"text":"NP_568161","term_id":"18415088","term_text":"NP_568161"}}NP_568161 (RBE), {"type":"entrez-protein","attrs":{"text":"CAB78784","term_id":"7268534","term_text":"CAB78784"}}CAB78784 (SAZ), {"type":"entrez-protein","attrs":{"text":"AAY78753","term_id":"67633658","term_text":"AAY78753"}}AAY78753 (SUP), and {"type":"entrez-protein","attrs":{"text":"ACU12847","term_id":"255529743","term_text":"ACU12847"}}ACU12847 (DST).

Supplemental Data

The following materials are available in the online version of this article.

• Supplemental Figure S1. Levels of the URO gene in different transgenic plants.

• Supplemental Figure S2.URO expression in different tissues of wild-type plants.

• Supplemental Figure S3.uro mutant has reduced number of lateral roots during early stage growing.

• Supplemental Figure S4. The p35S:URO/pDR5:GUS/W transgenic plants showed strong GUS staining in cotyledons and apex.

Supplementary Material