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Genetics, Vol. 174, 1841-1857, December 2006, Copyright © 2006
doi:10.1534/genetics.106.061044
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,1,1,2,3
,4
* Department of Biology, Harding University, Searcy, Arkansas 72149, Department of Biochemistry and Cell Biology, Rice University, Houston, Texas 77005 and Center for Plant Environmental Stress Physiology, Purdue University, West Lafayette, Indiana 47907
4 Corresponding author: Department of Biochemistry and Cell Biology, Rice University, 6100 S. Main St., MS-140, Houston, TX 77005.
E-mail: bartel{at}rice.edu
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSLITERATURE CITED |
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Arabidopsis screens have revealed mutants specifically resistant to root growth inhibition caused by IAA–amino acid conjugates (reviewed in WOODWARD and BARTEL 2005b). Through these screens, genes modulating IAA-conjugate sensitivity have been identified, including those encoding the amidohydrolases IAA–Leu resistant (ILR)1 (BARTEL and FINK 1995) and IAA–Ala resistant (IAR)3 (DAVIES et al. 1999) that cleave IAA–amino acid conjugates to release the active hormone. IAA–amino acid resistance screens have also uncovered the predicted membrane protein IAR1 (LASSWELL et al. 2000), the pyruvate dehydrogenase E1
subunit homolog IAR4 (LECLERE et al. 2004), and the novel protein ILR2 (MAGIDIN et al. 2003).
Triple-mutant seedlings deficient in three IAA-conjugate hydrolases (ILR1, IAR3, and ILL2) have reduced responsiveness to exogenous IAA conjugates and free IAA, display low-auxin phenotypes, and have decreased IAA levels compared to wild type, indicating that hydrolysis of endogenous IAA–amino acid conjugates by these enzymes contributes free IAA to the auxin pool during germination (RAMPEY et al. 2004). The hydrolases active on IAA–amino acids have putative N-terminal signal sequences and C-terminal ER retrieval signals (BARTEL and FINK 1995; DAVIES et al. 1999), suggesting localization in the ER lumen or an ER-derived compartment. The IAA-conjugate hydrolase genes are expressed in overlapping but distinct patterns not only during germination, but also at other growth stages (RAMPEY et al. 2004). IAR3 (TITARENKO et al. 1997; SASAKI et al. 2001) and ILR1 (ZIMMERMANN et al. 2004) transcripts are induced by jasmonic acid (JA), suggesting that these genes might play roles in JA conjugate hydrolysis or that IAA release may be JA inducible. However, proteins controlling hydrolase gene expression have not been identified.
In addition to transcriptional regulation, hydrolase activity may be controlled post-translationally via the availability of metal cofactors, because in vitro assays have shown that hydrolase activity requires Mn2+ or Co2+ (BARTEL and FINK 1995; DAVIES et al. 1999; LECLERE et al. 2002). The findings that several genes with roles in metal transport appear to regulate conjugate responsiveness suggest that the metal microenvironment affects hydrolase activity. For example, ILR2 appears to inhibit an unknown metal transporter (MAGIDIN et al. 2003). The ilr2 mutant is resistant to the inhibitory effects of IAA–amino acid conjugates as well as Mn2+ and Co2+ on root elongation, and ilr2 seedling microsomes transport more Mn2+ than wild type (MAGIDIN et al. 2003).
The IAA-conjugate-resistant iar1 mutant is defective in a predicted metal transporter with seven apparent transmembrane domains and many His-rich regions (LASSWELL et al. 2000). The mouse IAR1 homolog ZIP7/KE4 transports zinc from the Golgi apparatus into the cytoplasm (HUANG et al. 2005) and complements the iar1 mutant (LASSWELL et al. 2000), suggesting that IAR1 might efflux metals from a subcellular compartment, perhaps removing inhibitory metals from the compartment in which the hydrolases reside (LASSWELL et al. 2000).
Here, we describe the isolation and characterization of ilr3-1, a dominant mutation that confers resistance to IAA–Leu, IAA–Phe, and Mn2+. The gene defective in ilr3-1 encodes a basic helix-loop-helix (bHLH) leucine zipper transcription factor, bHLH105. We recapitulated several aspects of ilr3-1 phenotypes in wild-type seedlings by overexpressing an ilr3-1 mutant cDNA. Microarray and quantitative real-time PCR analyses identified five genes, including three encoding putative metal transporters, with decreased expression in ilr3-1 seedlings compared to wild type. Indeed, metal accumulation is altered in ilr3 mutants and the phenotypes of gain- and loss-of-function ilr3 mutant alleles depend on exogenous iron concentration, suggesting a role for ILR3/bHLH105 in metal homeostasis and reinforcing the importance of metal homeostasis for auxin metabolism.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSLITERATURE CITED |
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Plants grown for inductively coupled plasma–mass spectrometry (ICP–MS) analysis were seeded (n = 12) into 20-row plastic trays, stratified for 3 days at 4°, and allowed to grow for 5 weeks at 19°–22° under 90 µE m–2 sec–1 of photosynthetically active light provided by fluorescent bulbs (10 hr light/14 hr dark). The growth medium was Sunshine Mix LB2 (Carl Brehob & Son, Indianapolis) spiked with As, Cd, Co, Li, Ni, Pb, and Se (LAHNER et al. 2003). Plants were watered twice per week with 1/4 type 2 Hoaglands (LAHNER et al. 2003) in which the normal Fe was replaced with 0.5–30 µM Fe–N,N'-Di(2-hydroxybenzyl) ethylenediamine-N,N'-diacetic acid monohydrochloride hydrate (Fe–HBED). Fe–HBED was prepared by mixing HBED (Strem Chemicals, Newburyport, MA) with an equimolar amount of iron (III) nitrate, brought to pH 6.0 with KOH.
Mutant isolation and positional cloning:
The ilr3-1 mutant was isolated as described previously (BARTEL and FINK 1995; DAVIES et al. 1999) from the progeny of Col-0 seed mutagenized via fast-neutron bombardment (60 Gy). ilr3-1 was outcrossed to the Ws and Ler accessions for recombination mapping. F2 seeds were plated on 30 µM IAA–Leu, seedlings displaying wild-type sensitivity were selected for the mapping population, and the genome was examined for an area with linkage to the Ws or Ler parental ecotypes. The ilr3-1 mutation was localized to chromosome 5 using published markers (KONIECZNY and AUSUBEL 1993; BELL and ECKER 1994), markers posted on The Arabidopsis Information Resource (http://www.arabidopsis.org/), and the following new markers (see supplemental Table 1 at http://www.genetics.org/supplemental/ for primer sequences), including several dCAPs markers (MICHAELS and AMASINO 1998; NEFF et al. 1998): MBA10-2 and MBA10-3 yield a 171-bp product with an altered nucleotide in MBA10-3 that creates a BamHI site in Ws but not Col-0; F6N7-1 and F6N7-3 yield a 183-bp product with an altered nucleotide in F6N3-3 that creates a PvuII site in Col-0 but not Ws; ILL3-5B and ILL3-16 cut with NdeI yield a 360-bp product in Col-0 and 260- and 100-bp products in Ws; MDK4-4 and MDK4-5 yield an 
500-bp product with polymorphisms identified by sequencing with MDK4-4; At5g54510-5 and At5g54510-6 yield an 1.1-kb product with polymorphisms identified by sequencing with At5g54510-6; MRB17-17 and MRB17-18 yield an 1.1-kb product with polymorphisms identified by sequencing with MRB17-18; MRB17-23 and MRB17-24 yield an 1.1-kb product with polymorphisms identified by sequencing with MRB17-23; and K5F14-2 and K5F14-3 yield a 151-bp product with an altered nucleotide in K5F14-3 that creates a DdeI site in Ws but not in Col-0.
One candidate gene within the ilr3-1 mapping region, At5g54680, was sequenced using ilr3-1 mutant genomic DNA. DNA was isolated from a homozygous ilr3-1 line backcrossed three times and At5g54680 was PCR amplified with the following oligonucleotides: MRB17-27 and MRB17-28, MRB17-29 and MRB17-30, and MRB17-31 and MRB17-32 (supplemental Table 1 at http://www.genetics.org/supplemental/). The resulting products were sequenced directly with the corresponding oligonucleotides (SeqWright Laboratories, Houston).
The 859-bp region from chromosome 4 inserted in the At5g54680/ILR3 gene in the ilr3-1 mutant included 280 bp upstream of At4g22180 and the first 579 bp of the predicted At4g22180 coding sequence. At4g22180 is a hypothetical gene that lacks introns and is the third of three adjacent putative F-box genes (At4g22165, At4g22170, and At4g22180) on chromosome 4 that lack EST evidence for expression. No rearrangement of the sequence occurred upon insertion. We used PCR analysis with oligonucleotides flanking this region on chromosome 4 to determine that the At4g22180 locus was intact in the backcrossed ilr3-1 mutant.
ilr3-2 is a sequence-indexed Arabidopsis T-DNA insertion mutant (SALK_004997) isolated by the Salk Institute Genomic Analysis Laboratory (ALONSO et al. 2003) that we obtained from the Arabidopsis Biological Resource Center (ABRC) (Ohio State University, Columbus, OH). The position of the T-DNA insertion in ilr3-2 was verified using PCR analysis. PCR amplification with ILR3-12 and ILR3-13 (supplemental Table 1 at http://www.genetics.org/supplemental/) yielded a 689-bp product from wild-type genomic DNA, whereas amplification with MRB17-28 and LB1-Salk, a modified version of LBb1 (http://signal.salk.edu), yielded an 400-bp product from ilr3-2 genomic DNA. This product was sequenced, revealing that the T-DNA is located at position 186 of ILR3 (where 1 is the A position of the initiator ATG).
ilr1-5 is a mutant in the Col-0 accession isolated by screening progeny of 
-irradiated seeds on 50 µM IAA–Leu for auxin conjugate-resistant root elongation as previously described (BARTEL and FINK 1995). The mutant contains a C-to-T mutation at nucleotide 1309 of ILR1 (where 1 is the initiator ATG) that replaces a Thr residue with an Ile. The ilr1-5 mutant was backcrossed to Col-0 five times prior to analysis.
Reporter gene analysis:
A 1.8-kb potential ILR3 regulatory region (including –1863 to –1 bp from the ILR3 initiator ATG) was amplified from purified Col-0 DNA with Triplemaster polymerase mix (Eppendorf AG, Hamburg, Germany) using the oligonucleotides ILR3–GUS-1 and ILR3–GUS-2, and the resulting product was cloned into the pCR4-TOPO vector (Invitrogen, Carlsbad, CA). The insert of the resulting plasmid was sequenced to verify the absence of PCR-derived mutations. The ILR3 promoter fragment was removed from this plasmid with HindIII and BamHI and ligated into pBI101.2 (JEFFERSON et al. 1987) cut with the same enzymes to give pBI101.2-ILR3-prom, which was electroporated (AUSUBEL et al. 1999) into Agrobacterium tumefaciens strain GV3101 (KONCZ et al. 1992) for transformation into Col-0 plants (CLOUGH and BENT 1998). Transgenic lines containing the ILR3 promoter–ß-glucuronidase (GUS) construct were plated on medium containing 12 µg/ml kanamycin. Progeny of kanamycin-resistant T1 plants were grown for 1–8 days, and GUS localization was observed after staining for 4 hr with 0.5 mg/ml 5-bromo-4-chloro-3-indolyl-ß-D-glucuronide as previously described (BARTEL and FINK 1994). Thirty-seven- to 44-day-old adult plant parts were stained for 12–15 hr. The six independent transgenic lines that were observed had similar staining patterns with variable intensities.
ILR3 and ilr3-1 cDNA Isolation:
Col-0 and ilr3-1 seeds were surface sterilized (LAST and FINK 1988) and plated on filter paper on 150-mm plates containing PNS. Seedlings were grown for 7 days at 22° in yellow-filtered light. RNA was isolated using RNeasy Mini Kits (QIAGEN, Valencia, CA), and 1 µg of total Col-0 or ilr3-1 RNA was reverse transcribed with SuperScript III (Invitrogen) with oligonucleotide (supplemental Table 1 at http://www.genetics.org/supplemental/) ILR3-5 for ILR3 and ILR3-6 for ilr3-1. Each cDNA was PCR amplified with Triplemaster polymerase using oligonucleotides ILR3-4 and ILR3-5 for ILR3 and ILR3-4 and ILR3-6 for ilr3-1. PCR products were purified and cloned into the pCR4-TOPO vector. The inserts of the resulting plasmids, TOPO-ILR3 and TOPO-ilr3-1, were sequenced to verify the absence of PCR-derived mutations.
Overexpression analysis:
The ILR3 and ilr3-1 cDNAs were removed from TOPO-ILR3 and TOPO-ilr3-1 with SalI and NotI and ligated into 35SpBARN (LECLERE and BARTEL 2001) cut with XhoI and NotI. The resulting plasmids, 35S–ILR3 and 35S–ilr3-1, were sequenced using vector-derived oligonucleotides, 35S-F and NOS-R (LECLERE and BARTEL 2001), and transformed (CLOUGH and BENT 1998) into Col-0. T1 plants containing each construct were selected on PN containing 10 µg/ml Basta, and homozygous plants were identified in subsequent generations by following segregation of Basta resistance. For 35S–ILR3, nine transgenic lines were obtained, and two lines (D1 and K6) were arbitrarily selected for further study. For 35S–ilr3-1, only three transgenic lines were obtained, and two of these (C1 and E3) were arbitrarily selected for further study.
Microarray analysis:
Col-0 and ilr3-1 seeds were plated on filter paper overlaid on 150-mm plates containing PNS. Seedlings were grown for 7 days at 22° in yellow-filtered light. After 7 days, seedlings were frozen in liquid N2. Total RNA was isolated from three biological replicates of each genotype using RNeasy Mini Kits (QIAGEN), and 30–40 µg of total RNA from each sample was sent to the laboratory of Thomas McKnight at Texas A&M University where mRNA from Col-0 and ilr3-1 samples was converted to cDNA and amplified to produce biotin-labeled cRNA. The cRNA was hybridized to Affymetrix ATH1 Arabidopsis whole-genome (22,000 genes) microarray chips and analyzed with Microarray Suite 5.0 (Affymetrix). Transcripts with detectable signals (P < 0.05) on all three Col-0 chips or all three ilr3-1 chips or both (n = 14,065) were analyzed further in Excel (Microsoft) and are displayed in Figure 7 and supplemental Table 2 at http://www.genetics.org/supplemental/. For these transcripts, a two-tailed t-test assuming unequal variance was performed to test for significant differences between the three wild-type samples and three ilr3-1 samples.
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Quantitative real-time PCR was performed in triplicate or duplicate for each reverse transcription reaction using 10 µl diluted cDNA (from 30 ng total RNA) per PCR amplification. Each 25-µl reaction contained TaqMan Universal PCR Master Mix (Applied Biosystems), the appropriate forward and reverse primers (0.5 µM each), and the corresponding probe (0.2 µM). PCR conditions were 2 min at 50°, 10 min at 95°, and 40 cycles of 95° for 15 sec and 60° for 1 min. Amplification was monitored in real time utilizing the ABI Prism 7000 sequence detection system software. Template levels were normalized to APRT cDNA amplification using the comparative CT method (ABI Prism 7700 sequence detection system user bulletin no. 2, http://www.appliedbiosystems.com).
Ionomic analysis:
Medium-age rosette leaves (generally two leaves from opposite sides of the plant) from 5-week-old plants were harvested for ionomic analysis. Approximately 3 mg dry weight of each plant was sampled into Pyrex tubes (16 x 100 mm) and dried at 92° for 20 hr. After cooling, 7 of 108 samples from each tray were weighed. All samples were digested with 0.7 ml concentrated nitric acid (OmniTrace, VWR) and diluted to 6.0 ml with 18 M
water. Elemental analysis was performed with an ICP–MS (Elan DRCe; Perkin-Elmer, Norwalk, CT) for Li, B, Na, Mg, P, K, Ca, Mn, Fe, Co, Ni, Cu, Zn, As, Se, Mo, and Cd. Ten samples from each run were retained and rerun as a unit at the end of the experiment to facilitate cross-tray comparisons. All samples were normalized to calculated weights, as determined with an iterative algorithm using the best-measured elements, the weights of the seven weighed samples, and the solution concentrations, implemented in Microsoft Excel (LAHNER et al. 2003).
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSLITERATURE CITED |
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Because other IAA-conjugate response mutants suggest a link between metal homeostasis and IAA-conjugate sensitivity (LASSWELL et al. 2000; MAGIDIN et al. 2003), we tested ilr3-1 seedlings for root growth responses on medium supplemented with Mn2+, Co2+, Zn2+, Ca2+, Cd2+, or Fe. We found that ilr3-1 roots were clearly less sensitive to exogenous Mn2+ than wild type (Figure 4C) but had responses more similar to wild type to Co2+, Zn2+, Ca2+, Cd2+, and Fe (Figure 4C; data not shown). Because the metal response assays were conducted on medium lacking sucrose, this analysis also revealed that ilr3-1 had a somewhat short root on medium lacking sucrose (Figure 4C), but normal root elongation on sucrose-supplemented medium (Figure 4, A and B).
Ectopic ilr3-1 expression in wild type recapitulates ilr3-1 mutant phenotypes:
To confirm that the dominant lesion we identified in ilr3-1 was responsible for the ilr3-1 mutant phenotypes, we subcloned ilr3-1 and ILR3 cDNAs behind the cauliflower mosaic virus 35S promoter in the 35SpBARN plant transformation vector (LECLERE and BARTEL 2001). We transformed these constructs into wild-type plants and assayed homozygous lines derived from the transformants for root elongation on unsupplemented medium and medium containing IAA, IAA–Leu, IAA–Phe, or MnCl2.
Whereas we observed ilr3-1 root elongation defects only on medium lacking sucrose (Figure 4), we found that ectopic expression of the ilr3-1 cDNA in wild type reduced root elongation approximately fivefold on medium both with (Figure 5, A, C, and G) and without (Figure 5E) sucrose. Interestingly, the roots of these 35S–ilr3-1 plants were completely resistant to the inhibitory effects of IAA–Leu and IAA–Phe at concentrations that resulted in a fivefold reduction in wild-type root length (Figure 5, A and C). Similarly, exogenous MnCl2 did not inhibit 35S–ilr3-1 roots, but rather slightly promoted elongation (Figure 5E). To test whether this apparent insensitivity reflected a general inability to further reduce root elongation, we tested the response of 35S–ilr3-1 roots to IAA. We found that they responded by further reducing elongation (Figure 5G), consistent with the wild-type response of the ilr3-1 mutant to free IAA (Figures 4B and 5G). Resistance to IAA–Leu, IAA–Phe, and MnCl2 resulting from ectopic expression of the ilr3-1 cDNA in wild-type plants indicates that the lesion we identified in ilr3-1 is responsible for the phenotypes observed in the dominant ilr3-1 mutant. In contrast to 35S–ilr3-1 roots, roots of 35S–ILR3 plants more closely resembled wild type on medium with or without IAA–amino acid conjugates (Figure 5, B and D), indicating that ILR3 is not normally limiting for conjugate resistance. In contrast, ILR3 expression is limiting for MnCl2 resistance, as plants overexpressing wild-type ILR3 were resistant to MnCl2 (Figure 5F).
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19- to 24-fold more ILR3 transcript than wild type, whereas levels were increased only 3-fold in 35S–ilr3-1 seedlings (Figure 6A). This result suggests that the ilr3-1 mRNA is less stable than the ILR3 mRNA or that seedlings ectopically expressing high levels of ilr3-1 are compromised and not recovered following transformation. Indeed, even the modest overexpression of ilr3-1 that we observed was accompanied by a dramatic short-root phenotype that we did not observe in the ilr3-1 mutant or in 35S–ILR3 lines (Figure 5, A, C, and E), and we recovered fewer viable transformants using the 35S–ilr3-1 construct than the 35S–ILR3 construct (see MATERIALS AND METHODS).
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We examined root growth of heterozygous ilr3-2/ILR3 seedlings on medium containing IAA–Leu and found that ilr3-2 is recessive (data not shown). Because the ilr3-2 loss-of-function allele increases sensitivity to auxin conjugates, we conclude that ILR3 normally reduces responsiveness to IAA–Leu and IAA–Phe. This result implies that the dominant ilr3-1 mutant confers a gain of function rather than causing a dominant-negative effect, as it displays IAA-conjugate resistance phenotypes opposite to those of the ilr3-2 loss-of-function allele.
Genes with altered expression in ilr3-1 seedlings:
Because ILR3 encodes an apparent transcription factor, we sought to determine if the auxin conjugate resistance of ilr3-1 was accompanied by reduced expression of genes known to be necessary for conjugate responsiveness. We used qRT–PCR with gene-specific oligonucleotides and probes to assay expression of ILR2 and the auxin conjugate hydrolase genes ILR1 and IAR3 in RNA prepared from 7-day-old ilr3-1 and wild-type seedlings grown on unsupplemented medium. We found nearly wild-type mRNA levels in ilr3-1 (Table 1
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22,000 genes. We analyzed three biological replicates of RNA prepared from 7-day-old wild-type and ilr3-1 seedlings (Figure 7; supplemental Table 2 at http://www.genetics.org/supplemental/). This analysis revealed two transcripts potentially downregulated >10-fold in ilr3-1: ILR3 itself and At5g51720, which encodes an apparent C2H2 zinc-finger protein (BATEMAN et al. 2002). In addition, four transcripts appeared to be downregulated between 2.5- and 4-fold in ilr3-1, and these encode an oxidoreductase homolog (At3g12900) and three Ccc1p-like putative metal transporters (FU et al. 1994; LAPINSKAS et al. 1996; LI et al. 2001). The Arabidopsis genome contains six CCC1-like genes (Figure 8A); the three CCC1-like genes misregulated in ilr3-1 may be the more highly expressed members of the family (Figure 8B). In addition to identifying potential ILR3 targets, the microarray analysis confirmed that ILR1 mRNA levels were similar to wild type in ilr3-1 and revealed that IAR1 and IAR4, additional genes required for conjugate responsiveness (LASSWELL et al. 2000; LECLERE et al. 2004), had unchanged transcript levels in ilr3-1 (Table 1).
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To determine whether ectopic expression of the ilr3-1 cDNA, which recapitulated the root response phenotypes of ilr3-1 (Figure 5), also conferred gene expression changes observed in the ilr3-1 mutant, we measured levels of the putative ILR3-regulated mRNAs in the ilr3-1 and ILR3 overexpression lines. We found that two CCC1 transporter-like transcripts and the At5g51720 zinc-finger transcript accumulated to lower levels in 35S–ilr3-1 lines (Figure 6, B–D), confirming that the ilr3-1 lesion is responsible for the decreased expression of these putative target mRNAs. In contrast, we did not detect altered levels of messages misregulated in ilr3-1 plants when wild-type ILR3 was overexpressed (Figure 6, B–D), consistent with our finding that seedlings overexpressing ILR3 respond like wild type to IAA conjugates (Figure 5, B and D).
In the T-DNA insertion ilr3-2 allele, the transcripts misregulated in ilr3-1 were not dramatically affected. Expression of the CCC1-like gene At3g25190 resembled wild type in ilr3-2 seedlings (Figure 6C). At1g76800, another CCC1-like gene, had slightly higher (approximately twofold) expression in ilr3-2 compared to wild type (Figure 6B). At5g51720, encoding the zinc-finger domain protein, had slightly reduced (approximately twofold) expression in ilr3-2 seedlings (Figure 6D).
Altered ion homeostasis in ilr3 mutants:
To determine whether the observed CCC1-like transcript level changes were accompanied by metal ion level alterations in ilr3 mutants, we used inductively coupled plasma–mass spectrometry (ICP–MS) to quantify metal levels. Initial experiments suggested that supplemental Fe differentially altered the elemental profile of ilr3-1 plants, so we examined the effects of Fe nutrition in wild type, ilr3-1, ilr3-2 and wild-type plants transformed with 35S–ilr3-1 by treating plants with a range of supplemental Fe concentrations from 0.5 to 30 µM. As a control, we included the man1/frd3-3 mutant (DELHAIZE 1996; ROGERS and GUERINOT 2002), which has constitutively low shoot Fe accompanied by elevated levels of other ions (DELHAIZE 1996; ROGERS and GUERINOT 2002; LAHNER et al. 2003).
We found that supplemental Fe had a modest (1.3-fold) effect on Fe accumulation in wild-type leaves and that incremental increases in Fe in the fertilization solution resulted in decreased levels of leaf Cd, Co, Mn, and Zn (Figure 9A), consistent with the known Fe scavenging response of Arabidopsis (YI and GUERINOT 1996; VERT et al. 2002). man1/frd3-3 leaves accumulated less Fe and more Cd, Mn, and Zn than wild type at all supplemental Fe levels (Figure 9B; supplemental Figure 1 at http://www.genetics.org/supplemental/). Strikingly, we found that supplemental Fe resulted in 3-fold increases in Fe accumulation in 35S–ilr3-1 leaves (Figure 9B; supplemental Figure 1). In addition, the reduction of other metals in response to Fe fertilization was attenuated in 35S–ilr3-1 lines and the ilr3-1 dominant mutant. For example, Cd2+ levels declined 2.8-fold in Fe-treated wild type, but only 1.7-fold in ilr3-1 and 1.4-fold in 35S–ilr3-1 (Figure 9; supplemental Figure 1). We observed similar attenuation of the Fe-responsive decreases in Zn2+ and Mn2+ levels in ilr3-1 and 35S–ilr3-1 leaves (Figure 9; supplemental Figure 1). Conversely, the Fe response of the loss-of-function ilr3-2 allele displayed a heightened amplitude compared to wild type, showing increased levels of Cd, Co, Mn, and Zn in low-Fe conditions, accompanied by slightly reduced levels of some of these elements in Fe-replete conditions. Together, these changes approximately doubled the amplitude of Fe-responsive Cd, Co, and Mn diminution in the ilr3-2 mutant (Figure 9; supplemental Figure 1). We conclude that ILR3 plays a role in metal homeostasis in response to Fe nutrition, perhaps by regulating transcript levels of certain Ccc1p-like putative metal transporters.
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSLITERATURE CITED |
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Although there are only seven bHLH proteins with canonical leucine zippers in Arabidopsis (BUCK and ATCHLEY 2003; HEIM et al. 2003; TOLEDO-ORTIZ et al. 2003), bHLH–ZIP proteins are common in animals. Phylogenetic analysis suggests that juxtaposition of bHLH and leucine zipper domains occurred independently in the plant and animal lineages (BUCK and ATCHLEY 2003). ILR3 family members are widely conserved in flowering plants, with likely orthologs in rice, poplar, tomato, soybean, Medicago, and grape (Figure 2B and data not shown). The presence of monocot homologs in both bHLH–ZIP subgroups suggests the possibility of conserved, distinct functions for each group. Further, ILR3 homologs are present in pine, suggesting an ancient function for the bHLH–ZIP proteins in seed plants (Figure 2B).
ILR3 is expressed in many tissues during Arabidopsis development (Figure 2C). In particular, we noted ILR3 promoter-driven GUS expression in root tips, root and shoot vasculature, anthers, siliques, hydathodes, and stipules (Figure 3), suggesting that ILR3 functions at multiple developmental stages. These tissues include areas in which the IAA-conjugate hydrolase genes are expressed (RAMPEY et al. 2004), consistent with the possibility that ILR3 regulates hydrolase activity. As hydrolase transcript levels are nearly wild type in ilr3-1, this regulation is likely indirect (see below).
ilr3 mutants:
The dominant ilr3-1 allele (Figure 1A) confers decreased sensitivity to certain IAA–amino acid conjugates (Figures 4A, 5, A and C, and 10B) and to Mn2+ (Figures 4C and 5E). The recessive ilr3-2 allele contains a T-DNA inserted in the first intron (Figure 1C) and lacks intact ILR3 mRNA (Figure 6A). ilr3-2 seedlings have increased sensitivity to IAA–amino acid conjugates (Figures 5, A and C, and 10B), suggesting that the dominant ilr3-1 lesion confers a gain of function. The C-terminal domain missing in ilr3-1 does not contain recognizable motifs, but is conserved in other members of the ILR3 bHLH subgroup (HEIM et al. 2003), including those in rice (Figure 2A). Other bHLH proteins have similarly positioned transcriptional activation (FRANKS and CREWS 1994; GERBER et al. 1997; EMA et al. 1999) or repression domains (SATO et al. 1994; FISHER et al. 1996; FUJITANI et al. 1999). As the bHLH and leucine zipper domains remain intact in ilr3-1, it is tempting to speculate that the ilr3-1 protein still dimerizes and binds DNA via the intact bHLH and leucine zipper domains, but the missing C-terminal domain prevents proper modulation of gene expression. The dominant nature of the ilr3-1 mutation could result from altered activity or stability of ilr3-1 homo- or heterodimers.
Although expressing ilr3-1 from the 35S promoter recapitulated certain aspects of the ilr3-1 phenotype, such as conjugate resistance and gene expression changes, other 35S–ilr3-1 phenotypes were more severe. The striking root elongation defects (Figure 5) and dramatic Fe accumulation (Figure 9B) observed in 35S–ilr3-1 plants may result from increased ilr3-1 protein levels relative to the ilr3-1 mutant. Alternatively, 35S–ilr3-1 phenotypic severity could be enhanced by ectopic ilr3-1 expression in cells where ILR3 is normally not expressed. Either of these scenarios could cause ilr3-1 to interact with nontarget cis elements or to dimerize with normally unavailable partners, resulting in neomorphic phenotypes. We attempted to explore interactions between ILR3 and other Arabidopsis proteins, but both ILR3 and ilr3-1 proteins activate transcription in the yeast two-hybrid assay (data not shown), precluding use of this method to identify potential ILR3 dimerization partners.
Seedling phenotypes on exogenous IAA–amino acid conjugates and on exogenous metals do not correlate perfectly with ILR3 status. Although ilr3-1 and wild-type plants expressing 35S–ilr3-1 are resistant to IAA–Leu, IAA–Phe, and exogenous Mn, wild-type plants expressing 35S–ILR3 are resistant to Mn while remaining sensitive to IAA conjugates (Figure 5). Thus, resistances to IAA–amino acid conjugates and Mn are separable, and the latter appears more affected by changes in ILR3 level than does IAA-conjugate responsiveness. These results are consistent with our hypothesis that the primary function of ILR3 is in regulating metal homeostasis, which secondarily influences conjugate hydrolysis (see below). Future studies of ion homeostasis in 35S–ILR3 plants may allow dissection of which metals or what threshold metal levels are most relevant to IAA-conjugate sensitivity.
Transcripts misregulated in ilr3-1:
Analysis of whole-genome microarrays comparing gene expression in wild-type and ilr3-1 seedlings identified several genes potentially misregulated in ilr3-1 (Figure 7). Using quantitative real-time RT–PCR, we confirmed that five of these genes are misregulated in ilr3-1 (Table 1, Figure 6). At5g51720 is downregulated >10-fold in ilr3-1 (Table 1, Figure 6D) and encodes a 108-amino-acid protein with an apparent C2H2 zinc-finger domain (Pfam; BATEMAN et al. 2002), suggesting a role in DNA-binding or protein–protein interactions. Although apparent At5g51720 homologs are present in other plants, including pine, wheat, rice, poplar, cotton, and soybean (data not shown), the functions of these proteins have not been reported.
Three CCC1-like genes also are downregulated in ilr3-1 (Table 1, Figures 6 and 7). Yeast CCC1 has been isolated in several metal homeostasis screens (FU et al. 1994; LAPINSKAS et al. 1996; LI et al. 2001). Ccc1p has been localized to vacuolar (LI et al. 2001) and Golgi (LAPINSKAS et al. 1996) membranes, and CCC1 overexpression results in vacuolar Fe and Mn accumulation (LI et al. 2001), suggesting that Ccc1p transports Fe2+ and Mn2+ from the cytoplasm to intracellular stores.
Like yeast Ccc1p, the six Arabidopsis Ccc1p-like proteins contain five predicted transmembrane domains (Figure 8A). Plant homologs of Ccc1p have not been functionally characterized, although a soybean CCC1 family member is annotated as a nodulin (DELAUNEY et al. 1990). At2g01770 is 33% identical to yeast Ccc1p and shares an extended region between the second and third transmembrane domains; expression of this gene is not detectably misregulated in ilr3-1. Another group of five proteins are 21–24% identical to At2g01770 and yeast Ccc1p, 58–86% identical to one another, and lack the extended loop present in yeast Ccc1p and At2g01770 (Figure 8A). The three CCC1-like genes with reduced expression in ilr3-1 fall in this latter, more divergent group. The similarity between these potential ILR3 targets and the yeast Ccc1p Fe2+ and Mn2+ transporter suggests that ILR3 might modulate metal homeostasis by changing transporter levels.
As predicted by the altered Mn response and misregulation of CCC1-like genes, ion homeostasis is disrupted in ilr3 mutants (Figure 9; supplemental Figure 1). In the presence of low environmental Fe, the plant Fe scavenging response increases uptake and translocation of not only Fe, but also Mn, Cd, Co, and Zn (DELHAIZE 1996; YI and GUERINOT 1996; VERT et al. 2002). All of these Fe-coregulated metals are misregulated in ilr3 mutants. In particular, the amplitude of the reduced accumulation of Mn, Cd, Co, and Zn that follows Fe supplementation is dampened in the ilr3-1 gain-of-function mutant and increased in the ilr3-2 loss-of-function mutant, suggesting that ILR3 is involved in coordinating homeostasis of Fe and coregulated metals.
It remains to be determined which, if any, potential target genes identified here are directly regulated by ILR3 or which, if any, of these genes are involved in ilr3 phenotypes. ILR3 is in the subfamily of bHLH proteins expected to recognize both E- and G-boxes (TOLEDO-ORTIZ et al. 2003), and we found that each misregulated gene contains two to six E-boxes but no G-boxes within 1 kb of the initiator ATG (data not shown), indicating that the identified genes could be direct ILR3 targets. One possibility is that ILR3 directly regulates At5g51720 expression and that At5g51720 influences CCC1-like gene expression. As At5g51720 lacks close relatives in the Arabidopsis genome, it would be interesting to determine if plants defective in At5g51720 have altered responses to IAA conjugates or metals. However, no insertional mutants disrupting At5g51720 are currently available in public collections (http://signal.salk.edu/cgi-bin/tdnaexpress).
Transcript levels of putative ILR3 targets were only subtly affected in the ilr3-2 T-DNA insertional mutant compared to the dominant ilr3-1 mutant (Figure 6). It is possible that an ILR3 homolog, such as bHLH115 (Figure 2), partially compensates for loss of ILR3 and that future analyses of mutants defective in both of these putative transcription factors will reveal more dramatic transcript alterations in loss-of-function alleles.
Models for ILR3 function:
ILR3 is an apparent transcription factor important for IAA-conjugate responsiveness. The similar conjugate resistance profiles of ilr3-1, ilr1 (Figure 4A), and ilr2 (MAGIDIN et al. 2003) initially suggested that ILR1 or ILR2 might be ILR3 targets. However, ILR1 and ILR2 transcript levels were unaltered in ilr3-1 mutant seedlings. Similarly, transcript levels of the IAR1, IAR3, and IAR4 genes, which affect conjugate sensitivity (DAVIES et al. 1999; LASSWELL et al. 2000; LECLERE et al. 2004), were unaltered in ilr3-1 (Table 1). These data suggested that ILR3 might regulate ILR1 activity rather than message levels. Microarray analysis revealed that ILR3 may directly or indirectly target genes involved in metal homeostasis, supporting a model in which perturbed metal homeostasis affects ILR1 activity (Figure 11). In support of this hypothesis, ilr3-1 defects in IAA–Leu response and ion homeostasis are most apparent at high Fe, whereas ilr3-2 defects are most apparent at low Fe (Figures 9 and 10). The ILR1 amidohydrolase requires Mn or Co for activity and is predicted to be localized to the ER lumen (BARTEL and FINK 1995; LECLERE et al. 2002). In yeast, Ccc1p is suggested to transport Fe2+ and Mn2+ ions from the cytosol into the vacuole (LI et al. 2001). If the Ccc1p-like proteins misregulated in ilr3-1 are also metal transporters, we speculate that reduced CCC1-like transcript levels in ilr3-1 might limit metal cofactor availability in the compartment in which ILR1 resides, thereby reducing ILR1 conjugate hydrolase activity and conjugate responsiveness (Figure 11B). Moreover, the increased IAA-conjugate sensitivity of ilr3-2 seedlings suggests increased ILR1 hydrolase activity and conjugate responses, perhaps because CCC1-like expression is freed from ILR3 repression (Figure 11C). As either Mn or Co can serve as ILR1 cofactors (BARTEL and FINK 1995; LECLERE et al. 2002), the dependence of ilr3-1 and ilr3-2 mutant phenotypes on environmental Fe (Figure 10) could reflect Fe nutrition effects on Mn or Co levels (Figure 9) or localization. Further studies on the Arabidopsis CCC1-like genes are needed to test this model; analyses of CCC1-like mutant phenotypes and Ccc1p-like transport activity may be particularly informative.
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ILR3 is the second Arabidopsis bHLH transcription factor implicated in metal transport regulation. Expression of the FIT1 bHLH transcription factor gene is upregulated in roots of Fe-deficient plants (COLANGELO and GUERINOT 2004). fit1 mutants are inviable without Fe supplementation, and many transcripts normally induced during Fe starvation are no longer induced in the fit1 mutant, indicating that FIT1 is a positive regulator of Fe-responsive genes (COLANGELO and GUERINOT 2004). The ZIP metal transporter IRT1 (EIDE et al. 1996) is undetectable in fit1, although IRT1 transcripts remain present. FIT1 therefore may regulate expression of a gene that affects IRT1 turnover (COLANGELO and GUERINOT 2004). It appears that the FIT1 and ILR3 bHLH transcription factors have different targets, as none of the 72 transcripts misregulated in fit1 (COLANGELO and GUERINOT 2004) were also misregulated more than twofold in ilr3-1 (data not shown). [The only FIT1-regulated gene that initially appeared to be misregulated in the ilr3-1 microarrays was the putative oxidoreductase transcript (At3g12900), but we were unable to verify this transcript as misregulated in ilr3-1 using qRT–PCR.] It is intriguing that the FIT1 bHLH protein (Figure 2B) regulates a ZIP transporter and the ILR3 bHLH protein regulates a process (IAA-conjugate sensitivity) influenced by the ZIP-like IAR1 protein (LASSWELL et al. 2000). It will be interesting to determine whether further parallels exist between FIT1 and ILR3 functions.
The identification of three genes (ILR3, ILR2, and IAR1) implicated in metal homeostasis from auxin-conjugate sensitivity screens suggests that these screens are very sensitive to metal perturbations. Only single alleles of ilr3 and ilr2 (MAGIDIN et al. 2003) have been recovered from the screens, suggesting that additional components important for both metal homeostasis and auxin metabolism await discovery. These components might include other members of the bHLH–leucine zipper-encoding family of which ILR3 is the founding member.
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2 Present address: Center for Computational Biology and Bioinformatics, University of Texas, Austin, TX 78712.
3 Present address: Baylor College of Medicine, Houston, TX 77030.
0.05 in two-tailed t-tests assuming unequal variance) are shown as solid dots; genes with insignificant differences between ilr3-1 and wild type (P > 0.05) are shaded. The six transcripts with >2.5-fold reduction in ilr3-1 relative to wild type are labeled with gene names.
10. **, P < 0.001 in a comparison with wild type using a two-tailed t-test assuming unequal variance. (B) Recombination mapping localized ilr3-1 on chromosome 5 (thick line) between markers MRB17-17+18 and K5F14-3+2. DNA markers are shown above the line, and the number of recombinants over the number of chromosomes scored is shown below. Annotated genes in this region are represented by arrows, with arrowheads depicting the direction of transcription. (C) ILR3 contains five exons (boxes) separated by four introns (lines). The ILR3–bHLH domain (solid rectangle) and the leucine zipper domain (shaded rectangle) are both present in ilr3-1. The location of the ilr3-2 T-DNA insertion is indicated by the triangle. (D) The 859-bp insertion in ilr3-1 begins after position 1529 (where 1 is the A of the initiator ATG) in the fifth exon. RT–PCR analysis indicates that the ilr3-1 coding sequence includes seven codons after the insertion begins before a stop codon occurs. Nucleotides 1523–1538 in ILR3 are shown.
