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The TIR1 protein of Arabidopsis functions in auxin response and is related to human SKP2 and yeast Grr1p

The TIR1 protein of Arabidopsis functions in auxin response and is related to human SKP2 and... Downloaded from genesdev.cshlp.org on November 20, 2021 - Published by Cold Spring Harbor Laboratory Press The TIR1 protein of Arabidopsis functions in auxin response and is related to human SKP2 and yeast Grr1p 1 2 Max Ruegger, Elizabeth Dewey, William M. Gray, Lawrence Hobbie, Jocelyn Turner, and Mark Estelle Department of Biology, Indiana University, Bloomington, Indiana 47405 USA Genetic analysis in Arabidopsis has led to the identification of several genes that are required for auxin response. One of these genes, AXR1, encodes a protein related to yeast Aos1p, a protein that functions to activate the ubiquitin-related protein Smt3p. Here we report the identification of a new gene called TRANSPORT INHIBITOR RESPONSE 1 (TIR1). The tir1 mutants are deficient in a variety of auxin-regulated growth processes including hypocotyl elongation and lateral root formation. These results indicate that TIR1 is also required for normal response to auxin. Further, mutations in TIR1 display a synergistic interaction with mutations in AXR1, suggesting that the two genes function in overlapping pathways. The TIR1 protein contains a series of leucine-rich repeats and a recently identified motif called an F box. Sequence comparisons indicate that TIR1 is related to the yeast protein Grr1p and the human protein SKP2. Because Grr1p and other F-box proteins have been implicated in ubiquitin-mediated processes, we speculate that auxin response depends on the modification of a key regulatory protein(s) by ubiquitin or a ubiquitin-related protein. [Key Words: F-box protein; Arabidopsis; auxin response; TIR1; human SKP2; yeast Grr1p] Received September 10, 1997; revised version accepted November 14, 1997. Physiological studies have implicated the plant hormone appear to act in distinct pathways. AUX1 is a membrane indole-3-acetic acid (IAA or auxin) in the regulation of protein with similarity to amino acid permeases from diverse developmental processes including stem elonga- plants and fungi (Bennett et al. 1996). Because IAA is an tion, apical dominance, photo- and gravitropism, and lat- indolic compound structurally related to tryptophan, eral root initiation (Klee and Estelle 1991). At the cellu- AUX1 may function in cellular auxin uptake and not lar level, auxin acts to regulate these processes through response per se. In contrast, the axr1 and axr4 mutations changes in cell division and cell expansion (Evans 1984). display a synergistic interaction, suggesting that the Although the mechanism or mechanisms of auxin action wild-type genes function in the same or overlapping are largely unknown, the existing evidence suggests that pathways (Hobbie and Estelle 1995). Genetic experi- the hormone acts both at the plasma membrane and ments indicate that another gene, called SAR1, can also within the cell (Vesper and Kuss 1990; Venis and Napier be placed in this group. The sar1 mutants were isolated 1995). Auxin treatment results in rapid hyperpolariza- as suppressors of the axr1 mutations and have a pheno- tion of the plasma membrane and induction of specific type that is distinct from that of either axr1 or wild type gene expression (Barbier-Brygoo 1995; Abel and Theolo- (Cernac et al. 1997). In double-mutant combinations, sar1 is epistatic to axr1. This combination of suppres- gis 1996). In Arabidopsis, genetic studies have resulted in the sion and epistasis suggests that SAR1 functions after identification of a number of genes that are required for AXR1. Thus, AXR1, AXR4, and SAR1 form a genetically normal auxin response (Hobbie and Estelle 1994, Leyser related group of genes that appear to function together to 1997). For three of the genes, AXR1, AXR4, and AUX1, mediate auxin response. recessive mutations result in reduced auxin response as The AXR1 gene encodes a protein related to the ubiq- well as an array of auxin-related growth defects (Hobbie uitin-activating enzyme (E1), the first enzyme in the and Estelle 1994). By genetic criteria, AXR1 and AUX1 ubiquitin conjugation pathway (Leyser et al. 1993). The function of E1 is to form a thiol–ester linkage between the carboxyl terminus of ubiquitin and an internal cys- teine. The ubiquitin moiety is subsequently transferred Present addresses: Department of Biochemistry, Purdue University, West Lafayette, Indiana 47907 USA; Department of Biology, Adelphi to a ubiquitin-conjugating enzyme (E2). Finally, ubiqui- University, Garden City, New York 11530 USA. tin is attached to the target protein by an isopeptide link- Corresponding author. E-MAIL [email protected]; FAX (812) 855-6705. age between a lysine and the carboxyl terminus of ubiq- 198 GENES & DEVELOPMENT 12:198–207 © 1998 by Cold Spring Harbor Laboratory Press ISSN 0890-9369/98 $5.00; www.genesdev.org Downloaded from genesdev.cshlp.org on November 20, 2021 - Published by Cold Spring Harbor Laboratory Press An F-box protein that functions in auxin response Table 1. Segregation of CPD resistance in a tir1-2 × wild- uitin. This last step is catalyzed by a protein or protein type F population complex called a ubiquitin–protein ligase (E3). Proteins 2 related to AXR1 have now been identified in humans F root 2 F genotype (Chow et al. 1996) and fungi (Johnson et al. 1997; length 2 2 2 Shayeghi et al. 1997). These proteins all share extensive (mm) tir1/tir1 tir1/+ +/+ sequence similarity with the amino terminus of E1, but 17 2 lack the active-site cysteine required for E1 activity. Re- 16 5 cent studies in Saccharomyces cerevisiae indicate that 15 1 at least one member of this family, called Aos1p, func- tions as a dimer with a second protein called Uba2p (Johnson et al. 1997). Uba2p is similar to the carboxyl 12 1 terminus of E1 and includes the region containing the active-site cysteine (Dohmen et al. 1995). The Aos1p/ 10 6 Uba2p dimer forms a thiol–ester linkage with a ubiqui-96 87 6 tin-related protein called Smt3p. Thus, it is possible that 73 4 AXR1 and other members of the AXR1 protein family 62 4 each interact with a second protein to form a functional enzyme. The substrate for each heterodimer-type E1, ubiquitin and/or a ubiquitin-related protein, remains to be determined. Totals 8 27 14 To identify additional genes involved in auxin physi- ology, we have screened for Arabidopsis mutants that 1 Genotype determined by analyzing CPD resistance in F are resistant to the growth-inhibiting properties of the plants. auxin-transport inhibitors naphthylphthalamic acid Number of seedlings. (NPA) and 2-carboxyphenyl-3-phenylpropane-1,2-dione (CPD) (Ruegger et al. 1997). This screen resulted in the recovery of mutants that are altered in auxin transport as of the three alleles tested, heterozygous plants were well as auxin response. In this report, we show that the weakly resistant. The five mutants differ only slightly transport inhibitor response 1 (tir1) mutants are defi- with respect to CPD response. The tir1-2 allele is cient in auxin response. Molecular characterization in- slightly weaker, whereas tir1-1 and tir1-3 are slightly dicates that the TIR1 protein is, like AXR1, a member of stronger than tir1-6 and tir1-7. All of the phenotypic a family of proteins implicated in ubiquitin-mediated analysis described below was performed with the tir1-1 processes. mutant. The tir1 mutants are affected in auxin response Results Treatment of Arabidopsis seedlings with auxin-trans- Genetic characterization of the tir1 mutants port inhibitors such as CPD or NPA inhibits root elon- gation and promotes swelling of the root tip (Ruegger et Previously, we reported the isolation of 16 tir mutants by al. 1997). Both of these effects are thought to be caused screening for resistance to auxin-transport inhibitors by accumulation of auxin in this region as a result of the (Ruegger et al. 1997). Genetic analysis indicated that five inhibition of auxin transport away from the root tip (Mu- of these mutations were independent alleles at a locus day and Haworth 1994; Muday et al. 1995; Ruegger et al. we called TIR1. To determine the genetic basis for resis- 1997). Because auxin-transport inhibitors affect root tance, we examined segregation of CPD resistance in F growth by increasing auxin levels in the root tip, resis- and F populations. In our initial studies, seedlings that tance could result from a change in auxin transport or a were heterozygous for tir1 alleles were often signifi- cantly less affected by CPD than wild-type seedlings, suggesting that the mutations are at least partially domi- Table 2. CPD resistance in tir homozygous nant. When F populations were analyzed, a graded dis- and heterozygous plants tribution of phenotypes was observed. An example of a population scored on 5 μM CPD is shown in Table 1. The Root length Line (mm) ±S.E. No. genotype of individual F plants was determined by ex- amining the response of F progeny to CPD. This analy- +/+ 7.8 0.1 32 sis revealed that homozygous tir1 plants are clearly re- tir1-1/tir1-1 15.7 0.3 33 sistant to the compound and heterozygous plants tended tir1-3/tir1-3 15.8 0.4 34 to be somewhat resistant compared with homozygous tir1-7/tir1-7 13.0 0.5 33 wild-type plants. To confirm that tir1 mutations are tir1-1/+ 10.9 0.3 15 semidominant, we directly compared populations of tir1-3/+ 12.5 0.3 17 wild-type, tir1/+ and tir1/tir1 seedlings on medium con- tir1-7/+ 12.6 0.2 17 taining CPD. The results are shown in Table 2. For each GENES & DEVELOPMENT 199 Downloaded from genesdev.cshlp.org on November 20, 2021 - Published by Cold Spring Harbor Laboratory Press Ruegger et al. reduction in auxin response. To distinguish between these possibilities, we examined the growth of tir1-1 seedlings on media containing NPA, IAA, or the syn- thetic auxin, 2,4-dichlorophenoxyacetic acid (2,4-D). The results are shown in Figure 1. As expected, tir1-1 seedlings display increased root growth on NPA com- pared with wild type. Mutant seedlings, however, were also less sensitive to the growth-inhibiting effects of the auxins, suggesting that the primary effect of the muta- tion is on auxin response and not auxin transport. Con- sistent with this conclusion, polar auxin transport in tir1-1 stem segments is similar to wild type (Fig. 2). This contrasts with the behavior of the tir3 mutant, which is resistant to NPA but not auxin and has a clear defect in auxin transport (Ruegger et al. 1997). In addition, the response of tir-1 roots to abscisic acid and the cytokinin Figure 2. Polar auxin transport is similar in wild-type (solid benzyladenine was similar to wild type. bars) and tir1-1 (hatched bars) inflorescence stems. Two and one-half centimeters of stem was excised, and the apical end was placed in a nutrient solution containing 1 μM [ C]IAA for Cell proliferation in response to auxin-transport the times indicated. The amount of radioactive IAA transported inhibitors is reduced in tir1 seedlings to the basal end of the stem was assayed by liquid scintillation. Each column represents the mean of three replicates; the bar CPD treatment results in pronounced swelling of the represents the standard error of the mean. root tip and expansion of the zone of cell division away from the root tip (Ruegger et al. 1997). Root tip swelling is the result of extra periclinal cell divisions in the me- trast, tir1-1 roots grown on CPD displayed modest ristem. To determine whether these events occur in tir1 changes in cell number and organization, indicating that roots, we examined cross sections of wild-type and tir1-1 CPD-induced cell proliferation is reduced in tir1-1 seed- roots grown on media with or without CPD. The sec- lings. tions shown in Figure 3 were cut 300 μm back from the root tip at a position where the different cell layers of the The TIR1 gene is involved in lateral root formation mature root are clearly defined. The organization of Ara- Physiological and genetic studies have shown that auxin bidopsis roots is highly regular and consists of the epi- is required for lateral root initiation and growth (Wight- dermis, cortex, endodermis, pericycle, and vascular tis- man and Thimann 1980; Celenza et al. 1995; Hobbie and sue. The appearance of untreated tir1-1 roots was similar Estelle 1995). Formation of a lateral root primordium to that of untreated wild-type roots. When grown on starts with a local increase in anticlinal divisions in the CPD, wild-type roots exhibited a dramatic increase in xylem-radius pericycle cells. This is followed by radial cell number as expected (Ruegger et al. 1997). In con- expansion of these pericycle cells and several periclinal divisions to form a 4-cell-layer dome-shaped primordium (Malamy and Benfey 1997). When seedlings are treated with auxin, these early events occur in a synchronized fashion along the entire length of the root (Laskowski et Figure 1. The roots of wild-type (open bars) and tir1-1 (solid bars) seedlings are resistant to the growth-inhibiting properties of NPA, IAA, and 2,4-D. Seeds were germinated on nutrient medium. After 3 days, seedlings were transferred to media con- taining the indicated compound. Five days later, new root Figure 3. Cell proliferation in response to CPD is reduced in growth was measured and plotted as a percentage of root growth the roots of tir1-1 seedlings. Seedlings were grown for 7 days on on medium without compound. Bars represent standard errors. nutrient medium plus or minus 5 μM CPD, fixed, and embedded Absence of bar indicates error less than thickness of line in Spurrs (Ruegger et al. 1997). Root cross sections 300 μm from (n = 12). the root tip are shown. Bars, 20 μm. 200 GENES & DEVELOPMENT Downloaded from genesdev.cshlp.org on November 20, 2021 - Published by Cold Spring Harbor Laboratory Press An F-box protein that functions in auxin response al. 1995). Examination of seedlings grown on agar me- dium indicated that tir1 plants are deficient in lateral root formation. After 10 days of growth, wild-type seed- lings had 6.3 ± 0.4 lateral roots (n = 10), whereas tir1-1 seedlings had 1.3 ± 0.4 (n = 10). When tir1-1 seedlings were examined by confocal microscopy, no additional lateral root primordia were observed, suggesting that mutant seedlings are deficient in an early step in forma- tion of a lateral root (data not shown). To determine whether the TIR1 gene functions in auxin-induced lat- eral root formation, we exposed excised root segments from wild-type and tir1-1 seedlings to various concentra- tions of IAA or 2,4-D. The results for IAA are displayed in Figure 4A. Both compounds induced lateral root for- mation in wild-type and tir1-1 root segments. The num- ber of new lateral roots on tir1-1 segments, however, was Figure 5. Hypocotyl elongation in response to elevated tem- reduced compared with wild type. This effect can also be perature is reduced in tir1-1 seedlings compared with wild-type observed in Figure 4B. When grown on medium contain- seedlings. Seedlings were grown on vertically oriented agar me- ing 0.5 μM 2,4-D, a continuous row of lateral roots was dium for 9 days at the two temperatures. initiated along the length of the wild-type root. In con- trast, initiation of lateral roots on tir1-1 seedlings is spo- radic. These results indicate that the TIR1 gene func- the higher temperature. We have shown that this re- tions in auxin induction of lateral root formation. sponse is absent in the axr1-12 mutant and reduced in plants that are transgenic for the iaaLys gene and, con- sequently, have reduced IAA levels (W.M. Gray and M. The tir1 mutants are also deficient in a cell-elongation Estelle, unpubl.). In contrast, mutants deficient in gib- response berellin biosynthesis or ethylene response are unaf- Growth of Arabidopsis seedlings at 28°C under moder- fected. These results indicate that increased hypocotyl ate light conditions results in a dramatic increase in hy- elongation at elevated temperature is auxin dependent. pocotyl elongation compared with seedlings grown at To determine whether the TIR1 gene functions in this 20°C (Fig. 5; W.M. Gray and M. Estelle, unpubl.). After 9 response, we grew wild-type and tir1-1 seedlings at the days of growth, the hypocotyls of wild-type seedlings are two temperatures. The results shown in Figure 5 indi- 4.5-fold longer at 28°C compared with seedlings grown at cate that mutant seedlings are deficient in hypocotyl 20°C. This increase is primarily caused by increased cell elongation under these conditions. Mutant hypocotyls elongation because epidermal cells are 3.7-fold longer at are 2.6-fold longer at the higher temperature (compared with 4.5 for wild type). Similarly the epidermal cells of tir1-1 hypocotyls are 2.2-fold longer at 28°C compared with 20°C (wild-type cells are 3.7-fold longer). The tir1 and axr1 mutants display a synergistic interaction The AXR1 gene is also required for auxin response (Lin- coln et al. 1990; Timpte et al. 1995). To explore possible interactions between AXR1 and TIR1, we generated plants that were homozygous for both the axr1-12 and tir1-1 mutations and examined their phenotype. When grown on medium containing 0.1 or 0.5 μM 2,4-D, axr1- 12 tir1-1 seedlings are more resistant than axr1-12 seed- lings (Fig. 6A). Because this effect occurs in a concentra- tion range where tir1-1 does not confer resistance by it- self, the interaction between the two mutations is Figure 4. The tir1-1 mutant is deficient in IAA-induction of synergistic. A similar conclusion was reached when the lateral roots. (A) Eight-millimeter root segments were excised morphology of double-mutant plants was examined. In from 5-day-old wild-type (open bar) and tir1-1 (solid bar) seed- most respects, the tir1-1 rosette and inflorescence are lings grown on nutrient medium and transferred to medium identical in appearance to wild type (Fig. 6B). There was with IAA. Lateral roots were counted after 5 days by use of a a slight increase in the number of secondary inflores- dissecting microscope. Bars represent standard errors. Absence cences growing from the rosette (wild type 1.5 ± 0.2 com- of bar indicates error less than thickness of line (n = 10). (B) pared with tir1 2.3 ± 0.3; n = 10), suggesting an effect on Ten-day-old wild-type (left) and tir1-1 (right) seedlings grown on 0.5 μM 2,4-D. (Insets) Higher magnification images of roots. apical dominance. In an axr1-12 background, the tir1-1 GENES & DEVELOPMENT 201 Downloaded from genesdev.cshlp.org on November 20, 2021 - Published by Cold Spring Harbor Laboratory Press Ruegger et al. Figure 6. tir1-1 and axr1-12 have synergistic ef- fects on auxin response and plant morphology. (A) The tir1-1 mutation decreased auxin response in an axr1-12 background at a concentration at which tir1-1 has no effect by itself. Seedlings were grown and treated as described for Fig. 1. (Open bars) Wild type; (solid bars) tir1; (hatched bars) axr1; (stippled bars) axr1 tir1. Bars represent standard error and absence of bar indicates error less than thickness of line. (B) Seedlings (top row) were photographed af- ter 12 days of growth on 1 μM 2,4-D. Bar, 2.5 mm. Mature plants (bottom row) were photographed 35 days after germination in soil. Bar, 5 cm. mutation caused a further reduction in stature (Fig. 6B). placed adjacent to the CaMV 35S promoter and intro- This result indicates that, despite the lack of aerial phe- duced into tir1-1 plants. Of five independent transgenic notype, the TIR1 gene functions in aerial structures. Fur- lines examined, auxin response was restored to the wild- ther, it suggests that TIR1 is involved in AXR1-mediated type level in four, providing final proof that the candi- processes. date cDNA was TIR1 (Fig. 7C). To determine the map position of the TIR1 gene, a restriction fragment length polymorphism (RFLP) was Isolation of the TIR1 gene identified between the Columbia and Landsberg ec- otypes with the EcoRI–SalI fragment containing the The tir1-9 mutant was isolated from a population of T- TIR1 gene as a probe. The polymorphism was mapped by DNA transformed plants (Ruegger et al. 1997) generated use of 44 recombinant inbred lines (Lister and Dean by K. Feldmann and colleagues (Feldmann 1991). Ge- 1993) to position 128 on chromosome 3, close to nga128. netic analysis indicated that the tir1-9 line carried a single T-DNA insert that cosegregated with the tir1-9 The TIR1 protein contains an F-box domain mutation. The left end of the T-DNA insert and flanking and leucine rich repeats Arabidopsis sequences were cloned by plasmid rescue. Southern analysis revealed that a 5.5-kb EcoRI–SalI re- Examination of the TIR1 amino acid sequence revealed striction fragment recovered in this fashion detected a several features of interest. First, TIR1 contains an F-box polymorphism between wild type (WS) and the tir1-9 domain (Fig. 8A; Bai et al. 1996). This motif is present in mutant, confirming that this Arabidopsis DNA lies ad- a variety of regulatory proteins in mammals and yeast jacent to the T-DNA insert (data not shown). The EcoRI– including the S-phase kinase-associated protein (SKP2) SalI restriction fragment was used to isolate cDNA and Cyclin F proteins from humans and the Cdc4 and clones. The longest cDNA recovered was 2.2 kb in Grr1 proteins from yeast (Bai et al. 1996). Each of these length. DNA sequence analysis of the insert revealed an proteins binds a second protein called SKP1 in an inter- open reading frame (ORF) encoding a predicted protein of action that requires the F-box (Bai et al. 1996; Li and 594 amino acids (Fig. 7A). To confirm that this ORF cor- Johnston 1997). Recent evidence has also shown that the responds to the TIR1 gene, two tir1 alleles were se- UFO protein from Arabidopsis contains an F box and quenced by RT-PCR. The tir1-1 mutation is a glycine to binds Arabidopsis orthologs of SKP1 called Ask1 and aspartate substitution at position 147 and the tir1-2 mu- Ask2 (A. Samach, S. Kohalmi, G. Haughn, and W. tation is a glycine to aspartate change at position 441. Crosby, pers. comm.). Second, TIR1 has 16 degenerate Sequence analysis of a genomic clone spanning the leucine-rich repeats (LRRs) (Fig. 8B). Similar repeats have cDNA indicated that the gene contains two introns. The been identified in a wide variety of proteins although the tir1-9 allele has a T-DNA insert in the first intron. RNA repeat consensus sequence, length, and level of redun- blot experiments with RNA isolated from wild-type and dancy vary widely among different proteins (Kobe and mutant tissues detected a transcript of ~2.2 kb in wild- Deisenhofer 1995a,b). Data base searches indicate that type tissues. This transcript was absent in RNA from the the TIR1 repeats are most similar to those found in tir1-9 mutant (Fig. 7B). The TIR1 transcript was detected SKP2, Grr1p, and an uncharacterized ORF in Caenorhab- in all tissues examined including roots, rosettes, stems, ditis elegans called C02F5.7, a protein that also has an F and flowers (data not shown). The TIR1 cDNA was box. 202 GENES & DEVELOPMENT Downloaded from genesdev.cshlp.org on November 20, 2021 - Published by Cold Spring Harbor Laboratory Press An F-box protein that functions in auxin response Figure 7. The characterization of three mutant alleles and transformation rescue of the tir1-1 mutant with a candidate cDNA identify the TIR1 gene. (A) Predicted amino acid sequence of the TIR1 protein. The position of the tir1-1 and tir1-2 mutations, both (G → D), are indicated with an asterisk above the affected residue. The GenBank accession nos. for the cDNA and genomic sequences are AF005048 and AF005047, respectively. (B) RNA blot analysis of TIR1 transcript in tir1 mutants. Twenty-five micrograms of total RNA isolated from 13-day-old seedlings was loaded in each lane. (C) Transformation rescue of the tir1-1 mutant. The p35S:TIR1 plasmid was introduced into tir1-1 plants by vacuum infiltration. Transgenic lines were tested for auxin resistance by plating on medium con- taining 0.085 μM 2,4-D. The presence of an F box and the similarity of the quence of the third gene, LRF3, was determined by the LRRs in TIR1, Grr1p, SKP2, and C02F5.7 suggests that Arabidopsis Genome Initiative. LRF1 and LRF2 are 69% these proteins are related. A comparison of the four pro- and 60% identical with TIR1 respectively, whereas LRF3 teins is shown in Figure 7C. Although the biochemical is 34% identical to TIR1. There is also one highly related function of the F-box proteins and their presumed bind- rice EST present in the dBest database (accession no. ing partner SKP1 is uncertain, recent studies indicate D22807). that these proteins may function together as a ubiquitin– protein ligase complex called an SCF (Skp1–Cdc53–F-box protein) complex (Bai et al. 1996; Feldman et al. 1997; Li Discussion and Johnston 1997; Skowyra et al. 1997). TIR1 functions in auxin-dependent cell division and cell elongation TIR1 is a member of a family of leucine-rich repeat The tir1 mutants are deficient in several auxin-mediated F-box proteins in Arabidopsis responses including auxin-dependent hypocotyl elonga- In addition to the animal and fungal proteins mentioned tion, auxin inhibition of root elongation, CPD-mediated above, data base searches revealed the existence of a stimulation of cell proliferation in the root tip, and auxin number of TIR1-related genes in Arabidopsis and rice. stimulation of lateral root formation. In addition, tir1 We have called these genes LRF for LEUCINE-RICH RE- seedlings have reduced numbers of lateral roots when PEAT F-BOX. Two of the sequences (LRF1 and LRF2) they are grown on medium without auxin. These defects were identified as ESTs. We obtained these clones from strongly suggest that TIR1 functions in auxin response the Arabidopsis Biological Resource Center (ABRC) and and not auxin transport. Because both cell elongation screened cDNA libraries in an attempt to obtain full- (hypocotyl elongation) and cell division (cell prolifera- length clones. Sequence analysis indicated that for each tion at the root tip) are affected, we conclude that TIR1 gene, the longest cDNA we recovered did not contain the has a general role in auxin response. entire coding sequence. Because the LRF1 and LRF2 tran- The nature of the lesion in the tir1-9 allele, and the scripts are similar in size to TIR1 (data not shown), we absence of detectable full-length TIR1 RNA in this mu- estimate that ~50 nucleotides of coding sequence is tant, strongly suggests that homozygous tir1-9 seedlings missing from the 58 end of the two cDNAs. The se- completely lack TIR1 protein. Because this mutant still GENES & DEVELOPMENT 203 Downloaded from genesdev.cshlp.org on November 20, 2021 - Published by Cold Spring Harbor Laboratory Press Ruegger et al. LRRs. The F box was first identified by Bai et al. (1996), and shown to be required for binding to a conserved pro- tein called SKP1. This interaction has now been shown for the F-box proteins cyclin F and SKP2 from mammals, Cdc4p and Grr1p from yeast, and the UFO protein from Arabidopsis (Zhang et al. 1995; Bai et al. 1996; Li and Johnston 1997; A. Samach, S. Kohalmi, G. Haughn, and W. Crosby, pers. comm.). The function of SKP1 and the F-box proteins is uncertain. Several lines of evidence, however, suggest that they act as components of one or more ubiquitin–protein ligases (E3) to facilitate transfer of ubiquitin from an E2 enzyme to a target protein. In yeast cells, Skp1p binds the F-box proteins Cdc4p and Grr1p. These two proteins are essential for ubiquitin- mediated degradation of the Clb–CDK inhibitor Sic1p, and the G cyclins (Cln1p and Cln2p), respectively (Bai et al. 1996; Li and Johnston 1997). In skp1 mutants, both Sic1p and the G cyclins are stabilized. Taken together, these results suggest that Skp1p works in conjunction with an F-box protein to target the ubiquitin conjugation machinery to specific proteins. In two recent studies, Feldman et al. (1997) and Skowyra et al. (1997) shown that Skp1p, Cdc53p, and one of several F-box proteins form ubiquitin-ligase complexes called SCFs. The F-box protein is apparently involved in conferring specificity to the complex. There are at least two SKP1 homologs in Arabidopsis (Nadeau et al. 1996; A. Samach, S. Kohalmi, G. Haughn, and W. Crosby, pers. comm.) and at least one CDC53 homolog (Genbank accession no. AC002330). TIR1 may interact with these proteins or similar pro- teins and promote ubiquitin modification of one or more Figure 8. The TIR1 protein has an F box and 16 LRRs. (A) regulatory proteins required for auxin response. Alignment of F-box motifs from diverse proteins. Identical resi- dues are boxed. (B) Alignment of LRRs in TIR1. The affected residues in the tir1-1 and tir1-2 mutants are underlined. In con- AXR1 and TIR1 function together to mediate auxin sensus sequences listed in A and B, aliphatic residues are indi- response cated with an a. (C) Comparison of TIR1, SKP2, Grr1, and We have determined previously that the AXR1 protein is C02F5.7. Boxes represent the LRRs. similar to the amino-terminal half of the ubiquitin-acti- vating enzyme E1 (Leyser et al. 1993). Although AXR1 is missing key residues known to be essential for E1 activ- responds to auxin, the TIR1 protein is not essential for ity, recent studies in yeast indicate that AXR1 and re- auxin response. Somewhat paradoxically, the semidomi- lated proteins probably function to activate either ubiq- nant nature of the tir1 mutations indicates that Arabi- uitin or another small ubiquitin-like protein. In S. cer- dopsis plants are sensitive to TIR1 gene dose. One wild- evisiae, two AXR1 homologs (Aos1p and Enr2p) have type gene is not sufficient to confer a wild-type pheno- been investigated. Aos1p functions together with an- type, but complete loss of TIR1 function results in only other E1-related protein called Uba2p to activate a ubiq- a modest increase in the severity of the phenotype. One uitin-like protein called Smt3p (Johnson et al. 1997). possible explanation for this behavior is that the closely Enr2p is required for conjugation of a different ubiquitin- related LRF proteins are functionally redundant with related protein, called Rub1p, to the cell cycle protein TIR1. In the absence of TIR1, the LRF proteins may pro- Cdc53p (D. Lammer, N. Mathias, J. Laplaza, Y. Liu, J. vide sufficient function to maintain near normal growth Callis, M. Goebl, and M. Estelle, unpubl.). These obser- and development. vations suggest that AXR1 forms a dimer with a second protein that carries the active-site cysteine, similar to The TIR1 protein may function in ubiquitin–protein the situation with yeast Aos1p and Uba2p. This hetero- conjugation dimer would then activate ubiquitin or a ubiquitin-re- Among animal and fungal proteins, TIR1 is most closely lated protein. related to the SKP2 protein from human cells, the Grr1 The results of our genetic experiments suggest that the protein from S. cerevisiae, and an uncharacterized ORF AXR1 and TIR1 genes function in the same or overlap- in Caenorhabditis elegans called C02F5.7. Each of these ping pathways. This is consistent with our current un- proteins has a motif called an F box as well as a series of derstanding of the AXR1 and TIR1 proteins. AXR1 may 204 GENES & DEVELOPMENT Downloaded from genesdev.cshlp.org on November 20, 2021 - Published by Cold Spring Harbor Laboratory Press An F-box protein that functions in auxin response participate in the activation of either ubiquitin or a ubiq- a ubiquitin–protein conjugation pathway. Thus, our re- uitin-related protein and TIR1 could function later in the sults provide strong support for a role for ubiquitin (or pathway as part of an E3 complex. The nature of the related protein) modification in auxin response. Re- targeted protein is unknown. We hypothesize that auxin cently, D. Xie and J. Turner (University of East Anglia, response depends on ubiquitin (or ubiquitin-related pro- Norwich, UK) have independently shown that the LRF3 tein) modification of one or more regulatory proteins. gene described here is identical to a previously identified gene called COI1 (J. Turner, pers. comm.). The recessive Ubiquitination may then result in degradation by the proteosome (Hochstrasser 1996). This is consistent with coi1 mutants are completely insensitive to jasmonic a model proposed by Theologis and colleagues in which acid, suggesting that the gene plays a key role in jasmo- transcription of auxin-regulated genes is normally re- nate signaling or response (Feys et al. 1994). It appears pressed by the action of short-lived repressor proteins likely that the TIR1/COI1 genes represent the first (Ballas et al. 1995; Abel and Theologis 1996). Auxin may members of a novel class of signaling proteins in plants. relieve this repression by stimulating degradation of the repressors in an AXR1–TIR1 dependent manner. Alter- Materials and methods natively, the modification may alter the activity or cel- lular localization of a regulatory protein. For example, Plant materials and growth conditions ligand-dependent ubiquitin modification of the a-factor All mutant lines were from the Columbia ecotype. Plants were receptor in yeast results in endocytosis of the receptor grown at 21–23°C under continuous fluorescent illumination (Hicke and Riezman 1996). In mammalian cells, local- (100–150 μE/m per sec) in 13-cm clay pots containing Metro- ization of the protein RanGAP1 to the nuclear pore com- Mix (W.R. Grace & Co.) or an equivalent soilless mixture. Dur- plex depends on modification by the ubiquitin-related ing the first 3 weeks of growth, a mineral nutrient solution containing 5 mM KNO , 2.5 mM KPO (adjusted to pH 5.8), 2 protein SUMO1 (Mahajan et al. 1997). Clearly, we will 3 4 mM MgSO ,2mM Ca(NO ) ,50μM Fe–EDTA, 70 μM H BO ,14 4 3 2 3 3 need to identify the substrates of the AXR1–TIR1 path- μM MnCl , 0.5 μM CuSO ,1μM ZnSO , 0.2 μM Na MoO ,10μM 2 4 4 2 4 way(s) to establish a mechanism. One potential substrate NaCl, and 10 nM CoCl was supplied to the plants. For many is the product of the SAR1 gene. By genetic criteria, experiments, plants were grown under sterile conditions in petri SAR1 has a negative role in auxin response, and acts plates containing the above nutrient solution plus 0.7% agar, downstream of AXR1 (Cernac et al. 1997). 1.0% sucrose (wt/vol), and various hormones or inhibitors as How auxin affects the pathway is also uncertain, par- indicated in the text. Before plating, seeds were surface-steril- ticularly because neither the auxin receptor nor other ized by agitation for 10–20 min in 20% commercial bleach and signal transduction components have been identified. 0.02% Triton X-100, rinsed several times with sterile water, and held at 4°C for 3–4 days to enhance germination. The seeds were One attractive possibility is suggested by experiments of dispersed on the growth media with sterile water. The plates Li and Johnston (1997) on Grr1p. As mentioned above, were oriented vertically and held in an incubator containing Grr1p is required for ubiquitin-mediated degradation of fluorescent lighting (30–50 μE/m per sec; 16 hr photoperiod) the G cyclins Cln1p and Cln2p in a process that appears and a temperature of 20–21°C. In all experiments where plants to involve an SCF complex that contains Grr1p (Feldman are grown, day 0 is considered to be the time when seeds are first et al. 1997; Li and Johnston 1997; Skowyra et al. 1997). placed in the growth conditions described above. For hypocotyl Grr1p also plays a central role in glucose repression and and cell elongation studies, seedlings were grown under 24 hr glucose induction of genes encoding glucose transport- light at 85 μE/m per sec. ers, however, a function that also requires Skp1p. When wild-type cells are grown in glucose, there is a substan- Genetic analysis tial increase in the amount of Grr1p–Skp1p complex that For genetic analyses, resistance to inhibition of root growth by is immunoprecipitated relative to cells grown in glycerol CPD (a gift from G.F. Katekar, Commonwealth Scientific and even though the levels of Grr1p and Sk1p are similar in Industrial Research Organization, Canberra City, Australia) was the two extracts. Li and Johnston (1997) suggest that this assayed by the following procedure. Surface-sterilized, cold- difference may reflect a mechanism by which the cell treated seeds were plated on nutrient medium, as described links nutrient availability to cell cycle regulation. Be- above, supplemented with 5 μM CPD. On day 7 (unless other- cause TIR1 may bind a SKP1 homolog in Arabidopsis, it wise stated) the seedlings were straightened with forceps and is possible that auxin affects this interaction in some the root length (distance from the root/hypocotyl junction to way, thereby altering the level of ubiquitin modification the root tip) was measured. Resistance to 0.2 μM 2,4-D was of one or more targets. We are currently working to iden- assayed in a similar manner except that roots were scored vi- sually as either 2,4-D-resistant or wild type. tify TIR1-interacting proteins to test this model. To generate axr1-12 tir1-1 double mutant plants, crosses be- tween the single mutant lines axr1-12 and tir1-1 were per- Summary formed. The genotypes of individual F plants from these crosses were determined by test crosses to both of the parental Genetic studies in Arabidopsis have led to the identifi- lines and tests for CPD or 2,4-D resistance as described above. cation of eight genes that are required for normal auxin Linkage analysis was performed with a 9-kb EcoRI–SalI frag- response (Hobbie and Estelle 1994). To date, three of ment of Arabidopsis genomic DNA containing the coding se- these genes have been isolated. The structures of the quences of the TIR1 gene. This fragment detected a RFLP be- proteins encoded by two of the genes, AXR1 (Leyser et al. tween EcoRV-digested Columbia and Landsberg erecta (Ler) ge- 1993) and TIR1 (this study), suggest that they function in nomic DNA. Mapmaker I (S. Lincoln, M. Daly, J. Abrahamson, GENES & DEVELOPMENT 205 Downloaded from genesdev.cshlp.org on November 20, 2021 - Published by Cold Spring Harbor Laboratory Press Ruegger et al. A. Barlow, and L. Newburg; Massachusetts Institute of Tech- 3.8-kb region of D109ES containing the TIR1 coding region, nology, Cambridge, MA) was used to compile the results of the were subcloned into pBluescript and sequenced (Sequitherm, DNA blot analysis of 44 RI lines (Lister and Dean 1993) with Epicentre Technologies). this fragment as a probe. Characterization of tir1 mutant alleles Auxin-transport assays RT-PCR (Stratagene) was used to amplify the expressed se- quences of two tir1 alleles from total RNA prepared from seed- Auxin-transport assays were performed according to the lings grown in liquid culture. The PCR products were separated method of Okada et al. (1991). Stem segments (2.5 cm) of pri- in a low-melt agarose gel and sequenced directly by use of a mary inflorescence were incubated in a 1.5-ml Eppendorf cen- modified Sequenase protocol (B. Robertson, University of Col- trifuge tube containing 30 μl of nutrient solution with 1 μM orado). The position of the T-DNA insertion of tir1-9 was de- [ C]IAA (1.74 nCi/ml). The segments were incubated with termined by sequencing a subcloned fragment of the L3 plasmid their apical end in the solution for various times from 2 to 18 hr. that hybridized to probes made from the TIR1 cDNA and from After the incubation, a 5-mm section from the basal end of the the T-DNA left border (plasmid pBSH10). segment was excised and added to 3 ml of liquid scintillation cocktail (Bio-Safe II; RPI, Mount Pleasant, IL). The samples were shaken at 100 rpm for at least 2 hr and left overnight at room RNA blot analysis temperature before scintillation counting in a scintillation Total RNA was isolated from 13-day-old seedlings according to counter (model LS 6500; Beckman Instruments). The experi- Newman et al. (1993) with modifications by Timpte et al. ment involved three stem segments for each time point. Wild- (1995), and blotted as described in Timpte et al. (1995). type and tir1-1 plants were grown in 24 hr of light (105 μE/m per sec) for 36 days.The experiment was repeated three times with similar results. Stems incubated with 15 μM NPA or with Generation of transgenic plants the basal end of the stem segment in the solution transported The 2.2-kb TIR1 cDNA was cloned into the pBI121 vector (re- low levels of [ C]IAA. placing the GUS gene) to generate the p35S:TIR1 plasmid. This plasmid was introduced into the Agrobacterium strain GV3101 Induction of lateral root formation by IAA by electroporation. tir1-1 plants were inoculated with this strain by vacuum infiltration according to Bechtold et al. (1993). Seedlings were grown on nutrient medium for 5 days. Eight- Transformed seedlings were identified by selection on 50 μg/ml millimeter root segments were excised with a razor blade from kanamycin. Transgenic lines were tested for TIR1 function by mature root and transferred to nutrient medium supplemented plating on medium that contained 0.085 mM 2,4-D. with various concentrations of IAA. The number of lateral roots was determined by use of a dissecting microscope and expressed as the number of lateral roots per millimeter of primary root. Acknowledgments We are grateful to members of the Estelle laboratory and the Isolation of sequences flanking the T-DNA insert in tir1-9 Indiana University Arabidopsis group for stimulating discus- sion throughout the course of this work, to Roger Innes for Plasmid rescue of T-DNA sequences was done following the careful reading of the manuscript, to the ABRC for cDNAs, and procedure of Behringer and Medford (1992). Genomic DNA, pre- to L. Washington for assistance with DNA sequencing. This pared from rosette leaves of the tir1-9 mutant line, was digested research was supported in part by the National Science Foun- with either SalIor EcoRI to isolate DNA flanking the T-DNA dation (postdoctoral fellowship MCB-9008316 to L.H., left border or right border, respectively. The digested DNA was IBN-9307134 to M.E.) and National Institutes of Health (U.S. ligated with T4 DNA ligase, and electroporated into JS4 E. coli Public Health Service grant-GM43644 to M.E.) and an Eli Lilly/ (Bio-Rad). Four colonies derived from the SalI-digested DNA Indiana Institute for Molecular Biology predoctoral fellowship (L1–L4) and >100 colonies derived from the EcoRI-digested to M.R. DNA were recovered. Plasmid DNA from L1–L4 and from 14 of The publication costs of this article were defrayed in part by the EcoRI-derived colonies (R1–R14) was prepared and analyzed payment of page charges. This article must therefore be hereby by restriction analysis to eliminate clones derived entirely from marked ‘‘advertisement’’ in accordance with 18 USC section the T-DNA insert (Behringer and Medford 1992). Two left bor- 1734 solely to indicate this fact. der isolates (L3 and L4) had identical restriction digest patterns and potentially contained Arabidopsis DNA. When used as a probe in DNA blot analysis, a 5.5-kb EcoRI–SalI fragment from References L3 identified RFLPs between tir1-9 and wild-type WS genomic DNA. Abel, S. and A. Theologis. 1996. Early genes and auxin action. Plant Physiol. 111: 9–17. Bai, C., P. Sen, K. Hofmann, L. Ma, M. Goebl, J.W. Harper, and Isolation and characterization of genomic and cDNA clones S.J. Elledge. 1996. SKP1 connects cell cycle regulators to the The L3 5.5-kb probe was used to isolate six genomic clones ubiquitin proteolysis machinery through a novel motif, the (D1–D6; library, a gift from R. Davis, Stanford University, CA) F-box. 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Beach. 1995. Skp1 Skp2 process. Development 121: 3303–3310. p19 and p45 are essential elements of the cyclin A- Leyser, H.M.O., C. Lincoln, C. Timpte, D. Lammer, J. Turner, CDK2 S phase kinase. Cell 82: 915–925. GENES & DEVELOPMENT 207 Downloaded from genesdev.cshlp.org on November 20, 2021 - Published by Cold Spring Harbor Laboratory Press The TIR1 protein of Arabidopsis functions in auxin response and is related to human SKP2 and yeast Grr1p Max Ruegger, Elizabeth Dewey, William M. Gray, et al. Genes Dev. 1998, 12: Access the most recent version at doi:10.1101/gad.12.2.198 This article cites 40 articles, 16 of which can be accessed free at: References http://genesdev.cshlp.org/content/12/2/198.full.html#ref-list-1 License Receive free email alerts when new articles cite this article - sign up in the box at the top Email Alerting right corner of the article or click here. 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The TIR1 protein of Arabidopsis functions in auxin response and is related to human SKP2 and yeast Grr1p

Ruegger, M.; Dewey, E.; Gray, W. M.; Hobbie, L.; Turner, J.; Estelle, M.
Genes & Development – Jan 15, 1998
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Downloaded from genesdev.cshlp.org on November 20, 2021 - Published by Cold Spring Harbor Laboratory Press The TIR1 protein of Arabidopsis functions in auxin response and is related to human SKP2 and yeast Grr1p 1 2 Max Ruegger, Elizabeth Dewey, William M. Gray, Lawrence Hobbie, Jocelyn Turner, and Mark Estelle Department of Biology, Indiana University, Bloomington, Indiana 47405 USA Genetic analysis in Arabidopsis has led to the identification of several genes that are required for auxin response. One of these genes, AXR1, encodes a protein related to yeast Aos1p, a protein that functions to activate the ubiquitin-related protein Smt3p. Here we report the identification of a new gene called TRANSPORT INHIBITOR RESPONSE 1 (TIR1). The tir1 mutants are deficient in a variety of auxin-regulated growth processes including hypocotyl elongation and lateral root formation. These results indicate that TIR1 is also required for normal response to auxin. Further, mutations in TIR1 display a synergistic interaction with mutations in AXR1, suggesting that the two genes function in overlapping pathways. The TIR1 protein contains a series of leucine-rich repeats and a recently identified motif called an F box. Sequence comparisons indicate that TIR1 is related to the yeast protein Grr1p and the human protein SKP2. Because Grr1p and other F-box proteins have been implicated in ubiquitin-mediated processes, we speculate that auxin response depends on the modification of a key regulatory protein(s) by ubiquitin or a ubiquitin-related protein. [Key Words: F-box protein; Arabidopsis; auxin response; TIR1; human SKP2; yeast Grr1p] Received September 10, 1997; revised version accepted November 14, 1997. Physiological studies have implicated the plant hormone appear to act in distinct pathways. AUX1 is a membrane indole-3-acetic acid (IAA or auxin) in the regulation of protein with similarity to amino acid permeases from diverse developmental processes including stem elonga- plants and fungi (Bennett et al. 1996). Because IAA is an tion, apical dominance, photo- and gravitropism, and lat- indolic compound structurally related to tryptophan, eral root initiation (Klee and Estelle 1991). At the cellu- AUX1 may function in cellular auxin uptake and not lar level, auxin acts to regulate these processes through response per se. In contrast, the axr1 and axr4 mutations changes in cell division and cell expansion (Evans 1984). display a synergistic interaction, suggesting that the Although the mechanism or mechanisms of auxin action wild-type genes function in the same or overlapping are largely unknown, the existing evidence suggests that pathways (Hobbie and Estelle 1995). Genetic experi- the hormone acts both at the plasma membrane and ments indicate that another gene, called SAR1, can also within the cell (Vesper and Kuss 1990; Venis and Napier be placed in this group. The sar1 mutants were isolated 1995). Auxin treatment results in rapid hyperpolariza- as suppressors of the axr1 mutations and have a pheno- tion of the plasma membrane and induction of specific type that is distinct from that of either axr1 or wild type gene expression (Barbier-Brygoo 1995; Abel and Theolo- (Cernac et al. 1997). In double-mutant combinations, sar1 is epistatic to axr1. This combination of suppres- gis 1996). In Arabidopsis, genetic studies have resulted in the sion and epistasis suggests that SAR1 functions after identification of a number of genes that are required for AXR1. Thus, AXR1, AXR4, and SAR1 form a genetically normal auxin response (Hobbie and Estelle 1994, Leyser related group of genes that appear to function together to 1997). For three of the genes, AXR1, AXR4, and AUX1, mediate auxin response. recessive mutations result in reduced auxin response as The AXR1 gene encodes a protein related to the ubiq- well as an array of auxin-related growth defects (Hobbie uitin-activating enzyme (E1), the first enzyme in the and Estelle 1994). By genetic criteria, AXR1 and AUX1 ubiquitin conjugation pathway (Leyser et al. 1993). The function of E1 is to form a thiol–ester linkage between the carboxyl terminus of ubiquitin and an internal cys- teine. The ubiquitin moiety is subsequently transferred Present addresses: Department of Biochemistry, Purdue University, West Lafayette, Indiana 47907 USA; Department of Biology, Adelphi to a ubiquitin-conjugating enzyme (E2). Finally, ubiqui- University, Garden City, New York 11530 USA. tin is attached to the target protein by an isopeptide link- Corresponding author. E-MAIL [email protected]; FAX (812) 855-6705. age between a lysine and the carboxyl terminus of ubiq- 198 GENES & DEVELOPMENT 12:198–207 © 1998 by Cold Spring Harbor Laboratory Press ISSN 0890-9369/98 $5.00; www.genesdev.org Downloaded from genesdev.cshlp.org on November 20, 2021 - Published by Cold Spring Harbor Laboratory Press An F-box protein that functions in auxin response Table 1. Segregation of CPD resistance in a tir1-2 × wild- uitin. This last step is catalyzed by a protein or protein type F population complex called a ubiquitin–protein ligase (E3). Proteins 2 related to AXR1 have now been identified in humans F root 2 F genotype (Chow et al. 1996) and fungi (Johnson et al. 1997; length 2 2 2 Shayeghi et al. 1997). These proteins all share extensive (mm) tir1/tir1 tir1/+ +/+ sequence similarity with the amino terminus of E1, but 17 2 lack the active-site cysteine required for E1 activity. Re- 16 5 cent studies in Saccharomyces cerevisiae indicate that 15 1 at least one member of this family, called Aos1p, func- tions as a dimer with a second protein called Uba2p (Johnson et al. 1997). Uba2p is similar to the carboxyl 12 1 terminus of E1 and includes the region containing the active-site cysteine (Dohmen et al. 1995). The Aos1p/ 10 6 Uba2p dimer forms a thiol–ester linkage with a ubiqui-96 87 6 tin-related protein called Smt3p. Thus, it is possible that 73 4 AXR1 and other members of the AXR1 protein family 62 4 each interact with a second protein to form a functional enzyme. The substrate for each heterodimer-type E1, ubiquitin and/or a ubiquitin-related protein, remains to be determined. Totals 8 27 14 To identify additional genes involved in auxin physi- ology, we have screened for Arabidopsis mutants that 1 Genotype determined by analyzing CPD resistance in F are resistant to the growth-inhibiting properties of the plants. auxin-transport inhibitors naphthylphthalamic acid Number of seedlings. (NPA) and 2-carboxyphenyl-3-phenylpropane-1,2-dione (CPD) (Ruegger et al. 1997). This screen resulted in the recovery of mutants that are altered in auxin transport as of the three alleles tested, heterozygous plants were well as auxin response. In this report, we show that the weakly resistant. The five mutants differ only slightly transport inhibitor response 1 (tir1) mutants are defi- with respect to CPD response. The tir1-2 allele is cient in auxin response. Molecular characterization in- slightly weaker, whereas tir1-1 and tir1-3 are slightly dicates that the TIR1 protein is, like AXR1, a member of stronger than tir1-6 and tir1-7. All of the phenotypic a family of proteins implicated in ubiquitin-mediated analysis described below was performed with the tir1-1 processes. mutant. The tir1 mutants are affected in auxin response Results Treatment of Arabidopsis seedlings with auxin-trans- Genetic characterization of the tir1 mutants port inhibitors such as CPD or NPA inhibits root elon- gation and promotes swelling of the root tip (Ruegger et Previously, we reported the isolation of 16 tir mutants by al. 1997). Both of these effects are thought to be caused screening for resistance to auxin-transport inhibitors by accumulation of auxin in this region as a result of the (Ruegger et al. 1997). Genetic analysis indicated that five inhibition of auxin transport away from the root tip (Mu- of these mutations were independent alleles at a locus day and Haworth 1994; Muday et al. 1995; Ruegger et al. we called TIR1. To determine the genetic basis for resis- 1997). Because auxin-transport inhibitors affect root tance, we examined segregation of CPD resistance in F growth by increasing auxin levels in the root tip, resis- and F populations. In our initial studies, seedlings that tance could result from a change in auxin transport or a were heterozygous for tir1 alleles were often signifi- cantly less affected by CPD than wild-type seedlings, suggesting that the mutations are at least partially domi- Table 2. CPD resistance in tir homozygous nant. When F populations were analyzed, a graded dis- and heterozygous plants tribution of phenotypes was observed. An example of a population scored on 5 μM CPD is shown in Table 1. The Root length Line (mm) ±S.E. No. genotype of individual F plants was determined by ex- amining the response of F progeny to CPD. This analy- +/+ 7.8 0.1 32 sis revealed that homozygous tir1 plants are clearly re- tir1-1/tir1-1 15.7 0.3 33 sistant to the compound and heterozygous plants tended tir1-3/tir1-3 15.8 0.4 34 to be somewhat resistant compared with homozygous tir1-7/tir1-7 13.0 0.5 33 wild-type plants. To confirm that tir1 mutations are tir1-1/+ 10.9 0.3 15 semidominant, we directly compared populations of tir1-3/+ 12.5 0.3 17 wild-type, tir1/+ and tir1/tir1 seedlings on medium con- tir1-7/+ 12.6 0.2 17 taining CPD. The results are shown in Table 2. For each GENES & DEVELOPMENT 199 Downloaded from genesdev.cshlp.org on November 20, 2021 - Published by Cold Spring Harbor Laboratory Press Ruegger et al. reduction in auxin response. To distinguish between these possibilities, we examined the growth of tir1-1 seedlings on media containing NPA, IAA, or the syn- thetic auxin, 2,4-dichlorophenoxyacetic acid (2,4-D). The results are shown in Figure 1. As expected, tir1-1 seedlings display increased root growth on NPA com- pared with wild type. Mutant seedlings, however, were also less sensitive to the growth-inhibiting effects of the auxins, suggesting that the primary effect of the muta- tion is on auxin response and not auxin transport. Con- sistent with this conclusion, polar auxin transport in tir1-1 stem segments is similar to wild type (Fig. 2). This contrasts with the behavior of the tir3 mutant, which is resistant to NPA but not auxin and has a clear defect in auxin transport (Ruegger et al. 1997). In addition, the response of tir-1 roots to abscisic acid and the cytokinin Figure 2. Polar auxin transport is similar in wild-type (solid benzyladenine was similar to wild type. bars) and tir1-1 (hatched bars) inflorescence stems. Two and one-half centimeters of stem was excised, and the apical end was placed in a nutrient solution containing 1 μM [ C]IAA for Cell proliferation in response to auxin-transport the times indicated. The amount of radioactive IAA transported inhibitors is reduced in tir1 seedlings to the basal end of the stem was assayed by liquid scintillation. Each column represents the mean of three replicates; the bar CPD treatment results in pronounced swelling of the represents the standard error of the mean. root tip and expansion of the zone of cell division away from the root tip (Ruegger et al. 1997). Root tip swelling is the result of extra periclinal cell divisions in the me- trast, tir1-1 roots grown on CPD displayed modest ristem. To determine whether these events occur in tir1 changes in cell number and organization, indicating that roots, we examined cross sections of wild-type and tir1-1 CPD-induced cell proliferation is reduced in tir1-1 seed- roots grown on media with or without CPD. The sec- lings. tions shown in Figure 3 were cut 300 μm back from the root tip at a position where the different cell layers of the The TIR1 gene is involved in lateral root formation mature root are clearly defined. The organization of Ara- Physiological and genetic studies have shown that auxin bidopsis roots is highly regular and consists of the epi- is required for lateral root initiation and growth (Wight- dermis, cortex, endodermis, pericycle, and vascular tis- man and Thimann 1980; Celenza et al. 1995; Hobbie and sue. The appearance of untreated tir1-1 roots was similar Estelle 1995). Formation of a lateral root primordium to that of untreated wild-type roots. When grown on starts with a local increase in anticlinal divisions in the CPD, wild-type roots exhibited a dramatic increase in xylem-radius pericycle cells. This is followed by radial cell number as expected (Ruegger et al. 1997). In con- expansion of these pericycle cells and several periclinal divisions to form a 4-cell-layer dome-shaped primordium (Malamy and Benfey 1997). When seedlings are treated with auxin, these early events occur in a synchronized fashion along the entire length of the root (Laskowski et Figure 1. The roots of wild-type (open bars) and tir1-1 (solid bars) seedlings are resistant to the growth-inhibiting properties of NPA, IAA, and 2,4-D. Seeds were germinated on nutrient medium. After 3 days, seedlings were transferred to media con- taining the indicated compound. Five days later, new root Figure 3. Cell proliferation in response to CPD is reduced in growth was measured and plotted as a percentage of root growth the roots of tir1-1 seedlings. Seedlings were grown for 7 days on on medium without compound. Bars represent standard errors. nutrient medium plus or minus 5 μM CPD, fixed, and embedded Absence of bar indicates error less than thickness of line in Spurrs (Ruegger et al. 1997). Root cross sections 300 μm from (n = 12). the root tip are shown. Bars, 20 μm. 200 GENES & DEVELOPMENT Downloaded from genesdev.cshlp.org on November 20, 2021 - Published by Cold Spring Harbor Laboratory Press An F-box protein that functions in auxin response al. 1995). Examination of seedlings grown on agar me- dium indicated that tir1 plants are deficient in lateral root formation. After 10 days of growth, wild-type seed- lings had 6.3 ± 0.4 lateral roots (n = 10), whereas tir1-1 seedlings had 1.3 ± 0.4 (n = 10). When tir1-1 seedlings were examined by confocal microscopy, no additional lateral root primordia were observed, suggesting that mutant seedlings are deficient in an early step in forma- tion of a lateral root (data not shown). To determine whether the TIR1 gene functions in auxin-induced lat- eral root formation, we exposed excised root segments from wild-type and tir1-1 seedlings to various concentra- tions of IAA or 2,4-D. The results for IAA are displayed in Figure 4A. Both compounds induced lateral root for- mation in wild-type and tir1-1 root segments. The num- ber of new lateral roots on tir1-1 segments, however, was Figure 5. Hypocotyl elongation in response to elevated tem- reduced compared with wild type. This effect can also be perature is reduced in tir1-1 seedlings compared with wild-type observed in Figure 4B. When grown on medium contain- seedlings. Seedlings were grown on vertically oriented agar me- ing 0.5 μM 2,4-D, a continuous row of lateral roots was dium for 9 days at the two temperatures. initiated along the length of the wild-type root. In con- trast, initiation of lateral roots on tir1-1 seedlings is spo- radic. These results indicate that the TIR1 gene func- the higher temperature. We have shown that this re- tions in auxin induction of lateral root formation. sponse is absent in the axr1-12 mutant and reduced in plants that are transgenic for the iaaLys gene and, con- sequently, have reduced IAA levels (W.M. Gray and M. The tir1 mutants are also deficient in a cell-elongation Estelle, unpubl.). In contrast, mutants deficient in gib- response berellin biosynthesis or ethylene response are unaf- Growth of Arabidopsis seedlings at 28°C under moder- fected. These results indicate that increased hypocotyl ate light conditions results in a dramatic increase in hy- elongation at elevated temperature is auxin dependent. pocotyl elongation compared with seedlings grown at To determine whether the TIR1 gene functions in this 20°C (Fig. 5; W.M. Gray and M. Estelle, unpubl.). After 9 response, we grew wild-type and tir1-1 seedlings at the days of growth, the hypocotyls of wild-type seedlings are two temperatures. The results shown in Figure 5 indi- 4.5-fold longer at 28°C compared with seedlings grown at cate that mutant seedlings are deficient in hypocotyl 20°C. This increase is primarily caused by increased cell elongation under these conditions. Mutant hypocotyls elongation because epidermal cells are 3.7-fold longer at are 2.6-fold longer at the higher temperature (compared with 4.5 for wild type). Similarly the epidermal cells of tir1-1 hypocotyls are 2.2-fold longer at 28°C compared with 20°C (wild-type cells are 3.7-fold longer). The tir1 and axr1 mutants display a synergistic interaction The AXR1 gene is also required for auxin response (Lin- coln et al. 1990; Timpte et al. 1995). To explore possible interactions between AXR1 and TIR1, we generated plants that were homozygous for both the axr1-12 and tir1-1 mutations and examined their phenotype. When grown on medium containing 0.1 or 0.5 μM 2,4-D, axr1- 12 tir1-1 seedlings are more resistant than axr1-12 seed- lings (Fig. 6A). Because this effect occurs in a concentra- tion range where tir1-1 does not confer resistance by it- self, the interaction between the two mutations is Figure 4. The tir1-1 mutant is deficient in IAA-induction of synergistic. A similar conclusion was reached when the lateral roots. (A) Eight-millimeter root segments were excised morphology of double-mutant plants was examined. In from 5-day-old wild-type (open bar) and tir1-1 (solid bar) seed- most respects, the tir1-1 rosette and inflorescence are lings grown on nutrient medium and transferred to medium identical in appearance to wild type (Fig. 6B). There was with IAA. Lateral roots were counted after 5 days by use of a a slight increase in the number of secondary inflores- dissecting microscope. Bars represent standard errors. Absence cences growing from the rosette (wild type 1.5 ± 0.2 com- of bar indicates error less than thickness of line (n = 10). (B) pared with tir1 2.3 ± 0.3; n = 10), suggesting an effect on Ten-day-old wild-type (left) and tir1-1 (right) seedlings grown on 0.5 μM 2,4-D. (Insets) Higher magnification images of roots. apical dominance. In an axr1-12 background, the tir1-1 GENES & DEVELOPMENT 201 Downloaded from genesdev.cshlp.org on November 20, 2021 - Published by Cold Spring Harbor Laboratory Press Ruegger et al. Figure 6. tir1-1 and axr1-12 have synergistic ef- fects on auxin response and plant morphology. (A) The tir1-1 mutation decreased auxin response in an axr1-12 background at a concentration at which tir1-1 has no effect by itself. Seedlings were grown and treated as described for Fig. 1. (Open bars) Wild type; (solid bars) tir1; (hatched bars) axr1; (stippled bars) axr1 tir1. Bars represent standard error and absence of bar indicates error less than thickness of line. (B) Seedlings (top row) were photographed af- ter 12 days of growth on 1 μM 2,4-D. Bar, 2.5 mm. Mature plants (bottom row) were photographed 35 days after germination in soil. Bar, 5 cm. mutation caused a further reduction in stature (Fig. 6B). placed adjacent to the CaMV 35S promoter and intro- This result indicates that, despite the lack of aerial phe- duced into tir1-1 plants. Of five independent transgenic notype, the TIR1 gene functions in aerial structures. Fur- lines examined, auxin response was restored to the wild- ther, it suggests that TIR1 is involved in AXR1-mediated type level in four, providing final proof that the candi- processes. date cDNA was TIR1 (Fig. 7C). To determine the map position of the TIR1 gene, a restriction fragment length polymorphism (RFLP) was Isolation of the TIR1 gene identified between the Columbia and Landsberg ec- otypes with the EcoRI–SalI fragment containing the The tir1-9 mutant was isolated from a population of T- TIR1 gene as a probe. The polymorphism was mapped by DNA transformed plants (Ruegger et al. 1997) generated use of 44 recombinant inbred lines (Lister and Dean by K. Feldmann and colleagues (Feldmann 1991). Ge- 1993) to position 128 on chromosome 3, close to nga128. netic analysis indicated that the tir1-9 line carried a single T-DNA insert that cosegregated with the tir1-9 The TIR1 protein contains an F-box domain mutation. The left end of the T-DNA insert and flanking and leucine rich repeats Arabidopsis sequences were cloned by plasmid rescue. Southern analysis revealed that a 5.5-kb EcoRI–SalI re- Examination of the TIR1 amino acid sequence revealed striction fragment recovered in this fashion detected a several features of interest. First, TIR1 contains an F-box polymorphism between wild type (WS) and the tir1-9 domain (Fig. 8A; Bai et al. 1996). This motif is present in mutant, confirming that this Arabidopsis DNA lies ad- a variety of regulatory proteins in mammals and yeast jacent to the T-DNA insert (data not shown). The EcoRI– including the S-phase kinase-associated protein (SKP2) SalI restriction fragment was used to isolate cDNA and Cyclin F proteins from humans and the Cdc4 and clones. The longest cDNA recovered was 2.2 kb in Grr1 proteins from yeast (Bai et al. 1996). Each of these length. DNA sequence analysis of the insert revealed an proteins binds a second protein called SKP1 in an inter- open reading frame (ORF) encoding a predicted protein of action that requires the F-box (Bai et al. 1996; Li and 594 amino acids (Fig. 7A). To confirm that this ORF cor- Johnston 1997). Recent evidence has also shown that the responds to the TIR1 gene, two tir1 alleles were se- UFO protein from Arabidopsis contains an F box and quenced by RT-PCR. The tir1-1 mutation is a glycine to binds Arabidopsis orthologs of SKP1 called Ask1 and aspartate substitution at position 147 and the tir1-2 mu- Ask2 (A. Samach, S. Kohalmi, G. Haughn, and W. tation is a glycine to aspartate change at position 441. Crosby, pers. comm.). Second, TIR1 has 16 degenerate Sequence analysis of a genomic clone spanning the leucine-rich repeats (LRRs) (Fig. 8B). Similar repeats have cDNA indicated that the gene contains two introns. The been identified in a wide variety of proteins although the tir1-9 allele has a T-DNA insert in the first intron. RNA repeat consensus sequence, length, and level of redun- blot experiments with RNA isolated from wild-type and dancy vary widely among different proteins (Kobe and mutant tissues detected a transcript of ~2.2 kb in wild- Deisenhofer 1995a,b). Data base searches indicate that type tissues. This transcript was absent in RNA from the the TIR1 repeats are most similar to those found in tir1-9 mutant (Fig. 7B). The TIR1 transcript was detected SKP2, Grr1p, and an uncharacterized ORF in Caenorhab- in all tissues examined including roots, rosettes, stems, ditis elegans called C02F5.7, a protein that also has an F and flowers (data not shown). The TIR1 cDNA was box. 202 GENES & DEVELOPMENT Downloaded from genesdev.cshlp.org on November 20, 2021 - Published by Cold Spring Harbor Laboratory Press An F-box protein that functions in auxin response Figure 7. The characterization of three mutant alleles and transformation rescue of the tir1-1 mutant with a candidate cDNA identify the TIR1 gene. (A) Predicted amino acid sequence of the TIR1 protein. The position of the tir1-1 and tir1-2 mutations, both (G → D), are indicated with an asterisk above the affected residue. The GenBank accession nos. for the cDNA and genomic sequences are AF005048 and AF005047, respectively. (B) RNA blot analysis of TIR1 transcript in tir1 mutants. Twenty-five micrograms of total RNA isolated from 13-day-old seedlings was loaded in each lane. (C) Transformation rescue of the tir1-1 mutant. The p35S:TIR1 plasmid was introduced into tir1-1 plants by vacuum infiltration. Transgenic lines were tested for auxin resistance by plating on medium con- taining 0.085 μM 2,4-D. The presence of an F box and the similarity of the quence of the third gene, LRF3, was determined by the LRRs in TIR1, Grr1p, SKP2, and C02F5.7 suggests that Arabidopsis Genome Initiative. LRF1 and LRF2 are 69% these proteins are related. A comparison of the four pro- and 60% identical with TIR1 respectively, whereas LRF3 teins is shown in Figure 7C. Although the biochemical is 34% identical to TIR1. There is also one highly related function of the F-box proteins and their presumed bind- rice EST present in the dBest database (accession no. ing partner SKP1 is uncertain, recent studies indicate D22807). that these proteins may function together as a ubiquitin– protein ligase complex called an SCF (Skp1–Cdc53–F-box protein) complex (Bai et al. 1996; Feldman et al. 1997; Li Discussion and Johnston 1997; Skowyra et al. 1997). TIR1 functions in auxin-dependent cell division and cell elongation TIR1 is a member of a family of leucine-rich repeat The tir1 mutants are deficient in several auxin-mediated F-box proteins in Arabidopsis responses including auxin-dependent hypocotyl elonga- In addition to the animal and fungal proteins mentioned tion, auxin inhibition of root elongation, CPD-mediated above, data base searches revealed the existence of a stimulation of cell proliferation in the root tip, and auxin number of TIR1-related genes in Arabidopsis and rice. stimulation of lateral root formation. In addition, tir1 We have called these genes LRF for LEUCINE-RICH RE- seedlings have reduced numbers of lateral roots when PEAT F-BOX. Two of the sequences (LRF1 and LRF2) they are grown on medium without auxin. These defects were identified as ESTs. We obtained these clones from strongly suggest that TIR1 functions in auxin response the Arabidopsis Biological Resource Center (ABRC) and and not auxin transport. Because both cell elongation screened cDNA libraries in an attempt to obtain full- (hypocotyl elongation) and cell division (cell prolifera- length clones. Sequence analysis indicated that for each tion at the root tip) are affected, we conclude that TIR1 gene, the longest cDNA we recovered did not contain the has a general role in auxin response. entire coding sequence. Because the LRF1 and LRF2 tran- The nature of the lesion in the tir1-9 allele, and the scripts are similar in size to TIR1 (data not shown), we absence of detectable full-length TIR1 RNA in this mu- estimate that ~50 nucleotides of coding sequence is tant, strongly suggests that homozygous tir1-9 seedlings missing from the 58 end of the two cDNAs. The se- completely lack TIR1 protein. Because this mutant still GENES & DEVELOPMENT 203 Downloaded from genesdev.cshlp.org on November 20, 2021 - Published by Cold Spring Harbor Laboratory Press Ruegger et al. LRRs. The F box was first identified by Bai et al. (1996), and shown to be required for binding to a conserved pro- tein called SKP1. This interaction has now been shown for the F-box proteins cyclin F and SKP2 from mammals, Cdc4p and Grr1p from yeast, and the UFO protein from Arabidopsis (Zhang et al. 1995; Bai et al. 1996; Li and Johnston 1997; A. Samach, S. Kohalmi, G. Haughn, and W. Crosby, pers. comm.). The function of SKP1 and the F-box proteins is uncertain. Several lines of evidence, however, suggest that they act as components of one or more ubiquitin–protein ligases (E3) to facilitate transfer of ubiquitin from an E2 enzyme to a target protein. In yeast cells, Skp1p binds the F-box proteins Cdc4p and Grr1p. These two proteins are essential for ubiquitin- mediated degradation of the Clb–CDK inhibitor Sic1p, and the G cyclins (Cln1p and Cln2p), respectively (Bai et al. 1996; Li and Johnston 1997). In skp1 mutants, both Sic1p and the G cyclins are stabilized. Taken together, these results suggest that Skp1p works in conjunction with an F-box protein to target the ubiquitin conjugation machinery to specific proteins. In two recent studies, Feldman et al. (1997) and Skowyra et al. (1997) shown that Skp1p, Cdc53p, and one of several F-box proteins form ubiquitin-ligase complexes called SCFs. The F-box protein is apparently involved in conferring specificity to the complex. There are at least two SKP1 homologs in Arabidopsis (Nadeau et al. 1996; A. Samach, S. Kohalmi, G. Haughn, and W. Crosby, pers. comm.) and at least one CDC53 homolog (Genbank accession no. AC002330). TIR1 may interact with these proteins or similar pro- teins and promote ubiquitin modification of one or more Figure 8. The TIR1 protein has an F box and 16 LRRs. (A) regulatory proteins required for auxin response. Alignment of F-box motifs from diverse proteins. Identical resi- dues are boxed. (B) Alignment of LRRs in TIR1. The affected residues in the tir1-1 and tir1-2 mutants are underlined. In con- AXR1 and TIR1 function together to mediate auxin sensus sequences listed in A and B, aliphatic residues are indi- response cated with an a. (C) Comparison of TIR1, SKP2, Grr1, and We have determined previously that the AXR1 protein is C02F5.7. Boxes represent the LRRs. similar to the amino-terminal half of the ubiquitin-acti- vating enzyme E1 (Leyser et al. 1993). Although AXR1 is missing key residues known to be essential for E1 activ- responds to auxin, the TIR1 protein is not essential for ity, recent studies in yeast indicate that AXR1 and re- auxin response. Somewhat paradoxically, the semidomi- lated proteins probably function to activate either ubiq- nant nature of the tir1 mutations indicates that Arabi- uitin or another small ubiquitin-like protein. In S. cer- dopsis plants are sensitive to TIR1 gene dose. One wild- evisiae, two AXR1 homologs (Aos1p and Enr2p) have type gene is not sufficient to confer a wild-type pheno- been investigated. Aos1p functions together with an- type, but complete loss of TIR1 function results in only other E1-related protein called Uba2p to activate a ubiq- a modest increase in the severity of the phenotype. One uitin-like protein called Smt3p (Johnson et al. 1997). possible explanation for this behavior is that the closely Enr2p is required for conjugation of a different ubiquitin- related LRF proteins are functionally redundant with related protein, called Rub1p, to the cell cycle protein TIR1. In the absence of TIR1, the LRF proteins may pro- Cdc53p (D. Lammer, N. Mathias, J. Laplaza, Y. Liu, J. vide sufficient function to maintain near normal growth Callis, M. Goebl, and M. Estelle, unpubl.). These obser- and development. vations suggest that AXR1 forms a dimer with a second protein that carries the active-site cysteine, similar to The TIR1 protein may function in ubiquitin–protein the situation with yeast Aos1p and Uba2p. This hetero- conjugation dimer would then activate ubiquitin or a ubiquitin-re- Among animal and fungal proteins, TIR1 is most closely lated protein. related to the SKP2 protein from human cells, the Grr1 The results of our genetic experiments suggest that the protein from S. cerevisiae, and an uncharacterized ORF AXR1 and TIR1 genes function in the same or overlap- in Caenorhabditis elegans called C02F5.7. Each of these ping pathways. This is consistent with our current un- proteins has a motif called an F box as well as a series of derstanding of the AXR1 and TIR1 proteins. AXR1 may 204 GENES & DEVELOPMENT Downloaded from genesdev.cshlp.org on November 20, 2021 - Published by Cold Spring Harbor Laboratory Press An F-box protein that functions in auxin response participate in the activation of either ubiquitin or a ubiq- a ubiquitin–protein conjugation pathway. Thus, our re- uitin-related protein and TIR1 could function later in the sults provide strong support for a role for ubiquitin (or pathway as part of an E3 complex. The nature of the related protein) modification in auxin response. Re- targeted protein is unknown. We hypothesize that auxin cently, D. Xie and J. Turner (University of East Anglia, response depends on ubiquitin (or ubiquitin-related pro- Norwich, UK) have independently shown that the LRF3 tein) modification of one or more regulatory proteins. gene described here is identical to a previously identified gene called COI1 (J. Turner, pers. comm.). The recessive Ubiquitination may then result in degradation by the proteosome (Hochstrasser 1996). This is consistent with coi1 mutants are completely insensitive to jasmonic a model proposed by Theologis and colleagues in which acid, suggesting that the gene plays a key role in jasmo- transcription of auxin-regulated genes is normally re- nate signaling or response (Feys et al. 1994). It appears pressed by the action of short-lived repressor proteins likely that the TIR1/COI1 genes represent the first (Ballas et al. 1995; Abel and Theologis 1996). Auxin may members of a novel class of signaling proteins in plants. relieve this repression by stimulating degradation of the repressors in an AXR1–TIR1 dependent manner. Alter- Materials and methods natively, the modification may alter the activity or cel- lular localization of a regulatory protein. For example, Plant materials and growth conditions ligand-dependent ubiquitin modification of the a-factor All mutant lines were from the Columbia ecotype. Plants were receptor in yeast results in endocytosis of the receptor grown at 21–23°C under continuous fluorescent illumination (Hicke and Riezman 1996). In mammalian cells, local- (100–150 μE/m per sec) in 13-cm clay pots containing Metro- ization of the protein RanGAP1 to the nuclear pore com- Mix (W.R. Grace & Co.) or an equivalent soilless mixture. Dur- plex depends on modification by the ubiquitin-related ing the first 3 weeks of growth, a mineral nutrient solution containing 5 mM KNO , 2.5 mM KPO (adjusted to pH 5.8), 2 protein SUMO1 (Mahajan et al. 1997). Clearly, we will 3 4 mM MgSO ,2mM Ca(NO ) ,50μM Fe–EDTA, 70 μM H BO ,14 4 3 2 3 3 need to identify the substrates of the AXR1–TIR1 path- μM MnCl , 0.5 μM CuSO ,1μM ZnSO , 0.2 μM Na MoO ,10μM 2 4 4 2 4 way(s) to establish a mechanism. One potential substrate NaCl, and 10 nM CoCl was supplied to the plants. For many is the product of the SAR1 gene. By genetic criteria, experiments, plants were grown under sterile conditions in petri SAR1 has a negative role in auxin response, and acts plates containing the above nutrient solution plus 0.7% agar, downstream of AXR1 (Cernac et al. 1997). 1.0% sucrose (wt/vol), and various hormones or inhibitors as How auxin affects the pathway is also uncertain, par- indicated in the text. Before plating, seeds were surface-steril- ticularly because neither the auxin receptor nor other ized by agitation for 10–20 min in 20% commercial bleach and signal transduction components have been identified. 0.02% Triton X-100, rinsed several times with sterile water, and held at 4°C for 3–4 days to enhance germination. The seeds were One attractive possibility is suggested by experiments of dispersed on the growth media with sterile water. The plates Li and Johnston (1997) on Grr1p. As mentioned above, were oriented vertically and held in an incubator containing Grr1p is required for ubiquitin-mediated degradation of fluorescent lighting (30–50 μE/m per sec; 16 hr photoperiod) the G cyclins Cln1p and Cln2p in a process that appears and a temperature of 20–21°C. In all experiments where plants to involve an SCF complex that contains Grr1p (Feldman are grown, day 0 is considered to be the time when seeds are first et al. 1997; Li and Johnston 1997; Skowyra et al. 1997). placed in the growth conditions described above. For hypocotyl Grr1p also plays a central role in glucose repression and and cell elongation studies, seedlings were grown under 24 hr glucose induction of genes encoding glucose transport- light at 85 μE/m per sec. ers, however, a function that also requires Skp1p. When wild-type cells are grown in glucose, there is a substan- Genetic analysis tial increase in the amount of Grr1p–Skp1p complex that For genetic analyses, resistance to inhibition of root growth by is immunoprecipitated relative to cells grown in glycerol CPD (a gift from G.F. Katekar, Commonwealth Scientific and even though the levels of Grr1p and Sk1p are similar in Industrial Research Organization, Canberra City, Australia) was the two extracts. Li and Johnston (1997) suggest that this assayed by the following procedure. Surface-sterilized, cold- difference may reflect a mechanism by which the cell treated seeds were plated on nutrient medium, as described links nutrient availability to cell cycle regulation. Be- above, supplemented with 5 μM CPD. On day 7 (unless other- cause TIR1 may bind a SKP1 homolog in Arabidopsis, it wise stated) the seedlings were straightened with forceps and is possible that auxin affects this interaction in some the root length (distance from the root/hypocotyl junction to way, thereby altering the level of ubiquitin modification the root tip) was measured. Resistance to 0.2 μM 2,4-D was of one or more targets. We are currently working to iden- assayed in a similar manner except that roots were scored vi- sually as either 2,4-D-resistant or wild type. tify TIR1-interacting proteins to test this model. To generate axr1-12 tir1-1 double mutant plants, crosses be- tween the single mutant lines axr1-12 and tir1-1 were per- Summary formed. The genotypes of individual F plants from these crosses were determined by test crosses to both of the parental Genetic studies in Arabidopsis have led to the identifi- lines and tests for CPD or 2,4-D resistance as described above. cation of eight genes that are required for normal auxin Linkage analysis was performed with a 9-kb EcoRI–SalI frag- response (Hobbie and Estelle 1994). To date, three of ment of Arabidopsis genomic DNA containing the coding se- these genes have been isolated. The structures of the quences of the TIR1 gene. This fragment detected a RFLP be- proteins encoded by two of the genes, AXR1 (Leyser et al. tween EcoRV-digested Columbia and Landsberg erecta (Ler) ge- 1993) and TIR1 (this study), suggest that they function in nomic DNA. Mapmaker I (S. Lincoln, M. Daly, J. Abrahamson, GENES & DEVELOPMENT 205 Downloaded from genesdev.cshlp.org on November 20, 2021 - Published by Cold Spring Harbor Laboratory Press Ruegger et al. A. Barlow, and L. Newburg; Massachusetts Institute of Tech- 3.8-kb region of D109ES containing the TIR1 coding region, nology, Cambridge, MA) was used to compile the results of the were subcloned into pBluescript and sequenced (Sequitherm, DNA blot analysis of 44 RI lines (Lister and Dean 1993) with Epicentre Technologies). this fragment as a probe. Characterization of tir1 mutant alleles Auxin-transport assays RT-PCR (Stratagene) was used to amplify the expressed se- quences of two tir1 alleles from total RNA prepared from seed- Auxin-transport assays were performed according to the lings grown in liquid culture. The PCR products were separated method of Okada et al. (1991). Stem segments (2.5 cm) of pri- in a low-melt agarose gel and sequenced directly by use of a mary inflorescence were incubated in a 1.5-ml Eppendorf cen- modified Sequenase protocol (B. Robertson, University of Col- trifuge tube containing 30 μl of nutrient solution with 1 μM orado). The position of the T-DNA insertion of tir1-9 was de- [ C]IAA (1.74 nCi/ml). The segments were incubated with termined by sequencing a subcloned fragment of the L3 plasmid their apical end in the solution for various times from 2 to 18 hr. that hybridized to probes made from the TIR1 cDNA and from After the incubation, a 5-mm section from the basal end of the the T-DNA left border (plasmid pBSH10). segment was excised and added to 3 ml of liquid scintillation cocktail (Bio-Safe II; RPI, Mount Pleasant, IL). The samples were shaken at 100 rpm for at least 2 hr and left overnight at room RNA blot analysis temperature before scintillation counting in a scintillation Total RNA was isolated from 13-day-old seedlings according to counter (model LS 6500; Beckman Instruments). The experi- Newman et al. (1993) with modifications by Timpte et al. ment involved three stem segments for each time point. Wild- (1995), and blotted as described in Timpte et al. (1995). type and tir1-1 plants were grown in 24 hr of light (105 μE/m per sec) for 36 days.The experiment was repeated three times with similar results. Stems incubated with 15 μM NPA or with Generation of transgenic plants the basal end of the stem segment in the solution transported The 2.2-kb TIR1 cDNA was cloned into the pBI121 vector (re- low levels of [ C]IAA. placing the GUS gene) to generate the p35S:TIR1 plasmid. This plasmid was introduced into the Agrobacterium strain GV3101 Induction of lateral root formation by IAA by electroporation. tir1-1 plants were inoculated with this strain by vacuum infiltration according to Bechtold et al. (1993). Seedlings were grown on nutrient medium for 5 days. Eight- Transformed seedlings were identified by selection on 50 μg/ml millimeter root segments were excised with a razor blade from kanamycin. Transgenic lines were tested for TIR1 function by mature root and transferred to nutrient medium supplemented plating on medium that contained 0.085 mM 2,4-D. with various concentrations of IAA. The number of lateral roots was determined by use of a dissecting microscope and expressed as the number of lateral roots per millimeter of primary root. Acknowledgments We are grateful to members of the Estelle laboratory and the Isolation of sequences flanking the T-DNA insert in tir1-9 Indiana University Arabidopsis group for stimulating discus- sion throughout the course of this work, to Roger Innes for Plasmid rescue of T-DNA sequences was done following the careful reading of the manuscript, to the ABRC for cDNAs, and procedure of Behringer and Medford (1992). Genomic DNA, pre- to L. Washington for assistance with DNA sequencing. This pared from rosette leaves of the tir1-9 mutant line, was digested research was supported in part by the National Science Foun- with either SalIor EcoRI to isolate DNA flanking the T-DNA dation (postdoctoral fellowship MCB-9008316 to L.H., left border or right border, respectively. The digested DNA was IBN-9307134 to M.E.) and National Institutes of Health (U.S. ligated with T4 DNA ligase, and electroporated into JS4 E. coli Public Health Service grant-GM43644 to M.E.) and an Eli Lilly/ (Bio-Rad). Four colonies derived from the SalI-digested DNA Indiana Institute for Molecular Biology predoctoral fellowship (L1–L4) and >100 colonies derived from the EcoRI-digested to M.R. DNA were recovered. Plasmid DNA from L1–L4 and from 14 of The publication costs of this article were defrayed in part by the EcoRI-derived colonies (R1–R14) was prepared and analyzed payment of page charges. 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Beach. 1995. Skp1 Skp2 process. Development 121: 3303–3310. p19 and p45 are essential elements of the cyclin A- Leyser, H.M.O., C. Lincoln, C. Timpte, D. Lammer, J. Turner, CDK2 S phase kinase. Cell 82: 915–925. GENES & DEVELOPMENT 207 Downloaded from genesdev.cshlp.org on November 20, 2021 - Published by Cold Spring Harbor Laboratory Press The TIR1 protein of Arabidopsis functions in auxin response and is related to human SKP2 and yeast Grr1p Max Ruegger, Elizabeth Dewey, William M. Gray, et al. Genes Dev. 1998, 12: Access the most recent version at doi:10.1101/gad.12.2.198 This article cites 40 articles, 16 of which can be accessed free at: References http://genesdev.cshlp.org/content/12/2/198.full.html#ref-list-1 License Receive free email alerts when new articles cite this article - sign up in the box at the top Email Alerting right corner of the article or click here. Service Cold Spring Harbor Laboratory Press

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