The IBR5 phosphatase promotes Arabidopsis auxin responses through a novel mechanism distinct from TIR1-mediated repressor degradation
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In Arabidopsis, INDOLE-3-BUTYRIC ACID RESPONSE5 (IBR5), a putative dual-specificity protein phosphatase, is a positive regulator of auxin response. Mutations in IBR5 result in decreased plant height, defective vascular development, increased leaf serration, fewer lateral roots, and resistance to the phytohormones auxin and abscisic acid. However, the pathways through which IBR5 influences auxin responses are not fully understood.
We analyzed double mutants of ibr5 with other mutants that dampen auxin responses and found that combining ibr5 with an auxin receptor mutant, tir1, enhanced auxin resistance relative to either parent. Like other auxin-response mutants, auxin-responsive reporter accumulation was reduced in ibr5. Unlike other auxin-resistant mutants, the Aux/IAA repressor reporter protein AXR3NT-GUS was not stabilized in ibr5. Similarly, the Aux/IAA repressor IAA28 was less abundant in ibr5 than in wild type. ibr5 defects were not fully rescued by overexpression of a mutant form of IBR5 lacking the catalytic cysteine residue.
Our genetic and molecular evidence suggests that IBR5 is a phosphatase that promotes auxin responses, including auxin-inducible transcription, differently than the TIR1 auxin receptor and without destabilizing Aux/IAA repressor proteins. Our data are consistent with the possibility that auxin-responsive transcription can be modulated downstream of TIR1-mediated repressor degradation.
KeywordsAuxin Response Unsupplemented Medium Auxin Response Factor Protein Auxin Influx Carrier Dual Specificity Protein Phosphatase
The phytohormone auxin is critical for plant growth and development, regulating vascular development, apical dominance, tropic responses, and organ patterning by modulating cell division and elongation [1, 2]. Changes in gene expression are among the earliest molecular responses to auxin. Many auxin-responsive transcripts fall into one of three classes: GH3-related, Aux in/ I NDOLE-3- A CETIC A CID (Aux/IAA), and S MALL A UXIN- U P R NA (SAUR) transcripts [3, 4, 5, 6, 7, 8]. Common to many of these auxin-responsive genes is a sequence in the upstream regulatory region termed the Auxin-Responsive Element (AuxRE; ).
AUXIN RESPONSE FACTOR (ARF) proteins are transcription factors that bind AuxREs (reviewed in ). Depending on the nature of the central domain, ARF family members can either activate or repress transcription [11, 12]. ARF proteins can form homodimers, dimers with other ARF proteins, or dimers with transcriptionally repressive Aux/IAA proteins [13, 14]. Many Aux/IAA proteins directly prevent transcriptional activation by interacting with activating ARF proteins [12, 15].
Many Aux/IAA transcriptional repressors are unstable  and are degraded even more rapidly following auxin application [16, 17]. Rapid Aux/IAA degradation following an auxin stimulus is thought to free activating ARF proteins from repression, allowing auxin-responsive gene expression. Mutant screens for decreased auxin sensitivity have identified several Aux/IAA proteins with stabilizing mutations (reviewed in ). Also isolated from auxin-response screens were trans-acting mutations that likewise stabilize Aux/IAA proteins, revealing the degradation mechanism for these repressors. Several auxin-resistant mutants have defects in the SCFTIR1 E3 ubiquitin ligase complex, as well as its regulatory components (reviewed in ).
TRANSPORT INHIBITOR RESPONSE1 (TIR1) and the other AUXIN SIGNALING F-BOX (AFB) family members are the substrate-recognition components of SCF complexes that bind auxin and promote the degradation of Aux/IAA repressor proteins [19, 20, 21]. Auxin is trapped in the TIR1 auxin-binding pocket by an interacting Aux/IAA protein . Subsequent 26S proteasomal degradation of Aux/IAA proteins relieves the repression of the ARF protein, allowing auxin-responsive transcription [16, 17, 23]. This novel receptor-ligand interaction allows a very short signal transduction chain that may facilitate rapid transcriptional responses to auxin. In addition, RUB (RELATED TO UBIQUITIN) modification of the CULLIN subunit of SCFTIR1 is necessary for auxin response [24, 25]. Mutations in AXR1 and ECR1, which encode subunits of the RUB-activating enzyme [26, 27], result in decreased auxin responses accompanied by slowed Aux/IAA protein degradation [16, 25, 28, 29, 30], presumably because of reduced SCFTIR1 efficacy in targeting these proteins for degradation.
Normal auxin responses require active movement of auxin through the plant, which is controlled by specialized influx and efflux carriers (reviewed in ). AUXIN RESISTANT1 (AUX1) is an auxin influx carrier protein that allows certain auxins to enter cells [32, 33, 34, 35]. Mutations in AUX1 result in resistance to IAA and 2,4-dichlorophenoxyacetic acid (2,4-D) , which are substrates of the AUX1 transporter .
A variety of natural and synthetic auxins and auxin precursors have activity in auxin bioassays . A mutation in IB A R ESPONSE5 (IBR5) was identified in a screen for resistance to the inhibitory effects of the auxin precursor indole-3-butyric acid (IBA) on root growth . Subsequent analyses revealed that ibr5 mutants are less sensitive not only to IBA, but also to all tested forms of auxin and to the phytohormone abscisic acid (ABA) . IBR5 encodes a putative dual-specificity protein phosphatase, and the ibr5-1 mutation causes a premature stop codon that would result in a truncated product lacking the conserved phosphatase domain . Here, we examined the role of IBR5 as a phosphatase in vivo by expressing a mutant version of IBR5 predicted to be catalytically inactive in the ibr5 mutant and found that phosphatase activity is likely required for full IBR5 function. Through double mutant analyses, we found that ibr5 enhanced most tir1 defects and a subset of axr1 and aux1 defects. Further, we demonstrated that ibr5 is defective in accumulation of an auxin-responsive reporter following auxin treatment. Because this reporter accumulates after degradation of Aux/IAA transcriptional repressors, we examined the effect of the ibr5 lesion on a reporter of Aux/IAA stability and an epitope-tagged Aux/IAA protein and, interestingly, found that these reporters were not stabilized in ibr5.
ibr5 enhances tir1auxin-response defects
In addition to auxin resistance, ibr5 mutant roots are resistant to the phytohormone ABA . We found that tir1 also exhibited ABA resistance, and that tir1 ibr5 roots were more ABA resistant than either single mutant (Figure 2C, Additional File 2). Because ibr5-1 is likely to be a null allele , these results support a model in which IBR5 and TIR1 act separately to affect auxin and ABA responsiveness.
ibr5 enhances certain axr1auxin-response defects
ibr5 enhances aux1root elongation defects
ibr5displays reduced auxin-responsive reporter accumulation
This reduced induction of DR5:GUS activity suggests that ibr5 misregulates at least some auxin-regulated transcripts. Thus, we examined basal levels and auxin responsiveness of endogenous IAA1, IAA2, and GH3.3 transcripts in wild-type and ibr5 seedlings. Although we detected subtle reductions in basal levels of IAA1 and IAA2 transcripts in ibr5 in several trials (data not shown), we did not detect dramatic differences in these experiments. In any case, IAA1, IAA2, and GH3.3 transcripts eventually reached similar maximal values in wild type and ibr5 (data not shown), suggesting that IBR5 is not required for full response to high auxin levels.
ibr5does not accumulate an AXR3/IAA17 reporter protein
Gain-of-function mutations that stabilize any of several Aux/IAA proteins can confer dominant auxin resistance (reviewed in ). Moreover, Aux/IAA repressor proteins or Aux/IAA-reporter fusion proteins are stabilized in numerous other auxin-resistant mutants, including tir1 , axr1 , ecr1 , afb1, afb2, and afb3 , cul1 , eta2/cand1 , eta3/sgt1b , and aar1 .
The reduced AXR3NT-GUS activity in ibr5 was apparent immediately following the 2-hour heat shock used to induce reporter expression (data not shown). Because a heat-responsive promoter drives the AXR3NT-GUS construct, we tested whether the lack of AXR3NT-GUS activity in ibr5 could be explained by a reduced transcriptional response to heat in the mutant. We introduced a construct altered to contain the stabilizing axr3-1 mutation (HS:axr3-1NT-GUS; ) into ibr5-1 by crossing. The proline to leucine substitution in axr3-1  confers reporter stability by decreasing axr3-1NT-GUS interaction with SCFTIR1 . We found similar axr3-1NT-GUS activity in wild type and ibr5 with or without auxin treatment (Figure 6C), suggesting that the transgenes were efficiently transcribed in response to the heat stimulus, and that the decreased AXR3NT-GUS activity in ibr5 was not caused by reduced transcription following heat shock. Moreover, treatment with the proteasome inhibitor MG132 restored AXR3NT-GUS activity in ibr5 and tir1 ibr5 to near wild-type levels (Figure 6D), again suggesting that the ibr5 defects in AXR3NT-GUS activity were not due to reduced transgene transcription.
ibr5does not accumulate an IAA28 reporter protein
An IBR5 substitution variant (IBR5C129S) does not fully rescue ibr5defects
Dual specificity protein phosphatase (DSP) proteins dephosphorylate both threonine and tyrosine residues of phosphorylated proteins, often thereby inactivating them (reviewed in ). DSP proteins contain a conserved aspartate residue and a separate, highly conserved signature motif of VxVHCx2GxSRSx5AYLM, with the cysteine and arginine residues participating with the conserved aspartate in catalysis. The cysteine of this signature begins the dephosphorylation process with a nucleophilic attack on the phosphorus atom of the phosphotyrosine or phosphothreonine substrate. Thus, disruption of this conserved cysteine results in catalytic inactivity in many DSP proteins (reviewed in ), including the IBR5 relative, DsPTP1 .
We found that IBR5 or IBR5C129S overexpression did not noticeably alter wild-type adult morphology (Figure 8B). Moreover, IBR5 or IBR5C129S overexpression in wild type did not alter any of the hormone-response phenotypes examined (Figure 8D, F). As expected, we found that ibr5-1 (35S:IBR5) plants were restored to wild-type height (Figure 8B) and had normal root responses to auxin and ABA (Figure 8E, F). ibr5-1 (35S:IBR5C129S) line A (low expressor) exhibited restored plant height but had similar leaf epinasty to ibr5-1, whereas ibr5-1 (35S:IBR5C129S) line B (high expressor) displayed rescue of ibr5-1 plant height (Figure 8B) and partial rescue of leaf epinasty (data not shown). Resistance to the inhibitory effects of 2,4-D and ABA on root elongation was not rescued in ibr5-1 (35S:IBR5C129S) line A and only partially restored in ibr5-1 (35S:IBR5C129S) line B (Figures 8E, F). The lack of full ibr5-1 rescue by IBR5C129Sis consistent with the possibility that IBR5 phosphatase activity is required for full auxin and ABA responsiveness. The partial ibr5-1 rescue observed when IBR5C129Saccumulates to high levels suggests that certain IBR5 functions do not require phosphatase activity. For example, IBR5C129S may bind and sequester its substrate(s), thereby dampening normal substrate activity.
Loss-of-function mutations in IBR5, which encodes a putative dual-specificity protein phosphatase (DSP), result in decreased auxin and abscisic acid responses . DSP proteins often regulate mitogen-activated protein kinase (MAPK) proteins. Arabidopsis has 20 predicted MAPK proteins  but only five predicted DSP proteins, suggesting that some DSP enzymes may regulate more than one MAPK. In Arabidopsis, two of the five predicted Arabidopsis DSP proteins have been demonstrated to regulate MAPK activity. DsPTP1 (At3g23610) dephosphorylates MPK4 , and MAPK PHOSPHATASE2 (MKP2; At3g06110) dephosphorylates MPK3 and MPK6 .
Although a MAPK regulated by IBR5 has not been reported, MAPK signaling has been implicated in both auxin and ABA responses, and both of these pathways are defective in ibr5. For example, transient expression in protoplasts of constitutively active MAPK kinase kinase protein ANP1 or the tobacco homolog NPK1 results in decreased auxin-responsive transcription and activation of AtMPK3 and AtMPK6 [55, 56], the targets of the MKP2 phosphatase . Additionally, auxin treatment activates a ~44-kD MAPK in Arabidopsis . The failure of IBR5C129S to fully restore ibr5-1 mutant phenotypes indicates that IBR5 phosphatase activity is required for full auxin and ABA responsiveness, and it will be interesting to learn if any of the MAP kinases implicated in hormone responsiveness are IBR5 substrates. The partial rescue of ibr5 defects observed when IBR5C129S was overexpressed might result from the IBR5C129S protein binding to and thus sequestering IBR5 substrate(s), as has been suggested for overexpression of the catalytically inactive MAPK phosphatase Pyp1C470S, which results in a phenotype similar to a loss-of-function allele of the substrate MAPK Spc1 in Schizosaccharomyces pombe [58, 59].
In an effort to clarify the pathways through which IBR5 affects auxin responses, we examined genetic interactions between ibr5 and the auxin-response mutants tir1, axr1, and aux1. The TIR1 F-box protein acts with Aux/IAA proteins as an auxin receptor [19, 20, 22]. tir1 appeared to enhance all ibr5 auxin-related physiological phenotypes examined, including response defects to applied natural and synthetic auxins (Figure 1C, D) and auxin transport inhibitors (data not shown). In addition, the tir1 ibr5 mutant displayed a longer primary root (Figures 1C, D) and fewer lateral roots than either single mutant on unsupplemented medium (Figure 2A), suggesting that the double mutant has enhanced resistance to endogenous auxin as well.
In addition to influencing auxin responses, IBR5 modulates certain ABA responses . We found not only that tir1 enhanced ibr5 ABA resistance, but also that tir1 itself exhibited substantial ABA resistance in the root elongation assay (Figure 2C). Although tir1 has not previously been reported to be ABA resistant, it has been characterized as glucose resistant , a phenotype common to many ABA-resistant mutants (reviewed in [61, 62]). Because tir1 ibr5 displays enhanced resistance to both auxin and ABA, and because the ibr5-1 allele is a likely null , these results are consistent with the possibility that TIR1 and IBR5 promote auxin and ABA responsiveness independently of one another.
AXR1 is a subunit of the RUB-activating enzyme involved in RUB modification of CULLIN proteins. CULLIN is the backbone for the over 600 putative SCF complexes in Arabidopsis , including SCFTIR1 ; therefore, axr1 mutants have pleiotropic phenotypes, including auxin resistance. The only examined phenotypes that appeared to be additive in axr1 ibr5 were the long primary root on unsupplemented media and aberrant vascular development (Figure 3). Unlike ibr5, tir1-1 enhances the root elongation resistance of axr1-12 to 2,4-D . The pleiotropic nature of the axr1 defect and the extreme auxin resistance of the axr1 single mutant complicate the interpretation of our results. Regardless, it is interesting that axr1 and ibr5 mutants, which are both less sensitive to the inhibitory effects of auxin on root elongation, seem to have opposite effects on AXR3NT-GUS stability (Figure 6A; ).
We also examined double mutants of ibr5 with aux1, a mutant defective in an auxin influx carrier [32, 34, 35]. The shoots of the aux1 ibr5 mutant resembled ibr5, whereas the roots of aux1 ibr5 were most similar to aux1. Because aux1 lacks shoot phenotypes and most aux1 root phenotypes are more dramatic than ibr5 root defects, these results are consistent with results expected from additive defects, although the extreme resistance of aux1 to 2,4-D and ABA prevented us from determining whether aux1 ibr5 had additive defects in response to these hormones.
ibr5 has decreased DR5:GUS activity when grown on unsupplemented media , and in this study we found defects in auxin-induced DR5:GUS activity in particular tissues (Figure 5). However, microarray analysis of mRNA accumulation in 7-day-old ibr5 and wild-type seedlings did not reveal any dramatic (> 2.5-fold) alterations in transcripts represented in the analysis . These results suggested that any gene expression changes in ibr5 might be subtle or local, as in the DR5:GUS analysis, and therefore not apparent in whole seedling RNA.
Because auxin-response transcripts are regulated by Aux/IAA protein stability, we sought to analyze Aux/IAA degradation in ibr5. Auxin promotes Aux/IAA protein degradation by mediating interaction of these repressors with SCFTIR1 [19, 20, 21, 65]. Several auxin-resistant mutants exhibit stabilized Aux/IAA proteins or Aux/IAA reporters, including mutants defective in the auxin receptors TIR1  and the related AFB1, AFB2, and AFB3 F-box proteins ; other SCF components such as CUL1 ; and proteins that modify SCF activity, such as AXR1 , ECR1 , ETA3/SGT1b , and ETA2/CAND1 . This stabilization probably accounts for the reduced levels of auxin-responsive transcripts reported in many of these mutants [21, 25, 42, 45, 46, 66]. Like previously characterized auxin-response mutants, ibr5 exhibits reduced accumulation of the auxin-responsive DR5:GUS reporter. Unlike these other mutants, however, AXR3NT-GUS activity was not increased in ibr5. Moreover, IAA28myc protein was less abundant in ibr5, which is not expected if Aux/IAA proteins are generally stabilized, also suggesting that IBR5 modulates auxin-responsive transcription without stabilizing Aux/IAA proteins. Of course, there are 29 Arabidopsis members of the Aux/IAA family , and it remains possible that ibr5 specifically stabilizes certain Aux/IAA family members to reduce transcriptional activation without affecting AXR3/IAA17 or IAA28 stability.
Regardless of whether Aux/IAA proteins that remain to be assessed turn out to be stabilized in ibr5, ibr5 is the only examined auxin-response mutant [16, 21, 28, 41, 42, 43, 44] that does not exhibit AXR3NT-GUS stabilization. This phenotypic bifurcation may be useful in dissecting the roles of additional auxin-response mutants. Recent experiments suggest that the MYB77 transcription factor promotes auxin responses and can dimerize with ARF proteins . It will be interesting to learn whether Aux/IAA proteins are stabilized in the myb77 mutant and whether IBR5 regulates this or some other factor needed to promote auxin-responsive transcription once the Aux/IAA repressors have been degraded. One of many possible scenarios is that IBR5 normally dephosphorylates and inactivates a MAPK that negatively regulates a transcription factor needed for auxin responses, providing a mechanism to fine-tune auxin responses without modulating Aux/IAA stability.
IBR5 resembles dual-specificity phosphatases, and in this work we provide evidence that IBR5 phosphatase activity is necessary for full auxin and ABA responsiveness. Analysis of double mutants between ibr5-1 and several other auxin-response mutants revealed that IBR5 appears to affect auxin responses independently of the TIR1 auxin receptor. Because transcriptional repression of auxin-responsive genes is relieved by Aux/IAA protein degradation, we examined the stability of two Aux/IAA reporters in ibr5 and found that these proteins were not stabilized in ibr5, suggesting that IBR5 acts downstream of auxin recognition by the SCFTIR1/AFB-Aux/IAA complexes. Future determination of IBR5 substrates may allow a more detailed understanding of how this apparent dual-specificity phosphatase is able to promote auxin responses without destabilizing Aux/IAA proteins.
Plant materials and growth conditions
Arabidopsis thaliana accession Colombia (Col-0) was the wild type used for all experiments. The ibr5-1 mutant contains a nonsense mutation at IBR5 amino acid 42, resulting in a truncated product lacking the catalytic domain . The aux1-7 mutant contains a missense mutation resulting in glycine 459 being replaced by aspartic acid . The tir1-1 mutant contains a missense mutation resulting in glycine 147 being replaced by an aspartic acid . The axr1-3 mutant contains a missense mutation resulting in cysteine 154 being replaced by a tyrosine .
Surface-sterilized  seeds were plated on PNS (plant nutrient medium with 0.5% [w/v] sucrose)  solidified with 0.6% (w/v) agar. Hormones used were from 0.1-, 1.0-, or 100-mM stocks in ethanol, with ethanol-supplemented media used as controls, and all treatments normalized to the same ethanol content (less than 0.1 μL ethanol/mL medium). Seedlings were grown at 22°C under continuous light. Unless indicated otherwise, plates were incubated under yellow long-pass filters to slow the breakdown of indolic compounds . Plants were grown in soil (Metromix 200; Scotts, Marysville, OH) at 22 to 25°C under continuous illumination by cool-white fluorescent bulbs (Sylvania, Danvers, MA).
The pKSIBR5c construct  was mutated using oligonucleotide-directed mutagenesis  to alter the presumptive catalytic cysteine at amino acid position 129 to a serine using a primer 5'-CTTTCCCAGACATCGAATGCACAAGAAC-3' (altered residues underlined) designed to contain a Taq1α restriction site. The mutant cDNA was then excised using NotI and subcloned into the plant transformation vector 35SpBARN  between the Cauliflower mosaic virus 35S promoter and the nos terminator. The 35S:IBR5C129S plasmid was electroporated  into Agrobacterium tumefaciens GV3101, which was used to transform wild-type Col-0 and ibr5-1. Transformants were identified on PNS medium supplemented with 10 μg/mL BASTA (glufosinate-ammonium) after 10 days under white light. Homozygous lines were identified in subsequent generations by examining the pattern of BASTA resistance.
The IAA28:IAA28myc construct  contains the IAA28 genomic region, including 3.1 kb of DNA 5' of the IAA28 coding sequence, subcloned into the pBIN19 plant transformation vector. The last exon of IAA28 has been modified in this construct to encode five copies of the c-Myc epitope immediately upstream of the IAA28 termination codon. The IAA28:IAA28myc plasmid was electroporated  into Agrobacterium tumefaciens GV3101, which was used to transform wild-type Col-0. Transformed seedlings were identified on PN medium supplemented with 12 μg/ml kanamycin after growth under white light. Homozygous single-insert lines were identified by examining the pattern of kanamycin resistance in subsequent generations. ibr5-1, tir1-1, and axr1-3 were crossed to a wild-type (Col-0) single-insert line to generate ibr5, tir1, and axr1 carrying the IAA28:IAA28myc construct.
All assays were conducted at least three times with similar results. For auxin-response root-elongation assays, seedlings were grown for 8 or 9 days on PNS with the indicated auxin concentrations and removed from the agar, and the lengths of the primary roots were measured. For ABA response root-elongation assays, seedlings were grown for 4 days on PNS to allow efficient germination, then were transferred to PNS supplemented with either ethanol or ABA. After an additional 4 days of growth, primary root lengths were measured.
In lateral root assays, seedlings were grown for 4 days on PNS, transferred to PNS supplemented with either ethanol or 10 μM IBA, and grown for an additional 4 days, after which lateral roots were examined under a dissecting microscope. Primordia emerged from the primary root were counted as lateral roots.
For hypocotyl elongation assays, seeds were plated on media supplemented with either ethanol or 20 μM IBA. After 1 day in the light, plates were wrapped with aluminum foil and incubated for an additional 4 days in the dark, after which seedlings were removed from the agar and hypocotyls lengths were measured.
To examine cotyledon vascular patterns, seedlings were grown for 8 days on PNS. Chlorophyll was removed using an ethanol series, and seedlings were cleared by incubating for one week at room temperature in chloral hydrate solution (80 g chloral hydrate, 20 mL glycerol, and 10 mL water). Cleared seedlings were mounted and photographed through a dissecting microscope.
Double mutant isolation
The ibr5-1 mutant was crossed to aux1-7 , axr1-3 , and tir1-1 , all in the Col-0 accession. Double mutants were identified by PCR analysis of F2 plants. Amplification of AUX1 with AUX1-3 (5'-CATGGGTCAACAAAGCTTTGGATTTTGTCC-3') and AUX1-4 (5'-TTCGTGACTTTTACTCCCTTCACGTATACG-3') yields a 464-bp product with two DpnII restriction sites in wild type and three in aux1-7. Amplification of AXR1 with the derived cleaved amplified polymorphic sequence primer [74, 75] AXR1-Acc1 (5'-AAACCAACTTAACGTTTGCATGTCG-3'; altered residue underlined) and AXR1-15 (5'-TCTCATATGTACTTTTCCTCGTCCTCTTCAC-3') yields a 185-bp product with one Acc1 restriction site in wild type and none in axr1-3. Amplification of TIR1 with TIR1-3 (5'-TTGAAGAGATAAGGCTGAAGAGGATGG-3') and TIR1-4 (5'-TACACCACCGTTAAATAAGACCCACCAGAAAG-3') yields a 488-bp product with one DpnII restriction site in wild type and two restriction sites in tir1-1. PCR-based identification of ibr5-1 was as described previously .
Surface-sterilized  seeds were plated on filter paper-lined PNS and grown under continuous illumination. After 6 days, the filter paper with seedlings was lifted off the agar surface. Seedlings were floated in liquid PN supplemented with mock (ethanol) or 200 nM IAA for 10 minutes. Seedlings were collected and ground with a mortar and pestle in liquid nitrogen. RNA was extracted using TriReagent (Sigma) according to the manufacturer's instructions. Total RNA (10 μg) was electrophoresed on a 1% agarose gel containing 0.37 M formaldehyde  and transferred to a positively-charged nylon 66 membrane (Roche Applied Science, Indianapolis, IN) in 20× SSC using capillary action. DIG-labeled probe was hybridized in DIG Easy Hyb buffer (Roche) overnight at 50°C and washed at moderate stringency according to the manufacturer's instructions. Probes were detected using an alkaline phosphatase-conjugated anti-DIG antibody (Roche) diluted 1:20,000 in Blocking Buffer (Roche), then visualized using a 1:200 dilution of CDP-Star (Roche).
DIG-labeled IAA28 probe was synthesized by PCR amplification using a PCR DIG Probe Synthesis Kit (Roche) according to the manufacturer's instructions from a cDNA template  using T1N24-20 (5'-CCATCGAACTGATGATTTTGGCC-3') and T1N24-21 (5'-CCTCCTTGTCACCAATTCACTTCC-3'), yielding a 525-bp product.
To visualize IBR5, protein was extracted from entire 2-day-old seedlings grown in 0.1% agar under white light by grinding frozen tissue with a pestle and adding one volume NuPAGE 2X LDS buffer (Invitrogen, Carlsbad, CA). Debris was pelleted by centrifugation for 4 minutes. The supernatant was heated to 100°C for 5 minutes. Protein extracts were separated by SDS-polyacrylamide gel electrophoresis beside Cruz markers (Santa Cruz Biotechnology, Santa Cruz, CA) using a NuPAGE 10% Bis-Tris gel and MES running buffer (Invitrogen).
Protein was transferred for 35 min at 24 V to a Hybond ECL nitrocellulose membrane (Amersham Pharmacia Biotech, Piscataway, NJ) using NuPAGE transfer buffer (Invitrogen). After blocking for 1 hour in 8% powdered milk in Tween Tris-buffered saline (TTBS; ), the membrane was incubated overnight at 4°C with affinity purified IBR5 antibody  diluted 1:20 in blocking buffer. The membrane was then washed three times with TTBS and incubated with HRP-conjugated goat anti-rabbit secondary antibody (Santa Cruz Biotechnology) diluted 1:2000 for 1 hour, washed again as described before, then visualized using LumiGLO reagent (Cell Signaling, Beverly, MA). Membranes subsequently were incubated with an antibody against spinach (Spinacia oleracea) HSC70 (Stressgen Bioreagents SPA-817) diluted 1:5000, followed by HRP-conjugated goat anti-mouse secondary antibody (Santa Cruz Biotechnology) diluted 1:2000, and visualized using LumiGLO reagent (Cell Signaling).
To visualize IAA28myc, 10-day-old light-grown seedlings were removed from PNS medium and floated in liquid PN supplemented with either ethanol (mock) or 200 nM IAA. At the indicated time points, roots were excised and protein was extracted. For MG132 treatment, 3-day-old light-grown seedlings were removed from PNS medium and suspended in water containing either DMSO (mock) or 300 μM MG132. After 1 or 2 hours, protein was extracted from whole seedlings. In both experiments, protein was separated by SDS-polyacrylamide gel electrophoresis and transferred to a membrane as described above. After blocking for 1 hour in 8% powdered milk in TTBS, the membrane was incubated overnight at 4°C with monoclonal 9E10 anti-c-Myc antibody (Santa Cruz Biotechnology SC-40) diluted 1:500 and anti-HSC70 antibody (Stressgen Bioreagents SPA-817) diluted 1:250,000 in blocking buffer. The membrane was then washed three times with TTBS and incubated with HRP-conjugated goat anti-mouse secondary antibody (Santa Cruz Biotechnology) diluted 1:2000 for 4 hours, washed again as described before, then visualized using LumiGLO reagent (Cell Signaling).
Wild-type (Col-0) lines carrying HS:AXR3NT-GUS and HS:axr3-1NT-GUS and axr1-3 carrying HS:AXR3NT-GUS were described previously . To obtain additional lines carrying these reporters, ibr5-1 was crossed to wild type carrying HS:AXR3NT-GUS and to wild type carrying HS:axr3-1NT-GUS. tir1 ibr5 was crossed to wild type carrying HS:AXR3NT-GUS to obtain tir1-1 HS:AXR3NT-GUS and tir1 ibr5 HS:AXR3NT-GUS. Mutants were identified by PCR analysis of the F2 plants, as described above. The presence of HS:AXR3NT-GUS or HS:axr3-1NT-GUS was assayed by resistance to kanamycin and confirmed by GUS staining.
For histochemical assays, 8-day-old light-grown seedlings were removed from PNS plates and floated in 0.5 mL liquid PN medium diluted six-fold with water (for hormone response assays) or sterile water (for MG132 assays) contained in a 12-well plate. Two milliliters of prewarmed (37°C) one-sixth liquid PN or prewarmed water was added to each well, and the plates were incubated at 37°C for 2 hours. For the hormone response assays, seedlings were transferred to room temperature for 20 minutes prior to 10- and 20-minute mock (ethanol) or 100 nM IAA treatments, or 40-minute mock (ethanol) or 10 μM IBA treatment. For MG132 assays, seedlings were incubated in the dark, and either DMSO (mock) or MG132 (to 50 μM) was added to each well 1 hour into the heat treatment. After heat treatment, plates were transferred to room temperature for 2 hours. Seedlings were stained for GUS activity as previously described .
We thank Luise Rogg for wild type transformed with the IAA28:IAA28myc construct, Ottoline Leyser and Stefan Kepinski for wild-type and axr1-3 lines carrying HS:AXR3NT-GUS and HS:axr3-1NT-GUS, Natasha Raikhel for the c-Myc epitope-tagging plasmid, Mark Estelle for axr1-3 and aux1-7, the ABRC for tir1-1 and cDNA clones, Mary Ellen Lane for microscope use, Kristen Rogers and Erin Beisner for technical assistance, and Matthew Lingard, Naxhiely Martinez, and Andrew Woodward for critical comments on the manuscript. This research was supported by the National Science Foundation (IBN-0315596), the Robert A. Welch Foundation (C-1309), and the National Institutes of Health (F32-GM075689 to LCS).
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