Figure 1.
a). Characterized alleles of IBR5. Arrows indicate the point mutations identified in IBR5. Triangles indicate the T-DNA insertions that alter or disrupt IBR5 expression. b) ibr5-4 has a G727 to A mutation in the third exon that changes G132 to E in the conserved dual-specificity phosphatase catalytic domain. ibr5-5 has a G to A mutation in the intron of the last intron-exon junction. c) IBR5 produces two mRNAs, one similar to the predicted splice variant AT2G04550.3 and AT2G04550.1 containing the last intron. d) mRNAs produced by ibr5-5. Total RNA was isolated from four day old ibr5-5 and wild type Col-0 seedlings and cDNA was synthesized. IBR5 was amplified using two different primer combinations (Table 1). e) Presence of IBR5.3 in wild type Col-0. IBR5 was amplified from cDNA synthesized from Col-0 seedlings as described in (d). PCR products were subjected to BsrI digestion up to four hours for complete digestion. IBR5.3 amplified from IBR5.3 clone was used as the positive control.
Figure 2.
Catalytic activity of IBR5.1-Myc protein.
a) Immuno-precipitation of IBR5.1-Myc and ibr5-4-Myc proteins. Total protein was isolated from transgenic plants over-expressing IBR5.1-Myc and ibr5-4-Myc. Tagged proteins were immuno-precipitated using anti-Myc antibody. 10% of the immuno-precipitate was visualized by western blotting using anti-Myc antibody. “*” indicates a non-specific protein that immuno-precipitates with anti-Myc antibody. b) The effect of ibr5-4 mutation on IBR5 activity was measured using OMFP assay. Similar amounts of immuno-precipitates were used for the OMFP assay. Reactions were carried out in triplicate. Error bars indicate standard deviations from the means.
Figure 3.
Aux/IAA degradation and auxin induced DR5::GFP expression.
a) Rapid degradation of AXR3NT-GUS in ibr5-4. Four day old light grown wild type Col-0 and ibr5-4 seedlings carrying HS::AXR3NT-GUS were heat shocked for two hours, fixed after the indicated time intervals and stained for GUS. b) Reduced DR5::GFP expression in ibr5-4. Four day old light grown wild type Col-0 or ibr5-4 seedlings carrying the DR5::GFP auxin inducible reporter were used. Seedlings were treated with mock (ethanol/DMSO), 100 nM 2,4D or 10 µM picloram for 3 hrs and imaged using Olympus FV1000 confocal microscopy. c) Expression of IAA12 and IAA28 were assessed by qRT-PCR using 4 day old Col-0 and ibr5-4 seedlings. UBA (AT1G04850) was used as the internal control. Expression levels were normalized against wild type Col-0. Error bars indicate standard deviations from the mean. Stars indicate that the means differ significantly from the respective control (ANOVA, P<0.05). d) Increased DR5::Venus expression in IBR5.1-Myc background. Four day old light grown wild type Col-0 or IBR5.1-Myc transgenic seedlings carrying DR5::Venus were used. Seedlings were imaged using Olympus FV1000 confocal microscopy. e) Quantitative analysis of Venus expression. Expression of Venus was quantified using ImageJ software. Error bars indicate standard deviation from the mean. Stars indicate that the means differ significantly from the control (n = 15, Student's t-test, P<0.05). f) Expression of IBR5.1-Myc in DR5::Venus lines. Total protein was isolated from homozygous seedlings carrying IBR5.1-Myc and DR5::Venus. IBR5.1-Myc was visualized by western blotting using anti-Myc antibody.
Figure 4.
Characterization of new ibr5 alleles.
a–b) Inhibition of primary root elongation by auxin. Seedlings were grown on unsupplemented ATS media for four days and transferred onto ATS containing the indicated concentrations of a) picloram, b) IAA. Seedlings were grown for four additional days, and the length of the primary root was measured. Results were normalized against unsupplemented media. Error bars indicate standard error of the mean. Stars indicate the means that differ significantly from the control (n = 15). c) Number of lateral roots in ibr5 mutants. Seedlings were grown on unsupplemented ATS media for 12 days, and the number of primordia emerged from the primary root were counted as lateral roots using a dissecting microscope. Error bars indicate standard deviations of the means (n = 20). d) Adult plant morphology of six week old ibr5 alleles grown in continuous light at 25°C. e) Seed size of ibr5 mutants. Dried mature seeds were photographed and the lengths of the seeds were measured using ImageJ software. Error bars indicate standard deviations of the means (n = 20). Stars indicate the means that differ significantly from the control; letters indicate the samples that differ significantly from each other (ANOVA, Tukey's HSD, P<0.05). f) Interdigitation of leaf epidermal pavement cells of ibr5 alleles. Propidium iodide stained lower epidermis of seven-day old cotyledons were imaged.
Figure 5.
Complementation of ibr5 mutants by overexpression of IBR5.1 and IBR5.3.
a) Complementation of auxin resistant primary root elongation of ibr5 mutants by IBR5.1-Myc but not IBR5.3-Myc over-expression. The root growth assay was performed as described in figure 4a. Error bars indicate standard error of the mean. b) Complementation of reduced lateral root phenotype of ibr5 mutants by IBR5.1-Myc and IBR5.3-Myc overexpression. The experiment was performed as described in figure 4c. Error bars indicate standard deviations from the means. Stars indicate that the means differ significantly from the control (n = 20, ANOVA, P<0.05).
Figure 6.
Subcellular localization and tissue specific expression of IBR5.
a) Sub-cellular localization of IBR5.1 and IBR5.3 isoforms. 35S::IBR5.1-GFP and 35S::IBR5.3-GFP reporter constructs were transiently expressed in Nicotiana benthamiana leaves and images were acquired using Olympus FV1000 confocal microscopy. b) Expression of IBR5.1-GFP in Arabidopsis seedlings. The IBR5::IBR5.1-GFP translational reporter construct was stably expressed in Arabidopsis. Expression of IBR5.1-GFP in root cells was detected using Olympus FV1000 confocal microscopy. (c–j) Tissue specific expression of IBR5.1-GUS in Arabidopsis seedlings. The IBR5::IBR5-GUS translational reporter construct was used to examine tissue specific expression of IBR5. Seven day old light grown seedlings were fixed and stained for GUS. IBR5 expression in the (c) cotyledons and top region of the hypocotyl, (d) elongation zone of the root tip, (e) shoot-root junction, (f) pericycle and endodermis of the mature region of the root, (g–j) adjacent pericycle and endodermis cells of lateral root initiation sites.
Figure 7.
ABA and stress responses of ibr5 alleles.
a) inhibition of primary root elongation by ABA. Four day old seedlings were transferred on to ATS containing 10 µM ABA, and the length of the primary root was measured after 4 day incubation at 21°C under continuous illumination. Error bars indicate standard error of the mean (n = 15). Stars indicate that the means differ significantly from the control, letters indicate the samples that differ significantly from each other (ANOVA, Tukey's HSD, P<0.05). b) Post germination inhibition by ABA and stresses. Seeds stratified at 4°C for two days were grown on media containing indicated amounts of ABA, NaCl or mannitol. Seedlings with green cotyledons were counted after 7 days of growth, and percentage was calculated. c–d) Expression of IBR5::IBR5.1-GUS in response to ABA and stresses. Four day old IBR5::IBR5.1-GUS transgenic seedlings treated with various concentrations of ABA, NaCl or mannitol for 18 hrs were used to perform quantitative GUS assays. Each data point indicates the mean value of 3 replicates. e–f) Expression of IBR5 in response to ABA and stresses. Four day old Col-0 seedlings treated with various concentrations of ABA, NaCl and mannitol for 18 hrs were used. Expression of IBR5 was assessed by qRT-PCR. UBA (AT1G04850) was used as the internal control. g) Response of ibr5 alleles to oxidative stress in post germination growth. The experiment was performed as described in (b) in the presence of 1 µM methyl viologen. Seedlings with green cotyledons were counted after 7 days of growth, and percentage was calculated. Error bars indicate standard deviation from the mean. Stars or letters indicate that the means differ significantly from the respective control (ANOVA, P<0.05).
Table 1.
Primers used in the study.