Constitutive auxin response in Physcomitrella reveals complex interactions between Aux/IAA and ARF proteins
- Howard Hughes Medical Institute, University of California, San Diego, United States
Figures
Figure 1 with 2 supplements
The aux/iaaΔ mutant displays a severe phenotype.
(A) Scheme representing the auxin-signaling pathway in WT plants (left panel) and in the aux/iaaΔ mutant (right panel). (B) GUS expression in PIAA1B:GUS and aux/iaaΔ plants carrying the PIAA1B:GUS …
Figure 1—figure supplement 1
The aux/iaaΔ is a null mutant that displays constitutive auxin response.
(A) PCR validating the absence of the genomic sequence of IAA1A, IAA1B, and IAA2 after CRE recombination. Primer pairs used: PML 749, 751 (IAA1A), PML 750, 751 (IAA1B), PML753, 754 (IAA2). (B) qPCR …
Figure 1—figure supplement 2
Early stages of filamentous growth of WT and aux/iaaΔ mutant.
(A) Six WT and aux/iaaΔ mutant plants were transferred to BCD medium four days after protoplast recovery and measured for 5 days. Error bars represent s.e.m. (B) Protonemal filaments of WT and aux/ia…
Figure 2 with 3 supplements
Loss of the Aux/IAA genes results in dramatic changes in gene expression at the filamentous developmental stage.
(A) Confocal images of WT and aux/iaaΔ mutant protonemata seven days after tissue homogenization (Left panels-chloroplasts are visualized in green. Right panels-cell structures are visualized with …
Figure 2—figure supplement 1
Protonemal tissue grown under the growth conditions used for the RNAseq experiment.
WT and aux/iaaΔ grown on BCDAT plates with cellophane overlays for seven days after tissue homogenization. Scale bar: 0.5 cm.
Figure 2—figure supplement 2
qPCR showing the expression levels of auxin responsive genes in WT plant and aux/iaaΔ mutant in mock- or 10 μM IAA-treatment for one hour.
https://doi.org/10.7554/eLife.13325.008
Figure 2—figure supplement 3
Graphical representation of enriched cellular components associated with auxin-responsive genes.
Colors of cells represent significance levels. The significance values are designated within each cell. Green arrows represent negative regulation.
Figure 3 with 2 supplements
Repressing ARFs target auxin-induced genes.
(A) WT, aux/iaaΔ and two aux/iaaΔ lines overexpressing ARFb4 (ARFb4OE_aux/iaaΔ) grown for one month on BCDAT. (B) GUS expression in aux/iaaΔ and two ARFb4OE_aux/iaaΔ lines carrying the PIAA1B:GUS …
Figure 3—figure supplement 1
Phylogeny of Land Plant ARFs.
The relationships between land plant ARFs were inferred using an alignment of amino-acid sequences and the MrBayes 3.2.2 program. The text colors indicate the plant whose genome encodes the protein: …
Figure 3—figure supplement 2
The Arabidopsis repressing ARF1 has similar effects on plant growth as ARFb4.
This figure is an extension of Figure 3A,B and includes two independent AtARF1 overexpression lines in aux/iaaΔ mutant background. (A) WT, aux/iaaΔ and two ARFb4 overexpression in aux/iaaΔ (as shown …
Figure 4
Repressing and activating ARFs display opposite effects on the same target genes.
(A) ARFb4OE_aux/iaaΔ (line #1 in Figure 3A), and two ARFa8-GR ARFb4OE_aux/iaaΔ lines grown on BCDAT or BCDAT supplemented with 10 μM DEX for a month. Scale bar: 0.5 mm. (B) Enlargement of …
Figure 5 with 3 supplements
Both repressing and activating ARFs affect auxin response in the same direction in the presence of the Aux/IAAs.
(A) qPCR showing the expression levels of auxin responsive genes in WT, arfb2 arfb4Δ (arfb2,4), iaa1b iaa2Δ (iaa1b,2), and arfb2 arfb4 iaa1b iaa2Δ (iaa1b,2 arfb2,4), mock- or 10 μM IAA-treated for …
Figure 5—figure supplement 1
PCR detecting the insertion position of different arfb2 arfb4Δ mutants.
(A) Validating the absence of the genomic sequence of ARFb2 and ARFb4PCR. Upper panel: validating the transgene integration in arfb2 arfb4Δ mutant. Primer pairs used to confirm the presence of …
Figure 5—figure supplement 2
Arfb2 and arfb4 Knockout in aux/iaaΔ mutant background (arfb2 arfb4Δ aux/iaaΔ) does not result in phenotypes comparable to reduced auxin sensitivity.
(A) qPCR showing the expression levels of auxin responsive genes in WT, aux/iaaΔ, and arfb2 arfb4Δ aux/iaaΔ. (B) arfb2 arfb4Δ aux/iaaΔ and aux/iaaΔ mutants grown for one month on BCD. Scale bar: 0.5 …
Figure 5—figure supplement 3
Phenotypic analysis of arfb2 arfb4Δ in WT and iaa1b iaa2Δ mutant background.
WT, arfb2 arfb4Δ, iaa1b iaa2Δ, and arfb2 arfb4Δ iaa1b iaa2Δ lines grown for one month on BCDAT medium without auxin or with different NAA concentrations. Scale bar: 0.5 cm.
Additional files
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Supplementary file 1
(A) Differentially expressed genes between mock- and IAA treated WT protonemata, (fold change ≥1.5; padj <0.01). (B) Differentially expressed genes between mock treated WT protonemata and the aux/iaaΔ mutant (fold change ≥1.5; padj <0.01). (C) Differentially expressed genes between IAA treated WT protonemata, and the aux/iaaΔ mutant (fold change ≥1.5; padj <0.01). (D) Differentially expressed genes between mock- and IAA treated aux/iaaΔ mutant (padj <0.05).
- https://doi.org/10.7554/eLife.13325.018
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Supplementary file 2
(A) GO terms of biological processes (P), molecular function (F), and cellular component (C) associated with auxin-regulated genes. (B) GO terms of biological processes (P), molecular function (F), and cellular component (C) associated with genes regulated between WT and the aux/iaaΔ mutant.
- https://doi.org/10.7554/eLife.13325.019
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Supplementary file 3
Primer sequences used in this study.
- https://doi.org/10.7554/eLife.13325.020
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Supplementary file 4
Backbone vectors used in this study.
- https://doi.org/10.7554/eLife.13325.021
