Fig 1.
Auxin signaling factors in M. polymorpha.
(A) Diagrams of domain structures of MpIAA and MpARFs. DBD: DNA-binding domain, Q-rich: glutamine-rich region, Roman numbers: domains I through IV. (B-D) Phylogenetic positions of MpIAA (B), MpARFs (C) and MpTIR1 (D) using the amino acid sequences from M. polymorpha (red), P. patens (green), S. moellendorffii (blue), and Arabidopsis (black). See S2 Table for sequence information. Numbers on the branches indicate bootstrap values. Bar in (A): 100 amino acid residues in length. Bars in (B-D): number of amino acid changes per branch length.
Fig 2.
Effects of mutations in domain II of MpIAA on auxin sensitivity.
(A) Diagram of proMpEF1α:MpIAA and proMpEF1α:MpIAAmDII. Two conserved proline residues in domain II were substituted with serine. (B) Resistance to auxin by domain II-modified MpIAA expression. Photographs of WT, proMpEF1α:MpIAA and proMpEF1α:MpIAAmDII plants cultured with exogenous 3 μM NAA or under mock conditions for 2 weeks. Bars: 5 mm. The values indicate area ratios of 2-week-old thallus grown in the presence of NAA to that grown under mock conditions with SE (n = 12).
Fig 3.
DEX-inducible system for repressing auxin responses.
(A, B) GUS staining (A) and quantitative fluorometric assays (B) of proGH3:GUS and proMpIAA:MpIAAmDII-GR/proGH3:GUS transgenic plants. Each plant was treated with 10 μM NAA and/or 10 μM DEX for 12 h. Error bars: SE (n = 3).
Fig 4.
Effects of exogenous auxin on the morphology and cell shape.
WT (A-I) and proMpIAA:MpIAAmDII-GR plants (J-R) were grown for 12 days in the absence of both NAA and DEX, then grown under mock condition (A, D, G, J, M, P), with 10 μM NAA (B, E, H, K, N, Q), or with 10 μM NAA and 10 μM DEX (C, F, I, L, O, R). At day 7 post treatment, photographs (A-C, J-L; bars: 10 mm) and scanning electron micrographs (D-I, M-R; bars: 500 μm) were taken.
Fig 5.
Expression pattern of MpIAA throughout the life cycle.
GUS staining of proMpIAA:GUS plants. (A) 3-week-old thallus. Arrow: gemma cup. (B) Longitudinal section of gemma cup. (C) Magnified view of the region shown by dotted line in (B). Arrows: developing gemmae. (D, E) Overview (D) and longitudinal section (E) of antheridiophore. The arrows represent antheridia. (F, G) Overview (F) and longitudinal section (G) of archegoniophore. Arrows: archegonia. (H-K) Sporophytes generated by crossing WT female and proMpIAA:GUS male. (H) Overview of fertilized archegoniophore containing developing sporophytes (arrows). (I-K) Isolated developing sporophytes. The apices of the sporophytes are directed downward. Scale bars: 2 mm (A, H), 0.5 mm (B, I-K), 0.1 mm (C, E, G), 5 mm (D, F).
Fig 6.
Morphological defects of proMpIAA:MpIAAmDII-GR plants.
(A-D) proMpIAA:MpIAAmDII-GR plants grown for 14 days without DEX. (A) SEM of the dorsal side of the thallus. Air pores are visible as black dots. (B, C) SEM (B) and longitudinal section (C) of a gemma cup. (D) SEM of the ventral side of the thallus. Arrow: ventral scale. (E-H) proMpIAA:MpIAAmDII-GR gemmalings grown in the presence of 10 μM DEX for 14 days. SEM images are shown. Arrows: serrated structures reminiscent of gemma cup. Arrowheads: air pores. (I-L) proMpIAA:MpIAAmDII-GR plants grown in the absence of DEX for 7 days and then subsequently in the presence of 10 μM DEX for 7 days. (I, J) SEM images of the dorsal side of the thallus (I) and a gemma cup (J). (K) Longitudinal section of a gemma cup. (L) SEM image of the ventral side of the thallus. (M-P) Antheridiophores (M, N) and archegoniophores (O, P) of male and female plants, respectively, grown for 2 weeks under mock (M, O) or DEX-treated (N, P) conditions. (Q-T) Fertilized archegoniophores at 4 weeks (Q, R) or 2 weeks (S, T) after crossing without (Q, S) or with DEX treatment (R, T). DEX treatment was performed every one or two days, beginning the day after crossing. Arrows: developing sporophytes. Scale bars: 1 mm (A, D, E, I, L), 0.5 mm (B, F-H, J), 0.1 mm (C, K), 5 mm (M-R), 0.2 mm (S, T).
Fig 7.
Protein-protein interactions between MpIAA and MpARFs in yeast.
Yeast two-hybrid assays with the HIS3 reporter. Ten-fold serial dilutions of overnight cultures were spotted on either nonselective +His (-Trp/-Leu) or selective-His (-His/-Trp/-Leu) media and grown for 2 days at 22°C. AD: proteins fused to VP16 activation domain. BD: proteins fused to lexA DNA-binding domain.
Fig 8.
Protein-protein interactions between MpIAA and MpARFs in planta.
BiFC assays of MpIAA and MpARF using N. benthamiana leaves. Confocal images of YFP (yellow) and chloroplast auto-fluorescence (red) were merged with bright-field images. Vectors containing only nYFP or cYFP was used as negative controls. Bars: 50 μm.
Fig 9.
Tranactivation assay for MpARFs.
(A) Diagrams of the constructs for dual luciferase assay. (B) Relative luciferase activity elicited by effector plasmid. The vector expressing only Gal4 DBD was used as a control. A virus-derived activation domain, VP16, was used for a positive control. See Material and Methods for effector vectors. Error bars: SE (n = 3).