Auxin-Mediated Transcriptional System with a Minimal Set of Components Is Critical for Morphogenesis through the Life Cycle in Marchantia polymorpha

Abstract

The plant hormone auxin regulates many aspects of plant growth and development. Recent progress in Arabidopsis provided a scheme that auxin receptors, TIR1/AFBs, target transcriptional co-repressors, AUX/IAAs, for degradation, allowing ARFs to regulate transcription of auxin responsive genes. The mechanism of auxin-mediated transcriptional regulation is considered to have evolved around the time plants adapted to land. However, little is known about the role of auxin-mediated transcription in basal land plant lineages. We focused on the liverwort Marchantia polymorpha, which belongs to the earliest diverging lineage of land plants. M. polymorpha has only a single TIR1/AFB (MpTIR1), a single AUX/IAA (MpIAA), and three ARFs (MpARF1, MpARF2, and MpARF3) in the genome. Expression of a dominant allele of MpIAA with mutations in its putative degron sequence conferred an auxin resistant phenotype and repressed auxin-dependent expression of the auxin response reporter proGH3:GUS. We next established a system for DEX-inducible auxin-response repression by expressing the putatively stabilized MpIAA protein fused with the glucocorticoid receptor domain (MpIAA(mDII)-GR). Repression of auxin responses in (pro)MpIAA:MpIAA(mDII)-GR plants caused severe defects in various developmental processes, including gemmaling development, dorsiventrality, organogenesis, and tropic responses. Transient transactivation assays showed that the three MpARFs had different transcriptional activities, each corresponding to their phylogenetic classifications. Moreover, MpIAA and MpARF proteins interacted with each other with different affinities. This study provides evidence that pleiotropic auxin responses can be achieved by a minimal set of auxin signaling factors and suggests that the transcriptional regulation mediated by TIR1/AFB, AUX/IAA, and three types of ARFs might have been a key invention to establish body plans of land plants. We propose that M. polymorpha is a good model to investigate the principles and the evolution of auxin-mediated transcriptional regulation and its roles in land plant morphogenesis.

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 _pro MpEF1α:MpIAA and _pro MpEF1α:MpIAA ^mDII. Two conserved proline residues in domain II were substituted with serine. (B) Resistance to auxin by domain II-modified MpIAA expression. Photographs of WT, _pro MpEF1α:MpIAA and _pro MpEF1α:MpIAA ^mDII 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 _pro GH3:GUS and _pro MpIAA:MpIAA ^mDII -GR/ _pro GH3: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 _pro MpIAA:MpIAA ^mDII -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 _pro MpIAA: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 _pro MpIAA: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 _pro MpIAA:MpIAA ^mDII -GR plants.

(A-D) _pro MpIAA:MpIAA ^mDII -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) _pro MpIAA:MpIAA ^mDII -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) _pro MpIAA:MpIAA ^mDII -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).