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The roles of the sole activator-type auxin response factor in pattern formation of marchantia polymorpha

Cell division patterning is important to determine body shape in plants. Nuclear auxin signaling mediated by AUXIN RESPONSE FACTOR (ARF) transcription factors affects plant growth and development through regulation of cell division, elongation and differentiation. The evolutionary origin of the ARF-mediated pathway dates back to at least the common ancestor of bryophytes and other land plants. The liverwort Marchantia polymorpha has three phylogenetically distinct ARFs: MpARF1, the sole ‘activator’ ARF; and MpARF2 and MpARF3, two ‘repressor’ ARFs. Genetic screens for auxin-resistant mutants revealed that loss of MpARF1 function conferred auxin insensitivity. Mparf1 mutants showed reduced auxin-inducible gene expression and various developmental defects, including thallus twisting and gemma malformation. We further investigated the role of MpARF1 in gemma development, which is traceable at the cellular level. In wild-type plants, a gemma initial first undergoes several transverse divisions to generate a single-celled stalk and a gemma proper, followed by rather synchronous longitudinal divisions in the latter. Mparf1 mutants often contained multicelled stalks and showed defects in the execution and timing of the longitudinal divisions. While wild-type gemmae finally generate two meristem notches, Mparf1 gemmae displayed various numbers of ectopic meristems. These results suggest that MpARF1 regulates formative cell divisions and axis formation through auxin responses. The mechanism for activator ARF regulation of pattern formation may be shared in land plants and therefore important for the general acquisition of three-dimensional body plans

Auxin-mediated transcriptional system with a minimal set of components is critical for morphogenesis through the life cycle in Marchantia polymorpha

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

DEX-inducible system for repressing auxin responses.

<p>(A, B) GUS staining (A) and quantitative fluorometric assays (B) of _<i>pro</i><i>GH3</i>:<i>GUS</i> and _<i>pro</i><i>MpIAA</i>:<i>MpIAA</i>^<i>mDII</i><i>-GR/</i>_<i>pro</i><i>GH3</i>:<i>GUS</i> transgenic plants. Each plant was treated with 10 μM NAA and/or 10 μM DEX for 12 h. Error bars: SE (n = 3).</p

Protein-protein interactions between MpIAA and MpARFs <i>in planta</i>.

<p>BiFC assays of MpIAA and MpARF using <i>N</i>. <i>benthamiana</i> 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.</p

Expression pattern of <i>MpIAA</i> throughout the life cycle.

<p>GUS staining of _<i>pro</i><i>MpIAA</i>:<i>GUS</i> 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 _<i>pro</i><i>MpIAA</i>:<i>GUS</i> 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).</p

Tranactivation assay for MpARFs.

<p>(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 <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005084#sec015" target="_blank">Material and Methods</a> for effector vectors. Error bars: SE (n = 3).</p

Effects of exogenous auxin on the morphology and cell shape.

<p>WT (A-I) and _<i>pro</i><i>MpIAA</i>:<i>MpIAA</i>^<i>mDII</i><i>-GR</i> 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.</p