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First published online 3 October 2007
doi: 10.1242/dev.009654

Development 134, 3849-3859 (2007)
Published by The Company of Biologists 2007
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The gene MACCHI-BOU 4/ENHANCER OF PINOID encodes a NPH3-like protein and reveals similarities between organogenesis and phototropism at the molecular level

Masahiko Furutani1,*, Takahito Kajiwara1,*, Takehide Kato1, Birgit S. Treml2, Christine Stockum2, Ramón A. Torres-Ruiz2 and Masao Tasaka1,{dagger}

1 Graduate School of Biological Sciences, Nara Institute of Science and Technology, Nara 630-0101, Japan.
2 Lehrstuhl für Genetik, Technische Universität München, Wissenschaftszentrum Weihenstephan, Am Hochanger 8, 85350 Freising, Germany.


Figure 1
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  		Fig. 1. Organogenesis in wild type, pid, mab4, pin1 pid, pid mab4 and pid enp. (A-F) Seedlings (5-day old) of wild type (A), pid-3 (B), mab4-1 (C), pin1-201 pid-3 (D), pid-3 mab4-1 (E) and pid-15 enp (F). Arrows and arrowheads in D-F indicate the development of leaves and the loss of cotyledons, respectively. (G-N) Inflorescence of wild type (G,M), pid-3 (H), mab4-1 (I,N), pin1-201 pid-3 (J), pid-3 mab4-1 (K) and pid-15 enp (L). (O-Q) Scanning electron micrographs of gynoeciums of wild type (O) and mab4-1 (P,Q). Asterisks in M and N indicate sepal primordia. Orange arrows indicate fusion between floral organs. Scale bars: 50 µm in M,N; 200 µm in O-Q.

 

Figure 2
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  		Fig. 2. Identification of the MAB4/ENP gene. (A) Map-based cloning. The MAB4/ENP locus was mapped to lie between the F3L17 and F10M6 markers on chromosome 4. The number of recombinants are indicated under these markers (recombinant chromosomes/analyzed chromosomes). The mab4-1 mutation is a deletion of 9 bp in At4g31820, which loses three amino acids, G,L and Y. The enp mutation is a point mutation, C to T, which creates a stop codon. In mab4-2 mutants, T-DNA is inserted at the 5' untranslated region of the gene. ARE, auxin-responsive element; WUS, WUS-binding site. (B) The deduced amino acid sequence of MAB4/ENP, shown in comparison with the sequence of NPH3 and RPT2. Asterisks denote residues deleted in the mab4-1 mutant. Black circle indicates the mutated residue in the enp mutant. Gray and black shadings indicate residues identical in the two and three sequences, respectively. (C) Phylogenetic tree of Arabidopsis MAB4/ENP homologs and the two related rice homologs and CPT1. The tree was obtained by the neighbor-joining method using 1000 bootstrap replicates, generated with CLUSTALW. Black circles indicate rice NPH3-like protein.

 

Figure 3
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  		Fig. 3. MAB4/ENP mRNA expression. (A-F) MAB4/ENP expression in wild-type embryos at the 8-cell (A), 32-cell (B), globular (C), mid-heart (D), late-heart (E) and walking-stick stage (F). Arrows in B and C denote the hypophysis without any MAB4/ENP signal. (G-L) Localization of MAB4/ENP mRNA in postembryonic stage, at the vegetative stage in transverse section (G); in the inflorescence meristem in serial longitudinal sections (H) and in transverse section (I); during flower development in transverse (J) and longitudinal (K) sections; and during ovule development in transverse section (L). Arrowheads in H indicate the position of incipient floral primordia. IM, inflorescence meristem; FM, floral meristem; Se, sepals; ii, inner integuments; oi, outer integuments. Scale Bars: 20 µm in A-E; 30 µm in L; 50 µm in F,H-J; 100 µm in G,K.

 

Figure 4
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  		Fig. 4. MAB4/ENP expression in pin1 pid embryos. (A-D) Localization of MAB4/ENP mRNA in pin1-3 pid-2 embryos at the heart (A) and torpedo stage (B) in serial longitudinal sections. MAB4/ENP expression in the double mutant with rudimentary cotyledon development at the torpedo (C) and bending-cotyledon stage (D) in longitudinal sections. Scale bars: 50 µm in A-D.

 

Figure 5
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  		Fig. 5. PIN1:GFP and DR5rev::EGFP in wild-type and mab4/enp embryos. (A-D) PIN1:GFP localization in wild type (A,C) and mab4-1 embryos (B,D). (E-H) DR5rev::EGFP expression in wild type (E,G) and mab4-1 embryos (F,H). Heart-stage (A,B,E,F) and torpedo-stage embryos (C,D,G,H). White arrowheads in A and C point to PIN1:GFP localization in the plasma membrane. Orange arrowheads in B and D demonstrate the reduction of PIN1:GFP localization in the plasma membrane. Scale bars: 20 µm in A-H.

 

Figure 6
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  		Fig. 6. Spatial relationship between PIN1, PID and MAB4/ENP in cultured Arabidopsis cells. (A,C) PIN1-GFP was expressed under the control of its own promoter in Arabidopsis protoplasts (A). GFP-PID was expressed under the control of the CaMV 35S promoter in Arabidopsis protoplasts (C). GFP fluorescence images (left) and Nomarski image (right) were taken with an epifluorescence microscope. (B,D) Localization of PIN1-GFP (B), GFP-PID (D) (green; left) and mRFP-MAB4/ENP (red; middle). mRFP-MAB4/ENP and PIN1-GFP or GFP-PID were co-introduced into Arabidopsis protoplasts. Merged image (right) of mRFP-MAB4/ENP, and PIN1-GFP (B) and GFP-PID (D). An arrowhead in B indicates the close proximity of the PIN1-GFP and mRFP-MAB4/ENP florescence, which is shown enlarged in the inset. Arrows in D indicate the colocalization of GFP-PID and mRFP-MAB4/ENP florescence, which is shown enlarged in the inset. Scale bars: 10 µm in A-D.

 

Figure 7
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  		Fig. 7. Subcellular localization of MAB4/ENP. (A-D) Localization of MAB4/ENP-GFP (green; left) and mRFP or Venus-tagged subcellular marker genes (red; middle). MAB4/ENP-GFP and mRFP- or Venus-subcellular markers were co-introduced under the control of the CaMV 35S promoter into Arabidopsis protoplasts. Merged image (right) of MAB4-GFP and the late endosome marker ARA6-mRFP (A), the early endosome marker mRFP-ARA7 (B), the cis-Golgi marker Venus-SYP31 (C) and the trans-Golgi-network (TGN) marker Venus-SYP41 (D). Arrows in A indicate colocalization of fluorescence of MAb4/ENP-GFP and ARA6-mRFP. Scale bars: 10 µm in A-D.

 

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© The Company of Biologists Ltd 2007