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First published online 27 February 2008
doi: 10.1242/dev.017947

Development 135, 1335-1345 (2008)
Published by The Company of Biologists 2008
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Arabidopsis CAPRICE-LIKE MYB 3 (CPL3) controls endoreduplication and flowering development in addition to trichome and root hair formation

Rumi Tominaga, Mineko Iwata, Ryosuke Sano, Kayoko Inoue, Kiyotaka Okada* and Takuji Wada{dagger}

Plant Science Center, RIKEN, 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama, Kanagawa 230-0045, Japan.


Figure 1
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  		Fig. 1. Gene structure and amino acid sequences of Arabidopsis CPC homologs. (A) Sequence alignment of CPC-homologous MYB proteins (CPL3, CPC, TRY, ETC1, ETC2, Os01g43180 and Os01g43230). Red outlined letters indicate identical residues. (B) Phylogenetic tree based on the amino acid sequences. Numbers above branches are genetic distances based on 10,000 bootstrap replicates. The tree was obtained by the neighbor-joining method using Genetyx ver. 11.2.7 software (Genetyx, Tokyo, Japan). (C) Structure of CPC-homologous genes and positions of mutations. The locations of start and stop codons are indicated. Three exon (boxes) and two intron (lines) positions were determined by comparing the genomic sequences with the cDNA sequences. Positions of T-DNA insertions and the identity of mutations are indicated (cpc-2, try-029760, etc1-1, etc2-2 and cpl3-1). (D) Semi-quantitative RT-PCR analysis of CPL3. EF (At1g07930) was used as a control.

 

Figure 2
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  		Fig. 2. Phenotypes associated with CPC-homolog gain- and loss-of-function mutants. (A-L) Root hair formation of a 5-day-old Arabidopsis seedling showing no root hair phenotype (G,H), reduced number of root hairs (B,C) and increased number of root hairs (I-L). (M-X) Trichome formation on the 2-week-old third leaves showing increased number of trichomes (N-T). No trichome formation was observed in gain-of-function plants (U-X). Scale bars: 100 µm in A for A-L; 1 mm in M-X.

 

Figure 3
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  		Fig. 3. cpl3 cpc try etc1 quadruple mutant phenotype. (A) The adaxial surface of rosette leaf of a cpl3 cpc try etc1 quadruple mutant Arabidopsis was entirely covered by trichomes. (B) Pistil and stamen of a cpl3 cpc try etc1 quadruple mutant were surrounded by trichomes. (C) Adaxial epidermis of a rosette leaf of the cpl3 cpc try etc1 quadruple mutant. (D) Trichome phenotypes of a cpl3 cpc try etc1 quadruple mutant. Trichome phenotype of Col-0 (E) and cpl3 mutant (F). Scale bars: 200 µm in A; 100 µm in B-F.

 

Figure 4
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  		Fig. 4. CPL3 gene expression. (A) Real-time PCR analysis of CPL3 gene expression in Arabidopsis organs. Total RNA was isolated from the indicated tissues. (B-E) In situ hybridization patterns of CPC-homologous genes at the shoot apex. Arrowheads in B and C indicate CPC and ETC1 signals evident in young trichomes. No signal for either ETC2 or CPL3 was detected in D and E. (F-M) Activity of CPL3p::GUS reporter in 2-week-old leaves (F), in 5-day-old cotyledons (G), hypocotyls (H) and roots (I), in 4-week-old inflorescence (J,K) and silique (L,M). (N) Localization of CPL3-GFP fusion protein. Fluorescence from the GFP fusion protein (green) and propidium iodide (red) was observed with confocal laser scanning microscopy. CPL3p::CPL3:GFP signal localization in 2-week-old leaves. Scale bars: 50 µm in B for B-E and in G,H,N; 100 µm in I; 200 µm in K,M; 1 mm in F,J,L.

 

Figure 5
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  		Fig. 5. Expression of flowering-related genes in the cpl3 mutant and transgenic Arabidopsis plants expressing CPL3. Real-time PCR analyses of FT (A), SOC1 (B), CO (C) and CPL3 (D) genes at three developmental stages. Expression levels were normalized to ACT2 expression. Relative expression levels: expression levels of each gene in cpl3, 35S::CPL3 and CPL3p::CPL3 relative to wild type at 7 days. RNA was isolated from 7-, 14- and 21-day-old rosette leaves grown under long-day conditions. The experiment was repeated four times. Error bars indicate s.d.

 

Figure 6
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  		Fig. 6. Phenotypes of cpl3 mutant and transgenic plants expressing CPL3. (A) Soil-grown rosettes from 4-week-old Col-0, cpl3, etc2 cpl3, 35S::CPL3, CPL3p::CPL3#1 and CPL3p::CPL3#2 Arabidopsis plants. (B) Fresh weight of rosette leaves per plant was calculated from the means (±s.d.) of a minimum of five rosettes from each line. (C-E) Microscopic analysis of leaf epidermis of Col-0, cpl3 and CPL3p::CPL3. The study was carried out in the middle region of the leaf blade of 2-week-old plants. (F-H) Hypocotyls from 2-week-old Col-0, cpl3 and 35S::CPL3. (I-K) Hypocotyl epidermis of Col-0, cpl3 and 35S::CPL3. Scale bars: 5 mm in A; 100 µm in C,I; 200 µm in F.

 

Figure 7
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  		Fig. 7. Loss of CPL3 function increases polyploidy levels. (A) Relative ratios of each cell ploidy of 8- and 16-day-old first leaves of Col-0, cpl3, 35S::CPL3 and CPL3p::CPL3 Arabidopsis plants. (B) Relative ratios of each cell ploidy of 3-week-old third leaves of Col-0, cpl3, 35S::CPL3 and CPL3p::CPL3. Approximately 5000 nuclei were counted in Col-0, cpl3, 35S::CPL3 and CPL3p::CPL3 tissues.

 

Figure 8
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  		Fig. 8. CYCA and SIM expression in cpl3 mutant and transgenic Arabidopsis plants expressing CPL3. Real-time PCR analysis of CYCA2;1 (A), CYCA2;2 (B), CYCA2;3 (C), CYCA2;4 (D), CYCA1;1 (E) and SIM (F) in wild type, cpl3, 35S::CPL3 and CPL3p::CPL3 at four developmental stages. Expression levels of each gene were normalized to ACT2 expression. Relative expression levels: expression levels of each gene relative to wild type at 12 days. The experiment was repeated four times. Error bars indicate s.d. Student's t-test, *P<0.020 versus wild type.

 

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