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First published online 22 February 2006
doi: 10.1242/dev.02301


Development 133, 1241-1252 (2006)
Published by The Company of Biologists 2006


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EARLY IN SHORT DAYS 1 (ESD1) encodes ACTIN-RELATED PROTEIN 6 (AtARP6), a putative component of chromatin remodelling complexes that positively regulates FLC accumulation in Arabidopsis

Mar Martin-Trillo1,*, Ana Lázaro2,*, R. Scott Poethig3, Concepción Gómez-Mena1,{dagger}, Manuel A. Piñeiro2, Jose M. Martinez-Zapater1 and Jose A. Jarillo2,{ddagger}

1 Departamento de Genética Molecular de Plantas, Centro Nacional de Biotecnología, C/ Darwin 3, Madrid 28049, Spain.
2 Departamento de Biotecnología, Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria, Ctra. de A Coruña, km 7, Madrid 28040, Spain.
3 Plant Science Institute, Department of Biology, University of Pennsylvania, PA 19104, USA.


Figure 1
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Fig. 1. The flowering phenotype of esd1 mutants. (A) Wild-type Ler and esd1-2 2-week-old plants grown under LD. (B) Ler and esd1-2 4-week-old plants grown under SD. (C) Histogram comparing the number of juvenile, adult and cauline leaves in Ler and esd1 mutants. Plants were grown under both LD and SD. (D) Ler and esd1-2 3.5-week-old plants grown under LD.

 

Figure 2
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Fig. 2. The inflorescence phenotype of esd1 mutants. (A) Apex of 3-week-old Ler plants. (B) Apex of 2.5-week-old esd1 mutant plants, showing open flowers with extra sepals and petals. (C) Detached flowers showing the increased number of sepals and petals in esd1 mutant flowers. (D,E) Scanning electron micrographs of Ler (D) and esd1 (E) flowers of plants grown under SD. s, sepal; p, petal. (F) A comparison of silique shape and length in Ler, esd1-1 and esd1-2 plants. (G) Number of sepals, petals, stamens and carpels in Ler and esd1 mutants. Plants were grown under both LD and SD. Bars represent the standard error.

 

Figure 3
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Fig. 3. Suppression of FLC-dependent late flowering by esd1 mutations. (A) Photograph illustrating the flowering phenotype of double mutant esd1 fve and esd1 fca plants grown under LD. (B) Flowering phenotype of lines where an active allele of FRI is combined with esd1 grown under LD. (C) Analysis of the expression of FLC in the late-flowering genotypes FRI, fve and fca combined with esd1. RNA blot hybridizations were performed using total mRNA from 9-day-old Col, esd1-3, FRI, esd1-3 FRI, Ler, esd1-2, fve-1, esd1-2 fve-1, fca-1 and esd1-2 fca-1 plants grown under LD. (D) Analysis of the expression of FT and SOC1 genes in esd1 mutants. RT-PCR assays comparing FT and SOC1 expression in 9 day-old Col and esd1-3 plants. The samples were taken at the time of the day with the maximum expression; for FT expression analysis, before dusk, and for SOC1 analysis, 8 hours after dawn. (E) Flowering phenotype of esd1 flc double mutant plants grown under LD. (F) Analysis of the expression of MAF genes in esd1 mutant plants. Total RNA was extracted from pools of 50 9-day-old seedlings grown under LD conditions. Expression was monitored by RT-PCR over 32 cycles for MAF1, 28 cycles for MAF2, and 35 cycles for MAF3, MAF4 and MAF5. For the UBQ10 control, we amplified during 22 cycles. RT-PCR products were blotted and hybridized with specific probes for each MAF gene.

 

Figure 4
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Fig. 4. Identification of ESD1. (A) Map-based cloning of ESD1. The genetic interval, molecular markers and BAC clones in the ESD1 region are shown. The number of recombinant events between molecular markers is given in parentheses. The centromere is located between the T15D2 and T25F15 markers (http://www.arabidopsis.org/info/agicomplete.jsp). GAP indicates the existence of genomic regions of unknown size, where it was not possible to get overlapping BAC clones. Gray bars correspond to the deleted region in each esd1 allele. The ESD1 locus was delimited to a deleted overlapping genomic region between the 5F21A14 and 1T14A11 molecular markers. Mb, megabases. (B) Complementation of the esd1 mutant. Col, esd1-3 and TN 18.1, a transgenic esd1-3 plant containing the genomic region harbouring open reading frames At3g33520 y At3g33530, shown at the time of bolting initiation. (C) Flowering phenotype of esd1-10, a T-DNA insertion allele. Left, Col plant; right, a homozygous plant for the T-DNA insertion within the At3g33520 gene (Wisc Ds-Lox 289 line), showing an early flowering phenotype. RT-PCR analyses of the expression of At3g33520 in esd1-10 show no expression of this gene in the T-DNA mutant, indicating that it is a loss-of-function allele.

 

Figure 5
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Fig. 5. ESD1 encodes ARP6. (A) Scheme of the ARP6 gene showing the position of the T-DNA insertion in the esd1-10 mutant. Exons are shown as black boxes. The position of the start and stop codon are indicated. (B) Sequence comparison of AtARP6 with yeast (Sp), C. elegans (Ce), Drosophila (Dm) and human (Hs) ARP6s, and Arabidopsis Actin2. Amino acid residues in black are functionally similar in all sequences and those in gray are similar in at least four of them. Boxed regions indicate the two peptide insertions in ARP6s, which do not disrupt the conserved actin fold structure. GenBank Accession numbers are NP_566861 for AtARP6, AAF4849 for Dm ARP6, AAK14934 for Hs ARP6, CAA19116 for Sp ARP6, NP_495681 for Ce ARP6, and BAB01806 for AtACT2. (C) APR6 expression in different organs of Col plants. RT-PCR assays were performed with RNA prepared from different tissues. R, roots; S, main stems; F, flowers; FB, flower buds; CL, cauline leaves. RT-PCR products were blotted and hybridized with a specific probe for ARP6. UBQ10 was used as a loading control.

 

Figure 6
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Fig. 6. Histochemical ß-glucuronidase assays in fca-1 FLC:GUS and esd1-2 fca-1 FLC:GUS plants. (A-D) Gus staining is shown in the shoot apical meristem region (A,B) and the root tip (C,D) of representative fca-1 FLC:GUS (A,C) and esd1-2 fca-1 FLC:GUS (B,D) seedlings grown for 10 days under LD conditions.

 

Figure 7
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Fig. 7. Effect of esd1 mutation on histone H3 acetylation and methylation in the FLC genomic region by ChIP analysis. (A) FLC genomic region analyzed by ChIP. The white box corresponds to the promoter FLC region, gray boxes to exons and the black box to the first intron. The six FLC fragments analyzed by semi-quantitative PCR are depicted and numbered. (B) PCR products after 25 cycles of esd1-2, fve-1 and esd1-2 fve-1 mutants, using as a template DNA purified from chromatin inmunoprecipitated with antibodies against acetylated H3 (AcH3). UBQ10 was amplified during 22 cycles and used as control for DNA quantification. Fold enrichment in H3 acetylation of fve-1 over esd1-2 and esd1-2 fve-1 double mutant is shown. (C) PCR products after 25 cycles of Col, esd1-3, FRI, esd1-3FRI, Ler, esd1-2, fca-1, esd1-2 fca-1, fve-1, and esd1-2 fve-1 plants, using as a template DNA purified from chromatin inmunoprecipitated with antibodies against acetylated H3 (AcH3). UBQ10 was amplified during 22 cycles and used as control for DNA quantification. Fold enrichment in H3 acetylation of mutants over wild-type ecotypes is shown. (D) PCR products as in C, but using a as template DNA purified from chromatin inmunoprecipitated with antibodies against trimethylated H3-K4 (MeH3-K4). Fold enrichment in H3-K4 methylation of mutants over wild-type ecotypes is shown.

 





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