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First published online 23 April 2008
doi: 10.1242/dev.020743

Development 135, 2013-2022 (2008)
Published by The Company of Biologists 2008
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The Arabidopsis COP9 signalosome is essential for G2 phase progression and genomic stability

Esther M. N. Dohmann1, Mitchell P. Levesque2, Lieven De Veylder3,4, Ilka Reichardt1, Gerd Jürgens1, Markus Schmid5 and Claus Schwechheimer1,*

1 Tübingen University, Center for Plant Molecular Biology, Department of Developmental Genetics, Auf der Morgenstelle 3-5, 72076 Tübingen, Germany.
2 Max Planck Institute for Developmental Biology, Department of Genetics, Spemannstrasse 32, 72076 Tübingen, Germany.
3 Department of Plant Systems Biology, Flanders Institute for Biotechnology, Ghent University, Technologiepark 927, 9052 Gent, Belgium.
4 Department of Molecular Genetics, Ghent University, Technologiepark 927, 9052 Gent, Belgium.
5 Max-Planck Institute for Developmental Biology, Department of Molecular Biology, Spemannstrasse 32, 72076 Tübingen, Germany.


Figure 1
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  		Fig. 1. Differential expression of cell cycle regulatory genes in Arabidopsis csn mutants. (A) Phenotypes of 7-day-old wild-type and csn mutant alleles used in this study. Scale bars: 2 mm for wild type, csn5a and csn5b; 0.5 mm for csn3, csn4 and csn5ab. (B) Differential expression of cell cycle regulatory genes in 7-day-old dark- and light-grown wild-type and csn3, csn4 and csn5 seedlings (Benjamini Hochberg false discovery rate <0.05, 2-fold differential expression in all three csn mutants and in both growth conditions; see also Table S1 in the supplementary material).

 

Figure 2
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  		Fig. 2. Cell cycle marker activity in csn mutants. (A) CYCB1;1:GUS expression in primary roots (upper row) and root tips (lower row) of 7-day-old light-grown wild-type and csn3, csn4 and csn5ab Arabidopsis seedlings. (B,C) CYCB1;1:GUS expression in primary roots (upper row) and root tips (lower row) following 24 hours and 48 hours of treatment, respectively, with the synthetic auxin 2,4D. Arrowheads indicate lateral root primordia. (D) KN and CDKB1;1 accumulation in protein extracts (20 µg) of 2-day-old wild-type seedlings and 7-day-old csn3, csn4 and csn5ab mutant seedlings. A cross-reacting band was used as a loading control. Relevant bands are indicated by arrowheads. Wild-type and mutant plants of different ages were used for this analysis because 2-day-old wild-type and 7-day-old mutant seedlings are comparable in size and contain a comparable number of dividing cells. Scale bars: 1 mm in wild type; 0.5 mm in csn3, csn4 and csn5ab.

 

Figure 3
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  		Fig. 3. csn mutants accumulate cells in the G2 phase of the cell cycle. (A-H) Flow cytometric analyses of root tips from wild-type and csn mutant Arabidopsis seedlings as specified. 2C denotes the normal diploid DNA content of cells in G0/G1 phase, 4C denotes cells that have passed S phase, 8C and 16C denote endoreduplicated cells. The experiments were repeated three times (see Table S3 in the supplementary material), and one representative experiment is shown. The csn mutants used for this analysis were 7 days old.

 

Figure 4
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  		Fig. 4. Activation of the DNA damage response pathway in Arabidopsis csn mutants. (A) Differential expression of genes with a predicted role in DNA repair in response to DNA-damaging agents (1.5 µg/ml bleomycin + 22 µg/ml mitomycin) in the wild type, and their expression in dark- and light-grown csn mutants (Benjamini Hochberg false discovery rate <0.05, 2-fold differential expression; see also Table S2 in the supplementary material). The expression of BRCA1, PARP1 and RAD51 was independently verified by RT-PCR (see Fig. S1 in the supplementary material). (B) GUS-staining of wild-type and csn mutant seedlings expressing the DNA damage reporter PARP2:GUS. PARP2:GUS expression is induced in csn mutants and in the wild type following a 12-hour treatment with the DNA-damaging agent bleomycin (5 µg/ml). Scale bars: 2 mm.

 

Figure 5
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  		Fig. 5. Evidence for DNA damage in Arabidopsis csn mutants. (A) Accumulation of the phosphorylated H2AX variant {gamma}H2AX in a histone preparation (13 µg) from 2-day-old (same size) and 7-day-old (same age) wild-type seedlings and 7-day-old csn3, csn4 and csn5ab mutant seedlings as detected with a {gamma}H2AX-specific antibody (Friesner et al., 2005Go). A Coomassie-stained band serves as a loading control. H2AX transcription was not altered between the wild type and the csn mutants (data not shown). (B,C) Immunostaining of wild-type and csn4 mutant root tip cells with the anti-{gamma}H2AX antibody (green) identifies subnuclear {gamma}H2AX-specific foci that may mark sites of damaged DNA in csn4 mutants (C, left panel). The samples were counterstained using the DNA stain DAPI. Scale bars: 5 µm. (D) Activation of the stress-induced WEE1:GUS reporter construct in wild-type seedlings, bleomycin-treated (12 hours, 5 µg/ml) wild-type seedlings and in untreated csn mutants. Scale bars: 2 mm in wild type; 0.5 mm in csn3, csn4 and csn5ab. (E-M) Confocal images of primary roots of 7-day-old dark-grown wild type and csn mutants following the TUNEL assay (left column). Roots were counterstained with DAPI (right column). For the positive control, fixed root material was subjected to treatment with DNase I. For the negative control, terminal transferase was omitted from the TUNEL reaction. The experiment was repeated three times and a representative image from one experiment is shown. Scale bars: 1 mm in wild type, csn5a and csn5b; 0.5 mm in csn3, csn4 and csn5ab. (N) Confocal images of a 7-day-old bleomycin-treated (12 hours, 5 µg/ml) dark-grown wild-type seedling following the TUNEL assay (left panel). Roots were counterstained with DAPI (right panel). Scale bar: 1 mm.

 

Figure 6
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  		Fig. 6. DNA double-strand breaks might be responsible for the csn mutant growth arrest. (A) The IU.GUS transgene before and after DNA repair following a DNA double-strand break (DSB) in the interrupted GUS gene fragment. IU.GUS carries a GUS reporter gene, which is non-functional owing to an insertion in the GUS gene. In addition, this transgene construct carries a non-functional but uninterrupted 1087 bp GUS fragment, which corresponds to the GUS sequences that are upstream and downstream of the insertion in the first GUS gene. The rationale of the IU.GUS transgene is that a DSB within the interrupted GUS gene can be repaired by homologous recombination using the uninterrupted GUS fragment as template, thereby rendering the GUS gene active. LB, left border; RB, right border; P, promoter; T, terminator; Hygromycin, hygromycin resistance gene; GUS, intact GUS gene; GU*-U**S, interrupted GUS gene; U, uninterrupted GUS gene fragment. (B) Five-day-old light-grown wild-type and csn mutant Arabidopsis seedlings that contain IU.GUS following GUS-staining. GUS-positive cells (arrowheads) are detected in the csn mutants, but not in the wild type. Insets show GUS-positive cells at higher magnification. Scale bars: 1 mm in wild type; 0.5 mm in csn3, csn4 and csn5ab. (C) Quantitative analysis of the DNA repair events in wild-type and csn mutant seedlings. Both CSN5 genes reside on chromosome 1 and therefore the unlinked IU.GUS 7 (chromosome 5) was used for this cross. CSN3 and CSN4 are on chromosome 5, therefore IU.GUS 8 (chromosome 1) was used for this cross.

 

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