spacer gif spacer gif spacer gif spacer gif spacer gif
QUICK SEARCH:   [advanced]


spacer gif
     Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents    

First published online June 8, 2006
doi: 10.1242/10.1242/jcs.02967

Journal of Cell Science 119, 2486-2496 (2006)
Published by The Company of Biologists 2006
This Article
Right arrow Summary Freely available
Right arrow Full Text
Right arrow Full Text (PDF)
Right arrow Supplementary Material
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Kerzendorfer, C.
Right arrow Articles by Schlögelhofer, P.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Kerzendorfer, C.
Right arrow Articles by Schlögelhofer, P.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati   Add to Twitter  
What's this?

The Arabidopsis thaliana MND1 homologue plays a key role in meiotic homologous pairing, synapsis and recombination

C. Kerzendorfer1,2,*, J. Vignard3,*, A. Pedrosa-Harand1, T. Siwiec1, S. Akimcheva2, S. Jolivet3, R. Sablowski4, S. Armstrong5, D. Schweizer1,2, R. Mercier3,{ddagger} and P. Schlögelhofer1,{ddagger}

1 Department of Chromosome Biology, Max F. Perutz Laboratories, University of Vienna, A-1030 Vienna, Austria
2 Gregor Mendel Institute of Molecular Plant Biology, Austrian Academy of Sciences, A-1030 Vienna, Austria
3 Station de Génétique et d'Amélioration des Plantes, Institut National de la Recherche Agronomique, 78026 Versailles CEDEX, France
4 Department of Cell and Developmental Biology, John Innes Centre, Norwich, NR4 7UH, UK
5 School of Bioscience, University of Birmingham, Birmingham, B15 2TT, UK


Figure 1
View larger version (41K):

[in a new window]
  		Fig. 1. Molecular analysis of the AtMND1 T-DNA insertion mutant. (A) Schematic representation of the Atmnd1 mutant allele with the T-DNA insertion. The T-DNA insertion site as well as the orientation of left borders are indicated. The boxes represent exons, with UTRs in white and cDNA sequence in grey. The positions of the primers (arrows) and HindIII restriction sites (H) are marked. Expected DNA fragments after digestion are indicated by double arrows. (B) DNA sequence of the junction between T-DNA and genomic DNA. Capital letters indicate genomic sequences adjacent to the borders of the T-DNA. Lowercase letters indicate the deleted 85-bp genomic region. (C) DNA gel blot hybridisation of HindIII-digested genomic DNA with a genomic AtMND1 probe results in the production of ~0.8 kb and ~3.2 kb bands for wild-type (+/+), ~0.8 kb, ~2.2/2.3 kb and ~3.2 kb bands for heterozygous (+/-) and ~2.2/2.3 kb and ~3.2 kb for homozygous (-/-) mutant plants. (D) PCR assay to distinguish wild-type, heterozygous and homozygous mutant plants. Primers M2, M3 and LBa1 were used in the same PCR reaction. (E) RT-PCR experiment to analyse the expression of AtMND1. The AtMND1 transcript (M) was detected in various wild-type tissues but not in the Atmnd1 mutant line. Amplification of the ACTIN (A) transcripts was used as a control. C represents a control experiment with the corresponding RNA as template in the PCR amplification reaction, using primers directed against ACTIN. The expected band size for AtMND1 cDNA is ~0.7 kb and for ACTIN cDNA is ~0.4 kb.

 

Figure 2
View larger version (80K):

[in a new window]
  		Fig. 2. Atmnd1 mutant plants develop short siliques and no regular pollen grains. (A) Atmnd1 plants look like wild-type plants, except that they have shorter and empty siliques. The left panel shows a stem with full-grown siliques of a wild-type plant (wt). The middle panel shows the stem of an Atmnd1 plant of the same age, which failed to develop siliques (Atmnd1). The right panel shows an Atmnd1 homozygous mutant plant, transformed with a wild-type copy of the genomic AtMND1 region and showing restored fertility. (B) Anthers of wild-type (left panel) and Atmnd1 (right panel) plants stained according to Alexander (Alexander, 1969Go). The purple-stained cytoplasm indicates viable pollen grains. Atmnd1 plants did not develop regular pollen grains.

 

Figure 3
View larger version (54K):

[in a new window]
  		Fig. 3. Male meiosis is severely disrupted in Atmnd1 mutants. Meiosis in wild-type A. thaliana: (A) leptotene, (B) zygotene, (C) pachytene, (D) diplotene, (E) early diakinesis, (F) diakinesis, (G) metaphase I, (H) anaphase I, (I) telophase II. Disrupted male meiosis in the Atmnd1 mutant: (J) leptotene, indistinguishable from the wild type. (K) Zygotene-like stage. (L) Pachytene-like stage. The mutant failed to go through typical zygotene and pachytene stages, displaying no pairing and synapsis of chromosomes. (M) Early diakinesis-like stage, (N) late diakinesis-like stage and (O) metaphase-I-like stage show entangled chromosomes interconnected by chromatin bridges. (P) Progression through anaphase I with stretched chromatin and limited chromosome fragmentation. (Q) Metaphase II-like stage with chromosome fragments. (R) Late-anaphase-II-like stage with severe chromosome fragmentation. Chromosomes are stained with DAPI. Bar, 10 µm.

 

Figure 4
View larger version (99K):

[in a new window]
  		Fig. 4. Female meiosis is disrupted in Atmnd1 mutants. Female meiosis in wild-type A. thaliana: (A) pachytene, (B) metaphase I, (C) anaphase I. Disrupted female meiosis in the Atmnd1 mutant: (D) failed zygotene/pachytene, (E) metaphase-I-like stage with entangled chromosomes, (F) anaphase-I-like stage. Images show DAPI staining of the chromosomes. Bar, 10 µm.

 

Figure 5
View larger version (55K):

[in a new window]
  		Fig. 5. FISH analysis of Atmnd1 mutants reveals defects in pairing and chromosome disjunction. Preparations of wild-type (A-C) and Atmnd1 (D-F) meiocytes were hybridised with FISH probes directed against an interstitial region of chromosome 1 (BAC F1N21, green) and a sub-telomeric region of chromosome 2 (BAC F11L15, red). (A,D) Zygotene stage, showing consistent association of sub-telomeric regions in wild-type and occasional association in Atmnd1 cells. (B) Wild-type pachytene/diplotene transition with paired FISH probes. (E) Atmnd1 pachytene-like stage with unpaired FISH signals. (C,F) Anaphase I with a regular distribution of chromosomes and FISH signals in wild-type meiocytes, as opposed to the irregular chromosome disjunction and DNA fragmentation in Atmnd1 cells. Chromosomes are stained with DAPI. Bar, 10 µm.

 

Figure 6
View larger version (68K):

[in a new window]
  		Fig. 6. Chromosome entanglement and fragmentation observed in Atmnd1 mutants depends on SPO11-1. Comparison of meiotic progression in the spo11-1 mutant (A-F) and in the Atmnd1 spo11-1 double mutant (G-L). (A,G) Zygotene-like stage. No typical pachytene cells were detected, and only unsynapsed chromosomes were observed in the pachytene-like stage (B,H). In diakinesis (C,I), ten condensed univalents are visible. (D,J) Anaphase I. (E,K) Metaphase II. (F,L) Polyads. Chromosomes are stained with DAPI. Bar, 10 µm.

 

Figure 7
View larger version (70K):

[in a new window]
  		Fig. 7. Immunolocalisation of ASY1 and SCC3 in Atmnd1 mutant plants. In Atmnd1 mutants (lower panels), loading of the SCC3 cohesin protein (green) and of the axial-element associated ASY1 protein (red) is similar to that in wild-type plants (upper panels). However, no synapsis was observed in Atmnd1 in contrast to wild-type cells. Bar, 10 µm.

 

Figure 8
View larger version (61K):

[in a new window]
  		Fig. 8. Rad51 foci are formed normally in Atmnd1 mutants. Comparison of RAD51 focus formation in wild-type (A,B) and Atmnd1 mutant (C,D) plants. RAD51 foci (red) were observed in leptotene (A,C) of wild-type and of Atmnd1 mutants cells and in zygotene (B) and failed zygotene stages (D) of wild-type and Atmnd1 mutant cells, respectively. The abundance of RAD51 foci was similar in wild-type and Atmnd1 mutant meiocytes. Immunolocalisation of ASY1 is represented in green. Bar, 10 µm.

 

Figure 9
View larger version (52K):

[in a new window]
  		Fig. 9. AtMND1 and AHP2 interact in a yeast two-hybrid (Y2H) assay. Yeast strain AH109 was transformed with the indicated plasmids and grown on synthetic drop-out (SD) media lacking the amino acids leucine and tryptophan (SD -Leu/-Trp) or leucine, tryptophan and histidine (SD -Leu/-Trp/-His). Only the combination of plasmids containing AtMND1 fused to the GAL4-DNA binding domain and AHP2 fused to the GAL4 activator domain (pGBKT7/AtMND1, pGAD10/AHP2) or vice versa (pGBKT7/AHP2, pGAD424/AtMND1) enabled strain AH109 to grow on SD -Leu/-Trp/-His, demonstrating an interaction between AtMND1 and AHP2.

 

Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati   Add to Twitter Twitter    What's this?



© The Company of Biologists Ltd 2006