Functional analysis of the Arabidopsis thaliana meiotic proteins AtPCH2 and AtCHR24

In the past decade Arabidopsis thaliana has become an important system for studying meiosis in flowering plants. The identification of meiotic mutants has provided an important approach to studying plant meiosis. The availability of the Arabidopsis genome sequence together with developments in proteomics and bioinformatics provides an additional route for the identification of meiotic proteins and analysis of their functional interrelationships. This study has used a proteomics approach to identify a member of the SWI2/SNF2 chromatin remodelling gene family (Atchr24). Although a variety defects was observed in Atchr24 male meiocytes cytogenetic, at least two T-DNA insertion lines on this gene appear normal. Secondly, this research has also used a bioinformatics approach to identify a potential orthologue of Pch2/TRIP13 in Arabidopsis. PCH2 (Pachytene checkpoint 2) is a member of the AAA+ ATPase family of proteins. This study reveals that AtPCH2 plays an essential role in the controlled formation of meiotic crossovers (COs). Cytogenetic analysis of two Atpch2 T-DNA insertion lines revealed a high frequency of univalents at MI. The number of chiasmata (COs) is reduced to ~ 70% of wild-type (WT). Genetic analysis revealed that Atpch2 has significantly weaker CO interference than WT leading to a redistribution of COs along the chromosomes. The recombination defect is accompanied by incomplete chromosome synapsis. Immunolocalisation of the chromosome axis protein AtASY3 and cohesin, AtSYN1 appears normal. However in contrast to WT, AtASY1 co-localises with the synaptonemal protein AtZYP1 in ii Atpch2 rather than becoming depleted in regions of synapsis and the meiotic progression of Atpch2 is delayed during pachytene by ~5 hours. These observations suggest a defect in remodeling of the chromosome axes and highlight how this process is essential for normal CO control

Dual localization of ZYP1 and recombination pathway proteins in wild type Arabidopsis and <i>Atpch2-1</i> meiotic nuclei at mid/late prophase.

<p><b>(A-D)</b> Dual localization of ZYP1 (green) and HEI10 (red) on wild type <b>(A,C)</b> and <i>Atpch2-1</i><b>(B,D)</b> PMCs. Panels <b>C</b> and <b>D</b> show magnified sections of SC from <b>A</b> and <b>B</b> respectively. <b>(E-H)</b> Dual localization of ZYP1 (green) and MLH1 (red) on wild-type <b>(E,G)</b> and <i>Atpch2-1</i><b>(F,H)</b> PMCs. Panels <b>G</b> and <b>H</b> show magnified sections of SC from <b>E</b> and <b>F</b> respectively. DNA is stained with DAPI (blue). Bar = 10 μm. <b>(I)</b> Table showing the mean number of HEI10 and MLH1 foci in wild type and <i>Atpch2-1</i>.</p

Immunolocalization of ASY1 and ZYP1 in wild type and <i>Atpch2-1</i> during prophase I.

<p><b>(A-D)</b> Immunolocalization of ASY1 (green) on chromosome spread preparations from wild type <b>(A,C)</b> and <i>Atpch2-1</i><b>(B,D)</b> nuclei at leptotene. Panels <b>C</b> and <b>D</b> show magnified sections of axes from <b>A</b> and <b>B</b> respectively. White arrowheads mark the regions of ASY1 with higher signal intensity. <b>(E-J)</b> Immunolocalization of ASY1 (green) <b>(E,G,H,J)</b> and ZYP1 (red) <b>(F,G,I,J)</b> and merge images <b>(G,J)</b> on chromosome spreads from wild type at zygotene. Figs <b>H, I</b> and <b>J</b> show magnified sections of axes from <b>E, F</b> and <b>G</b> respectively. <b>(K-P)</b> Immunolocalization of ASY1 (green) <b>(K,L,N,P)</b> and ZYP1 (red) <b>(M-P)</b> and merge images <b>(N,P)</b> on chromosome spread preparations from <i>Atpch2-1</i> at mid/late-prophase I. Figs <b>L, O</b> and <b>P</b> show magnified sections of axes from <b>K, M</b> and <b>N</b> respectively. White arrowheads represent synapsed regions while yellow arrowheads represent unsynapsed regions. DNA is stained with DAPI (blue). Bar = 10 cm.</p

Meiotic stages from wild type Arabidopsis and <i>Atpch2-1</i> pollen mother cells.

<p>Chromosome spread preparations from wild type <b>(A,C,E,G,I,K)</b> and <i>Atpch2-1</i><b>(B,D,F,H,J,L)</b> PMCs. <b>(A,B)</b> leptotene; <b>(C,D)</b> pachytene (note in <i>Atpch2-1</i> cell is at late prophase I normal pachytene stage was not observed). Arrowheads mark unsynapsed regions. <b>(E,F)</b> metaphase I. Arrowheads mark univalent chromosomes; <b>(G,H)</b> metaphase I stage labelled with 5S (red) and 45S (green) rDNA probes to identify the individual chromosomes. Arrowheads mark univalent chromosomes; <b>(I,J)</b> dyad; <b>(K-L)</b> tetrad. DNA is stained with DAPI. Bar = 10 μm.</p

Immunolocalization of PCH2 in <i>B</i>. <i>oleracea</i> at the leptotene/zygotene transition.

<p><b>(A-C)</b> Dual localization of ASY1 (green) and PCH2 (red) on chromosome spread preparations from <i>B</i>. <i>oleracea</i> PMCs at the leptotene/zygotene transition. <b>(D-F)</b> Dual localization of ZYP1 (green) and PCH2 (red) at SC nucleation sites. Yellow arrows indicate examples of large ‘arrowhead’ shaped foci and white arrows indicate the smaller SC nucleation sites. <b>(G)</b> SIM images of arrowhead SC nucleation sites stained with ZYP1 (green) and PCH2 (red). <b>(H)</b> SIM image of dual localization of ZYP1 (green) and PCH2 (red) on a nascent stretch of SC. DNA is stained with DAPI (blue). Bar = 1 μm in <b>(G)</b> and 10 μm in all other images.</p

Dual localization of ASY1 and recombination pathway proteins in wild type and <i>Atpch2-1</i> meiotic nuclei at early prophase I.

<p>Dual localization of ASY1 (green) and RAD51 (red) on wild type <b>(A)</b> and <i>Atpch2-1</i><b>(B)</b> PMCs at mid-leptotene; ASY1 (green) and DMC1 (red) on wild type <b>(C)</b> and <i>Atpch2-1</i><b>(D)</b> PMCs at mid-leptotene; ASY1 (green) and MSH4 (red) on wild type <b>(E)</b> and <i>Atpch2-1</i><b>(F)</b> PMCs at leptotene/zygotene transition; ASY1 (green) and HEI10 (red) on wild type <b>(G-I)</b> and <i>Atpch2-1</i><b>(J-L)</b> PMCs at late-leptotene. DNA is stained with DAPI (blue). Bar = 10 μm.</p