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First published online January 12, 2006
doi: 10.1242/10.1242/jcs.02736

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PAIR2 is essential for homologous chromosome synapsis in rice meiosis I

Ken-Ichi Nonomura^1,2,*,, Mutsuko Nakano^1,*, Mitsugu Eiguchi¹, Tadzunu Suzuki¹ and Nori Kurata^1,2,

¹ Experimental Farm/Plant Genetics Laboratory, National Institute of Genetics, Yata1111, Mishima, Shizuoka 411-8540, Japan
² Department of Life Science, Graduate University for Advanced Studies/Sokendai, Yata1111, Mishima, Shizuoka 411-8540, Japan

View larger version (60K): [in a new window]   Fig. 1. Western blot analysis using anti-PAIR2 antibody (top), pre-immune serum as a control (middle) and anti--tubulin monoclonal antibody (bottom) against recombinant PAIR2 protein and crude extracts from anthers and vegetative tissues. Lanes M and 1 represent a molecular weight marker and 6xHis-tagged PAIR2, respectively. Lanes 2-8 show crude extracts from anthers of the following length ranges: 0.40.5 mm (2), 0.50.6 mm (3), 0.60.7 mm (4), 0.70.8 mm (5), 0.80.9 mm (6), 0.91.0 mm (7), 1.01.1 mm (8). Lanes 9 and 10 are from seedlings and roots, respectively.

View larger version (35K): [in a new window]   Fig. 2. PAIR2 accumulation in the meiocyte nucleus is induced following pre-meiotic DNA synthesis. (A) Immunofluorescence of incorporated BrdUs and PAIR2 proteins against pre-meiotic PMCs. Staining pattern of BrdU (magenta) and PAIR2 (green) was divided into five categories: (1) neither a BrdU nor a PAIR2 signal; (2) faint BrdU but no PAIR2 signal; (3) intense BrdU and faint PAIR2 signals; (4) faint BrdU and intense PAIR2 signals; and (5) no BrdU and filamentous PAIR2 signals. Chromosomes were counter-stained with DAPI (blue). Bar, 5 µm. (B) Frequency of each category of meiocyte in anthers 0.3, 0.4 and 0.5 mm long. The numbers above each bar correspond to each category of the meiocytes in A.

View larger version (40K): [in a new window]   Fig. 3. PAIR2 associates with axial cores of meiotic chromosomes in wild-type (A-G) but not in pair2-null mutant PMCs (H). Panels from left to right indicate the localization of histone pan antibody (representing chromatin regions), the localization of PAIR2, and merged image of histone pan antibody (red) and PAIR2 (green). In all images, an unstained circular body within the PAIR2 signals represents the nucleolar region. (A) Pre-meiotic S/G2. (B) Filamentous PAIR2 signals begin to elongate at early leptotene. (C) Late leptotene. (D) Zygotene. (E) Pachytene. (F) Diakinesis. Pairs of arrows indicate pairs of the PAIR2 foci whose bilateral localization on bivalents is clearly detected. (G) Metaphase I. (H) Immunofluorescence against a zygotene PMC of the pair2 mutant. Bars, 5 µm.

View larger version (44K): [in a new window]   Fig. 4. PAIR2 localizes on the meiotic chromosome axis and delocalizes after synapsis is completed in zygotene and pachytene. (A) Spread chromosomes (red) and PAIR2 localization (green) in the late zygotene meiocyte with destructed nucleus. (B) A magnified image of a box in A. (C) Another magnified image of early pachytene chromosomes. Arrows in B and C indicate synapsed regions of homologous chromosomes, and arrowheads show unsynapsed regions. (D) Spread chromosomes in pachytene. Bars, 5 µm.

View larger version (146K): [in a new window]   Fig. 5. EM observation of rice meiotic chromosomes. (A) Immunogold localization of anti-PAIR2 antibody in an ultrathin section of a wild-type meiocyte at zygotene. Arrows indicate the electron-dense and filamentous structure observed in the nucleus. Nu, nucleolus. Bar, 1 µm. (B) Magnified view of the box in A. Bar, 0.2 µm. (C) A negative control of A, excluding primary antibody. Bar, 1 µm. (D) EM observation of pair2 mutant meiocytes. Filamentous and electron-dense chromosome axes (arrows) were observed to be the same as in the wild type (not shown). Bar, 0.2 µm.

View larger version (60K): [in a new window]   Fig. 6. A pair of PAIR2 spots remains until diakinesis on centromeric regions of each homologous pair. Chromosomes were counter-stained with anti-histone pan antibody (blue). The centromeric chromatin and PAIR2 were stained with anti-OsCenH3 (magenta) and anti-PAIR2 antibodies (green), respectively. Bar, 5 µm.

View larger version (72K): [in a new window]   Fig. 7. PAIR2 function is not required for sister chromatid cohesion and kinetochore assembly in rice meiosis I. (A,B) Spread and Giemsa-stained meiotic chromosomes of the wild-type (A) and pair2-null mutant (B) at anaphase I. In both meiocytes, a diploid number (2n=24) of univalents was detected. (C-E) Spindle microtubules and chromosomes were stained with anti--tubulin antibody (green) and anti-histone pan antibody (red), respectively, in the wild-type (C) and pair2 mutant (D,E) meiocytes. Even lagging univalents at midzone were captured by spindle microtubules at mid anaphase I (two univalents showing bipolar attachment are magnified in the bottom right of D). In E, at late anaphase I, a single OsCenH3 focus attached to bipolar microtubules is elongated towards both poles (magnified in the bottom right), and a univalent shows monopolar microtubule attachment (magnified in the top left). Bars, 5 µm.

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