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Ahp2 (Hop2) function in Arabidopsis thaliana (Ler) is required for stabilization of close alignment and synaptonemal complex formation except for the two short arms that contain nucleolus organizer regions

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Abstract

A cytological comparative analysis of male meiocytes was performed for Arabidopsis wild type and the ahp2 (hop2) mutant with emphasis on ahp2’s largely uncharacterized prophase I. Leptotene progression appeared normal in ahp2 meiocytes; chromosomes exhibited regular axis formation and assumed a typical polarized nuclear organization. In contrast, 4′,6′-diamidino-2-phenylindole-stained ahp2 pachytene chromosome spreads demonstrated a severe reduction in stabilized pairing. However, transmission electron microscopy (TEM) analysis of sections from meiocytes revealed that ahp2 chromosome axes underwent significant amounts of close alignment (44% of total axis). This apparent paradox strongly suggests that the Ahp2 protein is involved in the stabilization of homologous chromosome close alignment. Fluorescent in situ hybridization in combination with Zyp1 immunostaining revealed that ahp2 mutants undergo homologous synapsis of the nucleolus-organizer-region-bearing short arms of chromosomes 2 and 4, despite the otherwise “nucleus-wide” lack of stabilized pairing. The duration of ahp2 zygotene was significantly prolonged and is most likely due to difficulties in chromosome alignment stabilization and subsequent synaptonemal complex formation. Ahp2 and Mnd1 proteins have previously been shown, “in vitro,” to form a heterodimer. Here we show, “in situ,” that the Ahp2 and Mnd1 proteins are synchronous in their appearance and disappearance from meiotic chromosomes. Both the Ahp2 and Mnd1 proteins localize along the chromosomal axis. However, localization of the Ahp2 protein was entirely foci-based whereas Mnd1 protein exhibited an immunostaining pattern with some foci along the axis and a diffuse staining for the rest of the chromosome.

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Acknowledgements

We would like to thank the reviewers for their insightful comments. We thank Dr. Jones for providing antibodies to Zyp1 and Asy1, Dr. Mercier for antibodies to Mnd1, and Dr. Makaroff for antibodies to Syn1 and Smc3. We would like to thank both Dr. Riggs and Dr. Siddiqui for their invaluable assistance in generating an antibody to the Ahp2 protein. We are also grateful to Dr. Sablowski for sending us seeds heterozygous for the ahp2 mutation. We also wish to thank Dr. Lysak for both FISH probes and valuable advice. This work was supported by a grant from the National Science and Engineering Research Council to CAH.

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  1. Department of Cell and Systems Biology, University of Toronto, 1265 Military Trail, Scarborough, M1C 1A4, Ontario, Canada

    P. Stronghill, N. Pathan, H. Ha, E. Supijono & C. Hasenkampf

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  1. P. Stronghill
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Correspondence to P. Stronghill.

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Communicated by: P. Shaw

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Supplemental Figs. 1–3

Supplemental Fig. 1. Representative negative control images from Ahp2 immunocytology experiments. Note: There was no labeling of wild-type and ahp2 chromosome spreads with preimmune serum and Ahp2 antibody, respectively. Scale bar 10 μm. Supplemental Fig. 2. Representative negative control images from Zyp1, Asy1, Syn1, and Smc3 immunocytochemistry experiments. Scale bar 10 μm. Supplemental Fig. 3. DAPI-stained Arabidopsis chromosome spreads from male meiocytes: a–c, g–i, and m, n wild type, d–f, j–l, and o, p ahp2 mutant. The prophase I substages are as follows: a, d mid-leptotene, b, e mid-zygotene, c, f mid-pachytene, g, j mid-diplotene, h, k diakinesis, i, l metaphase I, m, o anaphase I, and n, p anaphase II. Note: the ahp2 spreads were generated from buds of increasing size from the same inflorescence. Short white arrows indicate unpaired centromeres; long white arrows indicate paired centromeres and the arrowhead indicates the NOR. Scale bar 10 μm (GIF 2 kb)

High resolution image (TIFF 13208 kb)

Supplemental Figs. 4–6

Supplemental Fig. 4. Examples of chromatin bridging during anaphase II in an ahp2 meiocyte (see arrows). The chromosome spread was stained with DAPI. Scale bar 10 μm. Supplemental Fig. 5. S-phase BrdU pulse label time course experiment results for wild-type and ahp2 meiocytes: a A schematic summarizing both the average times for BrdU label to appear in tetrads and the average duration of meiosis in wild-type and ahp2 meiocytes. The bracketed times apply to the ahp2 meiocytes. L leptotene; TII telophase II (tetrads). b A table of the average times and the range of times measured from the mid-point of the BrdU pulse until peak BrdU signal was observed in the tetrads from the three wild-type and three ahp2 time course experiments performed. c A table of the raw data from the three wild-type BrdU experiments. d A table of the raw data from the three ahp2 mutant BrdU experiments. NTF no tetrads found, NA not assayed. Supplemental Fig. 6. Additional images demonstrating Mnd1 labeling of Arabidopsis ahp2 chromosome spreads and the nucleolus. Mnd1 antibody did not label mnd1 chromosome spreads. Note: All experimental parameters were kept constant for wild type and ahp2and mnd1 mutants; this included primary/secondary antibody dilutions and incubation times/conditions. Scale bar 10 μm (GIF 2 kb)

High resolution image (TIFF 8527 kb)

Supplemental Figs. 7–9

Supplemental Fig. 7. Fluorescent immunolocalization of Asy1 protein to Arabidopsis chromosome spreads: a–d wild type, e–h ahp2 meiocytes. The images shown are the result of merging the image of Asy1 protein (green) localization signal and the DAPI-counterstained (white) image of the same chromosome spread. The prophase I substages are as follows: a early leptotene, e late leptotene, b, f zygotene, c, g pachytene, and d, h diplotene. Scale bar 10 μm. TEM images of wild-type (i) and ahp2 (j) meiocyte sections provide representative examples of unpaired chromosomal axis (black arrows). Scale bar 1 μm. Supplemental Fig. 8 DAPI-stained pachytene ahp2 chromosome spread images. Arrows indicate the region of pairing that extends from the centromere to the NOR. Scale bar 10 μm. Supplemental Fig. 9. Fluorescent immunolocalization of the meiotic cohesin proteins Syn1 (a–j) and Smc3 (k–t) on Arabidopsis wild-type and ahp2 chromosome spreads counterstained with DAPI (white). The Syn1 antibody (green) was localized to both wild-type (a–e) and ahp2 nuclei (f–j) of the following substages: a, f leptotene, b, g mid-zygotene, c, h mid-pachytene, d, i diplotene, and e, j postdiakinesis. Scale bar 10 μm. The micrographs shown are merged DAPI and Syn1 images. The Smc3 antibody (green) was localized to both wild-type (k–o) and ahp2 nuclei (p–t) of the following substages: k, p leptotene, l, q mid-zygotene, m, r mid-pachytene, n, s diakinesis, and o, t postdiakinesis. The micrographs shown are merged DAPI and Smc3 images. Scale bar 10 μm (JPEG 258 kb)

High resolution image (TIFF 34829 kb)

Supplemental Fig. 10

A clustalW alignment of Ahp2 protein orthologs from several plant and animal species. Bolded sequence denotes the DNA minor groove binding motif (GIF 153 kb)

High resolution image (TIFF 2355 kb)

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Stronghill, P., Pathan, N., Ha, H. et al. Ahp2 (Hop2) function in Arabidopsis thaliana (Ler) is required for stabilization of close alignment and synaptonemal complex formation except for the two short arms that contain nucleolus organizer regions. Chromosoma 119, 443–458 (2010). https://doi.org/10.1007/s00412-010-0270-0

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