Rice Genetics Newsletter, Vol. 18

Rgn18

Literature Database | Rice Genetics Newsletters > Volume 18 Contents

37.	Detection of pre-meiotic DNA synthesis in pollen mother cells by immunofluorescence technique in rice
	K. MIYOSHI¹, N. KURATA^1,2 and K.I. NONOMURA¹ 1) National Institute of Genetics, Mishima, Shizuoka, 411-8540 Japan 2) School of Life Science, Graduate University for Advanced Studies, Mishima, Shizuoka, 411-8540 Japan

The pre-meiotic S phase is known to affect meiotic events such as homologous pairing, reductional chromosomal segregation and recombination in wheat (Martinez-Perez et al. 1999) and fission yeast (Watanabe et al. 2001). However, there is little information on the onset of meiosis in cereal plants. In order to establish the basis for analyzing the genetic machinery promoting early meiotic events in cereals, we identified pollen mother cells (PMCs) of rice (Oryza sativa L. cv. Nipponbare) at pre-meiotic S phase by a thymidine analog bromodeoxyuridine (BrdU) incorporation experiment according to the methods of Suzuki et al. (1992), with some modifications. Briefly, developing panicles were incubated with 100mM BrdU in the dark for five hours. After the incorporation of BrdU, panicles were fixed, dehydrated and embedded in a resin, Technovit 7100, and sectioned into 2mm thick by a rotary microtome. The samples were double-stained with DAPI and anti-BrdU mouse monoclonal antibody (Becton Dickinson Co.) followed by biotinylated anti-mouse IgG antibody and Alexa/streptavidine conjugate (Molecular Probe).

Forty-one developing flowers with 1.53 to 2.64mm flower lengths containing pre-meiotic or early meiotic cells were observed. Seven flowers ranging from 1.53 to 2.01mm, exhibited BrdU signals only in the anther wall cells (Fig. 1a, c). Nine flowers ranging from 2.00 to 2.53mm, incorporated BrdU in almost all PMCs and relatively fewer cell wall (Fig. 1b, d). No signals were detected in twenty flowers (2.10 to 2.64mm length) containing PMCs, wherein chromatin condensation at meiotic prophase was detected by DAPI staining (data not shown). The results showed that the PMCs at pre-meiotic S phase and those at meiotic prophase were present in flowers with almost the same length (2.10 - 2.53mm). This suggests that PMCs enter into meiosis as soon as pre-meiotic DNA synthesis finished. The anther wall of rice is composed of epidermal and three hypodermal cell layers. In all flowers examined, four layers of the anther walls were already formed. This indicates that four epidermal and hypodermal

layers were established in the anther walls before entering into pre-meiotic S phase (Fig. 1a, b). Our results will be useful information for the detailed analyses of pre- and early meiotic events in cereals.

Acknowledgement

This research was supported by Program for Promotion of Basic Research Activities for Innovative Biosciences.

References

Martinez-Perez E., P. Shaw, S. Reader, L. Aragon-Alcaide, T. Miller and G. Moore, 1999. Homologous chromosome pairing in wheat. J. Cell. Sci. 112: 1761-1769.

Suzuki T., S. Kawano, A. Sakai, M. Fujie, H. Kuroiwa, H. Nakamura and T. Kuroiwa, 1992. Preferential mitochondorial and plastid DNA synthesis before multiple cell divisions in Nicotiana tabacum. J. Cell. Sci. 103: 831-837.

Watanabe Y., S. Yokobayashi, M. Yamamoto and P. Nurse, 2001. Pre-meiotic S phase is linked to reductional chromosome segregation and recombination. Nature 409: 359-363.

Last modified: Thu Oct 31 00:11:51 2002