MNU-induced mutant pools and high performance TILLING enable finding of any gene mutation in rice.

Mutant populations are indispensable genetic resources for functional genomics in all organisms. However, suitable rice mutant populations, induced either by chemicals or irradiation still have been rarely developed to date. To produce mutant pools and to launch a search system for rice gene mutations, we developed mutant populations of Oryza sativa japonica cv. Taichung 65, by treating single zygotic cells with N-methyl-N-nitrosourea (MNU). Mutagenesis in single zygotes can create mutations at a high frequency and rarely forms chimeric plants. A modified TILLING system using non-labeled primers and fast capillary gel electrophoresis was applied for high-throughput detection of single nucleotide substitution mutations. The mutation rate of an M(2) mutant population was calculated as 7.4 x 10(-6) per nucleotide representing one mutation in every 135 kb genome sequence. One can expect 7.4 single nucleotide substitution mutations in every 1 kb of gene region when using 1,000 M(2) mutant lines. The mutations were very evenly distributed over the regions examined. These results indicate that our rice mutant population generated by MNU-mutagenesis could be a promising resource for identifying mutations in any gene of rice. The modified TILLING method also proved very efficient and convenient in screening the mutant population.

PAIR2 is essential for homologous chromosome synapsis in rice meiosis I.

The PAIR2 gene is required for homologous chromosome synapsis at meiosis I in rice (Oryza sativa L.) and encodes a HORMA-domain protein that is homologous to Saccharomyces cerevisiae HOP1 and Arabidopsis ASY1. Immunocytological and electron microscopic analyses indicate that PAIR2 proteins associate with axial elements (AEs) at leptotene and zygotene, and is removed from the AEs of arm regions when homologous chromosomes have been synapsed. Immunocytology against a centromeric histone H3 variant revealed that PAIR2 remains at centromeres until diakinesis, by which time the homologous centromeres had already been synapsed. However, neither precocious segregation of sister chromatids nor kinetochore dysfunction is observed, and AEs are normally assembled in the mutant. In the pair2-null mutant, homologous chromosome synapsis is completely eliminated. This study provides the first description of AE-associated protein in monocot plants and indicates that PAIR2 plays an essential role in promoting homologous chromosome synapsis. However, PAIR2 does not play a role in AE formation, sister chromatid cohesion at centromeres or kinetochore assembly in meiosis I of rice.

Functional isolation of novel nuclear proteins showing a variety of subnuclear localizations.

Nuclear proteins play key roles in the fundamental regulation of genome instability, the phases of organ development, and physiological responsiveness through gene expression. Although nuclear proteins have been shown to account for approximately one-fourth of total proteins in yeast, no efficient method to identify novel nuclear proteins has been applied to plants. In this study, a trial to isolate nuclear proteins in rice was attempted, and several novel nuclear proteins showing a variety of subnuclear localizations were identified. The nuclear transportation trap (NTT) system, which is a modified two-hybrid system, isolated many nuclear proteins from rice (Oryza sativa) NTT cDNA libraries. Nuclear localization of the isolated proteins was confirmed by transient introduction of green fluorescent protein fusion constructs for a subset of protein genes into onion (Allium cepa) cells. The majority of these proteins, including novel proteins and proteins initially categorized as cytoplasmic proteins, were revealed to be localized in the nucleus. Detailed characterization of unknown proteins revealed various subnuclear localizations, indicating their possible association with chromatin and the nuclear matrix with a foci or speckle-like distribution. Some also showed dual distribution in the nucleus and cytoplasm. In the novel protein fraction, a protein was further identified for its chromatin-associated localization in a specific organ of rice by immunostaining. Thus, a variety of novel nuclear architectural proteins with chromatin or matrix associating abilities, which are important in nuclear organization by influencing certain organ developments or cell responsiveness, can be isolated using the NTT method. Because nuclear proteins other than transcription regulators have rarely been characterized in plants, such as matrix proteins and development-specific chromatin proteins, their identification and subsequent characterization could provide important information for genome-wide regulatory mechanisms controlled by nuclear organization.

The MSP1 gene is necessary to restrict the number of cells entering into male and female sporogenesis and to initiate anther wall formation in rice.

The function of the novel gene MSP1 (MULTIPLE SPOROCYTE), which controls early sporogenic development, was elucidated by characterizing a retrotransposon-tagged mutation of rice. The MSP1 gene encoded a Leu-rich repeat receptor-like protein kinase. The msp1 mutation gave rise to an excessive number of both male and female sporocytes. In addition, the formation of anther wall layers was disordered and the tapetum layer was lost completely. Although the mutation never affected homologous chromosome pairing and chiasma maintenance, the development of pollen mother cells was arrested at various stages of meiotic prophase I, which resulted in complete male sterility. Meanwhile, plural megaspore mother cells in a mutant ovule generated several megaspores, underwent gametogenesis, and produced germinable seeds when fertilized with wild-type pollen despite disorganized female gametophytes. In situ expression of MSP1 was detected in surrounding cells of male and female sporocytes and some flower tissues, but never in the sporocytes themselves. These results suggest that the MSP1 product plays crucial roles in restricting the number of cells entering into male and female sporogenesis and in initiating anther wall formation in rice.