;We have previously generated a large pool of T-DNA insertional lines in rice. In this study, we screened those T-DNA pools for rice mutants that had defective chlorophylls. Among the 1,995 lines examined in the T2 generation, 189 showed a chlorophyll-deficient phenotype that segregated as a single recessive locus. Among the mutants, 10 lines were b-glucuronidase (GUS)-positive in the leaves. Line 9-07117 has a T-DNA insertion into the gene that is highly homologous to XANTHA-F in barley and CHLH in Arabidopsis. This OsCHLH gene encodes the largest subunit of the rice Mg-chelatase, a key enzyme in the chlorophyll branch of the tetrapyrrole biosynthetic pathway. In the T2 and T3 generations, the chlorina mutant phenotypes are co-segregated with the T-DNA. We have identified two additional chlorina mutants that have a Tos17 insertion in the OsCHLH gene. Those phenotypes were cosegregated with Tos17 in the progeny. GUS assays and RNA blot analysis showed that expression of the OsCHLH gene is light inducible, while TEM analysis revealed that the thylakoid membrane of the mutant chloroplasts is underdeveloped. The chlorophyll content was very low in the OschlH mutants. This is the first report that T-DNA insertional mutagenesis can be used for functional analysis of rice genes.

We analyzed 6,749 lines tagged by the gene trap vector pGA2707. This resulted in the isolation of 3,793 genomic sequences flanking the T-DNA. Among the insertions, 1,846 T-DNAs were integrated into genic regions, and 1,864 were located in intergenic regions. Frequencies were also higher at the beginning and end of the coding regions and upstream near the ATG start codon. The overall GC content at the insertion sites was close to that measured from the entire rice (Oryza sativa) genome. Functional classification of these 1,846 tagged genes showed a distribution similar to that observed for all the genes in the rice chromosomes. This indicates that T-DNA insertion is not biased toward a particular class of genes. There were 764, 327, and 346 T-DNA insertions in chromosomes 1, 4 and 10, respectively. Insertions were not evenly distributed; frequencies were higher at the ends of the chromosomes and lower near the centromere. At certain sites, the frequency was higher than in the surrounding regions. This sequence database will be valuable in identifying knockout mutants for elucidating gene function in rice. This resource is available to the scientific community at http://www.postech.ac.kr/life/pfg/risd.Insertional mutagenesis is one of the most useful methods for analyzing gene function. When foreign DNA is inserted into a gene, it not only creates a mutation but also tags the affected gene, facilitating its isolation and characterization (Azpiroz-Leehan and Feldmann, 1997). Transposons and T-DNA have been used most widely as an insertional mutagen (Mathur et al., 1998;Wisman et al., 1998; Krysan et al., 1999;Parinov et al., 1999;Speulman et al., 1999;Tissier et al., 1999). It is believed that T-DNA insertion is a random event and that the inserted sequences are stable through multiple generations (Azpiroz- Leehan and Feldmann, 1997;Parinov and Sundaresan, 2000). Insertional mutant pools have been constructed in Arabidopsis and used for functional analysis of a number of genes (Feldmann, 1991; Koncz et al., 1992; Azpiroz-Leehan and Feldmann, 1997; Bechtold and Pelletier, 1998; Krysan et al., 1999; Galbiati et al., 2000;Parinov and Sundaresan, 2000; Bouché and Bouchez, 2001;Sessions et al., 2002;Szabados et al., 2002). The procedure for T-DNA insertional mutagenesis has also been applied to rice (Oryza sativa) using the Agrobacterium tumefaciensmediated transformation method (Hiei et al., 1994). Jeon et al. (2000) have reported the construction of over 20,000 T-DNA-tagged rice lines. A T-DNA insertional mutagen can be modified to trap a gene by inserting a reporter gene, such as gus (␤-glucuronidase), next to the T-DNA border (Sundaresan et al., 1995; Jeon et al., 2000;Springer, 2000). Approximately 5% to 10% of the mutagenized lines are GUS positive, demonstrating the efficiency of this gene-trapping system (Chin et al., 1999; Jeon et al., 2000).Completion of the genome sequencing for both Arabidopsis and rice has provided new reverse genetic means for assigning biological functions to sequenced genes (Kumar...

No abstract

Members in the YABBY gene family of proteins are plant-specific transcription factors that play critical roles in determining organ polarity. We have isolated a cDNA clone from rice that encodes a YABBY protein. This protein, OsYAB1, is similar to Arabidopsis YAB2 (50.3%) and YAB5 (47.6%). It carries a zinc-finger motif and a YABBY domain, as do those in Arabidopsis . A fusion protein between OsYAB1 and GFP is located in the nucleus. RNA gel-blot analysis showed that the OsYAB1 gene is preferentially expressed in flowers. In-situ hybridization experiments also indicated that the transcript accumulated in the stamen and carpel primordia. Unlike the Arabidopsis YABBY genes, however, the OsYAB1 gene does not show polar expression pattern in the tissues of floral organs. Our transgenic plants that ectopically expressed OsYAB1 were normal during the vegetative growth period, but then showed abnormalities in their floral structures. Spikelets contained supernumerary stamens and carpels compared with those of the wild types. These results suggest that OsYAB1 plays a major role in meristem development and maintenance of stamens and carpels, rather than in determining polarity.