SummaryWe have produced 22 090 primary transgenic rice plants that carry a T-DNA insertion, which has resulted in 18 358 fertile lines. Genomic DNA gel-blot and PCR analyses have shown that approximately 65% of the population contains more than one copy of the inserted T-DNA. Hygromycin resistance tests revealed that transgenic plants contain an average of 1.4 loci of T-DNA inserts. Therefore, it can be estimated that approximately 25 700 taggings have been generated. The binary vector used in the insertion contained the promoterless b-glucuronidase (GUS) reporter gene with an intron and multiple splicing donors and acceptors immediately next to the right border. Therefore, this gene trap vector is able to detect a gene fusion between GUS and an endogenous gene, which is tagged by T-DNA. Histochemical GUS assays were carried out in the leaves and roots from 5353 lines, mature¯owers from 7026 lines, and developing seeds from 1948 lines. The data revealed that 1.6±2.1% of tested organs were GUS-positive in the tested organs, and that their GUS expression patterns were organ-or tissue-speci®c or ubiquitous in all parts of the plant. The large population of T-DNA-tagged lines will be useful for identifying insertional mutants in various genes and for discovering new genes in rice.

Although genetic diversity has a cardinal role in domestication, abundant natural allelic variations across the rice genome that cause agronomically important differences between diverse varieties have not been fully explored. Here we implement an approach integrating genome-wide association testing with functional analysis on grain size in a diverse rice population. We report that a major quantitative trait locus, GLW7, encoding the plant-specific transcription factor OsSPL13, positively regulates cell size in the grain hull, resulting in enhanced rice grain length and yield. We determine that a tandem-repeat sequence in the 5' UTR of OsSPL13 alters its expression by affecting transcription and translation and that high expression of OsSPL13 is associated with large grains in tropical japonica rice. Further analysis indicates that the large-grain allele of GLW7 in tropical japonica rice was introgressed from indica varieties under artificial selection. Our study demonstrates that new genes can be effectively identified on the basis of genome-wide association data.

We have developed a new T-DNA vector, pGA2715, which can be used for promoter trapping and activation tagging of rice (Oryza sativa) genes. The binary vector contains the promoterless ␤-glucuronidase (GUS) reporter gene next to the right border. In addition, the multimerized transcriptional enhancers from the cauliflower mosaic virus 35S promoter are located next to the left border. A total of 13,450 T-DNA insertional lines have been generated using pGA2715. Histochemical GUS assays have revealed that the GUS-staining frequency from those lines is about twice as high as that from lines transformed with the binary vector pGA2707, which lacks the enhancer element. This result suggests that the enhancer sequence present in the T-DNA improves the GUS-tagging efficiency. Reverse transcriptase-PCR analysis of a subset of randomly selected pGA2715 lines shows that expression of the genes immediately adjacent to the inserted enhancer is increased significantly. Therefore, the large population of T-DNA-tagged lines transformed with pGA2715 could be used to screen for promoter activity using the gus reporter, as well as for creating gain-of-function mutants.Recent completion of the draft sequence for the rice (Oryza sativa) genome has resulted in an explosion of information on rice genes (Goff et al., 2002; Yu et al., 2002). The challenge for the post-sequencing era is to identify the biological functions for these genes. Of all the approaches used to discover gene function, the most direct is to disrupt the genes and analyze the consequences. Various methods have been developed in plants for this purpose. These include using ethyl methanesulfonate, fast-neutron treatment, or insertion of an element, such as a transposable element or T-DNA (Koornneef et al., 1982;Sundaresan, 1996;Krysan et al., 1999). Insertional mutagenesis has the advantage that the inserted element acts as a tag for gene identification. However, all gene disruption approaches also have some limitations. For example, it is difficult to identify the function of redundant genes, or of genes required in early embryogenesis or gametophyte development.To overcome those limitations, modified insertional elements have been developed. One of these modified designs is the gene trap system that involves creating fusions between the tagged genes and a reporter gene, such as ␤-glucuronidase (gus) or green fluorescent protein (gfp; Sundaresan et al., 1995;Springer, 2000). This system provides a way of identifying novel genes based on their expression patterns. Insertion of the promoterless reporter not only destroys normal gene function but also activates expression of the reporter gene. Because expression levels can be monitored in heterozygote plants, the gene trap system is useful for studying the patterns of most plant genes, including essential genes that cause lethal mutations. This system is convenient for observing mutant phenotypes because reporter activation indicates the location, condition, and time of expression for the disrupted gene. In Arabidopsis, ...

Knowledge about signaling in arbuscular mycorrhizal (AM) symbioses is currently restricted to the common symbiosis (SYM) signaling pathway discovered in legumes. This pathway includes calcium as a second messenger and regulates both AM and rhizobial symbioses. Both monocotyledons and dicotyledons form symbiotic associations with AM fungi, and although they differ markedly in the organization of their root systems, the morphology of colonization is similar. To identify and dissect AM-specific signaling in rice (Oryza sativa), we developed molecular phenotyping tools based on gene expression patterns that monitor various steps of AM colonization. These tools were used to distinguish common SYMdependent and -independent signaling by examining rice mutants of selected putative legume signaling orthologs predicted to be perturbed both upstream (CASTOR and POLLUX) and downstream (CCAMK and CYCLOPS) of the central, calciumspiking signal. All four mutants displayed impaired AM interactions and altered AM-specific gene expression patterns, therefore demonstrating functional conservation of SYM signaling between distant plant species. In addition, differential gene expression patterns in the mutants provided evidence for AM-specific but SYM-independent signaling in rice and furthermore for unexpected deviations from the SYM pathway downstream of calcium spiking.

RNA editing is the alteration of RNA sequences via insertion, deletion and conversion of nucleotides. In flowering plants, specific cytidine residues of RNA transcribed from organellar genomes are converted into uridines. Approximately 35 editing sites are present in the chloroplasts of higher plants; six pentatricopeptide repeat genes involved in RNA editing have been identified in Arabidopsis. However, although approximately 500 editing sites are found in mitochondrial RNAs of flowering plants, only one gene in Arabidopsis has been reported to be involved in such editing. Here, we identified rice mutants that are defective in seven specific RNA editing sites on five mitochondrial transcripts. Their various phenotypes include delayed seed germination, retarded growth, dwarfism and sterility. Mutant seeds from heterozygous plants are opaque. This mutation, named opaque and growth retardation 1 (ogr1), was generated by T-DNA insertion into a gene that encodes a pentatricopeptide repeat protein containing the DYW motif. The OGR1-sGFP fusion protein is localized to mitochondria. Ectopic expression of OGR1 in the mutant complements the altered phenotypes. We conclude that OGR1 is essential for RNA editing in rice mitochondria and is required for normal growth and development.