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, ...

SummaryWe have generated 47 932 T-DNA tag lines in japonica rice using activation-tagging vectors that contain tetramerized 35S enhancer sequences. To facilitate use of those lines, we isolated the genomic sequences flanking the inserted T-DNA via inverse polymerase chain reaction. For most of the lines, we performed four sets of amplifications using two different restriction enzymes toward both directions. In analyzing 41 234 lines, we obtained 27 621 flanking sequence tags (FSTs), among which 12 505 were integrated into genic regions and 15 116 into intergenic regions. Mapping of the FSTs on chromosomes revealed that T-DNA integration frequency was generally proportional to chromosome size. However, T-DNA insertions were non-uniformly distributed on each chromosome: higher at the distal ends and lower in regions close to the centromeres. In addition, several regions showed extreme peaks and valleys of insertion frequency, suggesting hot and cold spots for T-DNA integration. The density of insertion events was somewhat correlated with expressed, rather than predicted, gene density along each chromosome. Analyses of expression patterns near the inserted enhancer showed that at least half the test lines displayed greater expression of the tagged genes. Whereas in most of the increased lines expression patterns after activation were similar to those in the wild type, thereby maintaining the endogenous patterns, the remaining lines showed changes in expression in the activation tagged lines. In this case, ectopic expression was most frequently observed in mature leaves. Currently, the database can be searched with the gene locus number or location on the chromosome at http:// www.postech.ac.kr/life/pfg/risd. On request, seeds of the T 1 or T 2 plants will be provided to the scientific community.

SummaryWe have isolated a floury endosperm-4 (flo4) rice mutant with a floury-white endosperm but a normal outer portion. Scanning electron microscopic analysis revealed that this abnormal endosperm consisted of loosely packed starch granules. The mutant phenotype was generated by T-DNA insertion into the fifth intron of the OsPPDKB gene encoding pyruvate orthophosphate dikinase (PPDK). Plants containing flo4-1 produced no OsPPDKB transcript or the OsPPDKB protein in their developing kernels and leaves. We obtained two additional alleles, flo4-2 and flo4-3, that also showed the same white-core endosperm phenotype. The flo4 kernels weighed about 6% less than wild-type ones. Starch contents in both kernel types were similar, but the total protein content was slightly higher in the mutant kernels. Moreover, lipid contents were significantly increased in the flo4 kernels. Expression analyses demonstrated that the cytosolic mRNA of OsPPDKB was induced in the reproductive organs after pollination, and greatly increased until about 10 days after fertilization. This mRNA was localized mainly in the endosperm, aleurone, and scutellum of the developing kernel. Our results suggest that cytosolic PPDK functions in rice to modulate carbon metabolism during grain filling.

ADP-glucose pyrophosphorylase (AGP) catalyzes the first committed step of starch biosynthesis in higher plants. To identify AGP isoforms essential for this biosynthetic process in sink and source tissues of rice plants, we analyzed the rice AGP gene family which consists of two genes, OsAGPS1 and OsAGPS2, encoding small subunits (SSU) and four genes, OsAGPL1, OsAGPL2, OsAGPL3 and OsAGPL4, encoding large subunits (LSU) of this enzyme heterotetrameric complex. Subcellular localization studies using green fluorescent protein (GFP) fusion constructs indicate that OsAGPS2a, the product of the leaf-preferential transcript of OsAGPS2, and OsAGPS1, OsAGPL1, OsAGPL3, and OsAGPL4 are plastid-targeted isoforms. In contrast, two isoforms, SSU OsAGPS2b which is a product of a seed-specific transcript of OsAGPS2, and LSU OsAGPL2, are localized in the cytosol. Analysis of osagps2 and osagpl2 mutants revealed that a lesion of one of the two cytosolic isoforms, OsAGPL2 and OsAGPS2b, causes a shrunken endosperm due to a remarkable reduction in starch synthesis. In leaves, however, only the osagps2 mutant appears to severely reduce the transitory starch content. Interestingly, the osagps2 mutant was indistinguishable from wild type during vegetative plant growth. Western blot analysis of the osagp mutants and wild type plants demonstrated that OsAGPS2a is an SSU isoform mainly present in leaves, and that OsAGPS2b and OsAGPL2 are the major SSU and LSU isoforms, respectively, in the endosperm. Finally, we propose a spatiotemporal complex model of OsAGP SSU and LSU isoforms in leaves and in developing endosperm of rice plants.

A MADS family gene, OsMADS6, was isolated from a rice (Oryza sativa L.) young flower cDNA library using OsAMDS1 as a probe. With this clone, various MADS box genes that encode for proteinto-protein interaction partners of the OsMADS6 protein were isolated by the yeast two-hybrid screening method. On the basis of sequence homology, OsMADS6 and the selected partners can be classified in the APETALA1/AGAMOUS-LIKE9 (AP1/AGL9) family. One of the interaction partners, OsMADS14, was selected for further study. Both genes began expression at early stages of flower development, and their expression was extended into the later stages. In mature flowers the OsMADS6 transcript was detectable in lodicules and also weakly in sterile lemmas and carpels, whereas the OsMADS14 transcript was detectable in sterile lemmas, paleas/ lemmas, stamens, and carpels. Using the yeast two-hybrid system, we demonstrated that the region containing of the 109th to 137th amino acid residues of OsMADS6 is indispensable in the interaction with OsMADS14. Site-directed mutation analysis revealed that the four periodical leucine residues within the region are essential for this interaction. Furthermore, it was shown that the 14 amino acid residues located immediately downstream of the K domain enhance the interaction, and that the two leucine residues within this region play an important role in that enhancement.