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The DNA binding with one finger (Dof) proteins are plant-specific transcription factors that contain a particular class of zinc-finger DNA-binding domain. Thirty-different Dof genes are predicted to exist in the rice genome based on in silico analysis. A member of the Dof transcription factor family, Dof1 of rice (Oryza sativa), was identified from a dissociation (Ds) insertion mutant population. The Ds-tagged rice mutant line osdof1-Ds was identified from a population of rice tagged with promoterless β-glucuronidase (GUS) in T-DNA, and showed wound-responsive GUS expression. Genomic Southern analysis revealed that this mutant line contained a single copy of the Ds gene trap. Four wound-response-like cis-acting elements (PI-II, EIRE, W box, and G box-like elements) were found in the OsDof1 promoter region. To analyze the cis-acting elements, we constructed fusion genes between the OsDof1 promoter and the GUS reporter gene and transformed Arabidopsis and rice plants with these constructs. GUS gene expression was induced in leaf, seed, culm, and flower tissues of the osdof1-Ds mutant after wounding. Furthermore, histochemical analysis of the OsDof1 promoter::GUS fusion transgenic plants showed an early response to wounding. In addition, OsDof1 mRNA was induced by pathogen treatment. These results suggest that OsDof1 is a transcriptional activator that regulates gene expression in response to wounding.

Rice is the staple food of more than 50% of the worlds population. Cultivated rice has the AA genome (diploid, 2n=24) and small genome size of only 430 megabase (haploid genome). As the sequencing of rice genome was completed by the International Rice Genome Sequencing Project (IRGSP), many researchers in the world have been working to explore the gene function on rice genome. Insertional mutagenesis has been a powerful strategy for assessing gene function. In maize, well characterized transposable elements have traditionally been used to clone genes for which only phenotypic information is available. In rice endogenous mobile elements such as MITE and Tos (Hirochika. 1997) have been used to generate gene-tagged populations. To date T-DNA and maize transposable element systems has been utilized as main insertional mutagens in rice. A main drawback of a T-DNA scheme is that Agrobacteria- mediated transformation in rice requires extensive facilities, time, and labor. In contrast, the Ac/Ds system offers the advantage of generating new mutants by secondary transposition from a single tagged gene. Revertants can be utilized to correlate phenotype with genotype. To enhance the efficiency of gene detection, advanced gene-tagging systems i.e. activation, gene or enhancer trap) have been employed for functional genomic studies in rice. Internationally, there have been many projects to develop large scales of insertionally mutagenized populations and databases of insertion sites has been established. Ultimate goals of these projects are to supply genetic materials and informations essential for functional analysis of rice genes and for breeding using agronomically important genes. In this report, we summarize the current status of Ac/Ds-mediated gene tagging systems that has been launched by collaborative works from 2001 in Korea.

With 3 figures Abstract Male sterility is an important trait for rice breeding programmes based on heterosis. To generate male-sterile plants, we utilized RNAi technology with the promoters for two anther-specific genes, Osc4 and Osg6b, and one for rice allene oxide synthase (OsAOS). AOS catalyzes the biosynthesis of jasmonic acid and plays an important role in anther development. The promoters isolated from Osc4 and Osg6b were introduced into pK7gwiwgL, a promoter-less RNAi Gateway binary vector. Partial DNA fragments of OsAOS1 and OsAOS2 were combined into the Osc4::RNAi and Osg6b::RNAi cassettes by LR recombination. The resulting four RNAi constructs were transformed into rice calli by Agrobacterium. During the reproductive growth stage, 28% of OsAOS1 and 48% of OsAOS2 transgenic plants under the control of the Osg6b promoter exhibited a complete- or severe-sterility phenotype compared with the normal wild type. Here, we report that RNA interference of OsAOS2 with the Osg6b promoter is the most effective system for obtaining engineered genic male sterility in rice.

Although susceptibility to seed shattering causes severe yield loss during cereal crop harvest, it is an adaptive trait for seed dispersal in wild plants. We previously identified a recessive shattering locus, sh-h, from the rice shattering mutant line Hsh that carries an enhanced abscission layer. Here, we further mapped sh-h to a 34-kb region on chromosome 7 by analyzing 240 F(2) plants and five F(3) lines from the cross between Hsh and Blue&Gundil. Hsh had a point mutation at the 3' splice site of the seventh intron within LOC_Os07g10690, causing a 15-bp deletion of its mRNA as a result of altered splicing. Two transferred DNA (T-DNA) insertion mutants and one point mutant exhibited the enhanced shattering phenotype, confirming that LOC_Os07g10690 is indeed the sh-h gene. RNA interference (RNAi) transgenic lines with suppressed expression of this gene exhibited greater shattering. This gene, which encodes a protein containing a conserved carboxy-terminal domain (CTD) phosphatase domain, was named Oryza sativa CTD phosphatase-like 1 (OsCPL1). Subcellular localization and biochemical analysis revealed that the OsCPL1 protein is a nuclear phosphatase, a common characteristic of metazoan CTD phosphatases involved in cell differentiation. These results demonstrate that OsCPL1 represses differentiation of the abscission layer during panicle development.

본 연구는 동진벼 유래의 Ac/Ds 삽입변이집단의 GUS 분석을 통하여 뿌리 및 미숙종자에서 강하게 GUS가 발현한 개체를 선발하여 FST(flanking sequence tag) 분석 한 결과 Ds 전이 인자가 3번 염색체 zinc finger RING-H2 관련 Oszinc626 유전자의 첫 번째 exon 부위에 single copy로 삽입되어 있었으며, 선발변이체는 뿌리 및 종자 발달이 정상인 동진벼에 비해 매우 낮은 것으로 나타났다. Oszinc626 유전자는 RING-H2 type(C3-H2-C3)으로 $Cys-X_2-Cys-X_{28}-Cys-X-His-X_2-His-X_2-Cys-X_{14}-Cys-X_2-Cys$ 배열이 C-terminus 가장 말단에 위치하며, 49 kDa의 분자량을 가지고 있다. 또한 Southern blot 분석에서 Oszinc626 유전자는 벼 게놈상에 single copy로 존재하였다. RT-PCR을 통한 돌연변이 유전자의 발현분석 결과 250 mM의 염과, $4^{\circ}C$ 저온등과 같은abiotic stress에 의해 발현이 증가함을 보였고, 호르몬처리에 있어서 ABA와 IAA의 식물호르몬을 처리했을 경우 24시간까지 계속해서 발현양이 증가하는 것을 보이는 반면, 2,4-D 처리의 경우 30분 후에 발현이 일시적으로 증가되었으나 이후 발현이 급속히 감소한 것을 보였다. 벼의 조직 별 발현 검정에서 미성숙한 종자, 뿌리 분열조직 및 신초 등 주로 생장점 부위에서 강하게 발현되는 것을 보임에 따라 Oszinc626 유전자의 경우 식물의 생장에 관여하는 주동 유전자의 하나로 판단된다. 【Ac/Ds mutant lines of this study were transgenic rice plants, each of which harbored the maize transposable element Ds together with a GUS coding sequence under the control of a promoterless(Ds-GUS). We selected the mutants that were GUS expressed lines, because the GUS positive lines will be useful for identifying gene function in rice. One of these mutants was identified knock-out at Oszinc626(NP_001049991) gene, encoding a RING-H2 zinc-finger protein, by Ds insertion. In this mutant, while primary root development is normal, secondary root development from lateral root was very poor and seed development was incomplete compare with normal plant. RING zinc-finger proteins play important roles in the regulation of development in a variety of organisms. In the plant kingdom, a few genes encoding RING zinc-finger proteins have been documented with visible effects on plant growth and development. The consensus of the RING-H2(C3-H2-C3 type) domain for this group of protein is $Cys-X_2-Cys-X_{28}-Cys-X-His-X_2-His-X_2-Cys-X_{14}-Cys-X_2-Cys$ . Oszinc626 encodes a predicted protein product of 445 amino acids residues with a molecular mass of 49 kDa, with a RING-zinc-finger motif located at the extreme end of the C-terminus. RT-PCR analysis indicated that the expression of Oszinc626 gene was induced by IAA, cold, dehydration, high-salinity and abscisic acid, but not by 2,4-D, and the transcription of Oszinc626 gene accumulated primarily in rice immature seeds, root meristem and shoots. The gene accumulation patterns were corresponded with GUS expression.】

Insertional mutagen-mediated gene tagging populations have been essential resources for analyzing the function of plant genes. In rice, maize transposable elements have been successfully utilized to produce transposant populations. However, many generations and substantial field space are required to obtain a sufficiently sized transposant population. In rice, the japonica and indica subspecies are phenotypically and genetically divergent. Here, callus cultures with seeds carrying Ac and Ds were used to produce 89,700 lines of Dongjin, a japonica cultivar, and 6,200 lines of MGRI079, whose genome is composed of a mixture of the genetic backgrounds of japonica and indica. Of the more than 3,000 lines examined, 67% had Ds elements. Among the Ds-carrying lines, 81% of Dongjin and 63% of MGRI079 contained transposed Ds, with an average of around 2.0 copies. By examining more than 15,000 lines, it was found that 12% expressed the reporter gene GUS during the early-seedling stage. GUS was expressed in root hairs and crown root initials at estimated frequencies of 0.78% and 0.34%, respectively. The 5,271 analyzed Ds loci were found to be randomly distributed over all of the rice chromosomes.

A phosphate starvation-induced acid phosphatase cDNA was cloned from the rice, Oryza sativa. The cDNA encoding O. sativa acid phosphatase (OsACP1) has 1100 bp with an open reading frame of 274 amino acid residues. The deduced amino acid sequence of OsACP1 cDNA showed 53% identity to tomato acid phosphatase and 46-50% identity to several other plant phosphatases. OsACP1 expression was up-regulated in the rice plant and in cell culture in the absence of phosphate (Pi). The induced expression of OsACP1 was a specific response to Pi starvation, and was not affected by the deprivation of other nutrients. OsACP1 expression was responsive to the level of Pi supply, with transcripts of OsACP1 being abundant in Pi-deprived root. The OsACP1 cDNA was expressed as a 30 kDa polypeptide in baculovirus-infected insect Sf9 cells. In addition, the OsACP1 gene was introduced into Arabidopsis via Agrobacterium-mediated transformation. Functional expression of the OsACP1 gene in the transgenic Arabidopsis lines was confirmed by Northern blot and Western blot analyses, as well as phosphatase activity assays. These results suggest that the OsACP1 gene can be used to develop new transgenic dicotyledonous plants able to adapt to Pi-deficient conditions.

Two cDNA clones, pOS-ACO2 and pOS-ACO3, encoding 1-aminocyclopropane-1-carboxylate (ACC) oxidase were isolated from rice seedling cDNA library. pOS-ACO3 is a 1,299 bp full-length clone encoding 321 amino acids (Mr=35.9 kDa), while pOS-ACO2 is 1,072 bp long and is a partial cDNA clone encoding 314 amino acids. These two deduced amino acid sequences share 70% identity, and display a high degree of sequence identity (72-92%) with previously isolated pOS-ACO1 of deepwater rice. The chromosomal location studies show that OS-ACO2 is positioned on the long arm of chromosome 9, while OS-ACO3 on the long arm of chromosome 2 of rice genome. A marked increase in the level of OS-ACO2 transcript was observed in IAA-treated etiolated rice seedlings, whereas the OS-ACO3 mRNA was greatly accumulated by ethylene treatment. Results of ethylene inhibitor studies indicated that auxin promotion of the OS-ACO2 transcription was not mediated through the action of auxin-induced ethylene. Thus, it appears that there are two groups of ACC oxidase transcripts in rice plants, either auxin-induced or ethylene-induced. The auxin-induced OS-ACO2 expression was partially inhibited by ethylene, while ethylene induction of OS-ACO3 transcription was completely blocked by auxin. These results indicate that the expression of ACC oxidase genes is regulated by complex hormonal networks in a gene specific manner in rice seedlings. Okadaic acid, a potent inhibitor of protein phosphatase, effectively suppressed the IAA induction of OS-ACO2 expression, suggesting that protein dephosphorylation plays a role in the induction of ACC oxidase by auxin. A scheme of the multiple regulatory pathways for the expression of ACC oxidase gene family by auxin, ethylene and protein phosphatase is presented.

We have previously transformed rice (Oyrza sativa L.) with the maize ribosome-inactivating protein b-32 gene (Zmcrip3a) and the phosphinothricin resistance gene (bar). In the present study, Southern blot hybridization analysis of 56 primary fertile transformants resulted in distinct band patterns, indicating that all the transformants had been generated by independent integration events and 30% of them contained a single copy of the transgene. Segregation analysis of 15 R0 plants revealed that transgene was stably transmitted to their progenies and Southern blot band patterns of R1 progenies remained the same as the corresponding parents, suggesting that all the loci of multiple integration events are genetically linked. Also, in most of the lines, physical presence of the b-32 transgene co-segregated with the phosphinothricin- resistant phenotype, confirming that the transgene is behaving as a normal locus in the genome. However, some of R1 seedlings that contained multiple copies of the transgene became sensitive to phosphinothricin, indicating that its expression was silenced. Immunoblot analysis demonstrated that b-32 protein was produced and the levels of expression differed in different lines, estimated to be 0.5–1% of total soluble protein in the leaf tissues. In addition, the transgene-encoded protein was preferentially processed in germinating seeds and young leaves of R2 transgenic plants in a way similar to that in maize kernels, suggesting that the processing mechanism is conserved in the germination stage between rice and maize.

To mutagenize rice genomes, a two-element system is utilized. This system comprises an immobile Ac element driven by the CaMV 35S promoter, and a gene trap Ds carrying a partial intron with alternative splice acceptors fused to the GUS coding region. Rapid, large-scale generation of a Ds transposant population was achieved using a plant regeneration procedure involving the tissue culture of seed-derived calli carrying Ac and Ds elements. During tissue cultures, Ds mobility accompanies changes in methylation patterns of a terminal region of Ds, where over 70% of plants contained independent Ds insertions. In the transposon population, around 12% of plants expressed GUS at the early seedling stage. A flanking-sequence-tag (FST) database has been established by cloning over 19,968 Ds insertion sites and the Ds map shows relatively uniform distribution across the rice chromosomes.

During brown planthopper (BPH) feeding on rice plants, we employed a modified representational difference analysis (RDA) method to detect rare transcripts among those differentially expressed in SNBC61, a BPH resistant near-isogenic line (NIL) carrying the Bph1 resistance gene. This identified 3 RDA clones: OsBphi237, OsBphi252 and OsBphi262. DNA gel-blot analysis revealed that the loci of the RDA clones in SNBC61 corresponded to the alleles of the BPH resistant donor Samgangbyeo. Expression analysis indicated that the RDA genes were up-regulated in SNBC61 during BPH feeding. Interestingly, analysis of 64 SNBC NILs, derived from backcrosses of Samgangbyeo with a BPH susceptible Nagdongbyeo, using a cleaved amplified polymorphic sequence (CAPS) marker indicated that OsBphi252, which encodes a putative lipoxygenase (LOX), co-segregates with BPH resistance. Our results suggest that OsBphi252 is tightly linked to Bph1, and may be useful in marker-assisted selection (MAS) for resistance to BPH.

Root hairs are long tubular outgrowths that form on the surface of specialized epidermal cells. They are required for nutrient and water uptake and interact with the soil microflora. Here we show that the Oryza sativa cellulose synthase-like D1 (OsCSLD1) gene is required for root hair development, as rice (Oryza sativa) mutants that lack OsCSLD1 function develop abnormal root hairs. In these mutants, while hair development is initiated normally, the hairs elongate less than the wild-type hairs and they have kinks and swellings along their length. Because the csld1 mutants develop the same density and number of root hairs along their seminal root as the wild-type plants, we propose that OsCSLD1 function is required for hair elongation but not initiation. Both gene trap expression pattern and in situ hybridization analyses indicate that OsCSLD1 is expressed in only root hair cells. Furthermore, OsCSLD1 is the only member of the four rice CSLD genes that shows root-specific expression. Given that the Arabidopsis (Arabidopsis thaliana) gene KOJAK/AtCSLD3 is required for root hair elongation and is expressed in the root hair, it appears that OsCSLD1 may be the functional ortholog of KOJAK/AtCSLD3 and that these two genes represent the root hair-specific members of this family of proteins. Thus, at least part of the mechanism of root hair morphogenesis in Arabidopsis is conserved in rice.

With the completion of genomic sequencing of rice, rice has been firmly established as a model organism for both basic and applied research. The next challenge is to uncover the functions of genes predicted by sequence analysis. Considering the amount of effort and the diversity of disciplines required for functional analyses, extensive international collaboration is needed for this next goal. The aims of this review are to summarize the current status of rice mutant resources, key tools for functional analysis of genes, and our perspectives on how to accelerate rice gene discovery through collaboration.