Phosphate starvation constrains plant growth severely. To examine plant responses under phosphate deprived conditions, we carried out genome-wide gene expression analysis using Arabidopsis DNA array technology. Arabidopsis plants grown for 21 days in liquid medium were transferred to medium containing low concentrations of phosphate until 72 hours. Macro(JCAA) and micro(Agilent Tech., USA) DNA array carrying non-redundant 22,064 Arabidopsis genes were used for hybridization of mRNA isolated from the plants. Significant changes of transcripts were seen for 70 genes including AtPT2, and RNase1, SQD1 and SQD2. Induction of genes involved in glycolysis was also seen. Contrary, several photosynthesis genes and nuclear-encoded chloroplast targeted enzyme genes were down-regulated. Dynamic adaptation to phosphate deprivation in chloroplast and cytosol will be discussed.

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The objective of this study is to perform transcriptomic analyses of rice genes up- or down-regulated by the P-replenishment. cDNA microarray analyses were performed for total RNA of rice plants cultured hydroponically with P treatment (+P, -P, and P-replenishment). The number of genes regulated by P replenishment was about several times higher than that by the -P. OsPI1 gene, which was isolated as the most significant up-regulated gene by -P, was the most significant down-regulated gene by P-replenishment in both roots and shoots. Most of genes down-regulated by P-replenishment were up-regulated by -P, suggesting that their responses were specific in -P conditions. Contrary number of most transcripts up-regulated by the P-replenishment was also larger in P-replenishment than in +P. We considered that the genes up-regulated by P-replenishment played an important role on P acquisition from P deficient plants.

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Plants develop strategies to recycle phosphorus so that all organs receive adequate amounts, especially new growing organs. As many phosphorus compounds are closely related to carbon metabolism, the expression level of the following enzymes were evaluated by RT-PCR: Triose phosphote translocater, phosphoenolpyruvate/phosphate translocator (PEP/Pi translocator), phosphoenolpyruvate carboxylase, pyruvate kinase, NAD dependent glyceraldehydes 3-phosphate dehydrogenase, NADP dependent glyceraldehydes 3-phosphate dehydrogenase. Under phosphate deficient conditions, the level of inorganic phosphate decreased dramatically. Because the expression level of PEP/Pi translocator increased dramatically, the carbon flow from cytosol to chloroplast as PEP may play an important role in maintaining carbon metabolism under phosphate deficient condition.

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Living organisms respond to phosphate limitation by expressing various genes whose products maintain an appropriate range of phosphate concentrations within each cell. We identified a two-component system, which consists of histidine kinase Hik7 and its cognate response regulator Rre29, that was the dominant sensory system that controled gene expression in response to phosphate limitation in the cyanobacterium Synechocystis sp. PCC 6803.
The response regulators PhoB in E. coli and PhoP in B. subtilis bind specifically to conserved sequences, known as Pho boxes. In order to investigate the possibility that Synechocystis might also have a Pho box, we examined whether Rre29 can bind to the upstream flanking regions of the phosphate limitation-inducible genes. We found that Rre29 bound to the upstream flanking regions of several phosphate limitation-inducible genes at repetitive PyTTAAPyPy(T/A)-like sequences, suggesting that this repetitive sequence might be the Pho box in Synechocystis .

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A carrot mutant cell line, which was selected in Al-phosphate medium previously, could utilize the phosphate from Al-phosphate by enhanced citrate excretion. Physiological studies of citrate efflux from the mutant indicated that this system would be consisted of anion channel and PM H⁺-ATPase. The mutant releases electrochemically equivalent H⁺ during citrate excretion from the PM, which is possibly depended on its higher activity of PM H⁺-ATPase. In this study, we further characterized PM H⁺-ATPase in the mutant by molecular biological approaches. We isolated six kind of cDNAs encoding PM H⁺-ATPase by de-generate PCR. Transcriptional analysis revealed that DcPA1;1 gene, which is most abundant isoform in the mutant, was three times higher than that of wild type cells. Anti-sense reduction of DcPA1;1 in the mutant caused significant decrease of citrate excretion. These results indicate that higher PM H⁺-ATPase activity in the mutant was essential for its greater ability of citrate excretion.

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NOB is a line of transgenic Arabidopsis that expresses GFP according to sulfur status (*1). From NOB M2, nbm2(NOB mutant 2) -1 and 2 were isolated, in which GFP fluorescence increased. NBM2 locus was mapped to a 55 kb region on the chromosome 3, containing ten genes, including BIG. Phenotypes of the big mutants (*2) were similar to nbm2. Independent nonsense mutations were found in the BIG ORF of nbm2 mutants. In nbm2-2, concentrations of sulfate and glutathione did not significantly change, but concentration of nitrate decreased. Additionally, cysteine content was reduced, while γ-EC content was increased to some extents. Accumulation of mRNA corresponding to the γ-EC synthetase gene was elevated in nbm2. Polar auxin transport (PAT) is deficient in the big mutant. Possible relationship between PAT and sulfur deficiency response are being investigated.
*1 Ohkama et al. (2002) PCP 43,1493-1501: *2 Gil et al. (2001) Genes Dev. 15:1985-1997

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During sulfur starvation, the induction of sulfate uptake is associated with the increase of mRNA abundance of sulfate transporters. In addition, N and C status are reported to control sulfate uptake. Though phytohormons elicit critical cues for the regulation of nitrate and phosphate acquisition, their roles in sulfate uptake have not been investigated. In Arabidopsis, two high-affinity sulfate transporters SULTR1;1 and SULTR1;2 expressed in root epidermis and cortex contribute to the uptake of sulfate. We tested the effects of phytohormons on SULTR1;2 expression. Among the number of phytohormons, cytokinin significantly down-regulated the accumulation of SULTR1;2. Sulfate uptake was down-regulated by cytokinin, and the rate of down-regulation was moderated in cre1-1 mutant. The cytokinin dependent-regulation of the counterpart transporter, SULTR1;1, was proved in sultr1;2 knockout plants. However, the response of SULTRs to the external sulfur status was similar between Ler and cre1-1, suggesting that sulfur and cytokinin independently regulate sulfate uptake.

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The molecular mechanisms of plant responses to iron deficiency remain largely unknown. To identify the cis-acting elements responsible for iron-deficiency-inducible expression in higher plants, the barley IDS2 (iron deficiency specific clone no. 2) gene promoter was analyzed using a transgenic tobacco system. Deletion and linker-scanning analysis clearly identified two cis-acting elements: IDE1 (iron-deficiency-responsive element 1) at -153/-136 (relative to the translational start site) and IDE2 at -262/-236. IDE1 and IDE2 synergistically conferred the iron-deficiency-inducible and root-specific expression. Expression occurred mainly in the root pericycle, endodermis, and cortex. The principal modules of IDE1 and IDE2 were homologous. Sequences homologous to IDE1 were also found in many other promoters of barley, rice, and Arabidopsis iron-deficiency-inducible genes, suggesting the conservation of cis-acting elements in various genes and species. This is the first report identifying cis-acting elements responsible for micronutrient-deficiency-inducible expression in higher plants.

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The fern gametophytes are free-living unlike those of a seed plant, meaning that the gametophytes have to adapt to the external environment. In this work, we characterized the heavy metal tolerance and accumulation in the gametophytes of Athyrium yokoscense, because the sporophyte is known as a metal hyperaccumulator.
First, we examined the effects of Pb²⁺, Cd²⁺, Cu²⁺, Zn²⁺ and H₂AsO₄^- on fern spore germination. The germination of Athyrium spores was highly tolerant to these metals among several fern species, although Pteris vittata spores showed intensive tolerance to H₂AsO₄^-. Athyrium gametophytes were also found to accumulate lead up to 23,337 mg/kg dw when the gametophytes were cultured in the presence of 10 μM Pb²⁺ Furthermore, it was shown that the lead was accumulated in the rhizoid, as determined by histologic staining method with rhodizonate. At present, we are further analyzing the localization of lead using TEM.

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To investigate the mechanism of Ni tolerance in common plants, we selected cell lines of tobacco (Nicotiana tabacum L.cv.BY-2 ) tolerant to 700μM NiCl₂ (NIT cells). The NIT cells exhibited slight tolerance to excess of Cu and Al, and high tolerance to excess of Ni especially. Contents of Ni, Fe and Mg in NIT cells increased obviously under Ni treatment. Organic acids levels were constitutively high in NIT cells. Histidine level was 3 to 5 fold higher in the NIT cells with 700μM NiCl₂ than in the wild-type cells without Ni. Complexation with organic acids or histidine has been identified as an important mechanism for detoxifying Ni. We have also found the NIT specific proteins secreted to medium constitutively. To determine the localization of Ni, isolation of protoplasts and vacuoles from NIT cells is undergoing.

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Yeast PCA1, which is assigned a member of the heavy-metal-transporting P-type ATPase, is localized in plasma membrane and export copper. Yeast CAD2 that is identified as mutated PCA1 acquires the ability to transport cadmium as well as copper, conferring copper and cadmium resistance by exporting these metals. In this study, we analyzed the heavy metal resistance of transgenic Arabidopsis overexpressing yeast the PCA1 or CAD2 gene.
On MS medium, each transgenic plant carrying PCA1 orCAD2 showed retarded growth of roots, as compared with the wild type plants. Furthermore, we observed that on MS medium containing high concentration of each metal, the PCA1 transgenic plants showed resistance to copper and the CAD2 transgenic plants showed resistance both copper and cadmium. These results indicate that Yeast PCA1 or CAD2 protein function as efflux pump of heavy metal in plant cells and controlled intracellular heavy metal homeostasis and resistance.

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Nicotianamine synthase (NAS) and nicotianamine aminotransferase (NAAT) are the enzymes in the biosynthetic pathway of mugineic acid family phytosiderophores (MAs). MAs are metal chelators biosynthesized in graminaceous plants, and NA, a chelator of metals, is ubiquitously present in higher plants. NA is essential for reproductive growth, and the NA-less tobacco was sterile (Takahashi et al., 2003, 15, 1263-1280, Plant Cell).
In this study, we investigated NAS gene expression in inflorescences and seeds of Arabidopsis using the reporter gene (GUS) to illuminate the roles of NA at reproductive stage in dicot. Similarly, to illuminate the role of NA and MAs at reproductive stage in monocot, we investigated the gene expression of NAS and NAAT in inflorescences and seeds of rice. The results indicated that NA and MAs play a critical role in embryogenesis in addition to a role in metal translocation to inflorescence and seeds.

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There are organelles having complicated structures and highly differentiated functions in a plant cell. Organelles ,such as chloroplast and mitochondria, mainly consist of products that are encoded by the genes in nucleus genome.
A fuge information about these genes were provided from the genome projects, and next effort is needed to understand "where" "when" "how" and "which" gene product is expressd and localized in a cell.
In this study, We developed a system to visualize the gene products. This system distribute GFP gene with transposon, resulting insertional mutations which would express the fusion protein. This trick makes possible to find out the functional wild type proteins in organelles at the heterozygous generation. It also makes possible to observe phenotype of homozygote in the next generation. Here, we'd like to introduce some succeeded examples.

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The plastid has its own semi-autonomous genetic system. It has been known the expression of plastid genes depends at the translational level, rather than the transcription and the RNA accumulation levels.
The machinery of plastid genetics system is similar to that of prokaryotes, whereas the translation mechanism is distinguished between them. The translation of prokaryotic mRNAs is initiated through the Shine-Dalgarno (SD) sequence of the mRNAs. The tobacco plastid mRNA is comprised of 79 species, but, two-thirds of them do not contain SD-like sequences at a proper position. A question is what mechanism operates for the plastid mRNA translation.
We have characterized the translational level by our tobacco chloroplast DNA microarray with total RNA and polysome-associated RNA (translationally activated mRNA) isolated from tobacco plants. Then, conserved sequences within the 5' and 3' UTRs have been identified. We discuss possible translation mechanisms for plastid mRNAs.

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We isolated the pcb2 (pale-green and chlorophyll b reduced 2) mutant from the EMS mutant library of Arabidopsis. This mutant exhibits pale-green phenotype and contains only one third the amount of chlorophyll compared to wild-type. Especially the amount of chlorophyll b was reduced dramatically. Eelectron microscopic observation of pcb2 mutant mesophyll cells showed that their grana stacks were remarkably decreased, although both the size and the number of chloroplasts were almost the same as those of wild-type. The responsible gene was mapped to an area of 190kb length at the upper arm of the 5th chromosome, where 49 genes were found. Comparison of DNA sequences revealed a single nucleotide substitution causing nonsense mutation in one of the genes. This gene has similarity to genes for proteins from Oryza sativa and Chlorobium tepidum whose function has, however, not yet been determined.

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The cotyledons are thought to play crucial role in the development of the plant. They provide the essential energy to develop the true leaves, and recognize the environmental signals, such as light, water and organic compounds for adaptation. The function of cotyledons is not clear. The cotyledons are located in the first step of developmental stage of embryo, and are structurally and functionally different from true leaves.
The wco (WHITE COTYLEDONS) mutant was reported to exhibit an albinophenotype in the cotyledons, but develop normal true leaves. It was characterized to be defective in the maturation of 16S rRNA that is necessary for the translation of chloroplast genes.
There is a remaining question why wco mutation causes abnomality in chloroplasts in a cotyledon-specific manner. We performed genetical and physiological analysis on wco mutant to address the problem.

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In the higher plants, the nuclear genome encodes many proteins that are involved in the constitution of the chloroplast. A few genes such as ARC, FtsZ have been reported to be involved in the division of the chloroplast. Many other genes should be required to completely explain chloroplast development.
An Arabidopsis thaliana mutant line, 5-45, isolated from the EMS mutagenised population contained chloroplasts abnormal in both size and morphology. Sizes were not uniform, ranging from about one-fifth to several times that of wild type chloroplasts. Some were not round and some looked like buddings of yeast.
The mutation was mapped to the top arm of chromosome 5(8116kb~8118kb). The 5-45 failed to complement arc11, indicating that 5-45 is a new allele of arc11 regarding abnormality of chloroplast division.
We present the identification of the mutation of both 5-45 and arc11.

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The molecular mechanism of RNA degradation in Escherichia coli includes a series of sequential steps. The degradation starts with the initial endonucleolytic cleavage carried out primarily by RNase E. The cleaved products are then polyadenylated at their 3' ends and degraded exonucleolytically by polynucleotide phosphorylase (PNPase) and RNase II. In chloroplast, many characteristics of the gene expression system resemble those of bacteria because chloroplast is believed to have an evolutionally prokaryotic origin. Here, we isolated Arabidopsis mutants with disrupted homologus genes of bacterial RNases by T-DNA insertion. Based on the analysis of leaf color, growth rate and chlorophyll fluorescence, we identified putative chloroplastic RNase mutants; RNase E, PNPase and RNase II/R. PNPase mutant have etiolated leaves in proximal regions. RNase E and RNase II/R mutants could not grow photoautotrophically, indicating their essential roles in chloroplast RNA metabolism. We are now characterizing these mutants in detail.

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To identify Arabidopsis nuclear genes responsible for chloroplast development, we have identified a large number of albino and pale-green (apg) mutants by screening of Ds-tagged lines. One of the apg mutants, apg6, has Ds insertion in a gene encoding a Hsp101 homolog of heat shock protein. We have isolated its revertants, and also identified three alleles of apg6. All these three alleles showed same phenotype, pale-green. These results suggest a role of the Hsp101 homolog in chloroplast development. The APG6 protein contains a transit peptide that function chloroplast localization, but no transmembrane domain. Based on electron micrographic observation of chloroplasts of apg6 cells, apg6 plastids were smaller than those of wild type, and contained undeveloped thylakoid membrane. The expression of APG6 is strongly increased by heat stress. These results suggest that the APG6 protein may function as a chaperon in chloroplast stroma.

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PpRPOT1 and PpRPOT2 transcripts possess two in-frame AUG codons at the N-terminus. Observation of transient and stable transformants expressing the N-termini of PpRPOT fused with GFP suggested that both PpRPOT1 and PpRPOT2 are exclusively translated from the second AUG, and these are targeted to only mitochondria, although these proteins are potentially targeted to plastids when translation is forced to start from the first AUG codon. When the translation efficiency of the two AUG codons in PpRPOT1 and PpRPOT2 was evaluated using GUS reporter, the second AUG codon is used as the sole translation initiation site. One of the Arabidopsis homologs AtRpoT;2 that possesses two initiation AUG codons as do the PpRPOTs has been regarded as ′dually targeted′ protein. When the localization of AtRpoT;2 with GFP-fused constructs containing the 5′-UTR and N-terminal sequence containing two AUG codon was tested, GFP-AtRpoT;2 was localized to only mitochondria.

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C.merolae is a primitive red alga, that retains four transcripiton factors (Ycf27-30) in the plasitd genome. They could participate in the transcription regulation of the plastid genes corresponding to the various environmental stimuli. To make clear respective roles of Ycf27-30 in C. merolae will shed lights on the regulatory models of photosynthethic gene expression. Interestingly, RuBisCo genes in red algae are much more closely related to a-proteobacteria than to cyanobacteria because of lateral gene transfer during evolution. In the meeting, we will present evidence indicating that the transcription of RuBisCo operon is activated by plastid-encoded transcription factor, Ycf30. And Northen hybridization and RT-RCR analysis revealed the possibility that Ycf29 is involved in the transcription activation of the genes concerned with the phycobiliproteins during the acclimation from high light condition to low light condition. Analysis of the regulatory mechanism is in progress.

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Chloroplast contains a eubacteria-like multi subunit RNA polymerase (PEP) that is responsible for transcription of plastid-encoded photosynthesis-related genes. Exchangeable nuclear-encoded sigma factors confer promoter specificity of PEP. In bacteria, most of the housekeeping genes are transcribed by the RNA polymerase containing primary sigma factors, while alternative sigma factors are involved in transcription of stress responsive genes. Arabidopsis encodes six chloroplast sigma factors. However, general sigma factors important for transcription of standard photosynthesis-related genes have not been identified in chloroplasts. The AtSIG6 null mutant shows pale green phenotypes in early cotyledons. Accumulation of most PEP-dependent transcripts was diminished in young seedlings of the sig6 mutant. All of the AtSig6-dependent genes are preceded by the σ⁷⁰-type promoters. We concluded that AtSig6 is one of the general sigma factors in chloroplasts that recognize σ⁷⁰-type promoters and it plays an important role in early chloroplast development in cotyledons.

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The plastid genome contains more than one hundred genes and the expression is regulated depending on plastid types and environmental conditions. Sigma factors determine the specificity of the eubacteria-type plastid RNA polymerase (PEP), and the roles of sigma factors for chloroplast gene expression remain largely unknown. In this study, we analyzed the expression of the sigma factors (SIG1-6) under various stress conditions, and found that the SIG5 transcript was rapidly and drastically induced under various stresses conditions. Because this induction was well correlated with the activation of the light-responsive promoter (LRP) of psbD, and because the psbD-LRP activation was abolished in the newly identified sig5 mutant (sig5-2), we conclud that SIG5 is a stress-responsive sigma factor in Arabidopsis, and responsible for the psbD-LRP transcription. We also found that the restoration of PSII activity after high light irradiation was delayed in the sig5 mutant, suggesting roles of SIG5 under stress conditions.

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Photosynthesis occurs in chloroplasts in the light, where in-organello environment is drastically changed in the day/night cycle. Since active photosynthetic components such as D1 protein are rapidly replaced with newly synthesized ones, gene expression must be regulated through sensing the environmental change. Transcription of photosynthesis genes in chloroplasts is accomplished with an eubacterial-type RNA polymerase (PEP), which requires multiple nucleus-encoded sigma factors. We have made Arabidopsis transgenic with wild-type and putative phosphorylation sites-deleted SIG1 molecules, the major species of sigma factors, to express them ectopically. The plants were labeled with [³²P]orthophosphate in vivo and SIG1 was recovered with antibodies. Molecular mass of complexes associated with SIG1 was examined by sucrose density gradient centrifugation. The overall results lead to the conclusion that the phosphorylation of SIG1 with Ser/Thr protein kinase in the dark interferes with formation of active holoenzyme to transcribe the photosynthesis genes.

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Glutamine synthetase (GS) is one of the key enzyme that plays a central role in N-assimilation in plants. We have previously prepared transgenic tobacco with high level of GS2 by Agrobacterium-mediated nuclear transformation and showed a large capacity for photorespiration and high tolerance against high light in tobacco plants. In the present report, we tried to develop overexpressors of GS2 by means of chloroplast transformation.
A rice GS2 cDNA without a transit peptide is expressed from the plastid psbA promoter and 5'-untranslated region, known to be up-regulated in green leaves. The GS2-expressing cassette was introduced into tobacco chloroplast genome by homologous recombination. The expression of GS2 in the transplastomic tobacco was 30-40 times higher than that in wild-type plants. The enzymatic activity of GS in the transplastomic tobacco was 7-9 times higher than that in wild-type plants. The physiological feature of the transplastomic tobacco is under investigation.

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A growing body of evidence indicates that a RecA-mediated recombination system exists in chloroplast. However very little is known about mechanism for plastome homologous recombination. Using E. coli producing recombinant protein, we examined the enzymatic properties of chloroplast-targeted RecA homologue, recA-AT, encoded on the Arabidopsis nuclear genome (see Cerutti et al 1992). Although predicted mature recA-AT showed ATPase and DNA-binding activities, it had little strand exchange activity. More detailed characterization revealed that enzymatic properties of the recA-AT were distinct from those of authentic E. coli RecA: the effect of DNA on ATPase activity, the strength of DNA-binding activity and the migration pattern of DNA-protein complex in gel retardation assays. These differences between the recA-AT and the RecA raise a possibility that biochemical mechanism of the plastome homologous recombination is different from that of RecA-mediated homologous recombination in E. coli. Supported by METI/NEDO partly.

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Mitochondrial citrate synthase was overexpressed in Eucalyptus plant and its effects on organic acid excretion and low phosphorus tolerance were examined. The transformants and control plants were grown with a hydropoinc culture solution containing AlPO₄ as a sole phosphorus source. CS activity in roots of the transformants showed 1.4- to 2.5-fold increase in comparison with that in the control. Symptom of phosphorus deficiency was ameliorated in the transformants as judged form their phosphorus content. One transgenic line showed slightly higher (1.6-fold) citrate excretion than the control. This line was then grown with an acid soil containing insoluble phosphate and its growth tended to be slightly greater than that of the control. Therefore, it is suggested that overexpression of mitochondrial citrate synthase improves the growth performance of Eucalyptus plant under low phosphorus stress due to the high ability of phosphate-acquisition caused by the enhanced citrate excretion.

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Large DNA fragments of ~100kb can be introduced into plant genomes using the transformation-competent artificial chromosome vector TAC developed by Liu et al. (Proc. Natl. Acad. Sci. USA (1999) 96: 6535-6540). We are currently working on transferring many Arabidopsis genes into tomato to elucidate the possibility of transferring new traits from the model plant. We selected Arabidopsis genomic TAC clones that contained disease resistance related genes for transformation of a dwarf tomato, Micro-Tom. In this study, we analyzed 33 transgenic tomato plants carrying, in total, 11 disease resistance-related genes. RT-PCR primers were designed at the intron junctions for detecting splicing in the transgene transcripts. All transcripts from these genes detected in transgenic tomato were spliced as the same manner as in Arabidopsis. Alternative splicing detected in Arabidopsis was also observed in the transgenic tomato. We are also analyzing expression of these transgenes under disease-stress conditions.

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To investigate biological diversity between plants, which diverged more than 100 million years ago, we transferred Arabidopsis large genomic DNA fragments to tomato by using TAC vector for the expression analysis of various genes. Expression of genes on the DNA fragments were analyzed by using DNA array. No obvious effect on the internal tomato gene expression analyzed was seen in the transgenic tomato plants. By comparing expression patterns found in leaf, rachis (petiol) and root of these plants, transgenes were classified into two groups. The first group contains genes whose expressions were similar in two species, suggestting a conservation of transcription regulation in these plants. Contrary, the genes that exhibited distinct expression between plants are categorized in the other group. These result provides a landmark to understand biological diversity at molecular level in plants.

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A single amino-acid change (S653N) in the acetolactate synthase (ALS) protein of Arabidopsis confers resistance to the herbicide imazapyr. We have constructed a binary vector, which has a deleted, non-functional fragment from ALS gene, carrying the mutant site specifying herbicide imazapyr resistance. After in planta Agrobacterium-mediated transformation of Arabidopsis, we have selected imazapyr resistant plants. Four imazapyr resistant plants were obtained from 780,000 seeds. S653N mutation was found in two of these imazapyr resistant plants. This result showed that GT by homologous recombination had occurred between the endogenous ALS gene and the engineered ALS gene fragment. From extended analysis, one of the targeting events was concluded as true gene targeting (TGT) effable by double cross over model and another one was concluded as ectopic gene targeting (EGT) arising by modification of vector DNA by chromosomal template and its random integration into Arabidopsis genome.

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We have developed a rice gene targeting (GT) system by homologous recombination. As a model for an endogenous natural gene to be targeted, we have chosen the Waxy gene and achieved a reproducible GT without any detectable ectopic events. All the targeted plants were heterozygotes at the Waxy locus and the modified waxy allele segregated into their selfed progeny in Mendelian fashion (Annual Meeting of JSPP, 2002; 2003). Since our GT procedure with a strong positive-negative selection is in principle applicable to any rice gene, we have begun to apply one of multiple genes residing adjacent to multi-copy retroelements. We chose the Adh1 and Adh2 genes flanking a copia-like and a gypsy-like retrotransposon on chromosome 11. We outline our experimental design including the constructed vectors and describe PCR analysis of rice calli obtained for GT of these Adh genes.

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Rice (Oryza sativa L.) is an important staple food and a model plant for cereals. The gene targeting (GT) by homologous recombination is a powerful tool for elucidating gene function in rice. To demonstrate GT is applicable to other gene than Waxy in rice, we are trying to modify the Adh genes for alcohol dehydrogenase. Both Adh1 and Adh2 genes on chromosome 11 encode about 40 kDa proteins, which are functional as homodimers or heterodimers. By homologous recombination, the Hm^r positive marker connected with the transcriptional stop region of the En transposon is designed to be integrated into exon 1 of the Adh2 gene, upstream of the ATG initiation codon. PCR analysis of the calli obtained indicated that successful GT occurred at the Adh2 locus. Based on the results of Southern analysis, possible recombination events are discussed.

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The Wx gene encodes granule-bound starch synthase and is specifically expressed at pollen and endosperm. We constructed two RNAi vectors, pWRI-A and pWRI-B, which transcribe the sense and antisense Wx 5'UTR RNA with intron loop. We analyzed rice transgenic plants carrying these RNAi vectors and investigated both tissue-specific RNAi and the quantitative regulation of suppression efficiency. As a result, the 140bp of sense and antisense 5'UTR dsRNA from both constructs can be a trigger of RNAi and lead to silencing of Wx gene in the endosperm. Furthermore, the transgenic rice carrying pWRI-B which carry the substitution mutation at 5' splice site of the intron leads reduction of silencing effects on quantitative regulation of RNAi. However, in pollen, no severe suppression of Wx gene was observed.
In this report, further investigation of the effects of intron- or the foreign sequence-spacer on RNAi by introduction of various RNAi vectors are discussed.

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Transgenic plants exhibit various levels of expression of the transgene, believed to be due to "positional effects". We hypothesize that long sequences flanking to a transgene stabilize expression of the gene in plants. According to the hypothesis, we introduced the reporter gene GUS between flanking sequences of 32 kb and 41 kb derived from Arabidopsis thaliana in the binary vector Transformation-competent Artificial Chromosome (TAC). Without the long flanking sequences, many transgenic Arabidopsis exhibited gene silencing, that was evident in the presence of small RNA species (20-25 nt) detected by the transgene probe. On the other hand, a few transgenic plants carrying the transgene with the flanking sequences exhibited gene silencing only when multiple insertion of the transgene on a chromosome(s) occurred. These results indicate that the flanking sequences suppress gene silencing of the transgene. The long flanking sequences also facilitated production of transgenic plants carrying only a single transgene.

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Transgene expression is notoriously variable between different, independently generated, transgenic plants. The eukaryotic genome, organized in a complex, heterogeneous structure termed chromatin, is not homogeneous for transcriptional activity. The variation of transgene expression is due to random integration into the chromosome. Specialized DNA sequences known as insulators protect genes from both the positive and negative influences of nearby chromatin. We use an insulator from the sea urchin Ars gene to function in plant cells. Normally, expression of an introduced chimeric GUS (β-glucuronidase) gene is inactivated in approximately 60% of the transformed tobacco BY2 clones. Transgenes containing the Ars insulator, however, were expressed in all transformed tobacco BY2 cells. These results suggest that the insulator functions to suppress the variation of transgene expression in tobacco BY2 cells.

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Transcription factors control plant development and responses to environmental changes. Gene redundancy often interferes with many efforts for functional analyses of transcription factors using gene-knockout, antisense or RNAi technologies. We have developed a novel system, CRES-T, for functional analysis of redundant transcription factors. In this system, expression of the chimeric gene for a transcription factor fused to the EAR repression domain suppress the target genes and induce loss-of-function phenotypes in transgenic plants.
To clarify function of the TCP family in vivo, we made transgenic plants in which express the fusion gene for the chimeric TCP repressor. The transgenic plants had abnormal cotyledon and leaves with uneven shape and curvature. By contrast, the transgenic plants expressing TCP gene showed no visible phenotype. These results indicate that the TCP family has functions in development of cotyledon and leaf shape.

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Yeast two-hybrid (YTH) screen provides a powerful genetic approach to study protein-protein interactions. In order to apply YTH screening in genome scale analysis in Synechocystis sp. PCC6803, we have implemented the GAL4-based YTH method and developed a high throughput YTH system by introducing the improved vectors, yeast mating-based screening method, and culture system using the MultiScreen plate.
Using this system, we have examined possible protein-protein interactions with two-component signal transducers and Synechocystis genes whose orthologues are conserved in the Arabidopsis genome. As a result of the screening with 689 genes as baits, a total of 1,248 independent two-hybrid interactions were identified.
As an extension of this line, we initiated large-scale interaction analysis of the Synechocystis genes with no functional annotation. The latest status of the YTH screening and the interaction network thus obtained will be presented.

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To investigate the whole genetic system of legumes, we initiated
large-scale sequencing of the genome of a model legume, Lotus
japonicus. Using the information on ESTs and cDNA markers of
legume plants, genomic clones corresponding to the multiple seed
points of the genome have been chosen as initial targets. As the
accumulation of the seed sequences has progressed, clone selection by
walking has also initiated. Currently, a total of 1,550 clones have
been selected. One hundred and twenty seven of them are in the library
phase, 952 are in the finishing phase and 412 have been annotated. A
total of 3,975 protein coding genes were identified in 42.2
Mbp. Mapping of the seed clones has also been performed by generating
PCR-based markers from sequence information, and 1,140 clones have
been located on the linkage map. The sequence data, gene information
and mapping information are available through the WWW at
http://www.kazusa.or.jp/lotus/.

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Endosymbiotic origin of chloroplasts is believed as the most probable hypothesis. Many genes of endosymbiont origin must have been transferred to the initial photosynthetic eukaryote. We developed a software called 'Gclust' that clusters all protein sequences (about 110 thousand) of 17 representative organisms, such as eight cyanobacteria, three photosynthetic bacteria, two non-photosynthetic bacteria, two non-photosynthetic eukaryotes, as well as Arabidopsis and Cyanidioschyzon. Based on these results, the clusters that are shared by the eight cyanobacteria, plant and alga were extracted. We are trying to identify uncharacterized genes within these clusters. The 44 genes of Synechocystis are being disrupted. The 57 Arabidopsis genes are being characterized by analysis of tag lines, demonstration of chloroplast targeting and light-dependent expression.

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Arabidopsis is a model plant in which it is possible to carry out saturation mutagenesis for every coding gene. We constructed about 12,000 Ds transposon-mutated lines as a useful resource in Arabidopsis functional genomics, and sequenced the flanking region of Ds insertion for all independent lines. In our resource, we can investigate about 4,000 genes which would be disrupted by Ds insertion in coding regions. Recently, we have started a "phenome analysis", in which we observe various phenotypes systematically for each Ds insertional line. We are collecting phenotypic data for morphological observation on plant growth for 3,000 insertional lines so far. We classified the results into one hundred kinds of category of phenotypes.

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An endogenous retrotransposon of rice, Tos17, is inactive under normal conditions. Tos17 is activated by tissue culture and inactivated again in regenerated plants. These features of Tos17 make it suitable for the functional analysis of rice genes by gene disruption. More than 50,000 disruption lines have been produced. From 6,000 lines, 47,000 flanking sequences of Tos17 are determined.
Phenotype data from all disruption lines will be obtained in the next year. We constructed a relational database containing nucleotide sequences of flanking region of Tos17 insertions and phenotype data observed in the rice field. We will introduce in silico mutant screening using our web-based relational database (http://tos.nias.affrc.go.jp/) and some points for analyzing the mutant lines.

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Full-length cDNAs are essential for the correct annotation of genomic sequences and for the functional analysis of genes and their products. Using the biotinylated CAP trapper method, we have constructed full-length cDNA libraries from Arabidopsis plants and isolated 224,641 RIKEN Arabidopsis full-length (RAFL) cDNA clones. They were clustered into 18,127 nonredundant cDNA groups, about 70% of predicted genes¹⁾. We have determined full-length cDNA sequences of 13,181 RAFL cDNA clones²⁾. We have also used the RAFL cDNAs for the microarray analysis^{3), 4)} of expression profiles and the structure analysis of Arabidopsis genes. In this meeting, overview of our RAFL cDNA project will be presented.
1) Seki et al. (2002) Science 296:141. 2) Yamada et al. (2003) Science 302:842. 3) Seki et al. (2003) Topics Curr. Genet. 4:271. 4) Seki et al. (2004) J. Exp. Bot. (in press).

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About 10,000 non-redundant Arabidopsis full length cDNA (fl-cDNA) clones were mixed to make a normalized fl-cDNA over-expression T-DNA library. Agrobacterium was transformed with the normalized T-DNA library to give the corresponding Agrobacterium library. A. thaliana WT plants were dipped with the Agrobacteria library. About 1200 transformed plants were grown in green house to isolate 93 plants, which showed obvious phenotypes such as morphological abnormality, greening and pigmentation. PCR was performed to amplify 106 fl-cDNA fragments from these lines using vector specific primers. Average size of the fragments was 1,4 kb with the range between 0,3 kb and 4,2 kb. The PCR fragments were sequenced to find out that all such 43 fl-cDNA inserted newly in the genome was not redundant. We designated this activation tagging-like system as Fox Hunting System (fl-cDNA over-expressor gene hunting system) to develop it as a novel technology for the functional genomics of heterologous genomic materials.

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Here we report the complete nucleotide sequence of the chloroplast genome of Populus alba. This is the first report on angiosperm in terms of tree chloroplast genomes. The genome as a circular DNA is composed of 156,505 bp, in which a pair of 27,660 bp-inverted repeat are contained. The inverted repeat region disrupts the others into the large (85,017 bp) and the small (16,168 bp) single copy regions. The genome contains genes which are identified to have similarity to the chloroplast genes previously reported. Those are 85 potential protein-coding genes, 4 ribosomal RNA genes and 37 tRNA genes. The chloroplast genome of P. alba lacks rpl32 & rps16 though it resembles that of tobacco in overall structure. In addition to the structural organization, the expression pattern of each gene in the chloroplast genome of the P. alba will be discussed. This work was partly supported by METI.

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The complete nucleotide sequence of the chloroplast genome of Lactuca sativa (lettuce) has been determined. The chloroplast was isolated from 1-month-old seedlings by two stepwise percoll density gradient centrifugation. The Chloroplast DNA was extracted with CTAB and randomly cloned for DNA sequencing. Sequencing of the 5,000 individual clones by shotgun method covered the whole lettuce chloroplast genome. The chloroplast genome of lettuce is a circular double-stranded DNA of 152,765 bp. Two inverted repeat regions are separated by a large single copy (LSC) region of 84,103 bp and a small single copy (SSC) region of 18,595 bp. We have identified 121 genes including 80 potential-protein coding genes, 4 rRNA genes and 37 tRNA genes based on previously reported chloroplast genes. The gene content of total 80 potential-protein encoding genes in lettuce (compositae) chloroplast genome is identical with the chloroplast DNA of the different species tobacco (solanaceae). Supported by KEIHANNA/MEXT.

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GH3 gene was firstly isolated from Glycine max as early auxin inducible gene. In Arabidopsis genome, GH3 gene family was consisted of 20 members of GH3 gene. One GH3 mutant, ydk1-D was isolated from activation tagged lines as dwarfed mutant, and this mutant showed short hypocotyls, epinastic leaves, short primary root and reduced number of lateral roots. The expression level of YDK1 gene was increased by exogenously applied auxin and this induction was mainly regulated by ARF7. Here, we will report histochemical analysis of YDK1 gene expression using YDK1 promoter-GUS reporter gene construct. Strong YDK1 promoter activity was detected in lateral root branches in GUS transgenic seedlings. Our results showed good correlation between expression patterns of YDK1 gene and root phenotype that was observed in ydk-D seedlings, suggesting YDK1 might function inhibition in primary root elongation. In addition, YDK1 gene expression was down-regulated by blue and far-red light irradiations.

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Phosphorus is one of the most important nutrients and its deficiency induces several responses in plants. As one of the responses, glycolipid biosyntheses are activated during phosphate deprivation.
Monogalactosyldiacylglycerol (MGDG) is the most major lipid in thylakoid membranes of chloroplasts. In Arabidopsis, there are three MGDG synthases, which are classified into type A (MGD1) and type B (MGD2, 3). During phosphate deprivation, type B genes are activated particularly in roots. Whereas auxin efflux inhibitor inhibited the expression of type B genes in roots under phosphate-deprived condition, its expression was restored by auxin treatment, suggesting that under phosphate-deprived condition, auxin is necessary for the expression of type B genes in roots. In addition, we revealed that the induction of type B genes in that condition was inhibited by cytokinin treatment. Cytokinin may have antagonistic effects to auxin on type B expression under phosphate-deprived condition.

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In Arabidopsis, the SLR gene encodes IAA14, a member of Aux/IAA protein family. The solitary-root (slr) mutant, whitch has a gain-of-function mutation in IAA14 has no lateral roots, no root hairs, abnormality in gravitropic responses both in roots and hypocotyls, and has reduced sensitivity to auxin. In order to identify genes involved in auxin-regulated these root morphogenesis, we compared gene expression profiles between wild-type and slr mutant roots (with or without auxin treatment) by using DNA microarray (Agilent Arabidopsis-1, 14,403 genes). We will present the results on genes regulated by auxin and/or SLR gene from the microarray analysis. We are now analyzing the expression pattern of these genes.

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The solitary-root (slr) mutants in Arabidopsis, which have gain-of-function mutations in IAA14, have no lateral roots (LRs). It is suggested that the mutant IAA14 protein (mIAA14) might repress the activity of Auxin Response Factors (ARFs) that promote LR formation. To understand the relationship between tissue-specific auxin response and root morphogenesis, we analyzed the transgenic plants expressing the mIAA14-GR (glucocorticoid receptor) fusion protein under tissue-specific promoters. DEX-treated transgenic plants expressing mIAA14-GR under the IAA14 promoter showed the slr phenotypes, indicating that mIAA14-GR is functional. We found that expression of mIAA14-GR under the stele-specific promoter (SHORT-ROOT) suppresses LR initiation, indicating that auxin response in stele is important for LR initiation. On the other hand, expression of mIAA14-GR under the SCARECROW promoter (expressed in the specific cells during LR primordium development) caused the formation of LR primordia with abnormal organization. This result indicates that auxin response is also important during LR primordium development.

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Loss-of-function mutants of NPH4 are insensitive to auxin and have defects in hypocotyl tropism, hook formation, differential growth of leaf and lateral root formation. We carried out screening of suppressor mutants of nph4 from a M2 population of EMS-mutagenized nph4-103 by checking hyponastic growth of cotyledon and rosette leaf. In the second screening, we selected mutants that exhibited restored tropism of hypocotyl. Finally, we isolated 3 suppressor lines. All of them are recessive. One of them displayed epinastic leaves and restored gravitropism of hypocotyl; defects in phototropism were not restored. Another line was dwarf with dark green leaves and exhibited de-etiolated phenotype in the dark. Gravi- and phototropism were normal until 4 h from the start of stimulation. The other line was semi-dwarf with dark green leaves, and showed slight recovery of gravitropic defects, but no restoration in phototropism.

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