Anti-sense RNA, co-suppression (sense suppression) and RNAi (transcription of double strand RNA) methods have been widely used for post transcriptional suppression of a gene of interest in transgenic plants. We compared the efficiency and stability of these methods using flower color as the indicator in transgenic plants. When the expression of flavonoid 3'-hydroxyrase (F3'H) gene was successfully suppressed and rose dihydroflavonol 4-reductase was overexpressed in a red petunia, the color changed to orange. The phenotypic change frequencies were 27% and 77% for anti-sense and RNAi, respectively. When expression of the anthocyanidin synthase gene was suppressed by either of three methods in a blue torenia variety (Summerwave Blue), 50% of transgenic plants bloomed white with RNAi. In contrast less than 1% of transformants had white or pale colored flowers by anti-sense or co-suppression. RNAi is most effective to suppress the expression of the gene of interest.

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Verbena(Verbena hybrida), a Verbenaceae plant, is a popular garden plant that is suitable for ground coverage. Genetic transformation of verbena has been desired to achieve flower color modification, herbicide resistance, monitoring and remediation of polluted soil. In this study, we have established Agrobacterium mediated transformation for verbena cultivar Temari and Tapien.
Expression of butterfly pea flavonoid 3',5'-hydroxylase(F3'5'H) gene successfully changed the flower color of Temari Pink from pink to violet. The amount of delphinidin type anthocyanins increased in the transgenic plants. We have also cloned some flavonoid biosynthetic genes such as F3'5'H, flavonoid 3'-hydroxylase, dihydroflavonol 4-reductase. Their sequences showed reasonable homology to the counterparts of other plant species. Their biochemically and molecular characterization are in progress.

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To dissect the signal transduction of ABA, we studied on the expression of ABA-responsive rd29B gene, and isolated Arabidopsis mutants altered in the responses to ABA. We compared the expression of the rd29B gene and those of genes encoding transcription factors such as AREB1 and ABI5. The results indicate that ABA-responsive gene expression during germination is different from that in mature plants. We introduced a chimeric gene construct consisting of the luciferase under the control of the rd29B promoter (rd29B-LUC) into Arabidospis plants. Activation tagged lines of Arabidospis containing rd29B-LUC were obtained by transformation; mediated by Agrobacterium carrying the vectors pPCVICEn4HPT or pSKI015. We also obtained mutant seeds using EMS treatment. We screened mutants altered in ABA-response of the rd29B-LUC gene during germination or under the stress conditions in mature plants. Some mutants altered in the regulation of the rd29B-LUC gene were identified. Analysis of the mutants is in progress.

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To elucidate the signal transduction pathways between the initial drought / high-salinity-stress signal and gene expressions in Arabidopsis, we have analyzed the regulation of gene expression of rd29B in vegetative tissue. We have shown that ABA Responsive Elements (ABRE) play a crucial role in the dehydration-responsive induction of rd29B as regulatory cis-elements, and that bZIP transcription factors, ABA Responsive Element Binding Proteins (AREB), function as transcriptional activators in the ABA-inducible expression of rd29B.
In this study, to identify domains of the AREB1 protein involved in ABA-inducible transcriptional activation of rd29B, we analyzed trans-activation activity of AREB1 using a transient assay of Arabidopsis protoplasts. The analysis of mutants in this assay revealed that the N-terminal region can function as a transcriptional activation domain. Furthermore, the results of collective analyses suggested that conformational change in AREB1 via ABA-induced modification is required for the gene expression of rd29B.

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In Arabidopsis, the dehydration-responsive expression of rd29B gene is mainly mediated by abscisic acid (ABA). Promoter analysis indicated that two ABA Responsive Elements (ABREs) function as cis-acting elements, and cDNAs encoding bZIP-type ABRE binding transcription factors, AREB1 and AREB2 were isolated.
AREB1 and AREB2 require ABA for their activation as shown by their low transactivation abilities in protoplasts obtained from the aba2 mutant, suggesting that the AREB1 and AREB2 function as transcriptional activators in ABA-inducible rd29B expression, and ABA-dependent posttranscriptional modification is necessary for maximum activation. In-gel kinase assay revealed that specific ABA-activated protein kinase phosphorylates the conserved regions in the AREBs. Amino-acid substitution (Ser/Thr to Ala) of the putative target sites for CDPK in the conserved region resulted in suppression of both ABA-dependent phosphorylation and transactivation. Substitution of the Ser/Thr to Asp causes the high transactivation activity without ABA. These results suggest that ABA-dependent phosphorylation regulates the AREB transactivation activities.

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EIN2 is one of the central players in the ethylene response pathway. The amino-terminal portion of EIN2 consists of twelve putative membrane-spanning domains with similarity to Nramp metal-ion-transporters. Ion transporter activity of EIN2, however, has not been reported. By contrast, overexpression of the carboxyl-terminal portion was sufficient to constitutively activate ethylene responses, suggesting that this portion plays a crucial role in the ethylene response pathway. However, its molecular mechanism is unknown, either. Most ein2 mutants are null-type, making it difficult to identify the functional domains.
In order to identify the functional domains of EIN2, we randomly introduced a 15 bp insertion (translated to five amino acids in any frame) into the EIN2cDNA. Mutated EIN2cDNA was introduced into the ein2-5 plants, to identify the functional domains by examining the ability to complement the ein2-5 mutation. Currently, we are obtaining transgenic plants. Coming results will be discussed.

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A tobacco ERF2 binds to a GCC-box, involved in transcriptional control of defense genes in response to ethylene and elicitor. However, it has been still unclear whether ERF2 activates transcription of endogenous genes, such as a gene for chitinase, which contain GCC box in their promoter region. In this study, we examined the expression of a putative target gene of ERF2 in transgenic tobacco plants harboring CaMV35S::ERF2 or CaMV35S::ERF2:GR. In the transgenic plants, mRNA levels of BCHN and Hsr203J, which contains and lacks GCC-box in their promoter region, respectively, were examined. Most of 35S::ERF2 transgenic lines accumulated high level of mRNA for BCHN but not HSR203J. In 35S::ERF2:GR transgenic lines treated with DEX for 6 hours, BCHN mRNA accumulation was induced independently of ethylene but Hsr203J mRNA level was unchanged. These results indicate that ERF2 functions as an activator of the GCC-box mediated transcription of defense genes in tobacco cells.

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Tobacco ETHYLENE-RESPONSIVE FACTOR3 (ERF3) is a member of the ERF domain transcription factors and has an ability of downregulation of transactivation of other ERFs. To analyze the protein stability of ERF3 in vitro, we added the recombinant ERF3 into the crude extract from cultured cells and observed rapid degradation of the recombinant ERF3. To study the protein stability of ERF3 in plant cells, we prepared the transgenic callus in which over-expressed GFP-ERF3 gene but detected no fluorescence of GFP-ERF3. By contrast, the ERF domain and the repression domain were stable in vitro and in transgenic callus. These results suggest that ERF3 is an unstable protein and the ERF domain and the repression domain are not involved in the regulation of instability of ERF3 in consistent with the previous results that these two domains were not required for the interaction of ERF3 with NtUBC2.

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The hormone abscisic acid (ABA) regulates developmental processes and stress responses in plants. In this study we attention to phospholipid-based transduction mechanisms in seed germination. A physiological experiment showed PA triggers early signal transduction events that lead to the ABA responses during germination.
To test the possible role of PA in ABA signalling during germination, we measured PA production and revealed that PA increased transiently. Metabolic enzymes regulate lipid production. Therefore we investigate two kinds of metabolic enzymes, Phospholipase D (PLD) that is a PA producing enzyme and Phosphatidic acid phosphatase (PAP) that is a PA decreasing enzyme. To isolate the functional PLD gene and PAP gene during germination process, we performed expression analysis and obtained knockout plants for these candidates. The PAP knockout indicated a hypersensitive to ABA. The PLD knockout showed a reduced seed dormancy. These results suggest that PA is involved in seed dormancy and germination.

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RSG is a transcriptional activator, which regulates shoot growth by controlling endogenous level of gibberellins. Overexpression of dominant-negative mutant of RSG resulted in severe dwarfism. To understanding networking of RSG, we sought RSG-interacting proteins and succeeded in isolation of some cDNAs by tow-hybrid screen. One of them coded 14-3-3 proteins, which are conserved regulatory factors among eukaryote and known to regulate function of the target through binding to phosphorylated serine residue in it. Until now, 14-3-3 protein has revealed to regulate negatively RSG by sequester it in cytoplasm through binding with phosphorylated serine-114 of RSG. Furthermore, it has exhibited that RSG was not statically resided in cytoplasm but dynamically shuttling between nucleus and cytoplasm, by treatment of Leptomycin B that specifically inhibits nuclear export receptor Exportin1 . With the aim of fully understanding of significance of regulation of RSG by 14-3-3, we examine a signal which controls intracellularlocalization of RSG.

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Protein phosphorylation has pivotal roles in ABA and osmotic stress signaling in higher plants. Two protein phosphatase genes, ABI1 and ABI2, are known to regulate these signaling pathways in Arabidopsis. Here we demonstrate that two protein kinases, p44 and p42, are activated by ABA in Arabidopsis T87 cultured cells, and at least one protein kinase, p44, is activated not only by ABA but also by low-humidity in Arabidopsis plants. Analysis of T-DNA knockout mutants and biochemical analysis using a specific antibody revealed that the p44 is encoded by a SnRK2-type protein kinase gene, SRK2E. The srk2e mutation resulted in a wilty phenotype mainly due to loss of stomatal closure in response to a rapid humidity decrease. ABA-inducible gene expression was suppressed in srk2e. These results show that SRK2E plays an important role in ABA signaling in response to water stress.
Yoshida et al. (2002) Plant Cell Physiol. 43(12) in press

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Overexpression of PKABA1, a wheat ABA inducible Protein kinase(PK), has been shown to suppress the GA-induced promoter activity of α-amylase, suggesting that PKABA1 functions in the ABA-modulation of GA signaling. Guard cell-specific ABA-activated PK(AAPK), which control stomatal pores, has been shown to regulate plasma membrane ion channels. Both PKABA1 and AAPK belong to SnRK2 PK family. However, the functions of these PKs in ABA signaling pathway is still unclear. There are 10 members of SnRK2 family in the rice genome. We report here the expression and function of the ten rice SnRk2 members, designated SAPK(Stress and/or ABA regulated PK) 1 to 10. Transient expression of affinity and epitope-tagged SAPK1 to 10 revealed that all of these kinases are activated by osmotic stress. Three of these members are also activated by ABA treatment. These results suggest that rice SAPK family members are involved in both ABA and osmotic stress signaling cascades.

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Protein phosphorylation is involved in signal transduction pathway in blue light (BL)-induced stomatal opening and ABA-induced stomatal closure. However, the protein kinase involved in guard-cell signal transduction pathway is largely unknown. In this study, we tried to isolate and analyze protein kinase (vfPK1) from stomatal guard cells of Vicia faba L. The vfPK1 encoded a new type protein kinase that belongs to SnRK3 subgroup. Northern blotting analysis showed that the transcripts were predominantly expressed in guard cells and roots. Recombinant vfPK1 purified from E.coli showed autophosphorylation activity. To identify the protein that interact with vfPK1, we used vfPK1 as a bait in a yeast two-hybrid screening and identified a positive cDNA (vfCBL1) encoding a calcineurin B-like Ca²⁺-binding protein. The vfCBL1 is 88.3% identical to AtCBL2. These results suggest that vfPK1 may mediate Ca²⁺ signaling pathway in guard cells.

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In Arabidopsis thaliana, Histidine-to-Aspartate (His->Asp) phosphorelay is a paradigm of signaling system that is considered to be involved in response to plant hormones, including ethylene and cytokinin. In the current framework of His->Asp phosphorelay (AHK->AHP->ARR) in this higher plant, the AHP (histidine-contianing phosphotransfer) family members appear to act as regulatory intermediates of signal transduction. Although A. thaliana has 5 AHP members (AHP1 to AHP5), no information is available with regard to their biological functions. As the main objective of this study, we extensively characterized all of 5 AHPs, through searching for proteins that interact with AHPs, constructing a series of transgenic plants carrying each promoter::GUS fusion genes. Furthermore, we constructed a set of transgenic plants, aberrantly expressing each AHP. These results from such intensive analyses with regard to properties of AHPs are discussed with special reference to the His->Asp phosphorelay signal transduction network.

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In Arabidopsis thaliana, Histidine to Aspartate (His-Asp) phosphorelay is a paradigm of signaling system that is considered to be involved in response to phytohormones, including ethylene and cytokinin. In the current framework of His-Asp phosphorelay, the type-B ARR (response regulator) family members appear to act as DNA-binding transcriptional regulators. Although A. thaliana has 11 type-B ARR family members, except for ARR1 and ARR2, no biological information is available with regard to others. As the main objective of this study, we extensively characterized other 9 type-B ARRs, through constructing a series of transgenic plants carrying each promoter::GUS fusion genes. Furthermore, we constructed a set of transgenic plants, aberrantly expressing the corresponding C-terminal DNA-binding activation domain of each ARR (e.g., ARR11 and ARR14). These results with regard to properties of type-B ARRs are discussed in comparison with special reference to the cytokinin-responsive His-Asp phosphorelay signal transdution in this higher plant.

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In Arabidopsis thaliana, the AHK4/CRE1/WOL1 His-kinase and AHK2/3 homologs have been shown to function as cytokinin sensors. These cytokinin sensors presumably function as histidine kinase (His-kinase), involved in His-Asp phosphorelay signal transduction pathway. His-Asp phosphorelay pathway is typically made up of His-kinase, histidine containing phosphotransmitter (HPt) and response regulator (RR). Arabidopsis have 5 genes each coding HPt factor (AHP) and 22 genes each coding RR (ARR). ARRs form a gene family that includes the type-A and type-B. Thus, a multistep AHK2/3/4->AHP->ARR phosphorelay(s) was considered to be involved in a siglaling network in response to cytokinin.
In this study, we focused on a set of type-A ARRs and characterized their overexpressors and T-DNA insertion mutants. We also examined their expression patterns and intracellular localization in detail. These results will be discussed with special reference to cytokinin signal trunsduction and His-Asp phosphorelay network.

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Arabidopsis trichomes are branched-hair-like protruding giant cells on the leaf surface and each of them has a large nucleus caused by endoreduplication. Developing trichomes are known to express cyclin-dependent kinase CDKA (CDC2a). We expressed a fusion protein, CDKA-GFP, under the control of the CDKA promoter in Arabidopsis, and observed its expression period and intracellular localization. CDKA-GFP was expressed at early stages of trichome development and localized mainly in the nucleus, allowing us to expect that a certain cyclin is involved in the CDKA complex during trichome development.
To confirm this postulate, we expressed CDKA-GFP in Arabidopsis under the control of a developing-trichome-specific promoter (the CDKA promoter region of -591 to +4 bp from transcription initiation point) and CDKA-interacting proteins were co-immunoprecipitated with anti-GFP antibody. We will report the expected cyclin detected as a coprecipitated protein and discuss the function of the CDKA/Cyclin complex during endoreduplication in trichomes.

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*Endoreduplication is defined as nucleic DNA duplication without cell division. After some rounds of endoreduplicating cycle, cells contain multi-replicated DNA of a nucleus (polyploidy). Polyploid cells are often observed in various developed plant organs so that polyploidy is thought as a marker of differentiated cells. It is known that polyploidy occasionally correlate with cell enlargement. One mechanism is that plants control cell size through increasing polyploid level. However, little is known about molecular mechanisms of endoreduplication.
In order to identify genes involved in endoreduplication, we screened for mutants with enhanced polyploid level from activation-tagging lines using flow-cytometer. Until now, we identified 6 mutants as gain-of-function phenotypes from approximately 4500 lines. Here we report about precise screening method and phenotype of these mutants

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D-type cyclins (CYCD) respond to various signals and control the G1/S progression. We have previously demonstrated that one of CYCDs, CYCD4;1 might be involved in not only G1/S progression but also G2/M progression, suggesting that CYCD4;1 has unique function among the CYCD families. In the Aabidopsis genome, CYCD4;2 that shares about 55 % similarity to CYCD4;1 at the amino acid level is also present.
To elucidate the function of CYCD4 in plant development, we first studied the spatial expression patterns of both CYCD4;1 and CYCD4;2. The results showed that both genes were expressed in particular tissues, such as vasculature and anthers. Next, we isolated T-DNA tagged lines of both genes and found that early leaf senescence was seen in the line of CYCD4;2 although stem and flower development seemed to be normal.

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The cell cycle in plants, as in eukaryotes, is regulated by cyclin-dependent kinases (CDKs). CDK activity is controlled by various regulatory factors during the cell cycle and CDK inhibitors play a major role in controlling the G1/S transition through the control of CDK activity. Analysis of the complete Arabidopsis genome revealed the presence of seven genes belonging to CDK inhibitor, designated Kip-related proteins (KRPs). These genes encode proteins with distant sequence homology with an animal CDK inhibitor p27^Kip1. In this study, the function of KRP protein was analyzed in terms of an interaction with Cyclin/CDK.
With the exception of KRP7, all KRPs interact with Cyclin/CDK complexes but none interact with Cyclin and CDK protein alone using in vitro binding assay. We have purified three recombinant KRP proteins from E. coli. These proteins inhibit kinase activity of CyclinD2/Cdc2a complex, which is purified from insect cells, in a different dose-dependent manner.

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E2F transcription factors regulate the expression of genes required for the G1/S transition of cell cycle (e.g. DNA replication), and E2F family is negatively controlled by Rb (retinoblastoma) family proteins in mammalin cells. We have previously demonstrated that the tobacco NtE2F exhibits transactivation activity using a transient assay in tobacco BY-2 cells. To further analyze the regulation of transcriptional activity of NtE2F, we construct the experimental system using the PCNA gene expressed predominantly in late G1 to S phase. It is indicated that transcriptional activity of NtE2F is repressed by tobacco Rb (NtRb). In addition, the inhibitory activity of NtRb was blocked when it was further co-transfected with plasmid encoding cyclin D but not cyclin A or cyclin B.
Our results suggest that NtRb is a potent repressor of NtE2F activity and the control of the G1/S transition is likely mediated by E2F/Rb/cyclinD pathway in plant cells.

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Control of the G1/S transition is a crucial regulatory point of commitment to cell division in plants. Plant cell cycle regulators, acting at the G1/S transition, share a high degree of similarity to their animal counterparts. D-type cyclin (CycD) is involved in the integration of environmental stimuli in response to external signals through the formation with the regulatory subunit of cyclin-dependent kinase (CDK) complexes.
We have investigated the regulation of cyclin/CDKA activity when stationary phase of tobacco BY-2 cells were diluted into fresh medium. p13^Suc1-associated proteins contain cyclin/CDKA complexes and exhibit kinase activity against histone H1. Although kinase activity of p13^Suc1-associated proteins rapidly increased at 6 h with the highest levels reached at 10 h, the level of CDKA protein bound to p13^Suc1-beads were not changed during a 10-h period. This suggests that kinase activity of cyclin/CDKA complexes is regulated at the protein level.

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D-type cyclin plays essential roles in the G1/S transition. CycDs form complexes with CDKs (cyclin-dependent kinases) that contribute to the phosphorylation of the retinoblastoma protein (pRb) to control the onset of entry into S-phase. Recently new CycDs have been isolated from Arabidopsis and Tobacco in which these genes expressed preferentially in G2-M phase. We isolated two CycD3 genes (NtcycD3-2a, NtcycD3-2b) whose transcripts accumulated in G2-M phase. To investigate the role of CycD3, we performed a GST pull-down assay with tobacco CDK (Ntcdc2a, Ntcdc2b), tobacco Kip-related protein KIS1a and tobacco Rb and found that both CycD3 can bind to all these factors. Importantly, both CycD3/Cdc2a and CycD3/Cdc2b complexes exhibited kinase activity against histone H1 in vitro. Cdc2b represents a plant-specific CDKs involved in the control of G2/M progression, suggesting that these CycD3 may function as a regulatory partner of Cdc2b in G2/M progression.

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A proteinacious elicitor from a pathogenic fungus, cryptogein, induced hypersensitive cell death in suspension-cultured tobacco BY-2 cells. The elicitor also inhibited cell growth, suggesting that cell cycle arrest is induced prior to PCD. To elucidate the relationship between cell cycle regulation and PCD, we synchronously cultured BY-2 cells. Cell cycle arrest at specific checkpoint(s) was observed. Possible roles of cell cycle regulation on PCD, as well as possible molecular mechanisms for the elicitor-induced cell cycle arrest will be discussed.

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Transcription of a set of G2/M phase-specific genes is regulated by a common mechanism which involves promoter element called MSA (M-specific activator) and a group of Myb transcription factors related to mammalian c-Myb. Tobacco c-Myb like transcription factors, NtmybA1, NtmybA2, and NtmybB, bind to and modulate the activity of the MSA element. In transient expression assay, NtmybA1 and A2 function as transcriptional activators, and NtmybB functions as a competitive repressor. Here we analyzed in vivo functions of these Ntmyb genes by generating overexpression lines and suppression lines in BY2 cells, hairy roots, and tobacco plants. Overexpression and suppression of Ntmyb genes affect growth rate, DNA content, and cell size. The observed phenotypes of the transgenic lines support our model in which balance between Myb activator and repressor control MSA-mediated transcription, thus regulating cell cycle progression during the G2/M.

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Asymmetric cell division is of fundamental importance in the generation of the overall cellular pattern. Its molecular mechanism, however, remains unknown in plants. Regeneration of protoplasts in the moss, Physcomitrella patens assures a good system for the study of molecular mechanisms such as cell polarity, asymmetric cell division, and cell differentiation. We have devised a comprehensive system to screen genes affecting the regeneration step of protoplasts in P. patens.
We used full-length cDNA clones, whose sequences were determined from both ends, and >2,000 cDNAs were selected based on their sequence, subcloned under a constitutive promoter, then introduced into moss protoplasts for overexpression. We scored the phenotypes of regeneration of the protoplasts. About 5% of cDNAs caused abnormal regeneration at various degrees, some of which encode cytoskeletal proteins, signal transduction, unknown proteins and novel proteins. We will present the current status of this project.

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In plant DNA, cytosines in asymmetric CpNpN are frequently methylated, and genetic analysis suggested that the responsible enzyme is domains rearranged methyltransferase (DRM). We have obtained a tobacco cDNA, which encoded a novel protein consisting of 608 amino acids. Homology search indicated it to resemble DRMs found in maize and Arabidopsis, and designated as NtDRM1. The protein was localized exclusively in the nucleus. Upon expression in Sf9 cells, NtDRM1 exhibited de novo methylation activity towards synthetic as well as native DNA samples, yielding solely 5-methylcytosine. Further analysis for sequence specificity revealed NtDRM1 to preferentially methylate cytosines in CpNpN. Since methylation of CpNpN is proposed to function in epigenetic gene silencing, the present results suggest that NtDRM1, a plant-specific CpNpN-methylase, may play a critical role in regulation of gene expression in response to environmental cues.

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In a screen for morphological mutants from the T1 generation of approximately 50,000 activation tagging lines, we isolated four dominant mutants that showed hyponastic leaves. We designated them isoginchaku (iso). The iso-1D and iso-2D are allelic mutants caused by activation of the AS2 gene. Iso-3D and iso-4D are another allelic mutants caused by activation of the ASL1/LBD36 gene. These two genes belong to the AS2/Lateral organ boundaries gene family that is composed of 42 genes in Arabidopsis. The only recessive mutation isolated from this gene family was of AS2. Applying reverse genetics using a database of activation tagged T-DNA flanking sequences we found a dominant mutant that we designated peacock1-D (pck1-D) in which the ASL5/LBD12 gene was activated by a T-DNA. These results strongly suggest that activation tagging is a powerful mutant mining tool especially for genes that make up a gene family.

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Over 10,000 non-redundant Arabidopsis full length cDNA (fl-cDNA) clones were mixed to make a normalized fl-cDNA over-expression T-DNA library. Nearly all the cDNA was cloned in the sense orientation relative to the expression promoter. Agrobacterium was transformed with the normalized T-DNA library to give the corresponding Agrobacterium library. A. thaliana col. WT plants were dipped with the Agrobacteria library. 800 transformed plants were grown in green house to isolate 80 plants, which showed obvious phenotypes such as morphological abnormality, greening and pigmentation. PCR was performed from 40 of these lines using vector specific primers. The PCR fragments were sequenced to find out that all these 40 fl-cDNA inserted newly in the genome was not redundant. We designated this activation tagging-like system as Fox Hunting System (full-length cDNA over-expressor gene hunting system) to develop it as a novel technology for the functional genomics in plant science.

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Activation tagging system using a T-DNA vector that contains tetramer of the 35S enhancer followed by its minimum promoter was applied to rice. We have produced approximately 8,000 lines. Among them, one lesion mimic, eight stripe, and one needle-like leaved mutants were obtained in the T0 generation.
The lesion mimic mutant, which was dominant, was further characterized. The phenotype was linked to the T-DNA in the T1 generation. The genomic DNA adjacent to the T-DNA was cloned and sequenced. An open reading frame was located about 500 bp downstream of the T-DNA and the lesion mimic phenotype correlated with the enhanced expression of the mRNA in the T1 generation. The encoded protein shows sequence similarity to a protein whose expression is induced by hypersensitive reaction in tobacco. We are now investigating to confirm whether the overexpression of the gene is sufficient to induces the lesion mimic phenotype.

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We constructed more than 10,000 transposon-mutated lines in Arabidopsis so far. For every independent line, we sequenced the flanking region of Ds insertion, using a high throughput analytic method. Recently, we have started a "phenome project", in which we observe and screen in various phenotypes for each DS insertional line into coding regions in our resources. In a pilot experiment, we are selecting about 1,000 independent lines in which Ds element is located in coding regions of predicted Arabidopsis genes. We are planning to collect phenotypic data by two steps, morphological observation on plant growth and screening results under some environmental conditions. We report the present status of our phenome project and propose an international effort to develop a phenome analysis using gene knockout mutants for every functional gene in Arabidopsis.

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  Full-length cDNA is essential for identification of gene function in post sequencing era. Until now, we have collected RIKEN Arabidopsis full-length(RAFL) cDNAs and Ds transposon insertion lines. We have determined the full-length cDNA sequences of 12,189 clones in collaboration with the Arabidopsis SSP group of the USA as of December 2002. We also constructed promoter database by comparing the 5'-end sequence of the full-length cDNAs with the genomic sequence data. We are planning to construct the comprehensive basic database of expression profiles in Arabidopsis by combining microarray results with the promoter database. Furthermore, we constructed the database of 9,236 Ds transposon insertion lines and are applying it to functional analysis and phenome analysis.
  These data have been offered from the website (http://pfgweb.gsc.riken.go.jp/) of RIKEN GSC. In this meeting, we report our RIKEN Arabidopsis database.

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In order to establish a system for characterizing gene function by utilizing the genome sequence information, we have generated a large number of T-DNA insertion lines in Arabidopsis. The T-DNA contains a GUS reporter gene for gene trap mutagenesis. We found one line showing GUS staining in root tips reduced root growth compared with that of wild type plants. The tagged gene was a putative ABC protein gene related to the yeast GCN20 gene. GCN20 is known to interact with GCN1 in yeast, so we screened knockout lines of GCN1 orthologue from the Tag-lines. One out of eight knockout lines exhibited GUS staining in root tips, young leaves and carpels. Three knockout lines showed reduced root growth and sterile phenotype. The ABC protein interacted with the Arabidopsis GCN1 in the yeast two-hybrid system. These results indicated that the two proteins played an important role in growth and development in Arabidopsis.

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We have collected and completely sequenced 28,469 full-length cDNA clones of Oryza sativa ssp. japonica cv. Nipponbare until December, 2002. These clones were functionally annotated and many types of protein informatics analysis were performed. In spite of discontinuity of BAC/PAC genome clones with 94 % coverage from japonica, our clones were mapped at the about 19000 TU (Transcriptional Units) by Blat program. Multiple clones were mapped at the same TU with variant patterns such as differences of initiation, termination and internal exon sites. To elucidate of the regulatory mechanism of expression alternatively spliced genes, clone sets with internal exon skip pattern were selected. Some candidates of genes comprised of the genes such as the membrane protein and enzymes (hydorolase, transferase etc.). We have verified that candidate genes are alternatively spliced in rice. Three-dimensional structure change of these proteins was also checked by SOSUI and SWISSPORT programs.

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Serine/arginine-rich (SR) proteins play important roles in constitutive and alternative splicing in animals. They contain an N-terminal RNA recognition motif (RRM) and a C-terminal arginine/serine (RS)-rich domain for protein-protein interaction. We isolated 19 rice cDNAs encoding SR proteins, of which 7 contain plant specific charactaristics. To determine whether SR proteins modulate splicing efficiency and alternative splicing, we used transient assays with rice protoplasts. The results of assays showed that OsSR3, a plant-specific SR protein, enhanced splicing of Wx^b intron1. We are also determining binding sequences of each SR protein by using the SELEX method. Transgenic rice overexpressing each of the SR protein gene is being generated and they will be examined for possible changes in alternative splicing of pre-mRNAs in rice.

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The nuclear genome of tobacco cultivar Nicotiana tabacum consists of two-type sub-genome (S-genome and T-genome), and these are derived from ancestors of N. sylvestris (S-genome donor) and N.tomentosiformis (T-genome donor). On the other hand, old data suggested that the progenitor N. sylvestris is chloroplast genome donor. To compare the chloroplast genomes in Nicotiana speices, DNA was prepared from isolated intact chloroplasts from N. sylvestris and N. tomentosiformis green leaves and determined the complete nucleotide sequences of the each chloroplast genome using shotgun sequencing method. Comparison of Nicotiana chloroplast genomes confirms that the chloroplast genome of N. tabacum was certainly derived from that of N. sylvestris.

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The "Chloroplast Net" is a browser (.) based
on accumulating information on chloroplast genes and part of nuclear
genes encoding chloroplast components, like Lhc genes, in higher
plants. Chloroplasts in higher plants are estimated to consist of
2500 - 5000 proteins. Chloroplasts possess only about hundred
genes. Most chloroplast components are predicted to be encoded in
the nuclear genome. In October 2002, the draft genome sequencing of
Rice was achieved, therefore, the huge information about genomes in
Rice will be continued being supplied much more than now.
It is important to build up the relational data base linked
chloroplast genes with nuclear genes encoding chloroplast
components.
The systematical outline of a new relational data base named
"Navigator for Chloroplast/Photosynthesis-related Genes in Rice" is
discussed.
ChloroplastNet managed by NRPG (Nagoya Rice genome Project Group) is
supported by the MAFF of Japan.

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The genome of the higher plant mitochondria has been decided in Arabidopsis, sugar beet and rice plant by the present. The genotypic composition is not completely same between plant species, and there is a gene which is lacking to each other. This fact is indicating that the gene transfer to the nucleus is the developing on higher plant mitochondria at present. And, the transfer of some gene from the plastid is also observed. Thus, genome analysis of mitochondria are necessary, when transfer of gene and evolution of 3 kinds of genomes of plant cell are examined. In addition, it waits for the experimental analysis in in vitro of RNA editing mechanism and self-splicing mechanism of the group II intron.
We decided genome base sequence of the tobacco mitochondria. At present, the genome/genetic analysis are advanced in order to clarify many above-mentioned and unsolved problems.

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In order to monitor global gene expression changes during the nodulation process, we have initiated a SAGE analysis using the model legume, Lotus japonicus. In this study, comparison of gene expression profiles was made using 85,482 and 80,233 tags obtained from uninfected roots and nodule primordia. A statistical test has revealed 408 transcripts whose expression was induced in the nodulation process at the significance level of P <0.01, and 428 were identified as those which may be repressed. The gene identification of tags was done using the public as well as the in-house L. japonicus EST sequences. Extension of the SAGE tags into 3' cDNAs was performed for identification of tags with no matching EST by use of the reverse SAGE (rSAGE) technique. Genomic clones used in the genome sequencing project were PCR-screened to help annotate the novel 3' cDNA sequences.

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In the genome projects, protein coding genes are computationally predicted and annotated using many tools. On the other hand, annotation information of non-coding RNA (ncRNA) is not found in the most of published genome sequences. The reason is we don't have tools for gene prediction and analysis of ncRNA. tRNA is one of the ncRNAs and has significant function on translation. Their copy number is several hundreds and transcribed by Pol III. Since tRNA transcripts form rigid secondary (clover-leaf) structure and have highly conserved promoter motifs that we can easily find location of genes from genome sequences. However, we don't predict which tRNA gene is functional or pseudogene because of lacking gene characterization with experimental analysis. Therefore we have made genome-wide transcription assay from rice and Arabidopsis genes for increasing of information to build tRNA gene prediction algorithm. Furthermore, we have made comparison between rice and Arabidopsis tRNA gene organization.

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With the aim of understanding the whole genetic system of legume species, we initiated large-scale sequencing of the genome of a model legume, Lotus japonicus. Using the information on ESTs and cDNA markers of L. japonicus and other legumes, 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 been initiated. As of November 2002, a total of 1130 clones have been selected. Eighty-four of them are in the library phase, 6 are being sequenced, 570 are in the finishing phase, 68 are being annotated, and 237 have been annotated. Mapping of the seed clones has also been performed by generating PCR-based markers from sequence information, and 502 clones have been genetically localized. The detailed information on this project is available through the web site at http://www.kazusa.or.jp/lotus/.

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tonsoku (tsk) mutants of Arabidopsis thaliana show abnormal structure in both shoot and root apical meristems. In the meristem of tsk plants, expression of several meristem-specific genes show abnormal expression patterns, and cells dividing to abnormal direction are observed. It seems that abnormal direction of cell division in the meristem of tsk plants leads to disorganized meristem structure. Since TSK protein with 1311 amino acids has LGN repeats and Leucine-Rich repeats involved in protein-protein interactions, TSK might serve as a core of a protein complex with unknown cellular function. We expressed TSK protein fused with GFP at its N- or C-terminus in tobacco BY-2 cells and analyzed their cellular localization. Both of the TSK/GFP fusion proteins were found to be targeted to nucleus and localized in the nucleoplasm. Subcellular fractionation of transformed BY-2 cells and subsequent Western-blot analysis with TSK antibody also supported the nuclear localization of the fusion proteins.

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tonsoku (tsk) mutants of Arabidopsis thaliana show shorter roots and fasciated stems, which is due to abnormal structure and defective function of both shoot apical meristem and root apical meristem. TSK gene, expressed primarily in the meristem, encodes a novel protein which contains LGN repeats and Leucine-Rich (LR) repeats. Since these repeats are considered to be involved in the protein-protein interaction, it is crucial to identify protein(s) that interact with TSK to clarify the cellular function of TSK. We identified several protein(s) that interact with TSK by using yeast two-hybrid screening. One of them, POT3, was homologous to tobacco NtSIP1 that interacts with 6b protein encoded by T-DNA of Agrobacterium. The interaction between TSK and POT3 occurred through specific domain of each protein. 6b protein is a transcription factor and NtSIP1 has been shown to be targeted to the nucleus. These results suggest that TSK is involved in transcription regulation.

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OsEMF1 gene shows relatively low similarity to Arabidopsis EMF1 which plays an important role in maintaining vegetative stage by suppressing the expression of MADS-box genes in juvenile stage. Rice osemf1 mutant shows up-regulation of several MADS-box genes, resulting in dwarfism, abnormal flower development, indicating OsEMF1 is also involved in transcriptional regulation. Interestingly, two out of four allelic mutant lines also show longitudinal white sector consisting of mesophyll cells lacking mature chloroplasts, resulting in stripe leaf. The reproducibility of the phenotype in two lines indicates that stripe leaf is allele-specific phenotype caused by osemf1 mutation. Overexpression of the mutant osemf1 gene in wild type leads to stripe-leaf phenotype, indicating that stripe-leaf is caused by a gain-of-function mutation of OsEMF1 gene. The comparison of the expression profile obtained by microarray analysis between the mutant and wild type is also presented.

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Flowering in Arabidopsis is regulated by several interacting pathways which are integrated by genes such as FT, SOC1/AGL20, and LFY to finally execute the floral transition. We have been interested in factors acting downstream of FT. The late-flowering gene FD is a good candidate for such genes. The FD activity seems to be required for the precocious-flowering phenotype of 35S::FT, because otherwise very weak fd-1 mutation strongly suppresses 35S::FT phenotype. Suppression effect of fd-1 is specific to FT, because fd-1 does not strongly affect early-flowering phenotype caused by overexpression of LFY or SOC1/AGL20. FD encodes a bZIP transcription factor of the ABF/AREB/GBF4 subclass. FD is expressed at very low level in LD and SD conditions. FD protein can interact with FT protein in yeast cells. These results suggest that FT is likely to play a key role in regulation of FD function by protein-protein interaction.

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Late-flowering mutant fwa is a dominant epigenetic mutant that ectopically expresses a GL2-type HD-ZIP gene due to promoter hypomethylation (Soppe et al. 2000). In wild type, however, FWA is not expressed during vegetative phase, and loss-of-function mutants of FWA are indistinguishable from wild type in flowering time. These facts suggest that FWA per se is not a component of the regulatory mechanisms of flowering. Recent findings that over-expression of other GL2-type HD-ZIP genes, ANL2 and PDF2, also resulted in late-flowering phenotype (Weigel et al. 2000; Abe et al. in press) prompted us to hypothesize that ectopically-expressed GL2-type HD-ZIP proteins somehow interfere the regulatory mechanisms of flowering. Genetic analysis suggests that FWA blocks the pathway at FT and/or downstream of FT. We envisage that FWA may provide unique tools to dissect pathway from FT to flowering. We are currently investigating FWA-interacting proteins using yeast two-hybrid system. Results will be presented.

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In rice fzp mutant, spikelet meristems are transformed to branch shoots. Cloning of the FZP gene revealed that it is a member of ERF family which functions as a transcription factor. In situ hybridization analysis showed that FZP mRNA was expressed between rudimentary glumes and the spikelet meristem in young spikelets.
We asked if the FZP functions in a same way as ERF proteins that act as GCC box-mediated transcription factors. The FZP and a reporter gene containing the GCC box in promoter were introduced into leaves using particle gun. Expression of the reporter gene was increased by FZP. Next, FZP fused with GAL4 and a reporter gene containing GAL4 box in promoter were introduced into leaves. The rise of the reporter gene by FZP and GAL4 fusion protein was observed. These results demonstrated that the FZP acts as a GCC box-mediated transcription activator.

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To reveal the function of a MADS-box gene, AGL24, we generated and analyzed transgenic plants that overexpress AGL24 (AGL24ox). These transformants showed pleiotropic phenotypes, such as early flowering, conversion of floral organs and defect in organ abscissions, supposing that AGL24 is involved in flower development. AGL24ox plants flower earlier than wild-type plants both under LD and SD, but are sensitive to SD. Northern analysis of AGL24ox plants showed reduction of AP1 expression in comparison with wild type, and it would be resulting ap1-like flower phenotypes. We also investigated gene expression profile of AGL24ox plants by using cDNA microarray. As a result, expression of AP1 and several genes including a putative auxin-regulated gene, were decreased. Analysis of double mutants between AGL24ox and mutants that affect flowering time or floral organ identity, are currently undertaken. Here, we would discuss the probable functions of AGL24.

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Recent advances of molecular biological studies on gibberellin (GA) biosynthesis and signaling have succeeded to isolate some GA-related genes. Consequently, we can take new approach to know where bioactive GA is produced and GA is perceived through analysis on the expression pattern of GA-biosynthetic and GA-perception genes. For detecting the expression of these genes, we analyzed using promoter-GUS transgenic plants or in situ hybridization. Expression of the genes we used as marks was observed in young developing leaves, apical region of roots and elongating stems. At the inflorescence and floral stage, expression of the genes was observed in the shoot meristems just before flowering, floral meristems, stamen primordia and tapetum cells. The overlapped expression of GA-biosynthesis and GA-perception genes in these various tissues and organs indicates that active GA-biosynthesis occurs at the same site of GA-perception. This work was in part supported by PROBRAIN.

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Perilla frutescens var. crispa, a short-day plant, continues to flower under long-day if once photoinduced. This reminds us of vernalization where the low temperature effect is maintained for a long period of time. Such an analogy suggests the common mechanism functioning in both phenomena. Vernalization is regulated by the gene expression through DNA methylation and demthylation. Accordingly, seeds of green and red P. frutescens were treated with 250μM 5-azacytidine. The treatment induced flowering under long-day condition in both varieties. This is the first evidence indicating flower-induction by DNA-demethylating reagent in plant of which flowering is photperiodically regulated. Anything other than flowering was not influenced by 5-azacytidine although the vegetative growth was suppressed a little. 5-Azacytidine applied to shoot apex is also effective, and this treatment induced flower buds at the lower nodes, suggesting that 5-azacytidine induced the production of transmissible flower-inducing substance(s).

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