In cultivated rice, the Waxy gene which is involved in the amylose biosyntheses has two wild-type alleles, Wx^a and Wx^b. The activity of Wx^a is 10-fold higher than that of Wx^b at the level of mRNA. Wx^b has a G to T mutation at 5'splice site of the first intron.
Low amylose mutants "dull (du)" of Japonica rice have 5 loci and they are all different from the Waxy gene. We cloned the du3 gene by map-based cloning, found that it encodes Cap-binding protein 20kD subunit (CBP20) of the nuclear cap-binding complex. The du3 mutant exhibited the reduction of amylose contents, WX protein, and the splicing efficiency of Wx^b pre-mRNA compared with wild-type. Recently, it has been suggested that CBP20 may participate in pre-mRNA splicing events, involving the recruitment of U1 snRNP. Here, we suggest that CBP20 participate in some steps in splicing of Wx^b pre-mRNA.

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Alternative splicing profiles in higher plants seem to be affected by environmental stress conditions. 20 types of SR protein homologues as a splicing factor exist in Arabidopsis plants. We have shown that expressions of several SR protein homologues, RSZ22, RSZ22a, RSZ32, SR40, and SR34a are induced by the treatment with low temperature (4^.C). Here we analyzed the physiological roles of these SR proteins as a regulatory factor of stress-responsible alternative splicing events. The RT-PCR analysis showed that several splicing variants of RSZ22, RSZ22a and SR40 are produced by alternative splicing of their own pre-mRNAs. In order to identify a gene whose splicing efficiency is regulated by the SR proteins, we analyzed their target sequences by SELEX methods. Next, we generated the transgenic Arabidopsis plants overexpressing or suppressing of each SR protein. No phenotypic difference was observed in the stress tolerance between the transgenic and wild-type plants.

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Petunia pMADS3 is a class C homeotic gene that is specifically expressed in stamens and carpels. We previously reported that introduction of a part of the pMADS3 genomic sequence resulted in ectopic expression of endogenous pMADS3 (ect-pMADS3) in some lines of transgenic Petunia hybrida, while pMADS3 was silenced in other lines. The ect-pMADS3 phenotype is likely due to novel epigenetic transcriptional regulation (Kapoor et al., Plant J. 43:649-661, 2005). We have shown that inclusion of pMADS3 intron 2 (4 kb) in the transgene is essential to induce ect-pMADS3. ect-pMADS3 is occasionally observed even after the tansgenes are segregated away. We found that a 1-kb sub-region of pMADS3 intron 2 (region II) was responsible for ect-pMADS3 by deletion analysis. In the transgenic plants showing ect-pMADS3, the DNA sequences in region II was methylated, indicating a correlation between DNA methylation and ectopic transcriptional activation of pMADS3.

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The dioecious plant, S. latifolia contains two homologous genes of APETALA3 controlling floral morphogenesis. One of them is autosome-linked (SlAP3A) and the other is Y chromosome-linked (SlAP3Y). SlAP3A would be duplicated and transferred from an autosome to the Y chromosome. We here report genomic structures of these two SlAP3 genes. SlAP3A consists of 6 exons and 5 introns in 1.7kb of the whole gene. SlAP3Y consists of 7 exons and 6 introns, of which the 2nd intron stretches for 24 kb in 26kb of the whole gene. We are now in the process of locating SlAP3Y on the Y chromosome by fluorescent in situ hybridization (FISH). SlAP3A is expressed in petals of both male and female flowers whereas SlAP3Y is expressed specifically in only stamens of the male flower. These two SlAP3 genes would be regulated transcriptionally. We also report transcriptional regulation by DNA methylation in these two SlAP3 genes.

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Cytosine methylation is an epigenetic process that plays a role in regulating gene expression. Plants have at least three classes of cytosine methyltransferase, i.e., MET1 class of methyltransferase, Chromomethylases, and de novo DNA methyltransferase. In our study, three putative metyltransferase genes, BrMET1a, BrMET1b, and BrCMT were isolated from genome library of Brassica rapa. Identities of amino acid sequences between BrMET1a and AtMET1 and between BrMET1b and AtMET1 were 72.0% and 66.7%, respectively. Pfam analysis using deduced amino acid sequence showed the presence of BAH domain, CHROMO domain, and methyltransferase domain in BrCMT. RT-PCR analysis revealed the expression of BrMET1a, BrMET1b, and BrCMT. These results suggested that these three genes function as methyltransfarase in B. rapa.

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When plants were exposed to the herbivore-induced plant volatiles, HIPVs, the expression of defence-related genes were induced. However, molecular mechanisms of the HIPVs perception and the following signaling events have not been elucidated. Here we report that the HIPVs-induced increase in cytosolic Ca²⁺ concentration ([Ca²⁺]_C) in the Arabidopsis thaliana leaves. For the monitoring of [Ca²⁺]_C, aequorin-transformed A. thaliana (AQ plants) were used. When leaves were exposed a variety of HIPVs, increases in [Ca²⁺]_C were observed. Pharmacological assay revealed that the increase was induced by the efflux of Ca²⁺ from intracellular organelle. In addition, some of the HIPVs promoted the production of superoxide radicals but others did not.
Because the G-protein coupled receptor play as receptor of volatiles in animal, the possible involvement of Gα (GPA1) was also tested using the gpa1-knockout mutant of AQ plants (gpa1-AQ plants). Signal transduction pathway for the HIPVs-induced increase in [Ca²⁺]_C may also discussed.

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OsGI is one of the genes involved in the photoperiodic control of flowering in rice, however its biochemical function is unknown. To understand that, we attempt to identify members in the OsGI protein complex under native conditions by using the TAP method. The TAP method showed the rapid purification of complex without prior knowledge of the complex composition, activity or function.
In order to isolate the OsGI complex, we produced TAP tag constructs, N-terminal TAP (SGN) and C-terminal TAP (SGC) and transformed them into rice. OsGI and OsGI-TAP proteins were detected by western blot analysis with OsGI specific antibody. OsGI was localized in cytosol and nucleus by the analysis of GFP fusion genes in transient assay. OsGI complex from the cultured cell (SGC lines) were isolated by using the TAP method. Identification of proteins included in the OsGI protein complex is being carried out by massspectrometric method.

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Calmodulin (CaM), a ubiquitous Ca²⁺ sensor in eukaryotes, regulates a number of target proteins in a Ca²⁺-dependent manner. Previously, we identified NtMKP1, a tobacco MAP kinase phosphatase, as a novel CaM-binding protein. NtMKP1, produced by in vitro transcription/translation, dephosphorylated and inactivated SIPK, a tobacco stress-responsive MAP kinase. Deletion and site-directed mutagenesis revealed that Lys⁴¹ and Arg⁴³ in the N-terminal noncatalytic region of NtMKP1 are important to bind and inactivate SIPK, whereas the CaM-binding domain and C-terminal domain were dispensable. Moreover, hydrolysis of an artificial substrate by NtMKP1 was significantly stimulated by the SIPK-binding, but not by CaM-binding. Transient expression analysis in Nicotiana benthamiana indicated that Lys⁴¹ and Arg⁴³ in N-terminal noncatalytic region of NtMKP1, but not CaM-binding are important for the function of NtMKP1 in vivo. These results indicate that NtMKP1 activity is regulated by the binding of its physiological substrate SIPK, and CaM-binding to NtMKP1 might have other effect.

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COP9 signalosome (CSN) modulates signal transduction pathways involved in cell cycle, morphogenesis, and stress response, through its regulation on the ubiquitin-proteasome degradation pathway, in the animal and plant kingdoms. Null mutations for CSN results in lethal phenotypes, indicating its essential role in development.
Functional dissection of mammalian CSN revealed that the N-terminal portion of CSN1 (CSN1N) represses activated signals of the SAPK/JNK1 signaling pathway. CSN1N also reduces accumulation of JNK1, leading to suppression of c-Jun phosphorylation. To analyze this regulation, we have isolated proteins directly binding to CSN1N (NBPs). NBPs cover a novel spectrum of proteins, including SAP130, DDX15/hPrp43/mDEAH9, CFIm68, which are components of transcription/RNA processing complexes.
We are currently analyzing the Arabidopsis NBP orthologs and its knockout plants. Detailed biochemical analyses on CSN-NBP interactions together with in planta research should reveal this novel mechanism of CSN. Here we discuss the complex interaction between CSN, COP/DET/FUS, and their interacting partners.

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Under the poor availability of inorganic carbon (such as CO₂, HCO₃^- :Ci) for photosynthesis, Chlamydomonas or cyanobacteria induce the active uptake systems for Ci that allow accumulation of Ci within the cell (Carbon-Concentrating Mechanism: CCM). In Chlamydomonas, the regulatory factor CCM1 is indispensable to induce the CCM. CCM1 has five Cys residues conserved in the zinc-finger domain in its N-terminal region. In order to analyze the zinc atom occupancy, we generated E. coli expressed GST fusion N-terminal region of the CCM1. Zinc ion was detected in this fusion protein by the atomic absorption analysis. In order to detect the CCM1 protein, western blot analysis against soluble Chlamydomonas protein fraction was performed. A 95-kDa band was detected by anti-CCM1 antibody. Considering with the fact that the deduced molecular weight of the CCM1 was 70.073 kDa, some modifications of the CCM1 were suggested. The CCM1 was expressed constitutively independent of CO₂ availability.

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FtsH protease is an ATP-dependent protease localized on cytoplasmic membrane. This protease is a member of AAA superfamily. In higher plants, FtsH protease is localization on thylakoid membrane. Function of the FtsH protease was assumed to degrade membrane proteins incorporating as abnormal conformation. This protease also participate in the degradation of photo-damaged subunits of photosystem II.
In many cases, FtsH protease expressed in E.coli is deposited as inclusion bodies. Structure of FtsH protease contains an obvious transmembrane hydrophobic region. The region is responsible for the formation of inclusion bodies. In this study, therefore, it was eliminated from FtsH protease. For the results, though the formation of inclusion bodies was not improved, the expressed proteins were functionally refolded by the treatment of dialysis against the buffers containing the reduced reagents. The refolded FtsH protease has been applied to biochemical characterization.

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Senescence is a program to recover the nutrients from aged tissues. These nutrients are utilized in younger leaves or indispensable organs, such as shoots or flowers. Chloroplast is a major organelle to store the nitrogenous compounds of proteins. Mechanisms of protein degradation in chloroplast are one of the remaining problems to be solved.
FtsH protease is ATP-dependent protease which localized on thylakoid membranes. This protease degrades photo-damaged subunits in photosystem II. Therefore, this is a major candidate to manage the protein breakdown in chloroplasts.
Overexpression systems of FtsH protease from Arabidopsis were previously constructed, however the expressed proteins were deposited as inclusion bodies. This made difficult to promote biochemical investigations of FtsH protease. In this study, the inclusion bodies were solubilized, when the urea denatured proteins were dialyzed against the buffers containing the reduced reagents. We characterized the biochemical properties of FtsH proteases from Arabidopsis.

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In bacteria, there are a set of lipoproteins that are modified with lipid at their N-terminal cysteine residues. We have already identified two lipoproteins, PsbQ and Psb27, in Synechocystis sp. PCC6803. However, the structure of lipid modification of these lipoproteins has not been determined in detail. In this study, we have analyzed the structure of lipid modification of the lipoproteins using mass spectrometry. As a result, it was found that PsbQ was modified with a diacylglycerol and an acyl group at its N-terminal cysteine residue. Psb27 was also modified with a diacylglycerol but not with acyl group. To investigate the roles of N-acylation, we created a mutant defective in N-acylation of lipoproteins. The mutant showed growth retardation in a calcium-depleted medium and a different sensitivity to several sugars compared to the wild type. These findings indicate that N-acylation of lipoproteins plays important roles in Synechocystis sp. PCC6803.

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It is known that red light induces jasmonate (JA) synthesis in rice. On the other hand, elicitor-responsive genes are activated by cytoplasmic acidification in suspension-cultured rice (Oryza sativa cv. Nipponbare) cells. There is a possibility that the cytoplasmic acidification can be a key step (trigger) in the signal transduction leading to the expression of several genes. Therefore, we measured the cytoplasmic pH chenge induced by JA in suspension cultured rice (Oryza sativa cv. musasikogane) cells with ³¹P-NMR method. Jasmonate (4.5 nM-450 μM MeJA) induces transient cytoplasmic acidification in suspension-cultured rice cells. The patterns of pH change were similar to those induced by elicitor. However, 0.45 nM MeJA does not induce the transient cytoplasmic acidification. These results suggest that red light could be a trigger in the signal transduction events leading to the expression of some elicitor-responsive genes in rice.

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Jasmonates represent a useful tool in the investigation and engineering of natural bioproduction (secondary metabolism) in plants. In tobacco bright yellow-2 (BY-2) cells, treatment with methyl jasmonate (MJ) induces the rapid accumulation of alkaloids and various phenylpropanoids. We employed a comprehensive cDNA tobacco microarray to isolate novel signal transduction component, connecting the MJ-hormone signal to secondary metabolism-related gene expression. By overexpression analysis, we show that the tobacco MJ-inducible MYB-family transcription factor NtMYBJS1 positively regulates transcription of several early phenylpropanoid-related genes, causing accumulation of feruloylputrescine and caffeoylputrescine in cultured cells. These results are further supported by the gel mobility shift assay results, showing that this MYB gene can selectively bind to specific DNA motives in the tobacco PAL gene promoters. In conclusion, we propose that the NtMYBJS1 protein functions in tobacco MJ signal transduction, inducing phenylpropanoid biosynthetic genes and the accumulation of phenylpropanoid-polyamine conjugates during wound and/or pathogen stress.

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Results of recent studies on cytokinin responses in Arabidopsis thaliana, a His-Asp phosphorelay was revealed to play an essential role in the primary responses of plants to cytokinin. It is assumed that the downstream cytokinin-responsive signaling network must be very diverse and complicated. To gain insight into the downstream signaling network, we previously conducted a genome-wide DNA microarray analysis, which allowed us to identify a number of cytokinin-inducible genes. Amongst them, here we focused on a few such genes, and they were intensively characterized through both forward and reverse genetics. They included: (1) ASL9 (At1g16530) encoding a member of the AS2 (ASYMMETRIC 2) family, (2) CGA1 (At4g26150) encoding a light-responsive GATA-type transcription factor, (3) RADI1 (At2g21650) encoding a protein with a single SANT/MYB domain. The results will be summarized with special reference to possible links between these cytokinin-responsive genes and the cytokinin-mediated signal transduction in plants.

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Brz(brassinazole) was synthesized as the first specific inhibitor for brassinosteroid biosynthesis. In the dark germination, Brz-treated plant expressed chloroplastic genes that are expressed as very low level in dark-grown cotyledon without Brz. When the dark-germinated plant with Brz was transferred to the light conditions, chloroplast development was accelerated as compared to the cotyledon without Brz. In the light, Brz treated adult plant showed high-green leaves as compared to leaves without Brz. We screened a mutant that showed pale green phenotype with Brz in the light as bpg2 (Brz-insensitive-pale green2). A T-DNA insertion that related to mutant phenotype in any back crossed bpg2 mutants were identified in an ORF of a novel gene. Possible candidate gene of bpg2 possessed at least 4 hydrophobic regions, and localization of bpg2 into chloroplast was predicted by TargetP program.

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Brassinosteroids occurs both in dicot and monocot. Brassinosteroid-deficient mutants of dicot and monocot showed dwarfism, and major brassinosteroid biosynthesis genes and a receptor BRI1 are evolutionary conserved between dicot and monocot.
When seeds of rice wild type (Nipponbare) were germinated in the deep 1/2MS liquid medium under dark condition, the first leaf coleoptile elongated to the surface of the liquid medium. The rice coleoptiles grown in the medium including Brz showed dwarf phenotype. This dwarfism induced by Brz tratment was recovered by brassinosteroid treatment. In the dwarf coleoptile of Brz- treated rice, choloroplastic genes that are not expressed in the rice grown in the normal 1/2MS medium were expressed. These results suggested that Brz caused the de-etiolation in the dark to not only dicot but also monocot.

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A potential role for ABC transporters in the extrusion of xenobiotics has been suggested. Arabidopsis contains 15 genes encoding the pleiotropic drug resistance-type (PDR) ABC transporter, but the functions of most of them remain unknown. In this study, we isolated a semi-dominant mutant designated as atpdr9-1 conferring increased 2,4-D tolerance. The application of 2,4-D, IAA, or NAA to atpdr9-1 revealed that auxin-resistant phenotype is 2,4-D-specific. The root growth assay using T-DNA insertion line, atpdr9-2, showed clear 2,4-D specific hypersensitivity, confirming that isolated atpdr9-1 mutation is a spontaneous gain-of-function mutation. The site of atpdr9-1 is located in second nucleotide binding domain and the region is completely conserved in reported plant PDR-type transporters. Further combinational analysis using both atpdr9-1 and -2 revealed that AtPDR9 protein is implicated in the extrusion of not only 2,4-D but also related chlorophenoxy family of herbicides and the auxin transport inhibitor, NPA.

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The auxin primary-response proteins, Aux/IAA, usually have four conserved domains. However, Domains I and II are conserved imperfectly in the IAA20 subfamily proteins composed of IAA20, 30 and 31. Especially IAA20 and IAA30 lack Domain II, which has been thought essential for function of Aux/IAA proteins. In this study, we have examined phenotype of transgenic lines overexpressing IAA30 (IAA30 OX) and IAA31 (IAA31 OX) by the use of 35S promoter to understand function of the IAA20 subfamily. Both lines were defective in multiple auxin-related properties, such as gravitropism of hypocotyls and roots and lateral root formation. But only IAA31 OX displayed weaker apical dominance and late flowering. These results suggest that atypical Aux/IAA proteins also play a similar role in auxin signaling to the other Aux/IAAs, and have redundant and unique characteristics in the IAA20 subfamily.

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Like other winter annual species, germination of Arabidopsis seeds is inhibited by high temperature (thermoinhibition). By using Arabidopsis (Columbia) seeds as a model, we have shown that bioactive GA₄ production was repressed severely by high temperature, and their thermoinhibition was alleviated by exogenously applied GA₃. To evaluate the contribution of negative regulators of GA signaling on thermoinhibition, we studied germination phenotype of their loss of function mutants. The seeds of spy showed high germination percentages at supraoptimal temperatures. The seeds of rgl2 also showed tolerance to thermoinhibition. Quantitative RT-PCR analyses with wild type seeds indicated that SPY and RGL2 increased their transcript levels by imbibition and maintained their levels even after 24h at 34 ^oC but decreased to the low levels after 12h at 22 ^oC. These results suggest that GA response is suppressed at supraoptimal temperatures by maintaining negative regulator levels in combination with the repression of GA synthesis.

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RSG is a transcriptional activator that regulates endogenous amounts of GAs through the control of a GA biosynthetic enzyme. Functional repression of RSG reduced endogenous amounts of GAs and severely inhibited the process of cell elongation of stems, resulting in a dwarf phenotype. The 14-3-3 signaling proteins have been shown to bind to RSG through phosphorylated Ser-114 and suppress RSG by sequestering it in the cytoplasm. The intracellular localization of RSG is regulated by GA levels. We have identified calcium-dependent protein kinase (CDPK) as a protein kinase that specifically phosphorylates Ser-114 of RSG. We found that 14-3-3 proteins bind to CDPK as well as RSG. To know the binding domain of CDPK to RSG and 14-3-3, we performed pull-down assay. Furthermore, we examined the effects of overexpression of CDPK on the feedback regulation of the GA 20-oxidase gene in the transgenic plants.

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GAMYB is known as a transcriptional factor, which binds to the promoter of α-amylase gene and activates the GA-dependent gene expression. Recently we found GAMYB is also important in anther development from microscopic analyses of gamyb mutant.
We performed the microarray analysis using RNAs from gamyb anthers. The expression of many genes, related to fatty acid synthesis, flavonoid synthesis, cell wall degradation, and protein degradation, was down regulated in the mutant. Gel shift assay confirmed that some of them contain the GAMYB-interacting elements in their 5`-flanking regions. We also performed same experiment using anther RNA from GA-deficient mutant, oscps-1. Genes down regulated in cps, were almost overlapped with those in gamyb. This suggests that GAMYB works in down-stream of GA signaling in anther as well as in aleurone, and promotes the expression of genes related to various nutrient supplies from tapetum to pollen and cell death of tapetum.

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We have been collecting poplar full-length cDNAs. In 2004, we reported several thousands of clones from leaves of axenically grown poplar (Populus nigra var. italica) subjected to environmental abiotic stress treatments. The population of P. nigra expressed sequence tags (ESTs) has grown ever since, resulting in more than 13,000 non-redundant clones which originate from various organs of a field-grown stand and stress-treated in vitro leaves. Our poplar full-length cDNAs represent about 30% of the whole population of genes estimated in Populus. We will discuss similarity and specificity between trees and herbaceous model plants in the light of comparative genomics.

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Tomato and eucalyptus represent key species with great potential for conducting genomic research. With the aims of understanding their genetic systems, we decided to participate in the multinational tomato genome sequencing project, and also launched on a large-scale eucalypt genome analysis. The tomato genome is comprised of 950Mbp of DNA. The majority of genes are found in the 250Mbp euchromatin. In the tomato genome sequencing project, minimal tiling path of BAC clones on the euchromatin is going to be sequenced. Our allotted task is to sequence the chromosome 8. As of Nov. 2005, sequencing of nine clones has been finished. The genome size of eucalyptus is estimated to be 650Mbp. The Whole Genome Shotgun (WGS) sequencing strategy was applied to produce the draft sequence. To date, two million WGS reads have been produced. We are going to report the current status of genome projects of tomato and eucalyptus.

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With the aim of establishing the basic knowledge and resources needed for applied genetics, we investigated the genome structure of red clover Trifolium pratense L.. The average GC content of the nuclear genome was shown to be 34.2% by analysis of 1.4 Mb of random genomic sequences. A total of 26,356 ESTs, that were grouped into 9339 non-redundant sequences, were collected. A high-density genetic linkage map of the genome was constructed using two parent plants, HR and R130, and188 F1 plants from the mapping population. As a result, a total of 1434 loci detected by 1399 markers (1286 detected by microsatellite markers and 148 by RFLP markers) were successfully mapped onto seven linkage groups totaling 868.7 cM in length. Macrosynteny at the segmental level was observed between the genomes of red clover and two model legumes, Lotus japonicus and Medicago truncatula.

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The cultivated Brassica species are grown in many countries. They include oil crops and vegetables. Chinese cabbage (Brassica rapa) contains the A genome (2n=20). Although the physiology and developmental biology of Arabidopsis and Brassica are very similar, the A genomes of Brassica are 4 to 5 times larger than that of Arabidopsis thaliana. To identify biotic and abiotic stress-inducible genes in B. rapa, we prepared cDNA libraries (EST and full-length cDNA). We have isolated and functionally annotated ca. 2,000 ESTs using Arabidopsis genome information. We have initiated transcriptional monitoring of stress-inducible genes in response to biotic and abiotic stresses. In this report, we will present the analysis of transcriptional expression profiles of ca. 2,000 B. rapa cDNAs in response to a variety of biotic and abiotic stresses. We also discuss the similarity and difference of stress-inducible genes between B. rapa and A. thaliana.

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Phylogenetic analysis of photosynthetic prokaryotes contributes to our understanding not only of their phylogenetic relationship, but also of the origin of chloroplast. Current progress of the phylogenetic analysis of these organisms is achieved by using the conserved genes such as rDNA and rpoC1. However, the different branching patterns are obtained depending on the genes. This problem is reasonable because evolution is not reflected by a single or limited numbers of genes, but by whole genome sequences. Recently, a growing number of complete genome sequences are available and several methods for the phylogenetic analysis based on the whole genome sequences have been proposed. We developed a new method for the phylogenetic analysis based on the comparison of whole genome sequences and this method was applied to the analysis of the evolution of photosynthetic prokaryote and to the identification of the gene encoding chlorophyllide a oxygenase from Prochlorococcus genomes.

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To improve utility of tobacco BY-2 cells as an experimental tool we had been collecting ESTs and had analyzed expression profiles using a custom-made microarray made of the EST clones. However, there are limits in the usefulness of EST clones and sequences. To overcome this problem we are analyzing complete length cDNAs of BY-2. Library was made by V-cappping method using total RNAs prepared from log, stationary and jasmonate-treated cells. After sequencing of several thousand clones from the 5' terminus, we obtained approx. 2200 unique complete-length cDNAs. Using the sequence information of these clones, of previously analyzed EST and of other genes of tobacco found in public database, we are now constructing high-density oligo-nucreotide array using an on-chip synthesis method. In this presentation the result of the sequencing of full-length cDNA clones and the progress of the construction of oligo-array will be presented.

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Plants have evolutionally acquired many strategies to defend pathogens. The hypersensitive response (HR) is one of them. In the event, small defense signaling molecules such as salicylic acid, jasmonic acid and ethylene have been identified. Currently we propose that the polyamine spermine (Spm) acts as a signal transmitter during the HR induced by Tobacco mosaic virus.
To understand a generality of Spm-signaling pathway and reveal its pathway extensively, here we report on the identification of Spm-responsive genes in Arabidopsis thaliana using super serial analysis of gene expression technique. Most of the Spm-responsive genes respond during Cucumber mosaic virus-elicited HR in A. thaliana. Based on the results, we argue a physiological role of Spm-signaling pathway in Arabidopsis.

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All living organisms regulate their gene expression patterns in response to their internal conditions or environmental signals. Generally, in eubacteria, transcriptional patterns are determined by the replacement of sigma factors. The cyanobacterium Synechocystis sp. PCC 6803 that performs oxygenic photosynthesis (like algae and higher plants) has nine sigma factors. However, the function of these sigma factors is still largely unknown.
To identify the target genes regulated by each sigma factor, DNA microarray analyses were performed using knockout mutants of sigB, sigC, and sigD. The results indicated that psaB, psaJ and psbD, which encode components of photosystem I and II, reduced in these mutants. In addition, nitrogen assimilation-related transcripts, ntcA and glnN decreased specifically in sigB and sigD knockout mutants, respectively. I will report the details of gene expression profiles in these mutants and discuss the function of each sigma factor.

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Comparative genomics between lower and higher plants are very useful approach to understand the evolution and diversity of photosynthesis. Recently, genome sequences and expression profiles of photosynthetic organisms have been accumulated and become powerful tools for systematic analysis of photosynthesis. But most of these data has been examined by using major model organisms, such as Synechocystis and Arabidopsis. Chlamydomonas is referred as green yeast and recognized as a model organism to study important phenomena including photosynthesis, respiration, mating process and flagella apparatus. Establishment of genome resource of Chlamydomonas must lead to a better understanding of the photosynthetic evolution and diversity. Until now, we examined the expression profiles under high-light and low-CO₂ stress conditions by using cDNA macroarray containing 10,368 ESTs. And full-length cDNA libraries have been constructed from Chlamydomonas cells grown under various stress conditions. The progress of this project will be discussed.

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To elucidate functions of tomato genes that have no homology with Arabidopsis genes, we analyzed expression profiles during fruit maturation. We prepared DNA array using 10,905 cDNA clones that were selected from fruit and leaf cDNA libraries of a miniature tomato cultivar Micro-Tom, as representatives of non-redundant sequences of 37,972 Micro-Tom ESTs. Tomato gene expressions in fruit developmental stages were monitored using the DNA array. Patterns of gene expressions were arranged into a 4x4 array using a self-organizing map algorithm. We identified 1151 probes corresponding to tomato genes that have no sequence homology with Arabidopsis genes, namely, non-Arabidopsis genes, on the DNA array. The expression patterns of the non-Arabidopsis genes distributed evenly in the self-organizing map, without significant differences with those of the whole genes. This result suggested that the non-Arabidopsis genes participate in diverse physiological processes of fruit maturation.

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The salicylic acid, jasmonic acid and ethylene are known as signaling molecules on both localized and systemic resistant responses to infectious pathogens. In addition to these endogenous defense signals, several synthetic compounds that induce both systemic acquired resistance (SAR) and the expression of various SAR-associated marker genes have been identified previously. It is known that 2,6-dichloroisonicotinic acid, benzo(1,2,3)thiadiazole-7-carbothioic acid S-methyl ester and BABA protect plants against some pathogens through activation of defense mechanisms. Therefore, they are regarded as plant activators. To screen for candidates of plant activator, we performed Arabidopsis microarray analyses, consisting of ca. 1,200 full-length cDNA clones representing putative defense-related and regulatory genes. In present study, we suggest that gene expression profiling with a cDNA microarray can screen plant activators and predict the action of plant activators on plants.

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Total view of secondary metabolism would be explored best in model plants in which genome organization has been clarified and genetic approach. It has a limit in the secondary metabolism research, as the number of metabolites found in Arabidopsis is lower than those of other plants. Therefore, other model plants could be useful. The legume model Lotus japonicus could be one of the candidates as about 30% of the whole genes show no significant homology with the Arabidopsis genes. We have used suspension-cultured cells of L. japonicus for functional analyses of metabolism-related genes. Our previous study showed that yeast extracts induced several isoflavonoids and expression of related genes. Here we analyzed metabolites of elicitor-treated cultured cells were initiated via callus from hypocotyls of L. japonicus Gifu B129 and hypocotyls or cotyledon of L. japonicus Miyakojima MG20 using LC-MS. We observed different responses in suspension-cultured cells line.

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We established a metabolic profiling analysis system using FT-ICR MS (Fourier Transform Ion Cyclotron Resonance Mass Spectrometry) in combination with a data analysis tool (Dmass) and a metabolite-species database (KNApSAcK). The entire scheme was applied for the clarification of metabolic disorders in Arabidopsis seedlings treated with herbicides of different mode of actions. Arabidopsis extracts were directly infused into the ESI ion source on an FT-ICR MS system. Acquired metabolic data consisting of m/z values with ion intensity values were subjected to a principal component analysis. Metabolic disorders induced by the herbicide treatments were clearly differentiated from the herbicide-free metabolome. From each herbicide treatment, m/z values of ions representing the metabolic disorder were identified, and metabolite candidates search were then performed through a KNApSAcK database search. The database search and MSMS analyses manifested a dose dependent accumulation of shikimic acid 3-phosphate and several flavonoid glycosides in the specific herbicides treatment.

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We have developed a metabolomics experimental scheme comprising of an analytical tool for high-throughput processing of data from FT-ICR/MS analyses and a database system for searching available metabolite information (KNApSAcK, http://kanaya.naist.jp/KNApSAcK/). In this study, we applied our scheme for profiling Arabidopsis metabolites. After confirming data reproducibility (m/z values with intensities) among multiple FT-ICR/MS experiments, we found that about 10% of the total ion species detected from the FT-ICR/MS analyses have fluctuated representing different growth conditions for Arabidopsis. Furthermore, we were able to identify metabolite candidates corresponding to those m/z values using the KNApSAcK database. Some of these metabolites have been already found in Arabidopsis, while most of the m/z values of interest could not be assigned to any of known metabolites from Arabidopsis. For understanding plant metabolic functions as an integrated system, we are establishing further analytical data correction system including MSMS structural information together with a comprehensive metabolite database.

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Plant metabolomics has been recently growing rapidly. However most of their reports tend to rely on statistical analysis without molecular identification. Whereas, we are developing methodologies for metabolites of molecular identification by a combination of multi-dimensional NMR measurements of uniformly stable isotope labeled plants and signal assignment with our standard ¹H, ¹³C chemical shifts data base.
Firstly, we examined the culture conditions of Arabidopsis T87 cells for obtaining stable results of metabolic changes.Secondly, the T87 cells were uniformly labeled with [¹³C₆]glucose, and their ¹³C-HSQC spectra exhibited time-dependent changes of signal intensities of major metabolites reached maximum at one week. Furthermore, we found thatrelative ¹³C-¹H signal intensity changes within the assigned metabolites can be applied to metabolic flux analysis at the atomic level.
Thus, we are willing to develop the novel methodologies for the analysis of "dynamic change" of metabolism at atomic level.Future perspective of their capability will be discussed at the conference.

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The mass spectrometry-based measurement is major methodology in current metabolomics research. However, we are willing to introduce a novel concept in the metabolomics studies by using the potential ability of molecular identification and non-invasive measurement by multi-dimensional NMR.
Firstly, we are constructing of ¹H and ¹³C chemical shift database of standard compounds dissolved in both aqueous and organic solvents. Based on searching the database, our signal assignment strategy is to identify detectable metabolites before starting non-invasive measurements. So far we observed different amino acid levels in the tissue extracts of ¹³C uniformly labeled Arabidopsis and Populus.
Furthermore, we tried the direct sampling of stem and leaf tissues of Populus, and measured non-invasively by magic angle spinning (MAS) method, The MAS spectra exhibited higher signal intensities in lipids and carbohydrates regions than extracted samples, suggesting the ability to detect biomacromolecules. Potentialities of non-invasive measurements will be discussed in the conference.

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Excretion of organic acids, especially citrate, from roots is a tolerance mechanism to aluminum (Al) stress. The mechanism is suggested to be controlled by citrate metabolism and transport system on plasma membrane. A key enzyme in the metabolism is NADP-isocitrate dehydrogenase (NADP-ICDH), which is a member of citrate conversion pathway. We generated transgenic Eucalyptus, in which NADP-ICDH was down-regulated by RNA interference procedure. Two transgenic lines showing 50% and 20% of NADP-ICDH activity of wild-type plants were not changed activity of other enzymes. Citrate concentration in roots of the transgenic lines was 1.4-1.6 times higher than that of wild-type plants. In addition, transgenic lines excreted citrate 1.5-1.7 times more. Growth of transgenic line on an acid soil was greater than that of wild-type plant. We inferred that RNAi-mediated repression of NADP-ICDH is a useful technology to confer Al tolerance to woody plants.

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To understand mechanisms of cell differentiation, monitoring single transformant cells is important. In the cases of xylem cells, however, it is difficult to employ such a strategy due to the complex structure of vascular tissues. We are planning to develop an analytical method by which molecular mechanisms of xylem cell differentiation can be addressed at the cellular level. We introduced foreign genes into isolated single cells in Zinnia xylogenic culture and examined their effects with regard to two differentiation-related aspects. First, the auxin responsiveness of particular cells was assessed by the transfer of a DR5 promoter-containing reporter gene. Second, we examined the expression patterns, protein product localization and function of a tracheary element-associated lipase gene using this system. We may also discuss results of the trial to quantify the amount of cellular fluorescence signals derived from transgene-encoded marker proteins.

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For genetic improvement of plural traits in a short time, we tried to develop new breeding method. In this method, firstly, by combination of introduced chromosome region genotyping and phenotyping, superior chromosome region is left and inferior region is replaced. We identified the chromosome region affect various trait including inferior or superior traits (CRATs).
BIL (backcross inbred lines) were randomly substituted 'Koshihikari' chromosome with 'Kasalath' fragment. SNP analysis was used for genotyping. BIL lines were crossed with 'Koshihikari', next self-pollinate, we got BC1F2 progeny. Using the genotyping with results of CRAT analyses, four superior lines were selected among 500 BC1F2 plants and planted to observe their phenotype. We got a superior line, that was same as 'Koshihikari' to heading data and plant height. At a present, we examine the traits, ratio of filled grain, grain number, pushing resistance, et al., we confirm the effect of replacing.

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Somatic embryogenesis is an obvious experimental evidence of totipotency in plant cells. However, the mechanism by which somatic cells initiate embryogenesis remains unclear.
We established a convenient culture system for somatic embryogenesis in Arabidopsis. When young seedlings of ecotype Nossen were cultured on the medium containing 2,4-D, somatic embryos were formed on shoot apical meristems. The somatic embryos grew into young seedlings and finally into mature plantlets. We determined the optimal culture conditions for induction of somatic embryos to be as follows: starting materials, young seedlings 1 day after sowing; concentration of 2,4-D, 4.5 μM. The frequency of somatic embryo formation was around 70% under these conditions.
Using this culture system, we examined the effect of ecotype on somatic embryogenesis in Arabidopsis. The frequencies of somatic embryo formation markedly differed between ecotypes. The ecotypes with high embryogenic potential may be useful for analysis of totipotency in plant cells.

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The gene organization of chloroplast DNA is highly conserved between liverwort (Marchantia polymorpha) and higher plants. Chloroplast transformation is useful in the functional analysis of genes with unknown functions. We established a chloroplast transformation system for liverwort suspension cultures. The transformation vector, containing an aadA expression cassette as a selectable marker, was designed for integration into the trnI-trnA intergenic region of the chloroplast DNA sequence by homologous recombination. To obtain resistant calli, liverwort suspension culture bombarded with this vector was screened on media containing spectinomycin. Some resistant calli were confirmed to be chloroplast transformants by DNA gel-blot analysis and PCR. Rapid establishment of homoplasmic transformants is important for functional analysis of genes with unknown functions. Culture in media without sucrose efficiently made transformants homoplasmic.

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Previously, we constructed Dual Site Gateway Binary Vector for cloning two genes on separate position. In this paper we report the new Gateway Binary Vector (R4 pGWB) to connect two DNA fragment using a part of Multisite Gateway system. R4 pGWB has attR4-CmR-ccdB-attR2 acceptor site and by the LR reaction using L4-promoter-R1, L1-cDNA-L2 and R4 pGWB, we can make promoter::cDNA construct easily. We also made R4 pGWB seriese for tag/reporter fusion at C-terminal of cDNA. These new vectors were named Hybrid Gateway Binary Vector system and could be a powerful tool for promoter swapping experiments.

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Previously, we reported Dual Site Gateway Binary Vector System for cloning of two genes. The vector contains new recombination sequences attR3-attR4 in addition to attR1-attR2. The vectors used to convert the attL1-attL2 entry clone into the attL3-attL4 type were also made. Moreover, we made reporter/tag. With this system, reporter/tag sequence is independently fused to two genes. This system is very useful for plant research, because the biochemical and cellular analysis of interaction between two proteins are very important.
In the report we show the promoter analysis of the gene that expressed in guard cell, as example of application of this system.

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We have developed gene disruption lines with endogenous retrotransposon Tos17. Currently, our mutant collection is more than 50,000 lines. On the Rice Genome Project, Nipponbare insertion lines were shared to 7 laboratories to obtain phenotype data in the field. Stages of observation were chosen with seedling stage, tillering stage, flowering stage, and seed maturation stage. To enable search by computer, we categorized phenotypes with 54 IDs. Over a period of three years, we collected phenotype data from all Nipponbare insertion lines with phenotype category id, additional description and photograph image file. All data were stored into relational database PostgreSQL and merged with the flanking sequence database. With data of rice FoxHunting, our information will be useful for functional analysis of rice genes.

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For the genome-wide elucidation of gene functions in rice, we have been applying Full-length cDNA over-expresser gene (FOX) hunting system dealing with the analysis of gain-of-function phenotypes. So far, several thousands of independent FOX-rice lines (T1 regenerants) were generated through Agrobacterium-mediated transformation of cDNA expression library composed of approx. 14,000 rice full-lengh cDNAs. Identification of introduced cDNAs in individual transgenic plants is in progress using genomic PCR followed by direct DNA sequencing of the amplified cDNA fragments. Among more than 4,000 independent FOX-rice plants, a single PCR fragment was detected in approx. 81 % of them, and two or more in only 2 %. Genomic Southern analysis of several FOX-rice lines (T1 generation) revealed 2 copies in average of T-DNA insertions per diploid genome. Moreover, PCR and DNA sequence analyses showed a variety of cDNAs were integrated into our FOX-rice pools indicating their usefulness for uncovering rice gene functions.

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It has been reported that more than 37,000 genes exist in the rice genome. To uncover functions of a large population of rice genes and to search for agriculturally useful genes efficiently, we are taking advantage of Full-length cDNA over-expresser gene (FOX) hunting system. A binary vector pRiceFOX, which was constructed for rice FOX hunting, carries restriction sites enabling directional cloning of full-length cDNAs (FL-cDNAs) under the control of maize Ubiquitin-1 promoter. A cDNA library in Agrobacterium was made with approximately 14,000 cDNAs and pRiceFOX. The Agrobacterium library was then transformed into rice. Transgenic calli, regenerants, soil-grown plants were observed for their visible phenotypes such as cell and tissue proliferation, organ morphology, plant height, growth habit, heading, fertility, pigmentation, etc. At present we have observed phenotypes of approximately 6,000 independent transgenic rice plants, and more than 10 % of them showed aberrant phenotypes.

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FOX Hunting System (full-length cDNA over-expressor gene hunting system) is useful tool for large-scale functional gene analysis. By using this system, we attempt to isolate rice genes involved in tolerance to environmental stresses. So far, about 5,000 lines of transgenic Arabidopsis overexpressing rice full-length cDNA (rice FOX lines) were screened at T2 generation for isolation of mutant lines that show tolerance to high-salinity or high-temperature stress. By comparing the growth on agar medium containing 150mM of NaCl, 56 lines were selected as high-salinity tolerant mutants. 9 of them could grow on agar medium containing even 180mM of NaCl. Several lines were found to be tolerant to transient high-temperature (42 degree Celsius) stress. We will identify rice cDNAs introduced in these mutant lines and confirm their involvement in stress tolerance by re-introducing experiment.

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