Photosynthetic CO2 fixation takes place through the Calvin cycle in the stroma, and its activity is redox-regulated through the thioredoxin-ferredoxin system. We previously reported that glutathione is involved in the regulation of photosynthesis, and that, among isozymes of chloroplastic fructose-1,6-bisphosphate aldolase (FBA), one isozyme (FBA1) undergoes glutathionylation. It has been known that chloroplastic FBA tetramer catalyzes the two reactions in the Calvin cycle, but it remains unknown how the isozymes participate in the regulation of the Calvin cycle. Arabidopsis plants have three chloroplastic FBA isozymes, FBA1 (At2g01140), FBA2 (At2g21330) and FBA3 (At4g38970). Based on FBA sequences from various plants, those isozymes proved to be classified into two group, FBA1 and FBA2/3 groups. Analysis of FBA complex from Arabidopsis plants revealed that FBA1 was incorporated into the tetramer of FBA2 and FBA3. We will discuss the function of FBA1 in the FBA hetero tetramer.

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The small subunit of Rubisco is encoded by RBCS multigene family in the nuclear genome in higher plants. In this study, the mRNA levels of each member of RBCS multigene family were determined in different organs in rice. In a leaf blade, the mRNAs of OsRBCS2, 3, 4 and 5 were highly accumulated, suggesting that these four genes mainly contribute to Rubisco synthesis. In other organs with less or no Rubisco content, the amounts of mRNAs were low. Additionally, the members that accumulated in such organs were different from those in a leaf blade ; leaf sheaths, OsRBCS3, 4 and 5 ; seeds, OsRBCS3 and 4 ; roots, OsRBCS1. The results indicate that expression of each member of RBCS multigene family is different depending on the amount Rubisco synthesis in each organ.

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Eleocharis vivipara is an amphibious sedge which develops C₄ traits under terrestrial conditions and C₃ traits with non-Kranz anatomy under submerged conditions. The plant seems quite useful for the screening of genes and proteins which are indispensable for C₄ photosynthesis and for the elucidation of molecular mechanism for the integrated expression of C₄ genes. Since enzymatic properties of the C₄-form PEPC from this plant has not yet been studied, here we cloned a cDNA according to Agarie et al.(2002) and expressed in E. coli to prepare the recombinant PEPC (EvPEPC). A unique property of EvPEPC was that K_m for PEP was about 10-fold smaller than those for the C₄-form PEPCs from various C₄ species reported so far.
Several attempts were made to identify residues responsible for this unusual property by site-directed mutagenesis.

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Coccolithophorids (Haptophyta) are known to acquire their chloroplasts via secondary endosymbiosis and expected to have developed unique carbon metabolisms during evolution. In a coccolithophorid, Emiliania huxleyi, we already reported that C₄ compounds is produced via beta-carboxylation reactions in addition to the C₃ cycle and pyruvate carboxylase (PYC) is considered to be involved in the beta-carboxylation since PYC transcript remarkably increased under illumination. In this study, we detected PYC activity in the roughly isolated chloroplast fraction, but not in the crude extracts. Then, we examined the expression of PYC by using anti-PYC antibody raised against the partial peptide of a recombinant PYC produced in E. coli. PYC was immunologically detected in the crude extracts of light-grown E. huxeyi cells although the abundance of PYC did not show significant change in light- and dark-adapted cells. These data suggest the contribution of chloroplastic PYC to the active beta-carboxylation during photosynthesis in E. huxleyi.

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Acclimation to CO₂-limiting stress conditions needs the monitoring of the environmental CO₂ levels and the following induction of low-CO₂ responsive genes for the carbon-concentrating mechanism (CCM). We focus on understanding roles of the low-CO₂ responsive gene products in CCM. Using cDNA array, we previously identified two genes, LciB and LciC, encoding approximately 50 kDa soluble proteins, showing rapid induction under CO₂-limiting stress conditions in light. LciB complemented the Chlamydomonas mutant, pmp1, which shows reduced CO₂-transport activity and high-CO₂ requiring phenotype under low-CO₂ condition. We examined the subcellular localization of LCIB and LCIC protein by immuno-histochemistry and also found that LCIB and LCIC form a complex in vivo. Based on the above observations, function of the LCIB/LCIC complex will be discussed.

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In cyanobacteria, the genes related to nitrogen acquisition and metabolism are activated under nitrogen-limited conditions. In Synechococcus elongatus, one of the six genes encoding the group2 sigma factor of RNA polymerase, rpoD4, was also found to be upregulated by nitrogen limitation. The upregulation was partly (but not entirely) dependent on NtcA, the global nitrogen regulator protein. Under nitrogen-deficient conditions, the expression level of nblA, a gene required for adaption of the cells to N-deficent conditions, was low in a ΔrpoD4 mutant (<50% of wild-type), leading to complete loss of pigmentation after prolonged incubation. Regardless of the nitrogen availability, however, the mutant showed altered expression levels of a few genes related to cell division and showed abnormally elongated morphology, being about 10 times longer than the wild-type cells. These results indicated that RpoD4 takes part in adaptive response of the cells to N depletion as well as in regulation of cell division.

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In addition to the ABC-type nitrate/nitrite transporter, which has high affinity for both substrates (K_m ~1 μM), Synechococcus elongatus has an active nitrite transport system with an apparent K_m (NO₂^-) value of 20 μM. We found that this activity depends on the cynABD genes, encoding a putative cyanate (NCO^-) ABC-type transporter. Nitrite transport by CynABD was competitively inhibited by cyanate with a K_i (NCO^-) value of 0.025 μM. The transporter was induced under nitrogen deficiency conditions, and the induced cells showed a V_max value of 11-13 μmol mg^-1Chl h^-1 for cyanate or nitrite, which could supply ~30% of the amount of nitrogen required for optimum growth. Its relative specificity for the substrates and the expression pattern of the transporter suggested that the physiological role of the cyanate/nitrite transporter is to allow nitrogen-deficient cells to assimilate low concentrations of cyanate in medium.

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Nitrogen (N) is one of the most important macro elements for plant production, and urea is most frequently used N fertilizer worldwide. Recent studies suggested that urea can be directly taken up by plant in the form of urea.
We investigated AtDUR3, homologous gene for high-affinity urea transporter (DUR3) in budding yeast, in order to clarify its biological functions in Arabidopsis. The uptake of stable isotope labeled urea in T-DNA insertion lines for AtDUR3 was decreased compared with wild-type. Immunological analysis as well as promoter analysis showed the accumulation of AtDUR3 in the plasma membrane of roots under N deficient condition (Kojima et al., 2007). Recently Hem et al., 2007 reported identification of phosphorylation site in AtDUR3. We substituted potential phosphorylation site in AtDUR3 by site directed mutagenesis approach in order to mimic phosphorylation or dephosphorylation. Potential post-translational regulation of AtDUR3 will be discussed.

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Cyanobacteria are well-defined monophyletic oxygenic photoautotrophs, and are targets of intensive genomic analysis. Previous studies on the structural organization of genomes over various species revealed the genomic core with interspersed genomic islands. Here we show that a new genomic profile method successfully classified the conserved genes in marine cyanobacteria, including both Prochlorococcus and Synechococcus, into seven groups called 'virtual linkage groups' according to their respective distance relationships over all the genomes analyzed. The virtual linkage groups were used to define mosaic domain structure of the genomes. Surprisingly, the seven genomic domains were located at different distances from the replication origin in each genome. The current model of genomic mosaic domains can explain global evolution of the genomic core of the marine cyanobacteria. It also reveals islands of lateral gene transfer. The stability of such genomic organization during the evolution is discussed.

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Somaclonal variation is a phenomenon that results in phenotypic variation in plants regenerated from cell culture. Previously, we found that an endogenous retrotransposon, Tos17, is activated and transposed in cultured rice cells. Large scale phenotyping of 50,000 Tos17 insertion lines revealed that additional factors were likely to be responsible for somaclonal variation in these lines. We analyzed whole genome sequences of Tos17 insertion lines by next generation sequencing with a new strategy using a relational database. We found that many single point mutations, insertions and deletions occurred in the rice genome during cell culture. These mutations are a likely cause of the somaclonal variation. Our strategy could be applied to determine whether these types of mutations occur at high frequencies in cultured cells of other organisms, including the stem cells of animals. This strategy can also be applied to gene mapping and isolation, and to the diagnosis of disease.

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Peptides encoded by short open reading frame (sORF) are reported to be involved in various functions in yeast and Drosophila. We identified 7,159 sORFs ranging from 90 to 300 bp in the intergenic regions of the Arabidopsis genome. We developed an agilent custom microarray including 5,921 sORFs and 26,192 AGI genes (TAIR7) in Arabidopsis to identify sORF function. Expression of AGI genes and intergenic transcripts were detected in about 70% and 56% of total spot number, respectively. On the other hand, expression of about 9% sORF was detected. Some sORF expressions were confirmed by RT-PCR analysis to validate microarray results. RT-PCR analysis was preformed in various tissues (Leaf, Flower, Silique and Root). Some sORF showed tissue specific expression pattern. We will analyze gene expression profile during illumination to identify light-regulated sORFs and will report the microarray results in this meeting.

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It is well known that some of short peptides have hormone-like functions. However, small coding sequences may not be identified by current gene finders. In an earlier report, we identified coding sORF in A. thaliana genome by Bayes' theorem and validated the functionality as coding genes by selection pressure and/or RNA expression. In the present study, to examine whether functionality of sORF can be inferred by only selection pressure throughout comparative analysis, we compared the relationship between selective pressure and RNA expression because most of plant species do not have RNA expression data at genomic level. As a result, we found that sORFs with purifying selection tend to be more highly expressed in distant plant species than those in close plant species, indicating that comparative genomics substantially inferred functionality of sORF with respect to RNA expression. Finally, we will report novel coding sORFs with purifying selection in rice genome.

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Alternative usage of transcription start sites (TSSs) is one of the key mechanisms to generate gene variation. Here we show diversified evolution of TSSs in Oryza sativa and Arabidopsis thaliana by comprehensive analyses of full-length cDNAs and genome sequences. We determined 45,917 representative TSSs within 23,445 loci of O. sativa and 35,313 TSSs within 16,964 loci of A. thaliana. The nucleotide features around TSSs displayed distinct patterns when the most upstream TSSs were compared to downstream TSSs. In addition, this difference was commonly observed in the two species. Relative entropy analysis revealed that the most upstream TSSs had retained canonical cis-elements, whereas downstream TSSs showed atypical nucleotide features. These results indicate that plant TSSs were generally diversified in downstream regions, which resulted in the development of new gene expression patterns. We discuss the possibility that O. sativa and A. thaliana might have evolved novel TSSs in an independent manner.

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Cold and dehydration are adverse environmental conditions that affect plant growth and productivity. Many genes have been described that respond to both stresses at the transcriptional level, and their gene products are thought to function in stress tolerance and response even though these stresses are quite different. These genes include late embryogenesis abundant proteins, detoxification enzymes, chaperones, protein kinases, and transcription factors. The cis-acting elements that function in stress-responsive gene expression were analyzed to elucidate the molecular mechanisms of gene expression in response to these stresses. In this study, we searched for conserved sequences in the promoter regions of cold and/or dehydration inducible genes. The 1000-bp promoter regions upstream of putative transcriptional initiation sites located at the 5'ends of the full-length cDNA clones were used for this search. We found some highly conserved sequences including known cis-acting elements and novel conserved sequences in these promoter regions.

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Core promoter locates from -50 to +50 relative to the major transcription start site (TSS) in a promoter. It is recognized by the pre-initiation complex and determines direction and position of transcriptional initiation. We have collected large amount of TSS tags from Arabidopsis, and in combination with statistical analyses of promoter sequences, we have discovered novel plant-specific core promoter elements. Based on these findings, we further investigated relationship of the core promoter type to gene category and gene structure.

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An emergence of tiling arrays enables to make a comprehensive and a quantitative gene analyses. Toyoda and Sinozaki developed a genome wide gene prediction method, called ARTADE (ARabidopsis Tiling-Array-based Detection of Exons). The method had succeeded to detect numerous unknown non-coding genes from Arabidopsis. However, the method uses tiling arrays information independently for each probe. The conventional ARTADE is therefore weak for noisy probes. Moreover, if two genes are closed each other, it is difficult to divide the two genes from redundant tiling array sequence. For the problems, we have developed the new ARTADE(ARTADE2.0) based on multiple tiling array data. The method predicts genes by spatial probabilistic model based on correlation matrix score of multi-dimensional data by tilling arrays. ARTADE2.0 shows robustness for the noisy probes and a high performance for the start-end prediction of genes.

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Plants respond and adapt to drought, cold and high-salinity stresses in order to survive. We have applied Arabidopsis Affymetrix tiling arrays to study the whole genome transcriptome under drought, cold, high-salinity stress and ABA treatment conditions. The tiling array experiments showed that 7,719 novel transcription units (TUs) exist in the Arabidopsis genome. These include 1,275 and 181 TUs that are induced and downregulated, respectively, by the stress- or ABA treatments. Most of the novel TUs are hypothetical non-coding RNAs and about 90% of them are mapped on the antisense strand of the AGI code genes. Interestingly, high correlation of the expression ratios (treated/untreated) was observed between AGI code genes and the novel TUs on the antisense strand. We studied the biogenesis mechanisms of the stress- or ABA-inducible antisense RNAs and found that the expression of sense TUs is necessary for the stress- or ABA-inducible expression of the antisense TUs.

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Goldenrod (Solidago altissima L.) produces polyacetylenic compounds including cis-dehydromatricaria ester (cis-DME), which shows the allelopathic effect inhibiting the growth of other plants, in the root and rhizome. We studied the cis-DME production in goldenrod hairy root cultures, and previously reported that it was suppressed by light. To study the biosynthetic pathway of cis-DME and its regulation, we isolated 2 clones of gene segments homologous to FAD2-related fatty acid acethylenases (ACETs) from goldenrod, which were thought to be the first enzyme in the acetylenic compound production. Now, we have investigated the expression of the cloned sequences, named SaFAD2-A and B, in the goldenrod tissues by quantitative RT-PCR. Differences between the expressional patterns of the two sequences and their relevance to the cis-DME production will be discussed.

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Prenylated flavonoids show various physiological and pharmacological activities, therefore they have been drawn a large attention of researchers in the pharmaceutical, food and agricultural field. Detailed biochemical studies demonstrated that the prenyl chain plays a crucial role in their activities, while the gene coding for prenyltransferase of flavonoids has been unknown more than three decades until very recently. The first cDNA encoding flavanone-specific plant prenyltransferase, naringenin 8-dimethylallyltransferase (SfN8DT-1) was isolated from Sophora flavescens.
In this study, to figure out the overview of plant flavonoid prenyltransferase family, we have cloned several candidate genes coding prenyltransferases that accept other type of flavonoid as the substrate using S. flavescens EST information. Enzymatic characterization of these clones in yeast expression system has revealed that one of them encodes isoflavone-specific prenyltransferase, genistein 6-dimethylallyltransferase (SfG6DT). The gene expression analysis of SfG6DT and subcellular localization will be also reported.

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Plants produce a huge number of low molecular weight natural products with diverse structures as well as biological activities. These diversities are occurred by both a wide range of basic structures and multiple modifications of a common skeleton through glycosylation process. Although the glycosyltransferases (UGTs) catalyzing sugar transfer to small molecule aglycones have been extensively characterized, reports on sugar-sugar UGTs that glycosylate the sugar moiety of glycosides are limited.
We have recently cloned a cDNA encoding a novel sugar-sugar UGT (CaUGT3), which catalyzes glucosyl transfer to the 6"-hydroxyl group of the glucose moiety of flavonoid glucoside to form flavonoid gentiobioside, from cultured Catharanthus roseus cells. To gain an insight into the molecular structure governing the acceptor as well as donor specificity of this enzyme, a homology model of CaUGT3 was constructed. The conservation of primary and secondary structure in relation to substrate specificity of CaUGT3 is investigated.

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Precursors of nutraceuticals are synthesized through metabolic pathways common to higher plant species. Although light-emitting diode (LED) has recently been applied to "plant factories", we know little what enzyme genes or substances in metabolic pathways are enhanced in response to LED irradiance.
Arabidopsis thaliana and other Cruciferae plants were exposed to blue LED (470 nm), red LED (660 nm), blue LED + red LED, or blue LED (pulsed) at 110-240 μmol m^-2 s^-1, and subjected to DNA array analysis (Affymetrix GeneChip Arabidopsis ATH1) or metabolomics (Agilent 6510 Accurate-Mass Q-TOF LC-MS/MS and others). Expression of gene for biosynthesis of lignans and flavonoids have been enhanced by blue LED, leading to enrichment with kaempferol glycosides and anthocyanins. Gene for chalcone synthase (AtCHS) has been ectopically expressed and resulted in accumulation of kaempferol glycosides.

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Plants accumulate various aromatic secondary metabolites with biological activity. Biosynthetic pathways of those metabolites are branched at p-coumaric acid which is synthesized by phenylpropanoid pathway.
Our research objective is to modify aromatic secondary metabolism in Arabidopsis thaliana for production of useful metabolites. We have introduced the gene encoding tyrosine ammonia-lyase (TAL) from photosynthetic bacterium into Arabidopsis, aiming at bypassing the rate-limiting steps of phenylpropanoid pathway. Higher amount of some phenylpropanoids and quercetin glycosides were accumulated in TAL-expressing plants than in control plants. Transgenic Arabidopsis expressing the gene encoding flavone synthase (FNS) from parsley have been obtained for production of flavones. In FNS-expressing plants, apigenin was accumulated upon feeding of substrate naringenin.
We performed flavonoid-targeted analyses of TAL-expressing plants for understanding the effect of TAL expression more precisely. Transgenic plants harboring TAL and FNS were obtained, and secondary metabolites accumulated in those plants were analyzed.

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The production of amino acid-derived metabolites is thought to be comprised of many biosynthetic processes. Using transcriptomics database, the co-expression analysis allowed us to predict the transcriptional regulation factors, which regulate the specific expression patterns. In this study, we analyzed a metabolic system of methionine-derived glucosinolate (MET-GSL): transcriptional factors (PMG1, PMG2 and PMG3) and the MET-GSL biosynthetic genes. We previously showed that the double-knockout line, pmg1pmg2, are absent of MET-GSL. In transcriptomic data of pmg1pmg2, the expression levels of the PMG3, MET-GSL biosynthetic genes and functionally un-identified genes were significantly repressed. Thus, we assumed that the pmg1pmg2 is comparable to knockout line of three PMGs. Moreover, in the T-DNA insertion lines of the un-identified genes, biosynthetic genes and transporter genes, the MET-GSLs significantly decreased. These results suggested that these lines will be good models for elucidation of production of amino acid-derived metabolites.

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Flowers of Petunia axillaris subspecies axillaris emit isoeugenol and eugenol, as do flowers of P. hybrida, a hybrid of P. integrifolia and P. axillaris. However, the flowers of P. axillaris subspecies parodii emit neither isoeugenol nor eugenol but contain high levels of dihydroconiferyl acetate. We recently showed that in P. hybrida, isoeugenol synthase (PhIGS1) and eugenol synthase (PhEGS1) biosynthesize isoeugenol and eugenol, respectively, from coniferyl acetate. Here we show that P. axillaris subsp. parodii has a functional EGS gene, but its IGS gene contains a frame-shift mutation that renders the protein inactive. Despite the presence of an active EGS enzyme in P. axillaris subsp. parodii, the coniferyl acetate substrate is converted to dihydroconiferyl acetate. By contrast, suppressing the expression of PhIGS1 in P. hybrida by RNAi also leads to a decrease in isoeugenol biosynthesis, but instead of the accumulation of dihydroconiferyl acetate, the flowers synthesize higher levels of eugenol.

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Plant oxylipins are formed from linolenic acid 13-hydroperoxide. The hydroperoxide is the common substrate for the jasmonate and hexenal pathways. Jasmonates and hexenals have distinct physiological functions, thus, the two branches must avoid competition of hydroperoxide lyase (HPL) and allene oxide synthase (AOS) for the same substrate to adjust the amounts of the end products appropriately. When pHPL::GUS and pAOS::GUS plants were analyzed, pHPL was active in the stigmas, filaments, and calyxes, while pAOS was active specifically in anthers. The activity of pHPL was extensively suppressed in coi1 mutant, thus, it was suggested that jasmonates were essential for the activation. When the leaves were mechanically wounded, high activity of pHPL could be found at the rim of leaves while that of pAOS could be found at the mid vein. Accordingly, tissue specific expression of these two enzymes might be one way to avoid competition for the same substrate.

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It has been reported that the plasma membrane of animal cell has lipid microdomains in which particular lipids and proteins are assembled. Microdomain is likely to contribute for the signal transduction and membrane transport. Recently, also in plant cells, the presence of plasma membrane microdomain has been indicated.
In a plant cell, many kinds of membrane transporters are involved in various vacuolar functions, but the distributions of these transporters on the vacuolar membrane are remaining unknown.
In the present study, to analyze distribution of proton pumps on the vacuolar membrane, we isolated intact vacuole from Arabidopsis suspension cultured cells, and stained proton pumps with immunofluorescent method. We found that v-ATPase was assembled in small area on isolated vacuole, but v-PPase was dispersed. Detergent resistant membrane (DRM) prepared from the vacuolar membrane contained abundant v-ATPase. v-PPase was contained much in detergent soluble membrane (DSM) than in DRM.

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Hyperosmolality constitutes a major challenge to the growth of bacterial cells. It is known that the adaptation to the stress consists ofa biphasic response. During the first phase, cells accumulate additional K from the medium through their K uptake systems.During the second phase, cells synthesize high amounts of the non-ionic compatible solute. However, the detailed mechanisms are still unknown. In this study, to study the osmoadptaion mechanisms of a single Synechocystis cell, we produced the micro-channel device in combination with a small AWP cage to trap the cells. The cells are kept at the space when the medium is exchanged with another medium. Using the devices, the volume change of the wild-type cells and the the cells which has mutations of K transporter genes was monitored. The hyperosmotic shock introduced different rate of cell-volume change between them.

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Characean internodal cell generates a receptor potential when it is mechanically stimulated. We have assumed that the generation of receptor potential takes place only at the stimulated portion. An applied stimulus, however, should propagate to the everywhere of the cell through a change of intracellular pressure. In this study, we studied whether the receptor potential is restricted at the stimulated portion or not.
Surprisingly, a change in membrane potential was also observed at the non-stimulated portion with no delay. Furthermore, amplitude of the change was statistically larger at the non-stimulated portion than at the stimulated one. Thus, we strongly suggest that the response in membrane potential against the cell compression is induced by a membrane expansion which occurs allover the cell by an increase of intracellular pressure during the compression.

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Ca²⁺-permeable mechanosensitive channels are supposed to be involved in mechano-sensing in plants, but their molecular nature is basically unknown. Two candidates for such channels in Arabidopsis, designated as Mca1 and Mca2, have been recently reported. Here, we identified two MCA orthologues in tobacco BY-2 cells, named NtMCA1 and NtMCA2. The NtMca1/2-GFP fusion proteins appeared to be localized in the plasma membrane and were not observed in the vacuolar membrane. NtMCA1/2 complemented the lethal phenotype of the yeast mid1 mutant defective in a putative Ca²⁺-permeable mechanosensitive channel component, suggesting that both proteins have an activity to mediate Ca²⁺ uptake in yeast. Inhibition of growth of BY-2 cells under Ca²⁺ deficiency was significantly alleviated by overexpression of either NtMCA1 or NtMCA2. We will report ⁴⁵Ca²⁺ uptake characteristics of the overexpressors and discuss about possible roles of NtMca1/2 as well as their physiological relevance.

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Puccinellia tenuiflora (alkali grass) is a halophyte which is suggested as a good contrast to study salt tolerance mechanisms in monocotyledonous crops. It was shown that alkali grass can maintain its shoots potassium content remains unchanged during salt stress suggesting the involvement of ion transporter or channel in regulating ion homeostasis of the plant.
We isolated an HKT type transporter homologue from alkali grass and named it as PutHKT1. RT-PCR experiment showed that the expression level of PutHKT1 gene were induced by both salt stress and potassium starvation in roots, but only slightly regulated by those stresses in shoots. To characterize PutHKT1 functionally, PutHKT1 cDNA was expressed in yeast strain 9.3. The overall result of functional characterization in yeast suggested that PutHKT1 functions as a high affinity K⁺/Na⁺ co-transporter. Interestingly, the expression of PutHKT1 in Arabidopsis under the control CaMV35S promoter leads to salt sensitivity of the transgenic plants.

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Aluminum ion (Al³⁺) inhibits root growth in acidic soils. Nevertheless, a variety of plants have a capability to grow in acidic soils by excluding and/or detoxifying Al³⁺. In some Al-resistant plants, Al exposure triggers the secretion of organic acids such as malate or citrate from their root tips, and the organic acids chelate and detoxify Al³⁺.
Al-resistant cultivars of wheat release malate, which is controlled by the Al-activated malate transporter encoded by the ALMT1 gene. Heterologous expression of ALMT1 conferred Al³⁺-activated malate efflux in Xenopus oocytes. To clarify the underlying processes for Al³⁺-activated malate efflux via ALMT1, electrophysiological analyses were performed using Xenopus oocytes expressing ALMT1 with desired mutations. These results will be reported with a possible activation mechanism of ALMT1 malate transporter.

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Plant vacuole accumulates many solutes such as nutrients, proteins, second metabolites and ions. Uptakes of solutes from cytosol are maintained by solute-specific membrane transporters. However, most of membrane transporters have not been identified yet.
We attended to anthocyanin pigments, which are major second metabolites of plant. Interestingly, color developments of anthocyanins, red to blue, are depending not only on its structures but also on environmental status of vacuolar space. Vacuolar pH, metal-ions concentration, anthocyanin-binding small molecules and proteins influence colors of anthocyanins. On the contrary, anthocyanins are natural indicators of vacuolar solutes.
In this study, we prepared over-expression library of membrane transporter genes of Arabidopsis thaliana in order to screen genes that affect colors of anthocyanins. We transformed a mutant line of Arabidopsis thaliana that accumulates anthocyanin-pigments by Agrobacterium method using the library.

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Plasma membrane intrinsic protein is classified into two subfamilies, PIP1 and PIP2. The activities when co-expressing PIP1 and PIP2 were higher than sum of those when PIP1 and PIP2 expressing separately. It's thought that protein traffic and heterotetramer formation participate in this activation mechanism. Previous data of X-ray structural analysis of PIP2 tetramer showed that twenty and a few amino acid residues contribute to adhesion between neighboring monomers and most of them are located in transmembrane helices. Peptide sequence of the transmembrane domain of PIPs is so conservative that mixed complex with PIP1 and PIP2 is presumed to be able to form. As for protein traffic, a series of experiment using chimeric proteins of which N-terminal was exchanged showed that a peptide sequence which is needed for PIP2 protein to target to the plasmamembrane is located in the N-terminal. Determining the peptide sequence pattern of the motif is being continued.

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Three SIP members of A. thaliana are localized in the ER in a cell-specific manner (FEBS Lett. 2005). SIP1;1 and SIP2;1, but not SIP1;2, have water channel activity. Actual substrates and physiological role are unknown. Here we examined effect of various stimuli on expression of SIP genes. Database shows that SIP1;2 expression was enhanced by silver nitrate, an inhibitor of ethylene action. We also considered ethylene receptor in the ER.
Silver nitrate and silver thiosulfate enhanced SIP1;2 markedly. However the gene was not affected by an ethylene precursor ACC. There was no change in SIP mRNA levels in ethylene insensitive or ethylene-over producing mutant lines. SIPs may not be directly associated to ethylene action. SIP1;2 expression was reduced by the treatment with tunicamycin. Other reagents including paraquat, copper and zinc had no effect. SIP genes may be constitutively expressed and response to a few specific stimuli such as silver ion.

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When plants are exposed to various abiotic stresses, they respond and adapt to these stresses. DREB1A is one of cold inducible genes encoding transcription factors in Arabidopsis. Overexpression of DREB1A/CBF3 caused increased tolerance to freezing in Arabidopsis, suggesting that the DREB1A/CBF3 protein functions in cold stress-responsive gene expression. Many protein products regulated by DREB1A/CBF3 are probably responsible for the stress tolerance of plants.
In this study, we analyzed genes for chloroplast membrane localized proteins regulated by DREB1A. We tested the cellular-localization pattern of these proteins by using GFP fusion constructs. Under normal condition, these proteins were localized at chloroplasts of guard cells. Under cold condition, we detected GFP fluorescence not only at guard cells but also at chloroplasts of mesophyll cells. We generated transgenic plants overexpressing these proteins, and determined the metabolites of the chloroplasts of the transformants.

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Plants emit various kinds of volatile compounds after herbivore attack. In lima bean plants, it was known that an intact plant neighboring to an herbivore-damaged plant could induce some defense genes. It implies the existence of plant-plant interaction through herbivore-induced plant volatiles (HIPV). In this study, we observed induction of defense response against an herbivore, two-spotted spider mite, in HIPV exposed healthy plant. At first, we constructed a system of volatile exposure with air-flow. Secondly, we confirmed the induction of one of the defense genes, chitinase. Thirdly, we showed the decrease of the number of offspring laid on the HIPV exposed plant. We also observed slight decrease of proteins essential for photosynthesis such as oxygen evolving enhancer and ATPase. From these results, we concluded that the healthy plants neighboring to herbivore-damaged plants increased the resistance against the mite damage through HIPV perception and subsequent induction of the defense responses.

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In response to herbivory, plants emit specific blends of volatiles that attract carnivorous natural enemies of herbivores. These volatiles are called herbivore-induced plant volatiles (HIPVs). We hypothesized that microorganisms in two-spotted spider mites were involved in the induction of specific blend of HIPVs since accumulation of salicylic acid (SA) and expression of SA-inducible genes were observed in lima bean plants infested by the spider mites. To test this hypothesis, we prepared free spider mites free of microorganisms to compare the responses of lima bean plants to damage caused by either germ-free spider mites or normal ones. Amounts of HIPVs and levels of SA were higher in lima bean plants damaged by the germ-free mites. Expression of SA-inducible genes was also enhanced in plants damaged by germ-free mites. These results suggest that microorganisms are involved in the induction of specific blend of HIPV in lima bean leaves infested by spider mites.

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Ca2+ signals have evolved a wide spectrum of strategies as intra/intercellular signals. Even though Ca2+-binding sensory proteins like Ca2+-dependent protein kinase (CDPK) are expected to act as intracellular protein mediators that govern gene regulation networks in response to Ca2+ influx after herbivory, little is known about the mechanisms. To investigate whether CDPKs play certain roles in an herbivore response-signaling pathway, we screened the characteristics of Arabidopsis CDPK mutants damaged by a feeding generalist herbivore, Spodoptera littoralis. After herbivory, the cpk3 and cpk13 mutants showed lower transcript levels of plant defensin gene PDF1.2 compared to wild-type plants. in vitro Kinase assays of the CDPK proteins with defense-related transcription factors (TFs) demonstrated that CPK3 phosphorylates TFs and ATL2 as substrate targets. The results presented show that CDPKs are involved in controlling the herbivore-induced expression of plant defensin gene, and that they exert such control by interacting with transcription factors.

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Plants are exposed to many types of abiotic or biotic stresses. Many researchers have analyzed the mechanism of these stress responses using Arabidopsis plants. At the present day, creation of plants having tolerance for several abiotic or biotic stresses has reported. On the other hand, insect damage is very serious problem that decrease the crop yields. However, the mechanism of plant response to feeding damage has not been well understood.
We analyzed the interaction between Arabidopsis and western flower thrips (Frankliniella occidentalis), which is one of the most serious insect pests. Thrips is cell content feeding insect that penetrate single cells with stylet to suck out the contents. In addition, thrips transmit the virus from plant to plant. We focused on the function of the immunity-related plant hormones jasmonate (JA), ethylene (ET), and salicylic acid (SA) on plant tolerance to thrips feeding.

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Our previous works showed that cell division starts at 3 days after cutting, and tissue-reunion almost completes in 7days in Arabidopsis cut flowering stems and microarray analysis revealed that genes determined to be up-regulated at earlier stage include those that are related to phytohormone signaling and transcription factors (TF). In this study, we performed molecular analysis for tissue-reunion process. Auxin-responsible IAA gene was significantly up-regulated at 1day after cutting and mIAA transgenic plants, of which auxin response were suppressed, showed lower efficiency in the reunion process. ACC synthase and putative ethylene-responsive AP2-TF showed similar expression patterns. Ethylene-signaling deficient mutant also showed lower efficiency, and knockout plants for AP2-TF or NAC-TF using CRES-T methods did not show normal reunion process. These results suggest that auxin /ethylene signaling pathways and putative downstream TFs have an important role in the regulation of gene expression during tissue-reunion of Arabidopsis cut flowering stems.

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Systemic acquired resistance (SAR) is a potent innate immunity system in plants that is effective against a broad range of pathogen. SAR development in plants is mediated by salicylic acid (SA). Here, using different types of SAR-inducing chemicals, i.e., 1,2-benzisothiazol-3(2H)-one1,1-dioxide (BIT) and benzo(1,2,3)thiadiazole-7-carbothioic acid S-methyl ester (BTH) that act on upstream and downstream of SA in the SAR signaling pathway, respectively, we show that treatment with abscisic acid (ABA) suppresses the induction of SAR in Arabidopsis. Analyses using several mutants in combination with these chemicals revealed that ABA suppressed SAR induction by inhibiting the pathway both upstream and downstream of SA, independently of the jasmonic acid/ethylene mediated signaling pathway. Suppression of SAR induction by the NaCl-activated environmental stress response proved to be ABA dependent.

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Plants recognize pathogen attack by at least two distinct mechanisms and induce defense responses. One mechanism is for microbe-associated molecular patterns (MAMPs), such as flg22. We recently reported that flg22-induced resistance to a bacterial pathogen, Pseudomonas syringae pv. tomato DC3000 (PstDC3000) is partially dependent on SA signaling. Jasmonic (JA) acid and ethylene (ET) signaling are generally believed to inhibit SA signaling and negatively affect resistance against PstDC3000. We have explored the possibility that multiple signalings cooperatively contribute to the robust defense signaling network by constructing a dde2, ein2, pad4, sid2 quadruple mutant in which JA, ET and SA signaling are all blocked. Surprisingly, 80% of flg22-induced resistance was lost in the quadruple mutant. This result indicates that major parts of the resistance response are mediated by a signaling network controlled by these four genes and that JA/ET, as well as SA signaling, positively contribute to resistance against PstDC3000.

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The biosynthesis of camalexin, a major phytoalexin in Arabidopsis, is induced upon infection of a variety of pathogens, and abiotic stresses such as AgNO₃ treatment and UV-B irradiation are also known to stimulate the biosynthesis. Recent studies have almost elucidated the camalexin biosynthetic pathway including several cytochromes P450 and nitrilases, and the expression levels of such biosynthetic genes have been demonstrated to be greatly induced under those inducing conditions. However, possible mechanisms underlying the induction of camalexin biosynthesis remain elusive
Here we report our metabolic profiling study for identification of transcriotion factors involved in the induction of camalexin biosynthesis. One of T-DNA insertion events within transcrioption factor genes was revealed to have reduced the camalexin accumulation levels under the induction treatment. We discuss possible regulation mechanisms of the camalexin biosynthesis involving the induction of this transctiption factor gene in terms of transcriptome analyses.

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asymmetric leaves1 (as1) and as2 mutants of Arabidopsis show similar phenotype, such as the formation of asymmetrically lobed and downwardly curled leaves. It has been reported that as1 and as2 mutants also show the abaxialized leaves. The AS1 gene encodes a protein containing two myb repeats. The AS2 gene encodes a plant-specific protein with a domain containing a cysteine repeat and a leucine-zipper-like sequence. To elucidate the molecular function of AS1 and AS2 in the formation of adaxial-abaxial polarity, we isolated several enhancer mutants that enhance the abnormality in the adaxial-abaxial polarity in the leaves of the as1 mutant. One of the enhancer mutants, enhancer of asymmetric leaves2 and asymmetric leaves1 (eal) had abaxialized filamentous leaves in as1and as2 backgrounds. RT-PCR analysis revealed that the accumulation levels of transcripts of genes that affect the abaxialization of leaves were elevated in as2 eal and as1 eal double mutants.

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Loss-of-function mutations in the ASYMMETRIC LEAVES2 (AS2) gene in Arabidopsis result in various defects in leaf development, such as formation of asymmetrically lobed and downwardly curled leaves, formation of leaflet-like structures from petioles, generation of an abnormal venation system, shorter length of petioles and leaf laminas than WT, and ectopic expression of class 1 KNOX genes in the leaves. To uncover the function of AS2 in the leaf development, we screened enhancer and suppressor mutants of as2. We obtained several enhancer candidates. #16 mutant, one of enhancer candidates, showed filamentous leaves in as2 mutant background and pointed leaves in wild-type background. In high temperature condition, #16 mutant showed filamentous leaves in as2 mutant background and narrow leaves in wild-type background, suggesting sensitivity to high temperature. From map-based cloning, it was showed that #16 mutation mapped to the lower arm of chromosome 5.

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Guanine-rich DNA sequences often form G-quartets, planar arrays of four guanines stabilized by monovalent cations (K⁺ and Na⁺); which interact to form a G-quadruplex. In telomeres, G-rich sequences (repeats of TTTAGGG) form a G-quadruplex that interferes in the elongation reaction by telomerase. Recent bioinformatics analysis revealed that G-quadruplex-forming sequences exist ubiquitously in mammalian and bacterial genomes, and are enriched in promoters and near the translation start sites. Ligand binding of a G-quadruplex may decrease transcription of downstream genes. These findings support the current hypothesis that G-quadruplex may be a novel type of ubiquitous regulatory element. We studied the G-quadruplex sequence and its regulatory mechanism in Arabidopsis. Sequence analysis showed that the Arabidopsis genome possesses approximately 1,000 G-quadruplex sequences (approximately 700 genes). We also report that the similarity of G-quadruplex sequences and the changes of expression in genes nearest a G-quadruplex by the binding of its ligand.

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Auxin is synthesized primarily in young tissues, and is transported to other tissues by polar auxin transport where it is perceived by auxin receptors. Recent works on auxin transport and signaling showed how these two processes regulate plant development. However, it is still unclear how the genes related to auxin synthesis control plant development, because only a few genes related to auxin synthesis have been characterized in plants. Recently, we showed that the TRANSPORT INHIBITOR RESPONSE2 (TIR2)gene encodes the TRYPTOPHAN AMINOTRANSFERASE OF ARABIDOPSIS protein required for converting Trp to indole-3-pyruvic acid, a precursor of auxin. We show evidence that auxin synthesized by TIR2 is essential for root meristem development. Furthermore, we show that the expression of the TIR2 gene was controlled differently by different signals suggesting that the TIR2 gene has different role in different plant development.

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Pattern formation along the central-peripheral axis is a fundamental process in the development of vascular plans. Arabidopsis roots are consisted of central stele surrounded by ground tissue (endodermis and cortex), epidermis and lateral root cap. It has been shown that communication between stele and ground tissue layers is mediated by plant-specific GRAS-type transcription factors, SHORT-ROOT (SHR) and SCARECROW (SCR), and that this pathway is crucial for endodermis formation. SHR is produced in the stele and moves into the outer ground tissue, where it interacts with SCR. The SHR-SCR protein complex thus formed specifies single layer of endodermis.
Here, we report a novel function of SCR. SCR restricts expression domain of the class III HD-ZIP transcriptional factor, PHABULOSA (PHB), to vascular tissue via transcription of microRNA165/166. We will also discuss biological significance of the non-cell-autonomous function of miR165/166 in the Arabidopsis root radial patterning.

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Individual lateral root(LR) development including initiation step was positively regulated by auxin but, it was not clear how to distribute them. PUCHI, which negatively regulated the cell proliferation at early LR development, seemed to function in LR induction, as puchi had increased LRs. Some of pPUCHI::GFP expressed sites in WT differentiated into LR but the remaining lost the expression and never formed LR. PUCHI was expressed by auxin before initial cell division. Early expressed sites of auxin responsible marker DR5::GFP and Cell cycle G2/M phase marker pCyclinB1;1::GFP showed imperfect probability of LR formation. PUCHI expression marker showed the similar probability with DR5, lower than that of the cell cycle marker. It was increased in puchi as almost all PUCHI expressed sites formed LR. These results suggested that PUCHI was involved to select LR initiation sites from the auxin accumulation sites before cell division.

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rrd1, rrd2, and rid4 are temperature-sensitive mutants of Arabidopsis that were isolated by screening with adventitious root formation as an index phenotype and characterized by forming fasciated roots at the restrictive temperature. Temperature-shift experiments with a semi-synchronized lateral rooting system showed that lateral root primordia of these mutants develop into fasciated roots when exposed to the restrictive temperature at the initial stage. Detailed observation of arising primordia indicated that expansion of the area where cell division is reactivated to form a root primordium results in the fasciation phenotype.
The rrd2 mutation was mapped within about 140 kb at the 54-cM position of chromosome I. Sequence analysis of this region of the rrd2 genome detected a single base substitution in a gene encoding a pentatricopeptide protein, which is presumed to cause the fasciation phenotype. This is very suggestive in consideration of our previous finding that RID4 encodes another pentatricopeptide protein.

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