The NRT2 genes, which encode the transporters involved in nitrate uptake, comprise a small gene family in most plants. The moss Physcomitrella patens has eight NRT2 species, among which NRT2;1-NRT2;4 are the major components. Of the four NRT2 species, NRT2;1, 2;2 and 2;4 are more closely related to one another than to NRT2;3. NRT2;3 has significantly lower afinity for nitrate than the other three, and its mRNA comprises over 80% of total NRT2 mRNA in protonemata cultivated with high-consentrations (5-10 mM) of nitrate. Nitrate uptake by the high-nitrate grown protonemata was reversibly inhibited by addition of ammonium to the medium without an appreciable decrease of the NRT2;3 protein. This indicated that the activity of NRT2;3 is regulated at the posttranslational level without involvimg protein degradation. An S507A mutant of NRT2;3, in which one of the putative phosphorylation sites conserved in most plant NRT2 proteins was mutated, accumulated the NRT2;3 protein but was similar to the NRT2;3 deletion mutant in its high-level expression of the NRT2 genes other than NRT2;3. We infer that S507 of NRT2;3 is involved in functional expression of the NRT2;3 protein.

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Lotus japonicas is a model organism for leguminous plants and is used for a full genome sequencing project. To incorporate gene expression information in our metabolic studies, we investigated the biosynthesis of pyridine nucleotides and trigonelline in cell suspension cultures of L. Japonicas. The in situ metabolism of [³H]quinolinic acid and [¹⁴C]nicotinamide revealed that the activity of the de novo pathway for NAD synthesis was maximum in the exponential growth phase, whereas the salvage pathway was high in the lag phase. Transcript levels of NaPRT and NIC encoding salvage enzymes, were enhanced in the lag phase of cell growth whereas the maximum expression of NADS was found in the exponential growth phase. Correspondingly, the activities of the salvage enzymes, nicotinate phosphoribosyltransferase (EC 2.4.2.11) and nicotinamidase (EC 3.5.1.19), increased one day after transfer the stationary phase cells to the fresh medium. The greatest trigonelline synthesis was found in the stationary phase of cell growth. The role of trigonelline in leguminous plants is discussed.

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In Synechococcus elongatus PCC 7942, nitrate reductase (NR) is inhibited upon addition of NH₄⁺to the medium, but little is known about the regulatory mechanism except that it involves the PII protein. Since NR of Synechocystis sp. PCC6803 is insensitive to NH₄⁺ treatment, we determined the properties of the chimeras of NRs from S. elongatus PCC 7942 and Synechocystis sp. PCC 6803 to gain insight into the mechanism of regulation. Since NH₄⁺ inhibits the nitrate-nitrite transpoter (NRT) as well as NR in the wild-type Synechococcus cells, the chimeric genes were introduced into NC2ΔNR, an NR-deficient derivative of the Synechococcus NC2 mutant having NH₄⁺ -resistant NRT activity, making NR the rate-limiting step of nitrate assimilation. Examination of the effects of NH₄⁺ on nitrate uptake by these strains showed that the NR inhibition requires the N-terminal region of the enzyme. Cyanobacterial NRs having N-terminal regions similar to those of the NR proteins from the PCC7942 and PCC6803 strains, respectively, were selected and their response to NH₄⁺ treatment is being examined in the NC2ΔNR strain to elucidate the structure involved in the inhibition of NR activity.

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The nrtP gene from cyanobacteria encodes an MFS-type nitrate permease that is similar to NRT2 of algae and plants. The gene is found in marine strains of cyanobacteria, with an exception in the fresh-water strain Nostoc punctiforme ATCC29133. NrtP of N. punctiforme (NpNrtP) was previously reported to be a nitrate/nitrite permease with higher affinity for nitrate than for nitrite by heterologous expression in a Synechococcus elongatus mutant lacking the ABC-type nitrate/nitrite transporter. However, the S. elongatus mutant was recently found to have a cyanate/nitrite bispecic ABC transporter, making the nitrite transport activity of NrtP questionable. In this study, we used a double mutant of S. elongates, lacking both the ABC nitrate/nitrite- and cyanate/nitrite transporters to investigate the function of NrtP. The NrtP-expressing cells showed low but detectable activity of nitrite uptake as well as high activity of nitrate uptake, confirming its ability to transport nitrite. A strep-tagged version of NpNrtP was found only in the plasma membrane of S. elongatus, indicating occurrence of a common mechanism for targeting of the transporter to the plasma membrane in cyanobacteria.

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Chloroplastic 2-oxoglutarate/malate transporter (OMT) mediates between carbon and nitrogen metabolic pathways in concert with dicarboxylate transporter, and also constitutes malate valve together with NADP-malate dehydrogenase (MDH) isozymes. We produced Arabidopsis transformants overexpressing the OMT and NADP-MDH, and examined whether the tolerance to environmental stresses or ability of nitrate assimilation is improved in the transformants. The rate of oxaloacetate-dependent malate synthesis/efflux was higher in chloroplasts from the OMT/MDH double overexpressed plants compared with WT chloroplasts, and, therefore, we concluded that the capability of malate valve was improved in the transformants. Although growth rate of the transformants was similar with that of WT, the contents of some organic and amino acids were increased in the transformant's leaves. Additionally, a decrease in foliar Fv/Fm value in response to high light illumination was reduced in the transformants. Therefore, it is thought that the ability to avoid photoinhibition in response to environmental stresses is improved in the transformants.

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C4 plants have two types of photosynthetic cells, mesophyll (MC) and bundle sheath cells (BSC), that possess developed chloroplasts. Active metabolite transport is conducted across the chloroplast envelope membranes. We previously reported that chloroplastic 2-oxoglutarate/malate transporter (OMT) of maize, an NADP-ME-type C4 plant, is abundantly expressed in MC of leaf blades but not in BSC and root tissues. In this study, we investigated the cell-specific expression of OMT genes in other C4-type plants and its nitrogen-responsiveness. The expression of OMT in sorghum (NADP-ME-type), finger millet, proso millet (NAD-ME-type), rhodes grass and guinea grass (PCK-type) was MC-specific. The abundant expression of OMT in leaf blades was also confirmed in sorghum, finger millet and rhodes grass. Furthermore, the transcripts of OMT in maize leaf blades accumulated more rapidly in response to the nitrate supplementation compared to ammonium. Together with these results, there is a possibility that the common function of MC-specific OMT is supply of stromal reducing equivalents to cytosolic nitrate reductase.

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In the Brassicaceae, compatible pollen grains from same species germinate on the stigmatic papilla cells, while those from unrelated species are largely ignored. The molecules involved in this primary incompatibility (PI) recognition are largely unknown. There can also be an additional layer of recognition to prevent self-fertilization, the self-incompatibility (SI) recognition. It was shown that self-pollen recognition in SI is controlled by the multiallelic gene complex (S-haplotypes) at the S-locus, which contains both female determinant SRK (S-receptor kinase) and male determinant SP11 (S-locus protein 11). SP11 binding to its cognate SRK induces SRK autophosphorylation and initiates a signaling cascade leading to rejection of self-pollen. However, the downstream components involved in this SI signaling are also largely unknown. In order to identify the components involved in PI and/or SI, we performed global gene expression profiling of stigmatic papilla cells. Papilla cells and the beneath style cells were separately collected using laser micro-dissection and comparative cDNA microarray analysis was performed.

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Many flowering plants possess self-incompatibility (SI) system to prevent inbreeding. In Brassica self-incompatibility, self/non-self recognition is controlled by S-haplotype-specific interaction between the pollen-borne ligand, SP11 (S-locus protein 11), and its stigmatic receptor-kinase, SRK (S receptor kinase). Our genetic analysis of a self-compatible mutant revealed the involvement of a cytoplasmic protein kinase, MLPK (M-locus protein kinase), in the SI signaling. MLPK localizes to the papilla cell membrane and interacts directly with SRK on the plasma membrane to transduce SI signaling, but the regulation mechanism of MLPK kinase activity remains unclear.
To understand the activation mechanism between SRK and MLPK, we carried out an in vitro phosphorylation assay using E. coli-expressed recombinant MLPK and SRK protein, and found that N-terminal region of MLPK was specifically phosphorylated by SRK kinase domain.

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Many higher plants have genetically controlled to prevent self-fertilization, using specific interactions between pollen and pistil. These cell-cell recognition mechanisms (self-incompatibility, SI) are controlled by a multi-allelic S-locus. It is well known that SI of the Brassicaceae can be removed by a high-concentrated CO₂ treatment. Moreover, analyses of plants with various CO₂ sensitivity suggest the presence of other genetic-loci in the downstream signal transduction pathway of SI reaction to CO₂. However, many of the responsible genes remain unknown.
Model plant Arabidopsis thaliana belongs to the Brassicaceae but is self-compatible due to the non-functional pollen and pistil S determinants.
After being introduced S genes (SRKb and SCRb) of A. lyrata, a self-incompatible relative, A. thaliana cannot be self-fertilized but it could be overcome when treated with CO₂ gas. In this study, microarray analysis was performed using this SI A. thaliana with/without CO₂ treatment for hunting downstream components involved in SI signaling response to CO₂, which will give us a deeper understanding of mechanism of SI and knowledge for the breeding of the Brassicas.

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In the Brassicaceae, a compatible pollen grain adheres to and hydrates on a papilla cell of the stigma. The hydrated pollen grain germinates, and the pollen tube penetrates into the papilla cell wall to deliver the sperm cells to the embryo sac for fertilization. During cross-pollination, water and ions are transported from the papilla cell to the pollen grain. However, its mechanism is unclear. In this research, we characterized the water channels, which are essential for pollen hydration. First, pharmacological analysis using HgCl₂, an inhibitor of aquaporin, suggested that the stigmatic aquaporin(s) are involved in the water supply. Next, we identified aquaporins, which are highly expressed in stigmatic papilla cells, by micro array. We also examined the molecular dynamics of these aquaporins during pollination by expressing Venus fusions in the papilla cells.

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As an allo-recognition mechanisms to prevent self-fertilization, Solanaceae plants share S-RNase-type self-incompatibility (SI) systems, in which S-RNases and SLFs (S-locus F-box) have been identified as S-haplotype determinants of the pistil and the pollen, respectively. However, only one SLF gene, PiSLF2 in Petunia inflata, has been demonstrated to be the pollen determinant by transgenic experiment, and little is known about molecular details of self/non-self recognition mechanisms. In order to characterize the S-haplotype specific interactions between S-RNases and SLFs, we isolated new SLF alleles from Petunia hybrida.
We show that transformation of S_B2S_D2 plants with one of the new SLF alleles, S_D2SLF, causes breakdown of pollen SI function. This breakdown is consistent with 'competitive interaction' phenomenon, which explains the SI-breakdown observed in tetraploid plants, and therefore supports that S_D2SLF is a pollen determinant. The in vitro protein binding assays show the non-self specific interaction between S_B2-RNase and S_D2SLF. These results support the model in which SLFs are able to recognize and inactivate the non-self S-RNase, but not the self S-RNase.

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Phytochrome is one of the major photoreceptor families in plants and regulates the flowering mechanism. It is known that phytochrome translocates from the cytoplasm into the nucleus in a light-dependent manner and transmits a signal. However, the detail of the phytochrome-mediated signaling mechanism in the late-vegetative phase remains unknown. The VOZ (Vascular plant One-Zinc finger) protein was isolated as a phytochrome-interacting factor by screening focused on flowering. Arabidopsis possesses two VOZ genes, VOZ1 and VOZ2. Previous analysis revealed that VOZ functions downstream of phyB and upregulates the flowering promoting factor, FT. We analyzed functional intracellular localization of VOZ, using a nuclear localization signal (NLS) and a nuclear export signal (NES). Only VOZ-NLS complemented the late flowering phenotype of voz1 voz2 double mutants. The result indicates that VOZ functions in the nucleus. We also analyzed localization of GFP-VOZ. Functions of VOZ in phytochrome-signaling will be discuss.

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We reported that a fruit tree apple was able to accelerate the flowering by the overexpression of FT gene like as Arabidopsis model plant. This flowering efficiency highly depends on the source of FT gene and the expression tissues at all. The combination of 35S:MdFT(apple FT), rolCp:FT(Arabidopsis) and rolCp:Hda3(rice) set precocious flowers for 3 to 8 months. The other combinations were not successful. The GFP fused FT gene was detected at whole plant tissues under 35S promoter and at vascular tissues under rolC promoter. The 35S:FT fused GFP showed a slight suppression of flowering, but rolC:FT fused GFP promoted flowering as well as the no fusion rolC:FT. The FT fused GFP protein had an expected molecular weight with anti-GFP antibody. Could the FT fused GFP proteins move through from transgenic plants to non-transgenic plants by grafting, and could set flowers on non-transgenic plants? To analyze the graft transmissions, the precocious transgenic apples(6 months to 1 year) were grafted to seedlings from apple cultivar Tsugaru and Jonathan (1 year) on each stem. After one month, the graft-takes were checked and analyzed the movements with GFP fluorescence and antibodies.

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Germination and photomorphogenesis are fundamental for every higher plant species . The vast volume of information as to the molecular mechanisms underlying these processes have already come from the current Arabidopsis biology. In this regard, one of the current topics is a pair of the phyotchrome B (phyB)-interacting bHLH transcription factors, named PIH4/5, both of which play crucial roles in early photomorphogenesis. However, little is known about the factor(s), which acts immediately downstream of PIF4/5.
To uncover such an as yet unidentified hypothetical factor-X(s), here we took a new approach by taking advantage of that the current large-scale Arabidopsis datasets provided us with powerful Web-based querying in the context of hypothesis. First, we logically listed up a number of criteria, which the factor-X should fulfill. We then conducted Web-query-based in silico searches for the factor-X. As the result, we identified a homeodomain(HD-ZIP) type transcription factor, named ATHB2, as the most plausible candidate. Then, we will present a set of experimental evidence, which showed that ATHB2 is indeed the one functioning downstream of PIF4/5 in early photomorphogenesis.

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Photomorphogenesis and control of flowering time are fundamental for every flowering plant. The vast volume of information as to the molecular mechanisms underlying these processes has already come from the current Arabidopsis biology. From ecological viewpoint, however, the detailed mechanisms should be significantly diverse from one plant species from the other. Hence, it is timely and important to extend our understanding as to the mechanisms underlying photomorphogenesis and flowering toward the greatly diverse plant species other than Arabidopsis. In this respect, legume plants are economically important objects, because they yearly provide us with 1/3 of grain production in the whole world.
With the model legume Lotus japonicus, here we conducted genome-wide bio-informatics with special reference to the genes implicated in photomorphogenesis and control of flowering time including the genes involved in the photoperiodic and/or FLC-repression pathways, together with those implicated in the so-called ABC model. Furthermore, we will provide experimental evidence for that this legume had a florigen FT-like gene, which has an ability to promote flowering in Arabidopsis.

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Duckweeds (family Lemaceae) suit for physiological experiments in the laboratory because of the tiny plant bodies, rapid growth rates, strictly controllable environments under aseptic conditions, and so on. They also draw attention as a material for bioremediation. In 2009, a genome project for Spirodela polyrhiza (giant duckweed) began as a program in DOE JGI. Its small genome size (~150 Mbp) is a marked trait for the genome sequencing. However, physiological experiment techniques using S. polyrhiza are not developed especially in the study of the biological clock. Plants in Lemna genus instead have been widely used in physiological studies of photoperiodic flowering, circadian rhythms and so on. We analyzed clock-related genes from Lemna plants and also developed a convenient monitoring system of bioluminescent circadian rhythm that is introduced with a particle bombardment method. We then attempted to use S. polyrhiza for the study of biological clock. We first performed physiological characterization of this plant under a number of experimental conditions. We also tried to apply the bioluminescence reporter system to the monitoring circadian rhythm of S. polyrhiza.

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Lemna plants (duckweeds) have been used for the study of the circadian rhythm and photoperiodic responses. Recently, we have succeeded in monitoring the circadian rhythms of Lemna plants easily by introducing a bioluminescent reporter system transiently with a particle bombardment method. The bioluminescence of plants is measured by a photomultiplier tube, and thus the measured luminescence value is the sum of luminescence of those cells that bioluminescent reporter genes are introduced into. In order to clarify the relationships of circadian rhythms between cells (and the plant body) now we are attempting to establish a monitoring system for circadian rhythms of single cells in living Lemna plants. Lemna plants are very small and flat, and it is easy to observe a whole plant body at high magnification. First, we begin to develop a single-cell monitoring method by applying the bioluminescence reporter system with bright non-circadian reporters. We will report and discuss the conditions of gene transfection, the setup of camera system, imaging data analysis, and growth conditions.

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Circadian kaiBC expression in the Synechococcus elongatus PCC 7942, is generated by temporal information transmission from the KaiABC-based circadian oscillator to RpaA, a putative transcriptional factor, via the SasA-dependent positive pathway and the LabA-dependent negative pathway which is responsible for feedback regulation of KaiC. However, the labA/sasA double mutant has a circadian kaiBC expression rhythm, suggesting that there is an additional circadian output pathway. Here we describe a third output pathway, which is CikA-dependent. The cikA mutation attenuates KaiC overexpression-induced kaiBC repression and exacerbates the low-amplitude phenotype of the labA mutant, suggesting that cikA acts as a negative regulator of kaiBC expression independent of the LabA-dependent pathway. We propose a model in which temporal information from the KaiABC-based circadian oscillator is transmitted to gene expression through three separate output pathways.

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We previously reported that we tried to make bioluminescence reporters of nuclear clock genes to monitor circadian rhythmicities in the activities of the clock gene promoters in the unicellular green alga Chlamydomonas and that we encountered the problem. Two luciferase bioluminescence reporter strains having promoter regions of the clock genes ROC15 and ROC75, respectively, whose mRNA levels oscillate with an 8-hour phase-angle-difference, showed the circadian rhythms of luciferase mRNA levels oscillating with a phase-angle-difference similar to that of endogenous ROC15 and ROC75 mRNAs. But contrary to our expectations, they showed the bioluminescence rhythms with the same phase. Here, we report the results of analyses of the luciferase protein levels in the two clock gene reporter strains, and of effects of culture conditions and host strains on the bioluminescence rhythm. These analyses revealed that circadian rhythmicities of luciferase reporters are regulated at posttranscriptional steps in Chlamydomonas.

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Cyanobacterium Synechococcus elongatus PCC. 7942 is a simplest organism known to show circadian rhythm. Circadian clock of cyanobacterium can be reconstituted in vitro; robust circadian rhythm in KaiC phosphorylation is generated by mixing KaiA, KaiB, and KaiC. Phosphorylation state of KaiC is regulated by its autophosphorylation and autodephosphorylation activities. KaiC exists as a hexamer in the presence of ATP. ATP molecules bind at the interfaces of between subunits and phosphorylate S431 and T432 in the neighboring subunits. KaiC has two P-loops, which is conserved in catalytic sites of ATPases and protein kinases, however, KaiC shares no homology with protein phosphatases.
Autodephosphorylation of KaiC has not fully been studied so far. In order to examine whether KaiC dephosphorylates itself by simlar mechanism to known protein phosphatases, we phosphorylated KaiC in the presence of radioactive ATP and chased the dephosphorylation process. Based on the results, we will discuss the autodephosphorylation mechanism of KaiC.

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In angiosperms, a zygote generally divides into an asymmetric two-celled embryo consisting of an apical and a basal cell. The asymmetric cell division of the zygote is the possible first step for formation of the apical-basal axis of plants, and a fundamental feature of early embryogenesis and morphogenesis in angiosperms. In the study, using rice in vitro fertilization system and single-cell microarray analyses, we identified genes that are up- or down-regulated in the apical or basal cell of rice two-celled embryos. Approximately, 14,000 genes were expressed in rice zygotes and, among the transcripts from 14,000 genes, 0.5-1 % transcripts were detected only in apical or basal cell. These suggest that specific mRNAs are distributed with polar fashion in a zygote or degraded in one of the daughter cells of a two-celled embryo.

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How cell fate is specified according to its position during plant development is not well understood. To address mechanisms of position-dependent gene activation and cell fate specification, we analyzed the regulatory region of ATML1, which marks the outermost cell layer from the globular stage of embryogenesis. Expression studies suggested that surface position specific activity of the 3.4-kb ATML1 promoter was determined by positional signals independently of epidermal cell identity. Deletion experiments revealed that a 101-bp region, which contained an ATML1-binding site called L1-box, conferred all aspects of ATML1 expression, suggesting positive feedback regulation. Further overexpression experiments confirmed the L1 box-mediated autoregulatory activity of ATML1. Possible roles of autoregulation in determining epidermis-specific expression of ATML1 will be discussed. In addition, we will present a new yeast one-hybrid library that consists only of transcription factor clones, which allows the effective identification of transcription factors that bind to cis-regulatory sequences in the ATML1 promoter.

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Light and existence of shoot are essential for low pH (pH 4) induced root hair formation (RHF) in lettuce. In intact seedlings, the role of light could be substituted by chlorogenic acid (CGA). In dark-grown decapitated seedlings, addition of both CGA and sugar were required for RHF. These results suggested that sugar moved from shoot to root and promoted RHF in cooperation with CGA. To examine the relation between sugar and RHF, sugar contents (glucose and fructose) in lettuce root were determined.
RHF, which was induced by CGA in dark-grown intact seedlings at low pH, could be observed 4 hr after CGA application and ceased within 24 hr. Sugar contents in the roots also increased ca. twofold of the initial level at 4 hr and decreased to 1/2 of the initial level at 24 hr. Sugar contents in the roots of dark-grown intact seedlings at pH 6 and of dark-grown decapitated seedlings of pH 4 or 6 determined at 4 hr were approximately equal to the initial level. Seedlings cultured in these conditions showed no RHF by the application of CGA. These results indicated a strong correlation between RHF and sugar contents in lettuce root.

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The seeds of a T-DNA insertion mutant of Arabidopsis, nekojita1 (nkj1), shifted germinable temperature limits lower than wild type. Abscisic acid (ABA) content in nkj1 seeds decreased to low levels by imbibition even at the restrictive temperature, 30 ^oC, as in germinating wild type seeds. Interestingly, nkj1 seeds were hyper-sensitive to ABA, and the germination was recovered by ABA biosynthesis inhibitor, fluridone, at the restrictive temperature. These results suggest that NKJ1 is a negative regulator of ABA signaling and has a promotive role on germination at permissive temperature conditions. nkj1 was mapped on the bottom arm of chromosome 4, and the gene was identified by sequencing of the T-DNA border and by genetic complementation test. The deduced amino acid sequence was highly homologous to Sec23/Sec24 subunit of COPII coat protein which is involved in vesicular transport from ER to golgi. NKJ1 gene expression was detected from various organs, and it was induced during germination and suppressed by restrictive high temperature conditions. We are now analyzing subcellular localization of NKJ1 protein by using transgenic plants which express NKJ1-GFP fusion protein.

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The seeds of Arabidopsis germinate in autumn since the germination is suppressed by supra-optimal high temperature during summer. We have reported that high temperature increases abscisic acid (ABA) level in seeds through up-regulation of ABA biosynthesis genes (e.g. NCED), and ABA suppresses gibberellin biosynthesis genes (e.g. GA3ox). To dissect early heat induced and late ABA induced change, we analyzed gene expression in wild type and ABA deficient mutant (aba2-2) seeds imbibed at 22 and 34 ^oC by microarray. The genes were classified into 17 groups by their expression patterns using K-means clustering method. Their expressions were temperature dependent, ABA and temperature dependent, imbibition induced, germination induced, genotype specific, or others. The seeds of a T-DNA insertion mutant of a high temperature induced and ABA independent gene showed high temperature resistant germination phenotype. Such early heat induced genes may have a role in the process from temperature perception to NCED gene expression.

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ABA plays vital roles in the maintenance of seed dormancy. In Arabidopsis, fus3 seeds fail to undergo dormancy. However, little is known about the molecular mechanisms underlying this process. To this end, we mutagenized a transgenic line in which ectopic expression of FUS3 could be artificially induced by estrogen and screened for novel mutants defective in FUS3-imposed growth arrest at seedling stages. Among those, line 2-44 showed reduced induction of CRC and At2S3 by exogenous ABA, in the presence of FUS3, indicating that line 2-44 has a defect in ABA-sensitivity. Moreover, the mutant had reduced dormancy during seed development. Interestingly, however, dry mature seeds were hypersensitive to ABA upon germination. These observations suggest that the product of causative gene of the mutant may function on ABA sensitivity through different mechanisms between developing and dry mature seeds. To further explore the molecular roles of the product of the causative gene, map-based cloning of line 2-44 is being carried out. Based on these results, we will discuss the possible functions of the gene product in the regulation of embryo growth arrest and dormancy, and seed germination.

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Zostera marina is a kind of marine angiosperms (seagrasses), and is widely distributed in the Northern Hemisphere. Seeds of Z. marina develop in seawater. Here, we analyzed physiological phenomena during seed germination in Z. marina. Seed germination was promoted under low temperature (15 degree C), low salinity (below 22.5 permillage), continuous dark, and shaking. Higher germination rate of seed was also observed under lower osmotic pressure. During seed germination, the coleoptile elongation was accelerated under anaerobic condition (below 2 mg/l dissolved oxygen, DO.), whereas the first leaf formation was accelerated under aerobic condition (7 mg/l DO.). The coleoptile elongation and first leaf formation were inhibited by abscisic acid during seed germination, while seed germination was not promoted by gibberellin. Our results suggest that seeds of Z. marina have unique physiological features to adapt to the marine environment.

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The Arabidopsis SHEPHERD (SHD) protein is an Hsp90-like molecular chaperone resident in the endoplasmic reticulum, however, its target protein has not yet been revealed. The shd-1 mutant in Ws strain shows clavata (clv)-like phenotype, in which carpel number is increased. Therefore, we presumed that CLV2 in Ws strain (CLV2^Ws) required the SHD chaperoning activity for its function. In other words, we supposed CLV2^Ws was the direct target of SHD.
To verify this prediction, we tried to confirm the direct binding between CLV2^Ws and SHD. We detected it by pull-down assay and the surface plasmon resonance technique (Biacore system). Moreover, we also detected the binding between SHD and CLV2^Col.
The shd mutant shows pleiotropic phenotypes, such as retarded root growth and inhibited pollen tube elongation. Therefore it indicates that SHD has other targets. So we performed LC-MS/MS analysis to investigate other targets of SHD.

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The exine composing an outer wall of pollen grains is believed to be important for successful pollination because of its species-specific architecture. However, little is known either about the mechanism of exine formation or genes involved in it. We performed the screening of mutagenized Arabidopsis with SEM, and identified many mutants showing defective exine structure. Among them, kaonashi4 (kns4) mutant showed thin exine layer mainly due to the short baculae. It also showed low fertility and collapse of the whole structure of pollen grains. TEM observation revealed that the exine abnormality begins at the tetrad stage as an extremely thin primexine layer. The KNS4 gene encoding a protein which resembles beta-1,3-galactosyltransferase involved in glycan biosynthesis. There are 20 genes encoding similar protein in Arabidopsis, but their function is hardly understood. The KNS4 is specifically expressed in the tapetal cells at around tetrad stage and the KNS4 protein is localized in the Golgi apparatus. Our hypothesis is that the KNS4 functions in the Golgi apparatus of tapetal cell at tetrad stage and is involved in biosynthesis of the components of primexine.

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Cell cycle progression in eukaryotes is controlled by the protein kinase activity of cyclin-dependent kinase (CDK) complexes composed of catalytic CDK and regulatory cyclin subunits. D-type cyclins (CYCD) play key roles in the G1-to-S-phase transition. Although it has been shown that Arabidopsis CYCD4 promotes cell division in the stomatal lineage in hypocotyls, the molecular function of CYCD4 in stomatal pathway is poorly understood. Three related transcription factors, SPEECHLESS (SPCH), MUTE and FAMA, have been reported that control cell fate progression through the stomatal pathway. In this study, we examined the genetic interaction between CYCD4 and intrinsic factors in stomatal cell fate transitions. In the CYCD4-overexpressing seedlings, many small cells are generated in the nonprotruding cell files. We found that the phenotype of CYCD4-overexpressor was partially suppressed in several stomatal mutants. These results suggest that CYCD4 regulates stomatal pathway via modulation of several factors in stomatal cell fate transitions.

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In organ development the intercellular communications occur between shoot apical meristem (SAM) and lateral organ primordium. Recently several peptide ligands responsible for SAM organization or lateral organ development are reported, but it is not enough to understand all developmental events there. And it is indispensable to identify novel peptide ligands from SAM. Therefore we tried to identify peptide ligand from extracellular space of SAM. As a starting material, cauliflower or Arabidopsis ap1 cal mutant were used for identification. In cauliflower, proteins of 3000-18000 Da were analyzed by 2D gel electrophoresis following protein sequence, and using this method we identified 41 proteins. In Arabidopsis ap1 cal mutant, proteins of 500-4000 Da were separated by column chromatography and amino acid sequence was detected by LC-MS/MS. Here we identified 339 proteins categorized based on Arabidopsis database and selected 30 ligand candidates. We tested peptide activity by adding to Arabidopsis seedling and found abnormal structure in SAM and root.

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During leaf development, regulatory factors should localize correctly in adaxial- or abaxial-side of the primordia. Previously we reported that the expression domain of the abaxial-determining gene, FIL, is determined by its promoter function. In contrast, the expression domain of the adaxial-determining gene, PHB, is restricted by the miR165/166-mediated gene repression. We also revealed that six MIR165/166 genes are expressed only in the abaxial side, resulting in the side-specific repression of PHB. RT-PCR analysis indicated that expression levels of some MIR165/166 genes were reduced in fil yab3 double mutant seedlings. This result indicates that the expression is under control of FIL and YAB3. Then, using laser-microdissection technique, total RNA was prepared from the adaxial- or abaxial-side of the primodia, respectively. Semi-qRT-PCR analysis showed that intact PHB transcripts were detected in both side samples. Thus, we thought that PHB expression might be suppressed by miR165/166-meidated translation suppression mechanism only in the abaxial side of leaf primordia.

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Plant cells surrounded with rigid cell walls cannot move during development. Cell morphogenesis, which partly depends on directional cell elongation, plays an important role for polarized organ development. In plant, cortical microtubules, aligned at the cell cortex, are known to have an important role in the regulation of directional cell growth. To understand the mechanism that regulates the directional cell elongation, we isolated itosugi (itg) mutant of Arabidopsis, which shows defects in elongation of several organs, including stems, hypocotyls and roots. Cytological analyses of itg mutants revealed that longitudinal cell elongation was suppressed and that radial cell expansion was promoted in hypocotyls and roots. ITG-GFP was found in dot-like structures at the cell cortex, and co-localized with cortical microtubules. These results suggest that ITG may regulate anisotropic cell growth by way of its association with cortical microtubule.

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Postembryonic development in plants depends on the activity of shoot and root meristems. CLAVATA3 (CLV3), a putative ligand of Arabidopsis thaliana, regulates the stem cell population in the shoot apical meristem (SAM). Previously, we have reported that dodecapeptide with two hydroxy proline residues is a functional CLV3, and chemically synthesized CLV3 dodecapeptide function in our in vitro bioassay systems, resulted in reduced SAM size. In order to confirm the synthetic peptide acts through endogenous CLV pathway, we examined the SAM phenotype by treating the clv mutants with the CLV3 peptide. As a result, the clv1 and clv2 plants, but not the clv3 plants, showed resistance to the SAM defect caused by the CLV3 peptide. This suggests the synthetic CLV3 peptide acts through endogenous CLV1/CLV2 receptor complex(es). Together with results from the analysis of CLV3 peptide-resistant-mutants, we will discuss about CLV signaling pathway in the SAM.

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Shoot apical meristem (SAM) contains undifferentiated stem cells, and they divide to produce new tissues. SAM has important roles in shoot development and regulation of SAM is necessary for appropriate shoot formation. It is suggested that CLV3 regulates maintenance and differentiation of stem cells in SAM via receptors like CLV1, CLV2, and SOL2. At first, CLV3 is translated as a 96 amino acid containing peptide. This peptide is processed to be mature 13 amino acid containing CLV3 peptide, but this processing mechanism is still unknown. In order to know the molecular mechanisms of CLV3 peptide processing, we performed genetic and biochemical analyses. Here we will discuss about our recent work about it.

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In Arabidopsis, the CLAVATA (CLV) pathway operates in the regulation of the size of the stem cell population in the shoot apical meristem. CLV3 was shown to function as a dodecapeptide and is considered as a ligand for receptor complex(es) containing CLV1 and CLV2. Recent studies have identified a new member of receptor, Suppressor of Overexpression of LLP1-2 (SOL2)/CORYNE (CRN), capable of sensing the CLV3 peptide. Our current project aims to understand the precise molecular details of CLV3 signalling mediated by these receptor-like proteins in a target cell. We are conducting biochemical studies on these receptor functions. Our various mutational screenings for the CLV3 synthetic peptide insensitivity identified a number of potential candidates. Current progress in this study will be presented.

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In Arabidopsis, CLV3-CLV1 signaling system plays an important role in shoot apical meristem (SAM) maintenance. However, in legumes, such as Lotus japonicus, Glycine max and Medicago truncatula, the orthologs of CLV1 (HAR1, NTS1/NARK and SUNN) function in the regulation of nodulation but do not seem to involve in SAM maintenance. Thus, in legumes, whether the CLV3-CLV1-like intercellular signaling system functions in SAM maintenance is still unclear. Therefore, we focused on Lotus CLV3-like (LjCLV3) gene and performed its functional analysis. In situ hybridization analysis revealed that LjCLV3 is expressed in shoot apical, inflorescence and flower meristems. The calli transduced with LjCLV3 overexpression construct shows low efficiency in shoot regeneration. By contrast, the calli transduced with LjCLV3 RNAi construct shows higher efficiency than the control lines. Furthermore, LjCLV3-silenced plants show stem fasciation and the increase in the number of flowers per peduncle. Thus LjCLV3 is suggested to function in SAM maintenance.

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Organ regeneration in tissue culture involves de novo formation of apical meristems, which should provide a useful system to study how meristems arise. We have isolated a large number of temperature-sensitive mutants of Arabidopsis with defects in organ regeneration. Of these, the rid3 mutant is characterized by loss of the ability to form adventitious roots and shoots at the restrictive temperature, suggesting that rid3 has a defect in the regulatory mechanism of cell proliferation required for apical meristem neo-formation. Our previous analysis revealed that the RID3 gene encodes a novel WD-40 repeat protein and that, in tissue culture, its expression increases during callus development and decreases locally before the formation of the shoot apical meristem. Here we report some recent results on the function of RID3 in embryogenesis. We found that RID3 is expressed throughout embryogenesis and that the rid3 mutation interferes with expression of meristem-related genes and with embryonic pattern formation. Our results indicate that RID3 is involved in the formation of apical meristems during embryogenesis as well as during organ regeneration.

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rrd1, rrd2, and rid4 are temperature-sensitive mutants of Arabidopsis that are 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. Positional cloning revealed that RID4 and RRD1 encode a pentatricopeptide protein and a poly(A)-specific ribonuclease-like protein, respectively.
From the morphogenetic point of view, tissue organization in fasciated roots is of considerable interest. To assess this issue, we are now examining fasciated roots with tissue-specific reporter genes, which suggests an increase of cell files in the stele of fasciated roots.
Besides root development, embryogenesis is severely affected by any of the three mutations at the restrictive temperature. Double mutant analysis suggests that rrd2 enhances the embryo phenotype of rrd1. On the basis of these results, functional relations among RRD1, RRD2, and RID4 will be discussed.

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In Arabidopsis thaliana leaves, excessive cell enlargement is often triggered by decreased cell proliferation activity, a phenomenon that we named compensation. Compensation in fugu5 mutant is cancelled when sucrose is supplied in the growth media. Cloning of FUGU5 gene revealed that it encodes for the vacuolar H⁺-pyrophosphatase (H⁺-PPase). FUGU5 has two functions, cytosolic pyrophosphate (PPi) hydrolysis and vacuolar acidification. In this study, we attempted to analyze the two functions of FUGU5 separately. Since PPi is expected to be highly accumulated in the cytosol of fugu5 mutant cells, the cytosolic Inorganic Pyrophosphatase 1 (IPP1) gene of Saccharomyces cerevisiae was introduced to fugu5 under the control of FUGU5 own promoter. IPP1 hydrolyzes cytosolic PPi without contributing to vacuole acidification. Interestingly, our results revealed that the gross phenotype of fugu5 recovered and compensated cell enlargement was totally cancelled in the Pro_FUGU5::IPP1 transgenic lines. These findings convincingly demonstrated that PPi, produced during active postgerminative growth, is over-accumulated in fugu5 mutant cells and is the major cause of cell proliferation inhibition.

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Leaves in angiosperms generally show flattened morphology, which is established depending on the adaxial(Ad)/abaxial(Ab) identities of leaves. By contrast in unifacial leaves, some species develop flattened leaf blades, even though their leaf blades consist of only the Ab side. Thus, laminar outgrowth in unifacial leaves is regulated by a mechanism independent of the leaf Ad/Ab identities. To reveal the mechanism, we have adopted comparative analyses using closely related two species in Juncaceae, J. prismatocarpus and J. wallichianus, which exhibit flattened and radialized leaf blades, respectively.
In this study, we found that treatment of developing J. prismatocarpus seedlings with either exogenous auxin or auxin transport inhibitors eliminated flatness of leaf blades, leading them to radialized morphology. These treatments did not affect radialized leaf blades in J. wallichianus. Thus, we suggest that presumptive auxin gradient within leaves is necessary to promote the laminar outgrowth in unifacial leaves. We will discuss relationship of auxin gradient with expressions of the putative genetic factors, which are responsible for the laminar outgrowth in unifacial leaves.

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Recently, molecular basis of compound leaf morphogenesis has been increasingly understood. A series of the study have shown that leaflet initiation is highly similar to leaf initiation. On the other hand, when/where to generate leaflets still remains elusive. We mainly exploit two approaches in Eschscholzia californica to clarify the mechanisms underlying leaflet patterning. First, we used laser ablation system in order to perturb the leaf development physically. When the terminal leaflet was largely ablated, main axis of leaf is lost and reformed de novo. When ablated mildly, lateral leaflets were formed nearer to the leaf tip. These results suggest that the terminal leaflet serves both a main axis of leaf and an inhibitory effect on the incipient lateral leaflet primordia. Second, we analyzed expression patterns of several genes by in situ hybridization. In contrast to the general acceptance that acropetal leaflet-forming leaf keeps indeterminate tissue at its tip, we detected transcripts of indeterminate tissue marker and proliferating cell marker alongside leaf margin, and no apical-basal gradient was detected.

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AAL-toxin is a pathogenicity factor of Alternaria alternata f. sp. lycopersici. Little is known about the signaling pathway of AAL-toxin leading to programmed cell death. To investigate candidate genes involved in AAL-toxin-induced cell death, we employed virus-induced gene silencing (VIGS) and Nicotiana umbratica that is sensitive to the AAL-toxin. We showed that ethylene signaling pathway plays a pivotal role in AAL-toxin-induced cell death and identified NuERF4, which is AP2/ERF transcription factor, as a mediator for AAL-toxin-induced cell death. Overexpression of NuERF4 did not induce cell death, however, accelerated AAL-toxin-induced cell death. Moreover, disease symptom by A. alternata f. sp. lycopersici was more severe by the NuERF4 overexpression. We also indicated that NuERF4 is a transcriptional activator. These results suggest that the NuERF4 positively regulates some factors involved in AAL-toxin-induced cell death. At present, we are trying to hunt the targets of NuERF4 using PCR-select cDNA subtraction method to elucidate the downstream signaling of the NuERF4.

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Virus-Induced Gene Silencing (VIGS) with Tobacco rattle virus (TRV) vector, which is frequently used as a functional analysis tool for plant genes, especially in Solanaceous plants, is also able to be used for the suppression of endogenous gene expression in Arabidopsis. However the reported method is not suitable for uniform treatment of multiple samples of Arabidopsis plants. In this study, we tried to establish the simple and efficient method of large-scale phenotype-dependent screening by VIGS with TRV vector (TRV-VIGS) harboring normalized Arabidopsis cDNA library. Silencing phenotype was observed in all Arabidopsis ecotypes we examined by TRV-VIGS. The establishment of efficient experimental procedure that induces gene silencing in multiple plant samples using vacuum infiltration was succeeded. TRV-VIGS of CAD1, which is known as a negative regulator of cell death, induced similar cell death phenotype as in cad1 mutant. We suggested that this established TRV-VIGS screening method is applicable to isolate cell death-related genes efficiently. Using this screening method, we have isolated several novel Arabidopsis genes involved in cell death.

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Programmed cell death is crucial for developments and responses to various stresses in plants, and is highly regulated by a wide variety of factors. Bax Inhibitor-1 (BI-1) is a widely conserved cell-death suppressor. Arabidopsis BI-1 (AtBI-1) is a 7-times transmembrane protein localized in endoplasmic reticulum (ER), and suppresses cell death induced by oxidative stress, which generates reactive oxygen species (ROS) in plant cells. In addition, calmodulin interacts with the C-terminus of AtBI-1, which is essential for its function. However, the molecular mechanism of BI-1-mediated cell death is still unclear.
We suggested that AtBI-1 interacts with sphingolipid fatty acid metabolic enzymes via an electron transfer protein, cytochrome b₅. Sphingolipid has a 2-hydroxylated very-long-chain fatty acid (2-hydroxy VLCFA), and it is implied that AtBI-1 interacts with sphingolipid fatty acid 2-hydroxylases (AtFAH) and VLCFA condensing enzymes (AtELO). Here we show the relationship between AtBI-1 and sphingolipid fatty acid generated by AtFAH and AtELO.

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Generally, the bolting (stem elongation from rosette plants) of winter annuals is believed to be induced by an increase in the level of GA that occurs after a certain period of chilling (vernalization), and deficiency of GA make the plant keep rosette style. Lack of the direct evidence proving the above assumption in radish plants urged us to assume the presence of an anti-bolting compound; it actively keeping rosette habit through inhibition of growth of internode (bolting). Anti-bolting activity was detected in an extract of rosette shoot of radish plants by an assay using seedlings cultured in vitro. The causal compound that strongly inhibited bolting was isolated and identified as hexadecatrienoic acid monoglyceride. This compound did not inhibit leaf production at the apical meristem indicating that it merely inhibits the growth of internode. The compound disappeared completely after the vernalization and the bolting commenced thereafter. The results suggest that the release from the inhibition by the anti-bolting compound can trigger initiation of bolting.

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Winter annual plants keep vegetative growth in the first year and develop leaf rosette. After cold winter season the stem of the plant begin to elongate (bolting) under long days in spring, and flowers are formed on the stem. It is generally believed that deficiency of GA causes rosette style and an increase in the level of GA induces bolting. But such and increase was not observed in spinach plants and the commencement of bolting was explained by an increase in the sensitivity to GA. It is possible that some bolting-inhibiting (anti-bolting) compound that decides sensitivity to GA and controls bolting exists. Here we tried to detect and isolate the causal compound that controls bolting of spinach.
Spinach plants were grown in the experimental field, and the plants were subjected to ethanol extraction. After partial purification by solvent fractionation and chromatographies, the fractions were subjected to assay for anti-bolting activity by seedlings of spinach or qing-geng-cai cultured in vitro. Strong anti-bolting activity was detected in some fraction, suggesting that the bolting of spinach plant is controlled by the compound. Purification of the compound is now in progress.

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The endogenous auxin Indole-3-butyric acid (IBA) has been assumed to act via Indole-3-acetic acid (IAA) pathway to regulate the plant development. However, recent results suggest that cellular transport of IBA does not require either auxin influx carrier AUX1 or efflux carrier PIN2. In addition, IBA has been shown to effect the hypocotyl elongation and lateral root development differentially than of IAA, suggesting that IBA signaling and transport are possibly mediated by yet unidentified novel pathways. In the present work, by using available auxin signaling and transport mutants, we tried to elucidate the intracellular machinery that regulates the IBA response in Arabidopsis root. Our results suggest that IBA and IAA use a common response pathway in regulating the root meristem development, as all these auxin signaling and transport mutants showed resistance to both IAA or IBA induced root growth inhibition. In contrast, IBA but not IAA selectively increased the lateral root in all these mutants suggesting that IBA regulates the lateral root development through yet unidentified response pathway that works in parallel to known IAA pathway.

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KODA (9-hydroxy-10-oxo-12(Z),15(Z)-octadecadienoic acid), one of oxylipins formed with 9-lipoxygenase, has been found to be associated with flower induction of Lemna paucicostata [Yokoyama et al. (2000) Plant Cell Physiol. 41: 110-113; Yamaguchi et al. (2001) Plant Cell Physiol. 42: 1201-1209]. KODA biosynthesis is presumed to be formed with two enzymes, 9-lipoxygenase and allene oxide synthase [Feussner and Wasternack (2002) Annu. Rev. Plant Biol. 53: 275-297]. We here report the cloning of the 9-lipoxygenase from L. paucicostata for establishing the efficient production of KODA. Total RNA was extracted from L. paucicostata, SH and 441 strains, respectively. Partial sequences of the gene were obtained with degenerate PCR. Then, the complete cDNA sequences determined using 5' and 3' RACE gave one sequence from SH strain and two sequences from 441 strain. The lipoxygenase genes were introduced into E. coli and expressed. The recombinant proteins were examined in a reaction with α-linolenic acid as a substrate and confirmed to be 9-lipoxygenase.

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We earlier found that progesterone and allopregnanolone among mammalian steroid hormones strongly inhibit GA₄-Me induced antheridium formation of L. japonicum (Japanese climbing fern). We examined the effects of synthetic progestins and a progesterone receptor inhibitor on antheridium formation in L. japonicum. This paper reports that, among synthetic progestins, 5α-estran-17α-ethynyl-17β-ol-3-one and 5α-estran-17α-ethynyl-3α,17β-diol significantly suppressed GA₄-Me induced antheridium formation. By contrast, 4-pregnen-6α-methyl-17-ol-3,20-dione acetate and 4-pregnen-6α-methyl-17-ol-3,20-dione promoted GA₄-Me induced antheridium formation. These results suggested that L. japonicum can accept progestins. As a synthetic inhibitor of human progesterone receptor did not affect antheridium formation of L. japonicum, the receptor in this fern may have different characteristics from that of human.

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