The Arabidopsis seed coat is derived from the maternal parent and is composed of two integuments (the outer and inner integuments). The inner integument consists of three cell layers (ii1, ii2, and ii3). Recently, we reported that programmed cell death (PCD) occurs in the two cell layers (ii2 and ii3) of the inner integument at the early stage of seed maturation and that δVPE, one of vacuolar processing enzyme (VPE), is involved in the cell death. To clarify the molecular mechanism underlying the PCD of the two cell layers, we searched factors that express together with δVPE based on DNA microarray. Some of the candidates might collaborate with δVPE in cell death of limited cell layers, the purpose of which is to form a seed coat.

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In higher plants, transcriptional regulation by chromatin remodeling is suggested to be involved in the embryogenesis and transition of vegetative/reproductive phase.
We analyzed the effects of HDAC inhibitors on seedling growth after germination in Arabidopsis. Treatment with HDAC inhibitors suppressed the post-germination growth and resulted in the no-suppression of the expression of some embryogenesis related-genes. These results suggest that histone deacetylation is related to the repression of embryogenesis-related genes in transition from embryonic to post-embryonic phase.
To clarify which kind of HDAC factors is involved in transition to post-embryonic phase, we performed the screening of lines showing high sensitivity to HDAC inhibitor among some HDAC mutants. By this screening, HDA6:RNAi plant was identified as a line showing growth arrest after germination and ectopic expression of LEC1 under low concentration of HDAC inhibitor. This result suggests that HDA6 is involved in suppression of embryogenesis-related genes in transition to post-embryonic growth.

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It is well known that abscisic acid (ABA) is involved in acquisition of desiccation tolerance and dormancy in zygotic embryos. It has been reported that the VP1/ABI3 factor functions as a transcriptional factor on seed specific ABA signal transduction. We had suggested that two carrot homologs of the VP1/ABI3 factor, C-ABI3 and C-ABI3-L, are involved in the control of expression of some ABA-inducible genes in carrot zygotic and somatic embryos. We analyzed in detail the expression of C-ABI3 and C-ABI3-L in carrot somatic embryogenesis by quantitative real-time reverse transcription-PCR. The expression pattern of each gene, C-ABI3 and C-ABI3-L, during somatic embryo development was obviously different, although the expression of both genes was observed tissue-specifically in embryogenic cells, somatic embryos, and developing seeds. These results suggest that C-ABI3 and C-ABI3-L may function collaboratively on ABA signal transduction in carrot embryos.

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In maize, in vitro fertilization system, consisting of gamete isolation, fusion of isolated gametes and culture of produced zygotes, has been established, and the cells and embryos are successfully used as materials to investigate the molecular mechanisms of fertilization and early embryogenesis. In the preset study, we established in vitro fertilization system using rice gametes. Ovaries were first cut transversally in mannitol solution and subsequent pushing the lower part of the dissected ovaries resulted in release of egg cells. Sperm cells were isolated by bursting the pollen grains in mannitol solution. Using the isolated rice egg and sperm cells, in vitro fertilization was established via electrofusion. The produced zygote formed cell wall and divided into 2-celled embryo. Moreover, the 2-celled embryo developed into a cell cluster, via globular-like embryo. Currently, we are examining ability of the cell cluster to form shoot, coleoptile and root.

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Leaves of higher plants show diverse and complex patterns of leaf venation. Each species has its own characteristic vein pattern. Arabidopsis leaves have brochidodromous venation pattern. One primary vein without bifurcation runs through the middle of each leaf and secondary veins connect together in the lateral region of leaves. Tertiary and higher order veins bridge lower order veins, creating reticulate pattern.
To understand the mechanism of vein patterning, we have isolated Arabidopsis mutants defective in leaf venation pattern. 621C-27 show bifurcated primary veins, secondary veins that fail to connect each other, and reduction of higher order veins. These suggest that 621C-27 gene is involved in vein patterning in Arabidopsis. Analysis of expression pattern of procambium marker AtHB8 and auxin-distribution-marker DR5 in 621C-27 and map-based cloning of 621C-27 gene are now in progress.

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Leaf venation pattern is determined by pattern of the procambium that is the precursor tissue of the vasculature. To understand the mechanism regulating procambium patterning, we isolated and analyzed Arabidopsis mutant 3B-55 which has aberrant leaf venation pattern. In 3B-55, free-ending secondary veins are observed much more frequently than in wild type and the number of higher-order veins reduces prominently. Fragmented minor veins appear occasionally. Shapes of leaf laminas also exhibit abnormality. These suggest that 3B-55 gene is required both for leaf lamina development and for leaf vascular patterning. An analysis of the expression pattern of 3B-55 gene and subcellular localization of 3B-55 protein is now in progress. Expression patterns of procambium marker AtHB8 and auxin-distribution marker DR5 in 3B-55 mutant will also be presented.

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Toward the understanding the mechanisms of formation of leaf-venation pattern, we have isolated and analyzed Arabidopsis mutants defective in formation of leaf veins. no vein-1 mutant (nov-1) often produces leaves without veins. Expression patterns of ATHB8 and DR5 in nov-1 suggest that NO VEIN gene (NOV) is necessary for prepro/procambial cell formation and for proper auxin accumulation pattern that is prerequisite for vascular development in leaves. NOV encodes a protein with unknown function. Null alleles of nov-2~5 mutants exhibit embryo-defective phenotype. The phenotype of nov mutants and expression pattern of ATHB8 and DR5 in nov mutants will be presented.

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In Arabidopsis, the root vasculature contains two files of protoxylem and of protophloem elements, which are alternately located on the opposite sides of the vascular cylinder. Metaxylem develops centripetally from the protoxylem poles, creating the continuous xylem plate. Consequently, phloem is separated into two regions by the xylem plate. In order to clarify the mechanism of vascular development and the xylem-phloem patterning, genes involved in the formation of the procambium, precursor tissue of vascular elements, should be identified. We screened for enhancer-trap lines expressing reporter GFP in the procambium and obtained 376 procambium/vasculature lines. So far, three novel genes expressed in the root procambium in the cell-type-specific manner were identified. They are expressed in xylem and protophloem cell files, in metaxylem cell files, and in phloem and adjacent pericycle cell files, respectively. Expression patterns of GFP in the aforementioned lines, and of the isolated genes will be presented.

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The vascular system plays an important role in nutrient and signal transportation in land plants. Xylogen has been isolated and identified from zinnia tracheary element (TE) differentiation culture system as a factor directing continuous formation of xylem strands. Xylogen is an arabinogalactan protein (AGP) with non-specific lipid transfer protein (nsLTP) domain and glycosylated phosphatidylinositol (GPI) anchor domain. Homology search has revealed genes sequentially homologous to xylogen in Arabidopsis genome. These xylogen homologs have well conserved nsLTP domain and diverged AGP domain. In order to study their histological expression pattern, we have produced some transgenic lines harboring xylogen homologous gene promoter::GUS constructs. Histochemical localization of these reporter gene showed distinctive temporal and spatial expression patterns, indicating that the xylogen homologs may be functionally diversed in plant development.

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Recent studies revealed that endocytosis is involved in the many plant functions of higher order, although detailed molecular mechanisms of endocytosis remains almost unknown. We are studying how endocytosis participates in the morphogenesis or the response to the environment using Arabidopsis plants, especially focusing on the function of Rab GTPases. In plants, many Rab GTPases including Rab5 members are predicted to regulate endocytosis. There are three Rab5 members in Arabidopsis thaliana, conventional type of Rab5, Ara7 and Rha1, and plant-unique type of Rab5, Ara6. We have already reported that Ara6 is localized on the different population of endosomes from Rha1 and Ara7, and these two populations of endosomes are functionally differentiated. In this meeting, we are reporting the interesting genetic interactions between rab5s and atvam3/syp22, which suggest that RAB5s and AtVAM3/SYP22 participate in at least two different genetic pathways coordinately.

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It has been suggested the presence of the close relationship between the morphogenesis and defense responses in plant. But Molecular mechanism of its relationship is unknown. To make clear it, we analyzed Arabidopsis semi-dominant mutant uni-1d, which formed ectopic many lateral shoots and constitutively expressed defense response genes. UNI encoded a novel NBS-LRR family protein and uni-1d has missense mutation causing a gain-of-function. These results suggested that mtant UNI (mUNI) may activate signal transduction pathways of both morphogenesis and defense response. Analysis of double mutants between uni-1d and some defense response related mutants indicated that mUNI affected the morphogenesis without a part of defense response. In addition, a suppressor mutant restored only morphological phenotype in uni-1d was isolated. Here, we want to discuss a signal transduction pathway of mUNI-downstream.

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Cyclin-dependent kinases (CDKs) are key regulators of the cell cycle. Although CDK activities are regulated by phosphorylation and binding of regulatory subunits, such as cyclins, little is known about transcriptional, translational and other post-translational regulations of CDKs.
CDKA (CDKA;1) is a functional homolog of budding yeast CDC28, and its expression is cell cycle phase-independent. CDKB2 is unique to plants and is specifically expressed during G2 and M phase. For expression analysis, promoter::GUS transgenic plants have been generated and analyzed.

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Cyclin D and Cyclin E are known to regulate the G1/S phase progression during the cell cycle. However, the homolog of cyclin E has not been identified in plants. We investigated expression of Arabidopsis cyclin genes during the cell cycle by using the cell suspension culture MM2d, and revealed that the transcripts of two cyclin A3 genes (CycA3;1, CycA3;2) accumulated in the G1/S phase. In situ hybridization showed that CycA3;1 and CycA3;4 specifically expressed at the shoot apical meristem and leaf primordia. We investigated the hormonal response of CycA3 genes in seedling and cell suspension culture, and transgenic plants over-expressing CycA3s were also analyzed in terms of cell division and differentiation.

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The eukaryotic cell cycle is controlled by cyclin-dependent kinases (CDKs). We previously found that Thr-161 residue of T-loop in tobacco CDKA was phosphorylated after reentry to the cell cycle. To investigate the role of the T-loop phosphorylation in tobacco CDKA, we generated tobacco BY-2 cells expressing estrogen-inducible GFP-fused CDKA protein in which Thr-161 residue was substituted to unphosphrylatable amino acid, alanine (T161A). As a control, BY-2 cells expressing GFP-fused CDKA (D146N) were generated. Consistent with previous studies, BY-2 cells expressing CDKA (D146N) caused dominant-negative effects and ceased cell division, but elongated . Although immunoprecipitates with GFP antibody prepared from BY-2 cells expressing CDKA (T161A) showed virtually no histone H1 kinase activity, neither reduction of CDKA kinase activity bound p13^suc1 nor phenotypic change was observed. Further biochemical analysis is underway and will be presented and discussed.

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Plant cytokinesis is accomplished with the formation of phragmoplast, which is formed between two daughter chromatins during anaphase. The development of phragmoplast is shown to be regulated by the MAP kinase cascade. The member of MAP kinase cascade being identified, the targets of the cascade are little known. We applied the phosphoprotein purification with immobilized metal ion affinity chromatography and two dimensional-differential gel electrophoresis for the identification of the phosphorylated targets of MAP kinase cascade in Arabidopsis. Since some parts of proteomes obtained from nack1-1 and mpk4-2 mutants were found similar, it was suggested the MPK4 is one of downstream of MAP kinase cascade from AtNACK1. One of the up-regulated spots in nack1-1 mutant found to be the AtCDC48A, which seemed to raise a new target of MAP kinase cascade.

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Microtubule arrays are prominent molecular machineries managing proper regulations of cell number and shape. Spatial and temporal controls of microtubule arrays are essential for regular development. To understand the molecular mechanism of microtubule organization, we have analyzed function of a plant-specific kinesin-like protein, API1, which is expressed in dividing cells of the moss Physcomitrella patens.
Disruption of API1 or its sister gene, API1L alone did not result in any noteworthy phenotype, but double disruption of API1 and API1L caused formation of incomplete cell plate and multinucleated cells, and reduced and misoriented polar growth. In the double disruptants, the spindle was distorted and the midline of the phragmoplast was unusually expanded. The API1-GFP and API1L-YFP fusion proteins were localized in the equatorial plane of spindles and phragmoplasts, where the plus ends of microtubules overlap. Thus these proteins may crosslink antiparallel microtubules at the equatorial plane of spindles and phragmoplasts.

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Cyanidioschyzon merolae is a unicellular rhodophyte having a plastid and a mitochondrion. Division of these organelles precedes cell division. The organellar cycle as well as the cell cycle was analyzed in a synchronous culture triggered by a light/dark cycle (6h-light/18h-dark) combined with regulation of CO₂-supply. We examined changes in expression of various genes throughout the cell cycle by real-time RT-PCR. So far, the genes involved in the replication of the nuclear and organellar genomes, the DNA repair, the cell cycle progression (four cyclins and four cyclin-dependent kinases), the metabolism, the photosynthesis, and the division of organelles have been analyzed. Many genes including cyclins and CDKs showed a specific expression pattern during the cell cycle.

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Okazaki et al. (2005) reported induction of 2n pollen by arresting the meiotic process with nitrous oxide gas. However it has not been known mechanism by which 2n pollen was induced with nitrous oxide gas. In this study, we observed microsporegenesis in non-treated and N₂O-treated pollen mother cells (PMC) in lily, using fluorescence-labeled tubulin-specific-antibody and aceto-orcein stain.
In the prophase of non-treated PMC, most of the microtubules surrounded chromosomes instead of preprophase band formation. In the metaphase, normal meiotic spindle was formed, followed by formation of cell plates and the resulting tetrad. On the other hand, in the N₂O-treated PMC, depolymerization of microtubules was observed and resulted in arresting the meiosis, whereas cell plate was formed normally. Consequently, two copies of the genome were situated in one daughter cell of PMC and no genome in the other daughter cell, indicating formation of diploid pollen.

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Callus of a metal hypertolerant fern, Athyrium yokoscense (Ay), was exposed to 100 μM of Cd, and the molecular/physiological responses were compared to those in callus of Nicotiana tabacum (Nt). Ay could grow without any visible damage by the Cd exposure, while Nt reduced the growth severely. At that time, Ay and Nt differently accumulated Cd in cell wall fraction (about 90% and 40%,respectively), although each total cellular Cd content was almost the same. The Cd exposure reduced only Fe concentration in Nt. Phytochelatin synthase gene (PCS) expressions and the synthesis of phytochelatins, which plays a major role in Cd detoxification among common plant species, were stable even after the Cd exposure in Ay, while both of these increased significantly in Nt. These results suggest that the Cd tolerance of Ay is based on a phytochelatin-independent Cd-exclusion from inner cells and a preferential uptake of essential metals.

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The fern Athyrium yokoscense is known as a hyperaccumulator of cadmium(Cd). Although the tolerance to Cd and lead are reported, the mechanism of tolerance is not enough become cleared. Cd sets the upper-part of the plant in a state of Fe-deficiency in a usual plant. Recent molecular aspects in Arabidopsis indicated that Fe-transporters play a major role in Cd-uptake. Here, we isolated the three types of iron transporters from Athyrium yokoscense (AyIRT, AyYSL and AyNramp).
AyIRT and AyNramp showed differences in a specific amino acids, which are highly conserved between plants and considered to be important for the Cd tolerance. Furthermore, AyIRT is different from the major IRTs (AtIRT1) in number of transmembrane domain. When those genes were expressed in yeast cells, cells could grow well on 30μM Cd than the control. It is possible that these specific amino acids relate to the hyper Cd-tolerance in Athyrium yokoscense.

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Cd in soils, which can accumulate in agricultural product, threatens food safety. Phytoremediation is the attempt to clean up soils contaminated with Cd or other heavy metals using plants. To facilitate the attempt, we should know what happens in plant cells when they encounter heavy metals. We tried here to isolate Arabidopsis mutants that show altered responses to Cd, aiming to know what determines plant responses to Cd. In the screening, Cd-gradient agar plates were prepared, in which gradation of Cd concentration was formed. EMS-treated M2 seeds were planted onto the plates vertically set in growth chambers, so that roots elongate against the Cd gradient. Normal plants cease root elongation, as root tips proceed to Cd-rich regions. We successfully obtained some mutant lines: one elongated primary roots longer than WT against the Cd gradient, other stopped elongation of primary roots earlier and produced many lateral roots at Cd-free regions.

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We have reported the characterization of Ni-tolerant tobacco BY-2 cell line, NIT cells. Our previous study showed that the Ni tolerance of the NIT cells was highly related to the contents of organic acids and histidine (His), and vacuolar compartmentalization of Ni. Based on these findings, subcellular distribution of Ni and these metabolites was investigated by using protoplasts and intact vacuoles derived from the NIT cells. Studies on the intracellular localization of the Ni, organic acids and amino acids revealed that all of the Ni and citrate and approximately 60% of His were localized in the vacuole. This result indicates that Ni was possibly transported into the vacuoles in the form of chelate complex. Therefore, the vacuolar transport of Ni²⁺, Ni-citrate or Ni-His was investigated by measuring the fluorescence of cell-impermeable Newport green DCF, Zn/Ni specific indicator, in the tonoplast vesicles from the NIT cells.

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Arabidopsis thaliana metal tolerance protein 1 (AtMTP1, 43 kDa) is a member of the cation diffusion facilitator (CDF) family. We previously reported that AtMTP1 was localized in vacuolar membranes and play a key role for Zn tolerance. The transport mechanism of this attractive transporter is unclear. To analyze the transport mechanism, we established heterologous expression system of AtMTP1 in a Zn-sensitive zrc1cot1 double mutant strain of Saccharomyces cerevisiae. Expression of AtMTP1 complemented the zinc tolerance in the strain. Furthermore, spatial dynamics of Zn²⁺ concentration in yeast AtMTP1/zrc1cot1 by using the membrane-permeable fluorescent Zn²⁺ probe also indicate the Zn²⁺ accumulation in vacuoles. The analysis of Arabidopsis transformed with a promoter-GUS construct suggests that AtMTP1 is highly expressed in growing cells. AtMTP1 may sequestrate zinc in the cytosol to vacuoles in growing cells to maintain the zinc homeostasis.

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We have been isolating and characterizing heavy metal stress related three genes, bxa1, bmtA and bxmR, derived from O. brevis. Further studies for characterization of gene function of the bxa1 were performed using yeast transformants in this study.
Sensitivity test for various metal stresses (Cu, Co, Zn, and Cd) indicated that bxa1 transformant was more sensitive to Cd than a control strain carrying a vector DNA. Another sensitivity test for Cd using nine of metal stress sensitive yeast null mutants indicated that two mutants, smf3- and ycf1-, were sensitive to Cd. Both of these mutants carrying the bxa1 were more sensitive to Cd, compared with their parental null mutants. Our result implies that higher accumulation of Cd have occurred in these bxa1 transformants than their parental strains.
We are now constructing a transformant carrying bxa1::GFP fusion gene to determine the localization of this protein within yeast cell.

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Tobacco (Nicotiana tabacum L. cv. Xanthi) exposed to cadmium have been shown to excrete Cd/Ca-containing grains through their trichomes. In this work, tobaccos were exposed to various Zn and Ca concentrations. Growth tests showed that Ca has a protective effect against Zn toxicity, and the endogenous metal contents showed that Ca limited the uptake of Zn, but not the accumulation and translocation. Exposure to Zn induced an increase of the trichome density on the leaves. The grains produced by the trichomes were directly observed under variable-pressure scanning electron microscopy (VP-SEM) and isolated for further characterization. The mineralogical nature of the grains and the structural environment of Zn were determined by state-of-the-art synchrotron-based x-ray microanalyses: x-ray micro-fluorescence (XRF), x-ray micro-diffraction (micro-XRD), and micro-focused extended x-ray absorption fine structure spectroscopy (micro-EXAFS). This study shows that tobacco trichomes have an active role to discharge Zn by immobilizing it in biogenic minerals.

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Iron-deficiency tolerance of Gramineae plants is well correlated with the amount of mugineic acids secreted. However, not only the degree but also the pattern of iron-deficiency chlorosis is different between rice and barley. This phenomenon cannot be explained by only mugineic acid secretion. We suggested that barley retranslocated iron more efficiently from old leaves to the young leaves as compared with rice. In this work, we compared iron availability among rice, barley, sorghum under various iron-limiting condition. Under iron-limiting condition, growth of barley was relatively higher than that of rice and sorghum. However iron content of barley were lower than that of rice and sorghum. In addition, iron content of barley was almost constant regardless of the iron supply. These results suggest that iron availability is different among gramineae plant species. Correlation between available iron and chlorophyll index will be discussed.

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Plants absorb Zinc (Zn) from soil and distribute to whole plants. However, the molecular mechanism of Zn translocation is still unknown. We investigated whether phytosiderophores are involved in absorption and translocation of Zn in graminaceous plants using PETIS (Positron Emitting Tracer Imaging System) method.
(i) ⁶²Zn was supplied from Zn-deficient barley roots with deoxymugineic acid (DMA) or without DMA. Higher amount of Zn was absorbed when Zn was supplied with DMA.
(ii) ⁶²Zn was supplied from Zn-deficient rice roots with DMA or without DMA. Lower amount of Zn was absorbed when Zn was supplied with DMA. However, higher amount of Zn was translocated to newest leaf when Zn was supplied with DMA.
(iii) ⁶²Zn was supplied from Zn-deficient rice leaf with DMA or without DMA. Higher amount of Zn was translocated to the sheath or newest leaf when Zn was supplied with DMA.

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Iron is an essential nutrient for normal plant growth, and involved in many cellular activities. Graminaceous plants use the iron chelators known as mugineic acid family phytosiderophores (MAs) to acquire iron from the rhizosphere. The secreted MAs solubilize Fe(III) in the rhizosphere, and the resulting Fe(III)-MAs complexs are reabsorbed into the root cells. Nicotianamine (NA), an essential intermediate in the biosynthesis of MAs, was found in all plant, and chelates metal cations, including Fe(II) and Fe(III). Therefore, NA is thought to play a role in the internal transport of Fe and other metals. In this study, we examined the expression pattern of the genes encoding enzymes involved in the MAs biosynthesis and the iron transporters during seed germination by promoter-GUS analysis. All genes examined were expressed in germinating rice seeds prior to radicle protrusion. It was suggested that NA and DMA are produced and involved in iron transport during germination.

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Graminaceous plants secrete mugineic acid family phytosiderophores (MAs) in response to iron deficiency. Deoxymugineic acid (DMA) is the initial member of the MAs and serves as a precursor for the synthesis of other MAs.
We have cloned DMAS(s) genes from barley (HvDNAS1) maize (ZmDNAS1) and wheat (TaDNAS1). These genes belong to aldo-keto reductase super family and sequence of DMAS(s) is highly conserved among graminaceous plants. Based on nucleotide sequence all these genes were predicted to encode polypeptides of 314 amino acids and are highly homologous (82-96%) to each other. Fusion proteins, expressed in E. coli., showed the in-vitro enzymatic activity to synthesize DMA. Northern blot analysis revealed that DMAS(s) genes were up regulated in response to iron deficiency in root tissue, while no expression was detected in iron deficient or sufficient shoots. Subcellular localization of these genes will be characterized.

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Graminaceous plants solubilize Fe(III) by secreting phytosiderophores, which are chelators for Fe(III) and take up Fe(III)-phytosiderophores by roots via YS1 transporters. By studying the mutant of Zea maize the Fe(III)-phytosiderophore transporter, ZmYS1, was isolated. To understand the mechanism of Fe(III)-phytosiderophore transport we searched for YS1-like genes in the rice genome and identified 18 putative OsYSLs(OsYSL1-18). Northern blot analysis and quantitative real time PCR revealed that the expression of OsYSL18 was regulated by iron. OsYSL18:green fluorescent protein (GFP) was localized in the plasma membrane of onion epidermal cells. Yeast complementation assay and Xenopus oocytes uptake assay revealed that OsYSL18 transport Fe(III)-deoxymugineic acid. Now we are producing transgenic rice plants that express GUS genes under the control of OsYSL18 promoter.

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Rice plants use a well-known phytosiderophore-based system (Strategy II). Recently, we showed that rice also possesses direct ferrous transport system. In spite of possessing these two systems, rice plants are extremely susceptible to low iron supply, because of low secretion of phytosiderophores and low activity of ferric reduction. The mutational reconstructed yeast ferric chelate reductase gene, refre1/372, was fused to the promoter of iron-regulated-transporter OsIRT1 promoter, and was introduced into rice plants. The transgene was expressed in response to low iron nutritional status in roots of transformants. Transgenic rice plants expressing the refre1/372 gene showed higher ferric chelate reductase activity and took iron up at higher rate than vector controls under iron-deficient conditions. Consequently, the transgenic rice plants showed an enhanced tolerance to low iron availability, and 7.9 times greater grain yields than that of the nontransformant rice on calcareous soils.

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We have previously identified the novel cis-acting elements IDE1 (iron-deficiency-responsive element 1) and IDE2 through promoter analysis of barley iron-deficiency-inducible IDS2 gene. In this report, we carried out promoter analysis of barley iron-deficiency-inducible IDS3 gene, which encodes mugineic acid synthase. IDS3 promoter fragments were connected to GUS gene and were introduced into rice, tobacco and Arabidopsis plants. IDS3 promoter induced strong iron-deficiency-inducible expression in roots of the three plant species. Deletion analysis using tobacco and Arabidopsis transformants revealed that the region within -305 from the translation start site contains at least two cis-acting elements predominantly responsible for the inducible expression. This region possesses some sequences homologous to IDE1 and/or IDE2, which are expected to be the functional cis-acting elements. Results of gel-shift analysis using IDS3 promoter fragments and nuclear proteins extracted from leaves and roots of tobacco, rice and barley will also be presented.

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Phytochromes are well-known photoreceptors mediating photomorphogenetic responses in plants. Plant phytochromes and bacteriophytochromes covalently bind a linear tetrapyrrole as a chromophore, and show red/far-red reversible photoconversion. PixJ1 is a phytochrome-like photoreceptor necessary for positive phototaxis in a unicellular cyanobacterium Synechocystis sp. PCC 6803. His-tagged PixJ1 protein purified from the Synechocystis cells covalently bound an unknown linear tetrapyrrole and showed a novel blue (λ_Amax= 435 nm)/green (λ_Amax= 535 nm) reversible photoconversion. Assembly with red light-absorbing chromophore phycocyanobilin (PCB) well simulated the blue/green photoconversion, indicating that a PCB or PCB-like chromophore binds to PixJ1. In this study, site-directed mutations were introduced at the potential chromophore-binding site(s) to investigate the manner of the blue-shifted photoconversion. Mutation at the either conserved Cys or His residue bound a tetrapyrrole covalently, however, were deficient in reversible photoconversion. We will discuss the possible involvements of PCB to the blue/green photoconversion of PixJ1.

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A sensor of blue light using FAD (BLUF) protein is a new family of blue light sensory protein using FAD as a chromophore. It has been proposed that light signal acquired by FAD is transformed into structural changes of protein for the signaling state, through rearrangement of hydrogen bond network, involving FAD isoalloxazine ring. In this study, a BLUF domain of AppA was in vitro reconstituted from non-labeled apo-protein and isotope edited FAD, in which each C or N atom of the isoalloxazine ring was selectively labeled with ¹³C or ¹⁵N. The labeling induced the isotope shift of the specific IR bands in the light-induced Fourier transform infrared (FTIR) spectrum. The results enable us to assign respective IR bands, and, therefore, allow us to evaluate details of the light-induced structural changes for the signaling state in the BLUF domain.

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AppA is a member of a sensor of blue light using FAD (BLUF) protein family. In this study, effects of W104A mutation on spectroscopic properties of the AppA BLUF domain (AppA126) were investigated. The W104A mutant showed a nearly normal redshift in the UV-visible absorption of FAD upon illumination. However, the light state as revealed by the upshifted absorption relaxed to the dark state at a rate 150 times faster in the mutant than in the wild-type AppA126. The mutation led to 2cm^-1 upshift of the C4=O stretch bands in the light-induced Fourier transform infrared (FTIR) spectrum. Notably, prominent protein bands assigned to the light-induced change of the β-sheet structure were markedly affected by the mutation to be eliminated and/or modified. These results indicate that Trp104 is responsible for transforming the light signal into a specific β-sheet structure change for the signaling state in the apoprotein of AppA BLUF domain.

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FAD binding blue-light sensory domain BLUF is widely distributed among the genomes of photosynthetic bacteria, cyanobacteria and Euglena. PixD (Tll0078) protein of thermophilic cyanobacterium Thermosynechococcus elongatus BP-1 is composed of only BLUF domain. BLUF domain proteins show a red-shift in the absorption spectrum of flavin upon blue light absorption. The protein structure of the PixD protein was revealed in 2005. We studied the photo-reaction of purified Tll0078 protein and its mutant proteins on Tyr8 residues expressed in E.coli by time-resolved spectroscopy and molecular dynamics simulations. The transient absorption change of wild-type protein showed the formation of 10 nm red shifted form at 50 ns after the laser excitation at room temperature. On the other hand, Y8F and Y8A mutant protein showed a formation of triplet states.

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PixD is a blue light receptor which is involved in phototaxis in Synechocystis sp. PCC 6803. We determined the crystal structure of the dark state PixD of Thermosynechococcus elongatus BP-1 at 2.0A resolution. By site directed mutagenesis analysis, Tyr8-Gln50-N5/O4 network is critical for the photocycle.
We performed yeast two-hybrid screening with PixD of Synechocystis. PixD tightly interacted with PatA type response regulator PixE in the dark, but loosely interacted under blue light illumination. We performed pull down assay of His-PixE by Ni affinity chromatography. PixD was co-isolated with His-PixE in the dark, while only His-PixE was isolated under the blue light illumination. These results suggest that the dark form PixD interacts with PixE and the light form does not. We will discuss about light dependent structural changes and signal transduction mechanism of PixD and PixE.

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Synechocystis sp. PCC 6803 has been a model organism for molecular analysis of genes involved in such photoresponses. We have shown that SyPixJ1 is blue light receptor essential for positive phototaxis in Synechocystis. Previously, we reported properties of TePixJ_GAF. of T. elongatus that is homologous to the chromophore-binding domain of SyPixJ1. We expressed TePixJ_GAF with a His tag in Synechocystis. Purified TePixJ_GAF showed reversible photoconversion between the 430 nm and 530 nm-absorbing forms which is very similar to SyPixJ1. Here, we report detailed analysis of TePixJ_GAF. We identified a peptide fragment that binds the chromophore by using MALDI/TOFMS. Either spectral form of TePixJ_GAF did not show spontaneous dark reversion. Denaturation of TePixJ_GAF showed that the bound chromophore is significantly different from phycocyanobilin of phycocyanin. Therefore we suggested that structure and binding site of the chromoophore in TePixJ and SyPixJ1 are unique in comparison with plant phytochorome and bacteriophytochrome.

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The PAS domain is one of the important signaling modules that monitor changes in light, redox potential, oxygen, and so on. Especially, LOV-type flavin-PAS domains of diverse proteins serve as a blue light sensor. There are many signal transduction proteins carrying PAS domains in the genome of a nitrogen-fixing heterocyst-forming cyanobacterium Anabaena sp. PCC 7120. From these proteins, we detected three novel LOV-type flavin-PAS proteins. Here, we expressed and purified these three LOV-type flavin-PAS domains from Escherichia coli. Two flavin-PAS domains that are conserved in a related cyanobacterium indeed bound FMN. They showed photoreduction similar to plant phototropins. However, one was fully photoreduced and dark reversion was very slow, whereas the other was slowly photoreduced and dark reversion was very fast. The third PAS domain that is unique to Anabaena 7120 did not bind flavin at all. Possible biological roles of these PAS domains will be discussed.

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Phototropins, blue light receptors in plants, have two photosensory domains named LOV which bind FMN as a chromophore. We have studied the light-induced structural changes of LOV domains of Adiantum phytochrome3 (phy3) by means of low temperature FTIR spectroscopy. In order to identify IR signals, isotope-labeled LOV2 domains were prepared.
The reconstitution of [2-¹³C]FMN revealed that the hydrogen-bonding of C(2)=O group in FMN was weakened. The reconstitution of [1,3-¹⁵N₂]FMN revealed that N(3)-H group of FMN formed very strong hydrogen bond in the unphotolyzed state and stronger hydrogen bond upon the formation S390 intermediate. We could reveal the change of the hydrogen-bonding network between FMN and surrounding amino acid side chains. The changes of such hydrogen-bonding network may be more important than the direct change of adduct formation for the structural change in the α-helix and β-sheet regions.

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Phototropins (phot1 and phot2), membrane-associated protein kinases, act as blue-light photoreceptors for phototropism, chloroplast relocation, stomatal opening, and leaf flattening in Arabidopsis. Phototropins have three functional domains, two LOV domains in their N-termini and a Ser/Thr kinase domain in their C-termini. We have recently reported that phot2 associates with the Golgi apparatus in a light-dependent manner (Kong et al., 2005, Plant J., in press). Here we analyzed the structure/function relationship in phot2 using transgenic Arabidopsis. The phot2 C-terminal kinase domain fused to GFP (P2CG) was localized to the plasma membrane and the Golgi apparatus, whereas the N-terminus fused to GFP (P2NG) was uniformly localized in the cytoplasm. P2CG exhibited dominant-positive activity for the physiological responses such as chloroplast relocation and stomatal opening. By contrast, the P2CG with a mutation that abolishes the kinase activity was localized mainly to the plasma membrane and exhibited dominant-negative activity over the endogenous phot2.

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Phototropism of Arabidopsis seedlings in response to blue light source is initiated by phototropin1 (phot1) and phot2, blue light receptor kinases. Previous studies have shown that NPH3 and RPT2 are signal transducer in phototropin signaling. However, only few attempts have been made at biochemical analysis of NPH3 and RPT2. Here, we show that NPH3 in seedlings in dark or red light condition is phosphorylated in vivo, and NPH3 is de-phosphorylated under blue light. Accumulation of RPT2 in blue light condition was indicated. Both NPH3 de-phosphorylaion and accumulation of RPT2 which are induced by blue light are mediated by phot1 but not phot2. NPH3 and RPT2 make a complex depended on blue light. While we demonstrated cellular and subcellular localization of RPT2 in seedlings irradiated with blue light. On the basis of these results, we discuss the roles of NPH3 and RPT2 in the phot1 signaling pathways.

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Chloroplasts relocate in plant cells according to the ambient light conditions. Under the low-light conditions, chloroplasts accumulate in the light to capture weak light efficiently (accumulation response), while under the high-light conditions, they escape from the light to avoid photodamage (avoidance response). In a seed plant Arabidopsis thaliana, the accumulation response is mediated by two blue light receptors, termed phototropins (phot1 and phot2) that act redundantly, and the avoidance response is mediated by phot2 alone. Using a new screening method, we recently isolated a mutant, jac1 (J-domain protein required for chloroplast accumulation response 1), which lacks the accumulation response under weak blue light, but shows a normal avoidance response under strong blue light. Positional cloning of JAC1 gene reveals that this gene encodes a J-domain protein, resembling clathrin uncoating factor auxilin at its C-terminus. Amount of transcripts and encoded proteins of JAC1 gene were not regulated by light and phototropins.

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Plants curve their organs such as stems and petioles toward the incident light and intercept light efficiently. To regulate the light interception by respective leaves, the petiole curvature would be more effective than the stem curvature.
In our observation with Chenopodium album L., it was clarified that the petiole curvature was induced by continuous blue-light illumination from the abaxial side of the petiole, and the curvature rate tended to increase with the increase in blue-light intensity. On the other hand, the curvature increase was ceased when the adaxial side of the petiole was illuminated by blue or red light.
The blue-light receptor for the stem phototropism may be also concerned in the stimulation of the petiole curvature. In the cessation of the curvature, photosynthesis in the petiole may play an important role.

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CPD photolyase plays an important role in determining UV sensitivity in plants. This enzyme is grouped into two classes, class I and II. This study was aimed to characterize rice class II CPD photolyase. Crude enzyme from rice leaves was purified sequentially by ammonium sulfate precipitation, anion exchange chromatography, heparin chromatography and DNA containing CPDs affinity chromatography. CPD bound protein on affinity chromatography was released by blue-irradiation, and was used as a purified enzyme. Specific activity of this enzyme was 8,000 fold higher than that of crude enzyme. Western blot and TOF-MAS analyses of purified enzyme showed two proteins (55- and 57-kDa), while rice CPD photolyase expressed-E. coli protein did a 56-kDa protein. Both action spectra for photoreactivating CPDs of the purified enzyme and E. coli expressed enzyme showed a peak around 410 nm. Specific activity of the purified enzyme was several-times higher than that of E. coli expressed enzyme.

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Acaryochloris marina is a newly discovered and Chl d-dominated photosynthetic prokaryote. The absorption maximum of Chl d in organic solvents is located at wavelengths longer than that of Chl a by about 30 nm. In this study, the reversible absorption changes of Chl d by oxidation-reduction were investigated.
Chls were purified by column chromatography, and dissolved in aqueous micelles or MeOH. Reduction and oxidation was performed by adding dithionite or NaBH4 and ferricyanide, respectively.
The absorption maximum of reduced Chl d was blue-shifted by 40nm as compared with Chl d, and came back to the original position by oxidation. We determined a molecular structure of the reduced Chl d by MS spectroscopy to be 3-hydroxymethyl-Chl a. On the contrary, Chl a and b did not show a reversible change, indicating that electronic properties of Chl d are different from those of Chl a or Chl b.

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Cytb₅₅₉, β-carotene (Car), and redox-active accessory chlorophyll (Chl_Z) work as secondary electron donors to donate an electron to P680⁺ in PSII at low temperatures. Although this secondary electron donation plays an important role in photoprotection of PSII, its electron-transfer pathway remains unclarified. In this study, we have investigated the oxidation pathway of Chl_Z by means of FTIR spectroscopy. Two Car molecules in PSII membranes of spinach were selectively bleached by illumination at 250 K under an oxidizing condition. Even in this Car-bleached PSII, the FTIR signal of Chl_Z⁺/Chl_Z at 1713/1687 cm^-1 was developed by illumination at cryogenic temperatures. The kinetics of the ChlZ⁺ formation upon continuous illumination was mostly unchanged at 80 K, whereas the formation rate was appreciably reduced at 210 K. This result indicates that the oxidation pathway of Chl_Z without involving Car is dominant at 80 K, while the pathway via Car becomes significant at higher temperatures.

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According to the study of PSII particle action-spectra of Huzisige and Yamamoto (Plant & Cell Physil.,14:
953-963(1973)), Tris-inactivated chloroplasts were inhibited in O₂-evolving center and electron transport between Qa and Qb of PSII. This study detected the inhibition between Qa and Qb in Tris-washed and photo-reactivated chloroplasts by using fluorescence meter with powerful blue-light emitting diode, active to rise maximum stable emission in less than 50 μsec. The photo-eactivated chloroplasts recovered O₂-evolving activity 50%, and kept 70% of Qa-photoreducing activity (the chlorophyll fluorescence rise in 0-1 msec after blue-light spark in the presence of DCMU). While the Qb-photoreducing and fluores-
cence risig activity of reactivated chloroplsts at 20 msec without DCMU was depressed to 8-16% of normal chloroplasts caused by the inhibition between Qa and Qb.

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PsbL is one of subunits of PSII complex of which function is unknown. It consists of 37 amino acid residues and has one membrane penetrating -helix at C terminal region. In vitro reconstitution experiments have suggested that PsbL is involved in the pH dependent electron transfer in PSII, however, actual function of PsbL has not been clarified. In this work, we prepared transformants of tobacco plant with mutations at various positions in PsbL and investigated the effect of each mutation on the electron transfer activity of PSII by PAM. The introduction of mutation in the C terminal regions had no effect on the electron transfer behavior in PSII. In contrast, mutation in the N terminal region of PsbL resulted in the increase in F0 without any significant effect on Fm, suggesting a possible role of PsbL in the regulation of electron transfer at the acceptor side of PSII.

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The extrinsic PsbO protein which is conserved in all of oxygen-evolving organisms plays important roles in maintaining the stability and activity of Mn cluster for the water-oxidizing reactions. We examined the binding domains of spinach and red algal PsbO responsible for electrostatic interaction with PSII intrinsic proteins by chemical modification. The results showed that Lys residues in six domains of PsbO are commonly involved in the interaction. To determine which Lys residues in the six domains directly participate in the interaction, we constructed 8 mutant PsbO proteins in which positively charged Lys residues were substituted to non-charged Gly residues by site-directed mutagenesis. The results showed that K15G, K74G and K164G largely lost its binding ability, while K127G, K198G and K241G did not. Lys residues on PsbO responsible for the electrostatic interaction with PSII intrinsic proteins will be discussed in crystal structure of PSII.

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