Extracellular proteins play various functions in plants. Xylogen, an arabinogalactan protein (AGP) which is isolated from Zinnia mesophyll cell culture, is an extracellular arabinogalactan protein that promotes tracheary element differentiation. Xylogen is secretory protein whose membrane localization is supposed to be regulated by its C-terminus glycosylphosphatidylinositol (GPI)-anchor. In Arabidopsis, AtXYP2 is likely to be the ortholog of Zinnia xylogen. To understand the secretion and localization mechanisms of AtXYP2, we generated transgenic plants harboring GFP-fused AtXYP2 gene under the AtXYP2 promoter, and analyzed intracellular localization. In the root of transgenic plants, GFP signal was observed in the immature xylem and the procambium. Its subcellular localization differed dependently on differentiation stages. Together with the results using organelle marker and mutants of vesicular transportation, we will discuss on regulation of subcellular localization of xylogen.

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Yellow-stripe like (YSL) transporters belong to the oligopeptide transporter (OPT) family. In the present study, we examined an uncharacterized member of YSLs fin rice, OsYSL16. Knockout of this gene resulted in decreased accumulation of Cu in the shoots, but there was no effect on the accumulation of other metals including Fe, Zn and Mn. Further analysis showed that the knockout line contained a higher Cu concentration in the older leaves, but less in the younger leaves compared with the wild-type rice. Furthermore, when cultivated under copper-deficiency condition, the seed fertility of the knockout line was significantly reduced, but there was no difference in the fertility under high Cu condition. OsYSL16 was expressed both in the shoots and roots. At the reproductive stage, OsYSL16 was especially highly expressed in the node. Immunostainning showed that the protein was mainly localized in the phloem of the node. Yeast expressing OsYSL16 showed transport activity for Cu-NA. Taken together, we conclude that OsYSL16 is a Cu-NA transporter, which is involved in the translocation of Cu through phloem transport.

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Most blue color in flowers is due to anthocyanin, and considerable proportion of blue coloration can be attributed to metal-complexed anthocyanins. Recently, we reported vacuolar localized iron-transporter in blue petal cells of Tulipa gesneriana. However the mechanism of another metal ion transporters and subsequent flower color development has yet to be fully explored. In Hydrangea macrophylla, Al³⁺ is involved in blue coloration and the anthocyanin is formed Al³⁺-complex in vacuoles. To identify the molecular mechanism of blue coloration in hydrangea flowers, we tried to isolate the related genes transporting metal ion into vacuoles. From the sepal cDNA library we read the sequences of ca. 12000 genes, then a microarray analysis was carried out. From the sequences information, we chose several genes that might localize vacuolar membrane and transport ^Al3+. By using Al³⁺-sensitive yeast strain, we could identify the gene transporting ^Al3+ into vacuole. From the functional similarity and predicted localization, we could also identify the gene transporting Al³⁺ into cytosol. We will report Al³⁺ mobilization from out of cell into vacuole in the sepal of Hydrangea macrophylla.

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Magnesium (Mg) is essential nutrient in organisms. Although several functions of Mg in plants are known, Mg homeostasis mechanisms are not clear. In this work, to identify the genes involved in Mg deficiency response, we established screening methods to isolate mutants with altered response to Mg deficiency.
First, we examined conditions to see visual deficiency symptoms. When grown with agar medium, although Mg content in shoot was decreased under the deficiency condition, we could not get any symptoms/phenotypes. With the hydroponic culture, the growth of plants was severely inhibited under low Mg, indicating that agar is not suitable for screening based on visual symptoms.
Then, we established another screening method based on the expression of Mg-deficiency induced genes. We have identified Mg-deficiency induced genes by microarray. Those gene expression levels were high in mrs2-7 mutant, which has known to be involved in Mg homeostasis, compared with wild-type. This indicates that those genes could be used as marker for screening. Now we are generating transgenic plants carrying promoter-luciferase construct and confirming the response of luminescence to Mg.

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Magnesium (Mg) is an essential element for plant. Despite the importance of Mg function, the knowledge of Mg in regard to uptake and translocation manner in plants is limited. One of the reasons is lacking the available radioisotopes of Mg. In this study, we focused on ²⁸Mg (beta-ray emitter, half life: 21h) because ²⁸Mg can be prepared as carrier free. Nowadays, there is not any institute where ²⁸Mg is manufactured in the world. Therefore, we produced ²⁸Mg using an accelerator, CYRIC at Tohoku Univ. Irradiating α beam to pure aluminum target, ²⁸Mg was produced through ²⁷Al(α,3p)²⁸Mg reaction. After irradiation, the Al target was dissolved with hydrochloric solution and ²⁸Mg was purified by chromatography. Then, ²⁸Mg was supplied to the 3-week-old rice seedlings with 6 leaves. The plants were cultured with ²⁸Mg in a nutrient solution for 15 to 30 min. Then, the plant samples were washed with distilled water and each tissue was separated and exposed to an imaging plate (IP).The images of ²⁸Mg showed that ²⁸Mg was highly accumulated in basal part of the 5th leaf sheath, 20 mm in length. We will also present the feature of Mg uptake applying different Mg concentration.

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To study the transport mechanism of cadmium (Cd), the route of Cd from root to shoot was visually identified by employing a radioisotope tracer, ¹⁰⁹Cd. The radioactive Cd solution (2MBq/10ml 0.1μM CdCl₂) was supplied from the root of a rice seedling with emerging 6th leaf for 15 minutes and exposed to an imaging plate (IP). It was found that Cd was directly transported from root to the youngest 6th leaf. To compare the feature of Cd transport with those of P, S and Ca, radiographs of ³²P, ³⁵S and ⁴⁵Ca were also taken. The result showed that the initial accumulation pattern of Cd was similar to that of Ca. To understand the transport route of Cd more clearly, ¹⁰⁹Cd tracer was supplied to one root which was developed from one internode. The internode was successively sliced and was exposed to an IP to get sequential distribution of ¹⁰⁹Cd. From the radiographs, it was suggested that Cd was transferred to phloem in the very early stage of transport from the root. During the upward transportation in the stem, only a small amount of Cd was distributed to the small vascular bundles oriented to the leaf. The transport pathway of Cd will be explained and discussed.

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MCA1 and MCA2 are plasma membrane proteins that correlate Ca²⁺ influx and mechanosensing in Arabidopsis. MCA1 and MCA2 share 72.7% amino acid sequence identity and several common structural features, including putative transmembrane segments, an EF hand-like region in the N-half, a coiled-coil motif in the middle, and a PLAC8 motif in the C-half. To determine regions important for Ca²⁺ uptake activity, the activity of truncated forms of MCA1 and MCA2 was assessed using yeast expression assays. MCA1^1-173 and MCA2^1-173 had Ca²⁺ uptake activity, while MCA1^173-421 and MCA2^173-416 lacking the N-half had no Ca²⁺ uptake activity. MCA1^1-173 and MCA2^1-173 have a putative transmembrane region, and helical wheel analysis of the region showed that highly hydrophilic residues were lined up on one side of the helix. To test the possibility that Asp²¹ in the TM1 plays a role in Ca²⁺ uptake, Asp²¹ was replaced with Asn. MCA1^D21N had no Ca²⁺ uptake activity and MCA2^D21N had partial activity, although the levels of the mutant proteins were not reduced. These results suggest that the N-half of both proteins with the EF hand-like region is necessary and sufficient for Ca²⁺ uptake.

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The micronutrient zinc is essential for all organisms because it is required as a cofactor in over 300 enzymes. On the other hand, excess zinc is also toxic to cells, presumably due to competition with other biologically important ions, which in plants ultimately leads to reduced biomass leaf chlorosis, and root inhibition. To understand the effect of excess zinc on plant cells, we have performed iTRAQ based quantitative proteomics. Arabidopsis grown on MS medium were treated with high concentration zinc (300µM ZnSO4) for 0, 3, 6, 16 hr, and microsomal fractions were isolated from roots. Each fraction was used for iTRAQ analysis. About 1000 proteins including membrane proteins by zinc treatment were identified and quantified. The changed ratio of these proteins was calculated using software MASCOT. Based on the iTRAQ data, we would like to discuss mechanisms of early response proteins by excess zinc in Arabidopsis root.

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We investigated the zinc accumulation pattern and mechanisms in Lotus japonicus. L. japonicus is a model legume for molecular biology and the resources for genetics are well established. From our previous work, zinc accumulation in seed was different between two cultivars Gifu and Miyakojima, therefore, objectives of this study were to identify the zinc accumulation pattern in vegetative stage between these two lines and to determine the loci governing zinc uptake and translocation by using recombinant inbred lines (RIL; Gifu x Miyakojima).
In 4-weeks-old plants, zinc concentration in Gifu was higher than that in Miyakojima in all investigated tissues and, by comparing Zn-65 distribution images, zinc uptake and translocation activities were also higher in Gifu. The zinc accumulation in shoot was investigated with Zn-65 and 80 RILs. Using maximum likelihood method, J/qtl program identified several major QTL. The genes located on these QTL were investigated based on the L. japonicus genome sequence and several genes annotated zinc transporter, which gene expression levels were higher in Gifu, were found. The relationship between these genes and zinc accumulation will be discussed.

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Heavy metal ions are toxic to plants because they replace metal ions of enzyme-cofactor and act as inorganic catalyst of active oxygen in plant cells. Plant cells make resistance to heavy metals by use of plasma membrane heavy metal exporter and vacuolar membrane transporters. However, some of them are not well studied yet. In this study, We examined possibility of novel transporters for manganese (Mn), zinc (Zn), nickel (Ni) and cadmium (Cd).
We screened two-hundreds Arabidopsis thaliana lines those were transformed by membrane transporter over expression vectors. Then we obtained four lines of which heavy metal tolerance were enhanced. One line was more resistant to manganese and zinc than control line. Three lines were more resistant to nickel and cadmium. Some of vector-tagged genes of these lines were novel genes those are conserved in plant worlds. It is possible that these genes are novel transporter genes for heavy metals in plant.

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H⁺-pyrophosphatase (H⁺-PPase) generates a H⁺ gradient across endomembranes coupled with hydrolysis of pyrophosphate. In higher plants, a large amount of H⁺-PPase exists in vacuolar membranes and maintains the vacuolar acidic pH together with V-ATPase. However, it has been reported that H⁺-PPase is also localized in plasma membrane.
In this study, we inserted sGFP in a cytosolic loop of Arabidopsis type-I H⁺-PPase AtVHP1/AVP1 and transformed the construct with its own promoter into plant to determine intracellular localization. VHP1-sGFP expressed in plants had full enzyme activity and was fractionated into same fraction as endogenous VHP1 after sucrose density gradient centrifugation, indicating the adequate localization of VHP1-sGFP with function. VHP1-sGFP was detected in vacuolar membranes in all cells observed by CLSM. Strong fluorescence was observed in membranes of spherical structures within vacuoles, called bulbs. VHP1-sGFP was strongly expressed in almost all tissues, especially in growing tissues and vesicular bundles. In addition, there was a tendency that the expression level of VHP1-sGFP affect the number of bulbs.

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Phosphatidylglycerol (PG) is a phospholipid found in biological membranes. In plant cells, PG is mainly localized in thylakoid membranes and required for photosynthesis. To investigate the function of PG, we isolated pgp1 and pgp2 mutants of Arabidopsis thaliana, which have disrupted PGP1 and PGP2 genes for phosphatidylglycerophosphate synthases involved in the biosynthesis of PG. Glycerolipid profiles of the pgp1 and pgp2 mutants showed that PGP1 mainly contributes to PG biosynthesis in leaves, while PGP2 has only a minor contribution. To make the double mutant that cannot synthesize PG at all, the pgp1 mutant was crossed to the pgp2 mutant. Although the double mutant was not isolated, we found that 25% of seeds were albino and wrinkled when PGP1/pgp1 pgp2/pgp2 plants were self-fertilized. Embryo development in the albino and wrinkled seeds was delayed and not fully matured, and the embryos had short cotyledons. These findings suggested that PG has important roles in embryo development, growth of cotyledons and chloroplast differentiation.

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Soil waterlogging is an environmental stress that negatively affects crop productivity. Waterlogged soils are usually anaerobic and chemically reduced, because of slow diffusion of oxygen in water and rapid consumption of oxygen by soil microorganisms. Under these conditions, combination of large volume of aerenchyma and barrier to Radial Oxygen Loss (ROL) is two important traits.
The aim of our study was to better understand the adaptation to waterlogging condition in teosinte (Z. nicaraguensis) and Z. mays. Growth, root aerenchyma and ROL along the root were measured. When grown in stagnant deoxygenated conditions, formation of aerenchyma near the root tip and ROL barrier at the basal part of roots was observed only in Z. nicaraguensis. Our result demonstrated that Z. nicaraguensis is capable to form not only aerenchyma to near the root tip, but also ROL barrier for an efficient transport of oxygen to the root apex in stagnant condition. Furthermore, extensive histochemical analysis of apoplastic barriers in the root peripheral layers is in progress.
This work was supported by a grant from the PROBRAIN project. The authors thank Dr. Y. Mano and F. Omori for providing seeds.

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We reported previously that atmospheric nitrogen dioxide (NO₂) at an ambient concentration almost doubled plant growth and biomass¹⁾. Histological analysis of rosette leaves of Arabidopsis thaliana C24 revealed that the increase in shoot biomass by NO₂ is attributable to increase in cell size but not to that in cell number. In this study, we investigated possible involvement of the endoreduplication in the increase in cell size by NO₂. One-week-old seedlings grown in air without NO₂ were grown for 4 weeks more in air with (+NO₂ plants) or without (-NO₂ plants) 50 ppb NO₂, after which the ploidy level of leaf 8 of each of the plants was determined. We found that the ploidy level did not differ between +NO₂ and -NO₂ plants. This finding suggests that the endoreduplication is not responsible for the NO₂-induced cell expansion.
¹⁾ M. Takahashi et al. (2005) New Phytol 168: 149-154.

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In the world, large portions of fresh produces such as fruits are known to be lost during storage due to invasion by pathogenic microbes. In this study, we focused on the use of UV radiation at different storage temperature for promoting the storage quality of strawberry fruits. After the freshly harvested strawberry fruits were exposed to irradiation with UV-C, fruits were stored at different temperatures. During storage, the expression levels of some pathogenesis-related genes and secondary metabolism-related genes were examined as the indicators of resistance to pathogens. The expression level of some genes (ie. genes for glucanase and PAL) during low temperature storage was significantly increased by the UV radiation. This suggests that combination of UV radiation and low temperature could be effective method for protecting the strawberry fruits from pathogen attacks during storage.

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A thermophilic cyanobacterium, Thermosynechococcus vulcanus RKN, exhibits low tepmerature/light- induced cell aggregation. We previously reported that this aggregation was dispersed by cellulase treatment. Of three putative cellulose synthase genes, disrution of only Tvtll0007 abolished the cell aggregation. Accordingly, cellulose is suggested to serve as extracellular matrix to support the cell aggregation. Here, we established a method of cellulose quantitation, and measured its accumulation. Under normal growth conditions, the cellulose content was approximately 5 μg / 4 x 10⁹ cells. When cells were grown under the low temperature/light conditions, the level increased approximately two-fold in 24 h, and remained maintained during further growth. This accumulation appeared to slightly precede the cell aggregation. This cellulose accumulation under the induced conditions was abolished by disruption of Tvtll0007 but not by disruption of Tvtlr1795 or Tvtlr1930-33. It is thus concluded that under the low temperature/light conditions TvTll0007 produces cellulose, which is essential for the cell aggregation.

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Plants sense balance of carbon (C) and nitrogen (N) metabolites to regulate metabolism and development, called C/N response. To clarify the mechanisms of C/N response, we identified a novel C/N response regulator ATL31, an ubiquitin ligase. Ubiquitin ligase recognizes specific proteins for degradation by ubiquitin-proteasome system. Previous studies demonstrated that the ATL31 is involved in C/N response to degrade the polyubiquitinated protein. To identify the target protein of ATL31, we screened interaction proteins by using immunoprecipitation and MS analysis. Characterization of the identified proteins will be reported.

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The balance between carbon (C) and nitrogen (N) availability is an important determinant for different phases of plant growth. However detailed mechanisms regulating C/N response are not well understood. We previously revealed a novel ubiquitin ligase, ATL31, which functions in the C/N response in Arabidopsis thaliana (Sato et al., Plant J, 2009, 60:852-864). In addition, proteomics study with MS analysis demonstrated that 14-3-3 proteins were associated with ATL31 and it was shown that ATL31 has ligase activity against 14-3-3 proteins for direct ubiquitination. Now we are further evaluating the physiological function of 14-3-3 proteins in C/N response regulated by ATL31. The details of biochemical and phenotype analysis of atl31 and 14-3-3 mutants will be reported.

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26S proteasome is a large multisubunit complex that degrades aberrant cellular proteins. Most subunits of plant proteasome are coded by duplicated genes. However, specific functions of each paralogous subunit remain unclear. We have revealed that loss-of-function mutant of proteasome palalog shows different response to nutrient stress, suggesting each palalog has different functions.
Furthermore, it has been reported that peptidase activities of proteasome are altered responding to nutrient and oxidative stress. These results suggest that peptidase activities of plant proteasome are important in response to environmental stress.
To evaluate this hypothesis, we tried to detect alteration of peptidase activities and structural transformation.
We established affinity purification of proteasome from Arabidopsis and identification of each palalog using 2-DE/MS analysis. Change in peptidase activities under varies stress conditions will be reported.

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The Small Acidic Protein 1 (SMAP1) gene is responsible for the anti-auxin resistant mutation, aar1, and encodes a factor that mediates the synthetic auxin 2,4-D response. SMAP1 functions upstream of the AUX/IAA degradation step in auxin signaling and physically interacts to COP9 signalosome (CSN) in Arabidopsis extract. To investigate genetic relationship between CSN and SMAP1, the aar1 mutant was crossed with a weak csn mutant, csn5a-1. The double mutant aar1-1 csn5a-1 shows extremely short hypocotyls and slower root growth than the single mutants. The double mutant also showed relatively more auxin resistant than its parent lines and extremely dwarf phenotype in adult stage. The introduction 35S:SMAP1-GFP gene to the aar1-1 csn5a-1 background restored hypocotyl length and dwarf phenotype, suggesting that the luck of the SMAP1 gene results to the morphological defects of the double mutant. Although the aar1 mutant is morphologically indistinguishable to wild type except showing long hypocotyl, the phenotype of the double mutants unveiled the significance of SMAP1 for plant growth and development when CSN function is diminished.

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Reactive oxygen species (ROS) produced by NADPH oxidases have been shown to play many critical roles in stress responses and development in plants. Respiratory burst oxidase homolog (rboh) genes have been identified in various plants as NADPH oxidases. Among 10 rboh genes in Arabidopsis, AtrbohD and AtrbohF are involved in defense responses against pathogens and ABA signal transduction in stomatal guard cells. We have been analyzing the regulatory mechanisms for activation of Atrboh proteins by a heterologous expression system using the HEK293T cells. AtrbohD possessed ROS-producing enzyme activities, which were synergistically activated by phosphorylation and binding of Ca²⁺ to its EF-hand motifs (Ogasawara et al., JBC, 2008). We have characterized the activation mechanisms for AtrbohF. Similarities and differences of the ROS-producing activity and regulation between AtrbohD and AtrbohF will be discussed.

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Nitric oxide (NO) is a ubiquitous signaling molecule involved in diverse physiological process. Despite the wealth of information gathered over the last decade, the mechanisms of NO action are poorly known. We now assess the possibility that nitrated cGMP (8-nitro-cGMP) functions in guard cell signaling. At previous meeting, we reported that 8-nitro-cGMP is formed in an NO- and reactive oxygen species(ROS)-dependent manner and nitrated cGMP induce stomatal closure. At this meeting, we studied sorce of ROS to trigger the nitration of cGMP occur in guard cells, Immunocytochemical analyses were performed using a monoclonal antibody against 8-nitro-cGMP. The mitochondrial respiration cyclase inhibitor, photosynthesis inhibitor and the darek cause decrease of ABA-induced immunofluorescence. These results indicate that mitochondrial respiration and photosynthesis are the source of ROS-cGMP to form 8-nitro-cGMP.

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Rice has the canonical α(Gα), β(Gβ) and γ(Gγ) subunits, which mammals have. The deficient mutant for Gα gene, d1 exhibits dwarfism compared with WT. Previously we showed that the dwarfism in d1 was due to reduction in cell number. When we compared the responses of 7 plant hormones between WT and d1, the d1 mutant showed less sensitivity to brassinolide (24-epi BL). This result showed that a brassinosteriod (BR) signaling may be partially impaired in d1. In order to investigate relationship between the G protein signaling and the BR signaling, we analyzed a double mutant of d1-1 and d61-2 which was a deficient mutant of BR receptor kinase gene (OsBRI1).
Organ length of leaf sheath was synergistically reduced in d1-1d61-2 compared to d1-1 and d61-2. However, cell number of leaf sheath in d1-1d61-2 was not different from that in d1-1. This result suggests that d1-1 may be epistatic to d61-2 in regard to the regulation of cell number. We are carrying out the analysis whether the epistsis is observed in internode and lemma.

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Higher plants have α(Gα), β(Gβ), and γ(Gγ) subunits of the heterotrimeric G proteins as well as animals have. In rice, the deficient mutant of the Gα gene (RGA1), d1 exhibits altered phenotypes such as dwarfism, erect and dark-green leaves and small round seeds. The mutants for the Gβ gene (RGB1) and the Gγ gene (RGG1) were not isolated in rice. In order to estimate the function of Gβ, we produced the transformants that RGB1 expression was suppressed by RNAi in WT and d1-5, and named rgb1RNAi/WT and rgb1RNAi/d1-5. rgb1RNAi/WT and rgb1RNAi/d1-5 showed the abnormal phenotypes, namely dwarf, browning of lamina joint region and node, shorten seed and decreased fertility. In regard to cell proliferation, Gβ and Gα worked independently as a positive regulator. Browning of lamina joint regions and nodes in rgb1RNAi/WT and rgb1RNAi/d1-5 was a novel characteristic, which was not occurred in d1-5. These results indicate that Gβ has some independent functions, compared with Gα in rice.

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Rice is one of the most Al-tolerance species among small-grain cereals. Recent identification of a transcription factor ART1 revealed that this high Al tolerance in rice is achieved by multiple genes involved in detoxification of Al. ART1 is a C2H2-type zinc finger transcription factor and regulates the expression of 31 genes in the downstream. In this study, we attempted to identify a cis-acting element of ART1. We used the promoter region of STAR1, an Al tolerance gene in the downstream of ART1. With the help of gel shift assay, we were able to identify the cis-acting element as GGN[T/g/a/C]V[C/A/g]S[C/G]. This element was found in the promoter region of 28 genes among 31 ART-regulated genes. To confirm this cis-acting element in vivo, we transiently introduced this element one or five times tandemly repeated sequence with 35S minimal promoter and GFP reporter together with ART1 gene in the tobacco mesophyll protoplasts. The results showed that the expression of GFP reporter responded to Al. Furthermore, the expression increased with repetition of the cis-acting element. Our results indicate that the 5 residues identified are the target DNA binding sequence of ART1.

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Rice is the most Al-tolerant species among small-grain cereal crops. Recent identification of a C2H2-type zinc finger transcription factor ART1 for Al tolerance has revealed that multiple genes are implicated in high Al tolerance in rice. Here, we functionally characterized one of downstream genes of ART1, named OsCDT3. OsCDT3 encodes a predicted peptide of 53 amino acid residues, of which 14 (26.4%) are cysteine residues. OsCDT3 was mainly expressed in the roots and its expression was specifically induced by Al exposure. Furthermore, the Al-induced expression was not observed in art1 mutant. Analysis with transgenic plants carrying pOsCDT3::GFP showed that the signal was observed in all cells of the root tips. Both transient expression of GFP-OsCDT3 in onion epidermal cells and western blot analysis in rice roots showed that OsCDT3 was mainly localized to the plasma membrane. Knockdown of OsCDT3 resulted in increased sensitivity to Al in rice. Yeast expressing OsCDT3 showed increased tolerance to Al and decreased accumulation of Al, but the tolerance to Cd was not altered. Our results indicate that OsCDT3 is one of the components required for high Al tolerance in rice.

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Andropogon virginicus L. is a wild plant which shows a high tolerance to Al. Four tolerant mechanisms shown below were involved in this plant. 1) Low Al uptake in root tip. 2) High transportation of toxic Al from root to shoot not to accumulate it in root. 3) Accumulation of Al to trichome in leaf. 4) Induction of anti-peroxidation enzymes by Al stress to suppress oxidative damage. Especially 2) and 3) may be very unique mechanisms of this plant.
Nitric oxide (NO) generally has a function in signal transduction under various stresses in many organisms. Andropogon was investigated whether NO has a similar function under Al, Cd, Cr, Cu and Zn stress in this study. Production of NO was only observed by Al, but not by others. Moreover, NO was not detected in any other tested gramineae plants by the metal treatments. Furthermore, function of NO for Al tolerance in Andropogon was studied. Oxidative damage was suppressed under Al stress by an addition of cPTIO which is an inducer of NO production, while DNA fragmentation was not suppressed. These results suggested that NO production is an Al specific response in Andropogon, and is partially related to tolerance mechanism.

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The moss, Scopelophila cataractae (Mitt.) Broth., is a typical hyperaccumulator that is usually observed in copper-rich environments and which accumulates high concentrations of copper. However, many of the knowledge concerning copper response in S. cataractae remain unknown. In this study, we examined the effects of copper treatment on S. cataractae protonema culture cell. As a result, we found a copper-dependent response that switches between caulonema and gemma differentiation in S. cataractae. The effect was not observed by treatment with heavy metals other than copper, implying that the copper-specific regulatory mechanisms of cell differentiation might be related to the uneven distribution to the copper-rich environment in S. cataractae.

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When plants grow under low-P conditions, activities of external and internal acid phosphatase (APase) increase and contributes to P uptake and recycling, respectively. Proteaceae plants are well known as a low-P tolerant family, although the molecular mechanism has not been well understood. The aim of this study is to isolate and characterize APase homologs of Hakea laurina, which is a Proteaceae plant.
cDNAs for cluster roots of Hakea grown in low-P nutrient solution and a primer designed from conserved sequence among known APases were used for 3'-RACE. After sequencing for the clone library, 4 fragments for APase homologs were determined and designated as HlSAP1-4. These fragments were 40-76% similar to each other. HlSAP1 was identified most frequent from the library and shown the highest identity to an APase secreted from white lupin.
Localization of APase using a fluorogenic substrate was revealed that the higher APase activity was found in epidermis and root hairs of cluster roots. The activity of extracellular APase was dominant in cluster roots and contributed to P uptake from the rhizosphere.

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Oilseed rape (Brassica napus L) is one of the most important oil crops for food as well as for bio-fuel. The growth environment, especially availability of nutrient element plays a key role for the yield of oil production. In order to study the relation between the nutrient absorption manner and the quality of the oil, we measured the amount of each element absorbed during the developmental stage of B. napus (Westar), using ICP-AES and ion chromatography.
When hydro-culture was employed, the absorption amount of the major elements (N, P, S, Mg, Ca) per fresh weight was decreased during the development. The absorption of minor elements (Fe, Mn, Zn, B, Cu) per fresh weight showed characteristic curves around flowering stage. The element absorption manner was also measured for the plants grown in field.
To study the absorption manner of the elements more in detail, real-time RI imaging system, we developed, was used applying 32P labeled phosphate. The real-time uptake and distribution manner of the phosphate was visualized for both plants when grown in hydro-culture as well as in soil. This research was supported by Genesis Research Institute, Inc..

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Ionomic analysis is one of the powerful tools for screening of mutants related to elemental transporting systems. Previously, molybdenum transporter gene MOT1 has been identified in our laboratory by comparison of multiple mineral elements between two Arabidopsis accessions, Col-0 and Ler. To expand this concept to the wide-range of gene resources, we performed a large-scale ionomic analysis by using approximately 3000 lines of N-methyl-N-nitrosourea treated rice mutants (cv. Taichung-65). To minimize the vexatious complication for sample preparation such as acid digestion, xylem sap was collected from individual rice mutant lines grown in the 128-well tray and directly analyzed by ICP-MS as a primary screening. Both aerial parts and seeds were also collected in a single cultivation period for second and third screenings. This method allows effective screening by monitoring both the root-to-shoot and shoot-to-seed transport of essential and non-essential nutrients. Currently, we obtained several mutants affected by Cd content in rice seeds. Further investigations are also in progress for isolation of other mutants possessed altered elemental transporting systems.

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We have applied an approach based on activation tagging in de-differentiated cells to Arabidopsis for identification of genes governing salt response, being designated "salt tolerant callus (stc)" genes (JSPP meeting, 2007; 2009). We identified 18 potential mutants exhibiting tolerance to 150 to 200 mM NaCl, among which to stc5 attention has been paid. Three insertion loci of T-DNA for activation have been found in stc5 by TAIL-PCR. In these loci, gene for an ABC transporter assigned to AtWBC27 expressed approx. 2 times higher without salt stress or 4 times higher with the stress in stc5 calli in comparison with those in the wild-type as revealed by real-time PCR. Knock-out (KO) lines of AtWBC27 (hereafter WBC27) were more sensitive to NaCl, as well as to KCl, KNO₃ and LiCl. The KO lines were also sensitive to different levels of ABA, indicating that salt stress regulation by WBC27 may be linked to ABA signal. We have been developing over-expression lines to clarify the participation of WBC27 in salt tolerance. It is an intriguing question how WBC27 plays a role in a signal transduction network in salt stress and ABA response in Arabidopsis.

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We have previously reported finding of AtbHLH106 for salt tolerance by activation-tagging of Arabidopsis calli (JSPP meeting, 2009). We here show the evidences that AtbHLH106 (hereafter bHLH106) plays an important role not only in salt regulation but also in cold and ABA responses. An additional homozygous knock-out line newly generated by self-pollination, has shown sensitivity to salt stress to confirm our previous results. Knock-out lines were also sensitive to cold stress and different concentrations of ABA in a solid medium. Rescue lines have also been developed for bHLH106, showing the phenotype of enhanced tolerance to different levels of salt stress. Knock-out, rescue and over-expression lines were subjected to GeneChip analysis (Affymetrix), resulting in a number of genes up-regulated or down-regulated compared with wild-type with or without salt stress. Electrophoresis mobility shift assay (EMSA) has confirmed the interaction between bHLH106 synthesized in a cell-free system (CFS) and promoter sequences of at least five genes. These results provide proof for involvement of the transcription factor bHLH106 in a network of salt stress regulation in Arabidopsis.

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Recently we have reported that the transcription of 166 genes in ANAC078-overexpressing Arabidopsis plants (Ox-ANAC078) was up-regulated compared with the levels in wild-type plants under high-lihgt (HL) (Plant Cell Physiol., 2009). Interestingly, these genes included 13 members of the proteasome subunit genes. These findings suggest that the levels of proteasome are regulated by ANAC078. To explore the gene regulatory system of proteasome subunit genes via ANAC078 under stressful conditions, the transcript levels of proteasome subunit genes in response to HL stress (1200 μmol/m²/s) were analyzed. The transcript levels of several proteasome subunit genes in the Ox-ANAC078 plants were higher than those in the wild-type plants under normal conditions and HL stress. Furthermore, the levels of 26S and 20S proteasomes and chymotrypsin-like activity were highly increased in the Ox-ANAC078 plants compared with those in the wild-type plants. These data indicated that ANAC078 regulates the levels of 20S and 26S proteasomes under stressful conditions.

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The voltage-dependent anion channel (VDAC), a major outer mitochondrial membrane protein, is thought to play an important role in energy production and apoptotic cell death in mammalian systems. However, the function of plant VDACs is largely unknown. We reported that VDACs are involved in non-host pathogen resistance and also in Bax-mediated cell death using Nicotiana benthamiana as host plant. These data suggest that plant mitochondria are important for plant defense and cell death regulation. In this study, we performed the molecular and genetic characterization of all VDAC genes present in A. thaliana through sub-cellular localization of their genes products, histochemical monitoring of promoter-GUS activity, and functional analysis of T-DNA knockout mutants. Moreover, we examined the defense potential of mitochondrial vdac1 knockout plants against bacterial pathogens. Based on these results, we discuss the possible functions of individual Arabidopsis VDAC members in vegetative and reproductive growth, and in pathogen defense.

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Rice grassy stunt virus (RGSV) induces excess tillering. To elucidate the molecular basis of excess tillering in rice, the gene expression changes in rice by RGSV was analysed. The bud formation is controlled by LAS, RAX, REV, Ba1. Outgrowth of the bud is controlled by auxin, SL, and CK. Our results suggested excess tillering by RGSV infection is related to simultaneous suppression of genes involved in SL and GA synthesis. To examine the relationship between symptom severity and gene responses, the gene expression of rice under infection by the three RDV strains was done. Gene silencing-related genes were activated and the degree of activation was similar among the three RDV strains. Genes for hormone-regulated defense systems were also activated, and the degree of activation seemed to be correlated with the concentration of RDV in plants. Some development and morphogenesis processes were suppressed, but the degree of suppression was not correlated well with the RDV concentration. One host mutant fails to support RDV propagation rim1-1 has been isolated and function of RIM1 gene in rice has been characterized. Here we would like to discuss its contribution to host defense system.

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PpC24 is a gene that contains a region homologous to the nucleotide binding site (NBS) characteristic of TIR-NBS-LRR type of disease resistance genes (R genes), but kinase domain, not Toll and interleukin-1 receptor like (TIR) domain expected upstream of the NBS, is predicted in PpC (Akita and Valkonen 2002, J. Mol. Evol. 55:595-605). However, analysis of the genomic sequence of P. patens suggests that there could be a leucine rich repeat (LRR) downstream NBS, as in R proteins. The aim of this study was to obtain additional information about the transcripts produced by PpC24 and to characterize this gene. Analysis of cDNA sequences indicated that PpC24 could be read through until the downstream NBS. Alternative splicing of an intron located between NBS and LRR was also detected. However, there were several stop codons in all reading frames in this region, irrespective whether the intron was spliced or not. The P. patens genome contains at least four PpC24-like sequences, i.e. Kinase-NBS-LRR. We also generated knock-out (KO) mutants from which the kinase region of PpC24 was removed. The morphology of plantlets were not altered on the KO mutants.

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Among the plant-specific transcription factors, ethylene response factors (ERFs) conform one of the largest gene families in several plant species. ERFs are considered to have critical roles in the responses to the various environmental stresses and pathogens. So far, we revealed that the gene expression of a tobacco transcriptional repressor NtERF3 was up-regulated during the hypersensitive response (HR) induced by tobacco mosaic virus (TMV) infection in tobacco plants harboring the resistance gene N to TMV, and that transient overexpression of NtERF3 by the Agrobacterium-infiltration method induced HR-like cell death in tobacco. NtERF3 has an EAR repression motif in its C-terminal region. The cDNAs for some EAR-motif-containing ERFs were isolated from Arabidopsis, rice and tobacco, and many of the isolated genes induced HR-like cell death when overexpressed in tobacco. These results suggested that multiple ERF genes, which belonged to the same subgroup as NtERF3, might be involved in HR or cell death induction. Now, we are analyzing the effect of NtERF3 on TMV resistance by using the transgenic tobacco expressing a dominant negative type of NtERF3.

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Exogenous ABA application enhances rice susceptibility to M.grisea. We previously reported that ABA suppressed the expression of WRKY45 and OsNPR1, the two key regulatory factors in the rice SA signaling pathway. In this study, we analyzed the influences of reduced ABA levels and ABA signaling to defense responses around the penetration sites during early stages of M. grisea infection using transgenic rice plants that transiently express an ABA catabolic enzyme (OsABA8ox1) or a dominant-negative mutant form of ABA signaling component [OsABI(G-A)] under the control of DEX-inducible promoter.
ABA treatment of rice prior to, but not posterior to, M. grisea inoculation resulted the increase of lesion numbers. Similarly, DEX-induced expression of OsABA8ox1 or OsABI(G-A) prior to M. grisea inoculation reduced the lesion numbers but their expression after fungus inoculation did not. These results suggest that endogenous level of ABA in early stage of infection is important for the induction of defense responses. Microscopic observation revealed the suppression of hyphal invasion into host cells, enhancement of autofluorescence, and reduction of callose deposition at penetration sites.

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We clarified a novel regulatory mechanism of salicylic acid (SA) signaling by a model infection system using a combination of RCY1-overexpressed Arabidopsis and yellow strain of cucumber mosaic virus (CMV-Y). In this system, we can examine the RCY1-mediated extreme resistance (ER). The RCY1-overexpressed Arabidopsis were inoculated with CMV-Y by the carborundum method and kept for 10 min, 30 min, 1 h, 3 h, 6 h, 24 h, 36 h, 48 h and 72 h. Metabolites in the leaf samples were analyzed by ultra-performance liquid chromatography time-of-flight mass spectrometer (UPLC-TOF-MS). Principle component analysis of the metabolite data represented CMV-Y specific increase of several metabolites in comparison with the mock-induced metabolites. Significant changes of salicylic acid and its derivatives were observed in the CMV-Y inoculated leaves. Gene expression analysis showed CMV-Y specific repression of a salicylic acid glucosyltransferase gene UGT74F1 but not of salicylic acid biosynthesis and metabolizing genes. These results suggest that the suppression of SA glucosylation is a important regulatory step during RCY1-mediated resistance against CMV-Y.

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Salicylic acid (SA) is a key signal molecule in plant immunity and its synthesis is induced in response to pathogen infection. Chloroplasts are the site of SA biosynthesis; chloroplast–localized isochorismate synthase 1 (ICS1) is a key enzyme of SA biosynthesis in Arabidopsis. Furthermore it has been shown that flg22-induced SA synthesis and pathogen related 1 (PR1) gene expression are regulated by light. Recently, we demonstrated that chloroplast localized calcium binding protein CAS plays an important role in bacterially induced basal defense responses and Avr-induced hypersensitive cell death of Arabidopsis. In this study, we examined the role of CAS in flg22-induced synthesis of phytohormones. The results suggest that CAS is involved in SA biosynthesis.

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Pathogens deliver a number of effector proteins into plant cells to suppress PAMP (pathogen-associated molecular pattern)-triggered immunity (PTI). Resistant plants are able to recognize the effectors by the resistance (R) proteins and induce strong immune responses. AVR3a, an effector protein secreted from potato blight pathogen Phytophthora infestans, is translocated into plant cells and suppresses PTI induced by the recognition of INF1. However, its underlying mechanism is still unclear. Because the molecular function of AVR3a is not predictable from the amino acid sequences, we performed protein structural analysis. The NMR analysis revealed that AVR3a protein has a positively charged surface area, which is important for binding a phosphatidylinositol phosphate. AVR3a with a point mutation in the area was not able to suppress INF1-induced PTI, although it was still recognized by R3a, a potato R protein. These data suggest that the binding of a phosphatidylinositol phosphate plays an important role for the virulence functions of AVR3a. We will discuss the molecular relationship between the lipid-binding area and the virulence function.

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We are using a chemical biology approach to investigate Arabidopsis R-gene mediated disease resistance mechanism to the bacterial pathogen Pseudomonas syringae pv. tomato DC3000 avrRpm1. A high-throughput screening method for hypersensitive responses (HR) using suspension cells was established and seven compounds that enhanced HR cell death were identified from a library of 10,000 diverse chemicals. One of these HR potentiators, CB_6, induced expression of PR1 gene in Arabidopsis seedlings of both wild-type and sid2 mutant, indicating that CB_6 works as an analog of SA. CB_6 did not affect endogenous SA levels, suggesting that CB_6 is not involved in the positive feedback regulation of SA. Unlike SA, CB_6 did not suppress LOX2 gene expression induced by jasmonic acid (JA), suggesting that CB_6 does not possess antagonistic effect to JA signaling. To identify target protein of CB_6, we isolated 14 Arabidopsis sgi (SA agonist insensitive) mutants and their map-based cloning procedures are being carried out. We are also trying to employ two other different approaches for identification of CB_6 binding protein using biotinylated probe and yeast three-hybrid system.

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We employed chemical biology approach to understand salicylic acid (SA)-mediated disease resistance responses in plants. A high-throughput chemical screening that monitors hypersensitive responses (HR) of Arabidopsis suspension cells infected by Pseudomonas syringae pv. tomato DC3000 (Pst) avrRpm1, identified 7 chemicals named CB which enhance the HR cell death. Here we show the target identification of four of these CB compounds. CB_8 and CB_9 possess common structural skeleton and CB_11 and CB_12 share the same motif. These CBs conferred resistance to Arabidopsis plants against both virulent and avirulent Pst strains. They do not have SA-analog activity but enhanced defense responses during pathogen infection. Then, the chemical effects on SA-inactivating pathway were examined. We found that CB_8, CB_9, CB_11 and their derivatives inhibited the enzymatic activity of UGT74F1, a major SA glucosyltransferase (SAGT). The concentration ranges of them for SAGT inhibition coincided with those for HR enhancement. The knockout line of At2g43840 gene for UGT74F1 showed enhanced disease resistance, indicating the usefulness of SAGT as a target for plant protection technologies.

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The chloroplastic electron transport chain (ETC) plays an important role in the process of photosynthesis in the chloroplasts. Plastoquinone (PQ) is able to function as an electron carrier between photosystem II and cyctochrom b6/f complex in the chloroplastic ETC. It is reported that PQs involved leaf occurs in other chloroplast compartments different from thylakoid membranes. Although PQ redox depends on the light conditions, the redox state of non-carrier PQ independents on the states of chloroplastic ETC. We redesigned high performance liquid chromatography-based methods of determination of photoactive PQ as functional light response quinones in intact leaves. The range of oxidized states was from 35.2% to 77.7% in total PQs, and photoactive PQs was 42.5%. In intact leaf, non-oxidized and non-reduced forms showed 22.3% and 35.2%, respectively. Kruk et al. determined photoactive PQ in Arabidopsis leaves, However, the oxidized PQ decreased from 77.7% to 64.5% in spinach leaf segments without methyl viologen (MV). Therefore, non-photoactive PQ was overestimated in Arabidopsis leaves, because Kruk method lacked electron flow via stromal reductants.

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The uptake hydrogenase mutant (ΔHup) cells of heterocystous cyanobacterium Nostoc sp. PCC 7422 photobiologically accumulated H₂ to about 30% in the presence of O₂ evolved. N₂ gas concentration of 0.5-1% in Ar was optimal for photobiological H₂ accumulation in both the heterocyst induction (0-2 days after combined-nitrogen step-down) and the hydrogen production (thereafter) phases. We studied the effects of CO₂ concentration (0.03%, 1%, 5% CO₂) in 0.5% N₂ in Ar, and found that 5% CO₂ was optimal for H₂ accumulation in both phases. A culture could produce hydrogen for more than 60 days without changing the culture medium and with renewing the gas phase about once a week. H₂ accumulation experiments are routinely carried out using Fernbach flasks capped with butyl rubber stoppers, but the inner pressure rises with time by photobiological production of H₂ and O₂, and by absorption/emission of CO₂. The use of transparent gas-barrier Besela plastic bags (Kureha Co.) allows us to evaluate photobiological H₂ accumulation under the ambient pressure. We have produced gas-sampling devices for collecting sample gases for studies of the time course H₂ accumulation.

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Respiratory and photosynthetic electron transports share plastoquinone (PQ) pool in cyanobacteria, and PQ pool is either reduced by NADH dehydrogenase (NDH) complex or by photosystem II. Here we analyzed the photosynthetic characteristics of the disruption mutant of ndhF1, a gene encoding a subunit of NDH complex, in order to reveal the effect of respiratory electron transport on photosynthesis. The effective quantum yield of photosynthetic electron transport in the ndhF1 disruptant of Synechocystis sp. PCC 6803 is higher than that in the wild type, when measured by PAM fluorometer under growth light condition (20μmol/m²/s). However, electron transfer rates determined by oxygen electrode under saturating light showed little difference between the two strains. Chlorophyll emission spectra determined at 77 K revealed that the wild type was in state 2 in the dark while the ndhF1 mutant was fixed in state 1, which was canceled by the addition of KCN, an inhibitor of the terminal oxidase in respiratory chain. These results indicate that suppressed state transition due to more oxidized PQ pool is the cause of high photosynthetic quantum yield in the ndhF1 disruption mutant.

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Unicellular diazotrophic cyanobacteria synthesize insoluble α-glucans, instead of glycogen. In one of these species, Cyanobacterium sp. NBRC 102756, the cellular content of polysaccharide was increased from 15% to 40% per dry weight, under Fe deficiency. Effect of Fe nutrition and illumination conditions on the polysaccharide production in this strain was investigated. Within 24 h after dilution, the rate of polysaccharide synthesis in Fe-replete (0.47 ppm) medium was much lower than the growth rate, resulting in the decrease in the polysaccharide per cell. Under Fe-deplete conditions, on the other hand, a dramatic increase of polysaccharide was observed within 24 h of growth. To minimize the energy requirement for biomass production, we examined polysaccharide accumulation in different light conditions. In Fe-sufficient medium, the amount of polysaccharide after 120 h of the culture was comparable whether the cells were grown in continuous light or in 18 h L / 6 h D conditions. In Fe-deficient conditions, in contrast, the amount of polysaccharide was progressively decreased with increasing dark period. No significant turnover of polysaccharide was observed in the dark.

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In chloroplasts, polyunsaturated fatty acids in the thylakoid membrane lipids are readily oxidized by reactive oxygen species, which are produced primarily within or on the membrane. Lipid peroxides are then degraded to toxic reactive aldehydes (RAL). We have provided evidence for the toxicity of RAL in plants by showing (i) the formation of RAL in illuminated leaves, (ii) light- and methylviologen-tolerance of the transgenic plants that have RAL-scavenging capacity and (iii) high sensitivity of stromal thiol enzymes to RAL. In this study, we compared the toxicity to photosynthesis of several RAL species. When stroma fraction was incubated with various RAL, phosphoribulokinase, glyceraldehydes-3-phosphate dehydrogenase and dehydroascorbate reductase were most sensitive. Among tested species, 4-oxo-(E)-2-hexenal (OHE) inactivated these target enzymes as strong as did acrolein, the most toxic RAL known. OHE was produced in thylakoids from (Z)-3-hexenal, a wounding-responsive aldehyde, and was scavenged by glutathione very efficiently. These results indicate potential toxicity of OHE to photosynthesis in wounded leaves.

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Photosynthesis consists of two types of electron flow, linear (LET) and PSI cyclic electron transport (CET). CET is involved in both ATP synthesis and photoprotection. In higher plants, CET consists of two pathways, NDH-dependent and PGR5-dependent electron transport. Antimycin A (AA) inhibits mitochondrial electron transport and is also used as an inhibitor of PGR5-dependent CET. However, high concentration of AA is needed, causing several non-specific inhibitory effects. Furthermore, AA may not bind to the quinone-binding site of FQR, a putative enzyme involved in PGR5-dependent CET. In this study, we examined effects of a series of substituted phenols on CET activity in the wild type, and crr2-2 and pgr5 mutants, defective in NDH- and PGR5-deepndent CET, respectively. We found candidates which inhibit PGR5-dependent CET more efficiently than AA.

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