Reed (Phragmites communis) exhibits tolerance to salt stress, and keeps low Na concentration in the shoot under saline soil. It is thought that Reed has the mechanism to retranslocate Na to roots via the shoot-base ( basal part of the shoot ). In this study we measured the amount of ion transport through the shoot-base. Reed transported only 4.3% of Na absorbed by roots via the shoot-base, but rice(Oryza sativa L.cv.Nipponbare) transported 52.8%. These effects disappeared by the shoot-base hot girdling ( 3s with hot air ). On the other hand, the transport of KCl and Na2SO4 through the shoot-base was almost the same between reed and rice. These results suggest that Na specific transporter or channel may function in the shoot-base of reed.

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The pattern of iron deficiency symptoms is different between barley and rice. We examined whether this difference depends on iron acquisition ability due to differential secretion of mugineic acids from roots. Barley and rice, respectively, had an own iron distribution characteristic in plant. In barley the younger and older leaves had similar iron content, but in rice the older leaves had higher iron content than the younger leaves. The iron distribution characteristic was not change when both plants were grown in a same container to compensate mugineic acids secretion. ⁵⁹Fe pulse label experiment also revealed that rice absorbed a greater amount of iron than barley and that the iron distribution characteristic was not changed. Water soluble iron in leaves was also determined to show biologically available iron. It was considered that differential iron deficiency symptom depended not only on iron acquisition ability but on iron availability and distribution in plant.

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Cadmium (Cd) is a toxic heavy metal. Accumulation of Cd in the soil of cultivated field causes serious damage for crops. In order to eliminate Cd effectively from contaminated soil by plants, Cd should be transported to shoot organs. Cd is supposed to be transported to shoot organs via xylem. In this study, we investigated responses of proteins in the xylem exudate from Brassica napus L. to Cd treatment.
Plants were grown hydroponically in a greenhouse and were treated with Cd (10µM, 30µM). Xylem exudate was collected after cutting stems approximately 2 cm above the root. Proteins in the exudate were analyzed.
Protein concentration in the exudate was about 13µg/mL. The SDS-PAGE analysis demonstrates that the protein composition in these exudates was distinctly different from that in the extracts from leaves, roots, stems and in the phloem exudates. Proteins of 20kDa and 45kDa increased and 50kDa-protein decreased by Cd treatment.

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The Positron Emitting Tracer Imaging System (PETIS) is a non-invasive tool to capture movies of distribution of positron-emitting radioactive tracers in an intact plant body. This technique has been employed in studies of transportation of nutrients in living plants. In this study, we focused on cadmium which contamination of crops is an important problem in the world.
Rice (Oryza sativa L.) and oilseed rape (Brassica napus L.) plants were grown for 4 to 5 weeks and 2 to 4 weeks respectively, and used for the experiments. ¹⁰⁷Cd (half-life: 6.5 hours) was prepared as the radiotracer, with ion beam bombardment and chemical purification. We fed the tracer into hydroponic culture and carried out the imaging of the base of root and the aerial part of the plants for 11 to 37 hours. In this presentation, we show the first real-time images of cadmium transport in intact plants with PETIS.

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The photosynthates required for vegetative growth are transported mainly to the root and stem, while those required for reproductive growth are transported to the fruits and seeds. However, few studies have analyzed the transportation and distribution from captured moving image.
To analyze the behavior of photosynthates in an extending stem in real time, we used the positron-emitting tracer imaging system (PETIS). Hemp was employed because it grows very rapidly. With a mathematical analysis, we estimated the unloading rates of the tracer in the respective selected regions of interest in the obtained image. After supplying the largest expanded leaf with ¹¹CO₂, the ¹¹C-tracer was preferentially accumulated in the growing internode just below the leaf and to the shoot apex. When ¹¹CO₂ was supplied to a lower leaf, the ¹¹C-tracer was transported towards the root and distributed uniformly in the lower internodes along the pathway.

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The OsNRT2 gene has been isolated from rice as a putative NO₃^- transporter in the high-affinity nitrate transport system, which involves nitrate uptake at low external nitrate concentrations. In this study, we show the enhancement of nitrate uptake in transgenic rice plants overexpressing OsNRT2. The OsNRT2 was fused to a modified CaMV 35S promoter, and transformed into Oryza sativa cv. Yume-Ohmi. Five transgenic lines that stably overexpressed OsNRT2 were selected based on the results of RT-PCR from 100 drug resistant lines, and tested for NO₃^- uptake. The nitrate-starved seedlings of rice were incubated in a solution containing 200 μM KNO₃ for 4 to 16 hours, and the remaining NO₃^- were measured by using ion chromatography, revealing that the transgenic plants have significantly higher NO₃^- uptake than wild type. Similar results were also obtained by using the positron emitting tracer imaging system.

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Under phosphate deficiency, phosphate re-translocation from older organs to younger organs is one of the important adaptation mechanisms. In the present study, to understand the molecular mechanisms of phosphate re-translocation, we measured phosphate distribution in shoot and searched potential genes involving phosphate re-translocation in Arabidopsis.
First, we measured content of phosphate in each leaf under various phosphate levels in culture media. Second, re-translocation of ³²P was observed by an imaging plate system. During these studies, plants were cultured in a newly developed hydroponic culture system. We determined source and sink leaves based on the distribution analysis. Then, we performed microarray analysis of m-RNA isolated from source and sink leaves. We discuss the possible roles of genes which changed transcriptional levels between source and sink leaves in phosphate re-translocation.

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Molybdenum (Mo) is an essential trace element of many organisms including plants, and Mo deficiency and excess are agricultural problems. In plants, Mo cofactor (Moco) is an essential cofactor for several enzymes including nitrate reductase. Decrease of Moco amount under low Mo condition was reported, suggesting that Mo deficiency induce unusual nitrogen metabolism. Despite such importance, molecular mechanisms of Mo transport have not been understood. In the process of the analysis of an Arabidopsis thaliana mutant bor1-1, we noticed that Mo contents in leaves were several fold different between Col-0 and Ler. Genetic analysis indicated that the difference was determined by a single locus. The corresponding was identified as a putative membrane protein homologous to several known transporters. Activity of the membrane protein to transport Mo, phenotype of the T-DNA insertion mutants and expression patterns in plants will be presented.

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Boron is an essential element for plant growth with a very narrow window between deficiency and toxicity. Arabidopsis BOR1 is a boron exporter for xylem loading. Accumulation of BOR1 protein is regulated by boron availability at translational/post-translational levels. In this study, localization of BOR1-GFP in root tip cells of 35S:BOR1-GFP transgenic plants were followed after change in the medium B concentration. Under boron limitation, BOR1-GFP localized mainly to plasma membrane. After addition of a high concentration of boron, BOR1-GFP moved into dot-like structures in the cytoplasm prior to disappearance. Studies using various inhibitors suggested BOR1 transport via endosome and degradation in vacuole. These results suggest that endocytosis and degradation of BOR1 is regulated by B availability, to avoid accumulation of toxic levels of B in shoots under high B supply, while protecting the shoot from B deficiency under B limitation.

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Pumpkin Phloem Protein 16-1 (CmPP16-1) has been characterized as phloem-mobile single-stranded RNA-binding protein. In the whole phloem protein context, CmPP16-1 moves long-distance to shoot and root. We have recently found that root-ward movement of CmPP16-1 became inefficient when CmPP16-1 was purified, suggesting the presence of pumpkin phloem factors that is involved in determining movement destination. To understand how the long-distance movement of CmPP16-1 is regulated, we searched for the interacting proteins of CmPP16-1 from pumpkin phloem sap. Gel-filtration chromatography and co-immunoprecipitation, using anti-CmPP16-1 antibody, demonstrated that CmPP16-1 formed a complex with other phloem sap proteins. Root-ward movement of CmPP16-1 was restored in the presence of these interacting proteins, indicating that these proteins are involved in the movement regulation. This work was partly supported by Grant-in-Aid for Scientific Research in Priority Areas from the Ministry of Education, Culture, Sports, Science, and Technology of Japan (grant No. 15031227) to K.A

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We identified a 23 kDa protein (P23k) in barley scutellum, which is apparently associated with starch degradation in the endosperm during germination. The function of the protein is still unknown. Here we reported that this protein is barley-specific and coordinately regulated by the supply of sugars. We investigated the expression and localization of the protein at various developmental stages (seed development, germination, and vegetative stages),using Northern hybridization, Western blotting, in situ hybridization and immunocytochemistry. The P23k protein was specifically expressed in scutellum during germination. It was also expressed when leaf blades were fully developed and acted as a source tissues. Finally, it was specifically expressed in the nucellar projection, the vascular bundles and the aluerone cells during seed development (sink tissue). As these tissues are all involved in the supply of nutrients , we believe that the P23k is related to the translocation of nutrient.

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We have reported in the previous meeting that the application of recombinant precursor proteins on protein import into chloroplasts. Currently, we are developing the methods to visualize protein import into chloroplasts in vitro. We have obtained the encouraging results if recombinant precursor proteins were modified with fluorescent compounds. In brief, after import reaction, chloroplasts are solubilized with the SDS sample buffer, followed by the SDS-PAGE. The gel in wet condition was directly loaded on the fluorescent imaging-analyzer and scanned. In this way, the processing of recombinant protein, resulted by the precursor to be imported into chloroplasts, was visualized. The rate of import was also able to be analyzed. We will present these results and our current efforts at the meeting.

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The biosynthesis of iron-sulfur clusters is a highly regulated process involving several proteins. Among them, so-called scaffold proteins play pivotal roles in both the assembly and delivery of iron-sulfur clusters. Here, we report the characterizations of chloroplast-localized CnfU proteins from Arabidopsis whose cyanobacterial homologue was proposed to serve as a molecular scaffold. Deficient mutants of AtCnfU-V, one of three chloroplastic NifU-like protein homologues, exhibited a dwarf phenotype, impaired protein levels of both ferredoxin and photosystem I, and a decrease in the in vitro insertion activity of iron-sulfur cluster. We propose that AtCnfU has an important function as a molecular scaffold for iron-sulfur cluster biosynthesis and therefore is required for the biogenesis of ferredoxin and photosystem I. In addition to the results above, we would like to present the latest data of other possible protein factors involved in iron-sulfur cluster biosynthesis in chloroplasts.

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Proteins synthesized in the endoplasmic reticulum (ER) are correctly folded before translocation. If protein folding is incorrect, genes for the ER-resident chaperones such as BiP are induced. This phenomenon is known as the ER stress response. Studying molecular mechanism of the ER stress response in Arabidopsis, we identified a bZIP transcription factor AtbZIP60 induced by tunicamycin treatment. AtbZIP60 has a putative transmembrane domain in its C-terminal region. A truncated form without C-terminal region (AtbZIP60ΔC) fused with GFP localized to the nucleus. In addition, overexpression of AtbZIP60ΔC activated BiP promoters in a transient assay although full length did not, suggesting proteolysis triggers activation of the transcription activity. In a T-DNA tagged mutant of AtbZIP60, induction of one BiP gene was repressed. These results indicated that AtbZIP60 regulates the ER stress response in a manner specific to plants.

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The ZIG/VTI11 is Qb-SNARE involved in the vesicle transport from TGN to PVC/vacuole in Arabidopsis. The zigzag (zig) mutant is the VTI11 null allele and exhibit abnormal garavitropism and morphogenesis. To elucidate the molecular mechanism and physiological function of post-Golgi vesicle transport in higher plant, we have isolated several suppressor mutants of zig of Arabidopsis. Our previous study demonstrated that the dominant mutation zig suppressor1 (zip1) almost completely suppress the defects of zig-1.The recessive mutation zip2 and SC2-1 partially supress the defects of zig-1 mutant both in garavitropism and in morphogenesis. The position of ZIP2 and SC2-1 gene were mapped apploximately 10cM and apploximately 80cM of chromosome 1, respectively. These loci are different from the position of ZIP1/VTI12 gene. These genes are also expected to be involved in post-Golgi vesicle transport.

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SNAREs (*Soluble N-ethyl-maleimide sensitive factor attachment protein receptors*) play a critical role in membrane fusion process of vesicular transport system. Genome studies on Arabisopsis thaliana pointed out that there are 54 genes (much more than yeast and mammalian) which encode SNARE proteins. The abundance of SNARE genes indicates the complexity of endomembrane trafficking within the plant cell. SYP7-group containing three genes, SYP71, SYP72 and SYP73, has no homologues in mammalians and yeast, suggesting that SYP7 group has plant unique functions. We already reported that SYP71/72/73 (SYP7-group) was localized on the ER by transient expression system. However, localization studies of GFP-fused proteins by*using BY2 Cell, showed that the proteins were not only localized on the ER, but also on the plasma membrane. Furthermore, we are analyzing membrane trafficking in plant involving SYP-7s proteins by generating transgenic plant expressing GFP-fused SYP7-group proteins and performing promoter analysis by using GUS reporter.

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Myrosinases are β-glucosidases that are found mainly in the order Capparales. Myrosinases hydrolyze glucosinolates to produce the toxic compounds for the pests. The abnormal accumulation of myrosinases was found in Arabidopsis mutants of AtVAM3, which is a syntaxin homolog to yeast VAM3 involved in the vacuolar assembly. In this study, we characterized the abnormal accumulation of myrosinases in two AtVAM3 mutants; one expresses the insertion form of AtVAM3 with an additional peptide and another is a knockout mutant of the AtVAM3 gene. The organ specific expression and the localization to the myrosin cells of the myrosinases in these mutants were the same as in wild-type. Interestingly, the numbers of the myrosin cells were markedly increased in the mutants. The abnormal accumulation of myrosinases in the mutants might be caused by the excessive occurrence of the myrosin cells. These results suggest that AtVAM3 is involved in differentiation of myrosin cells.

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The preprophase band (PPB) of plants is a ring-like band of microtubules that underlies the plasma membrane in prophase. Although the PPB disappears in prometaphase, the newly formed cell plate attaches to the former PPB site at cytokinesis. The PPB plays an important role in determination of division site in a plant cell. Clathrin-coated vesicles (CCV) as well as structurally related non-coated vesicles (NCV) were frequently observed at the PPB. To understand the role of the vesicles in the PPB development, we quantitatively analyzed effects of cytochalasin D on the distribution and the morphology of these vesicles in high-pressure frozen onion epidermal cells by electron tomography. Our data suggest that the NCVs fuse each other and actin cytoskeleton is involved in formation of the CCVs and the fusion of the NCVs. These result support the idea that the NCVs are involved in the endocytic pathway.

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The vesicle transport systems in eukaryotes were found as secretion pathway starts from endoplasmic reticulum (ER). In transport vesicles, COPI (coat protein I) and COPII (coat protein II) work in the transport system between ER and Golgi apparatus. COPII coated vesicle that consists of the small GTP-binding protein, Sar1p and two coat protein complexes, sec23p/24p complex and sec13p/31p complex, are working in the transportation from ER to golgi apparatus. In this research, we examine the function of COPII components in development of Arabidopsis thaliana.
In these COPII components, we carry out the analysis of homologs of sec31p (ATSEC31), sec13p (ATSEC13), sec23p (ATSEC23) and sec24p (ATSEC24) that each ATSEC31, ATSEC13, ATSEC23 and ATSEC24. There are two homologs for each ATSEC31 and ATSEC13, are seven homologs for ATSEC23 and are three homologs for ATSEC24. In this research, we carry out the analysis of expression and localization for these homologs.

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The COPII vesicle, which is involved in the ER-to-Golgi transport, is generated by the assembly of Sar1p, Sec23/24p and Sec13/31p. Genes for these proteins are known to be essential in yeast. In plants, several genes for each COPII protein are present in the genome. To understand the role of this gene amplification, we chose Sec31 as a model because only two genes, At3g63460 (A) and At1g18830 (B) are found in the Arabidopsis genome. We made AaBb genotype plant and the progenies of this plant were analyzed. In the sheath of selfed AaBb plants, no dead or undeveloped seeds were observed. No aabb and aaBb plants were found in 110 progenines analyzed. In addition, no decrease in germination rate was observed. Therefore, the function of Sec31 in Arabidopsis essential from the early development, possibly formation of pollen or egg cells or germination of pollen

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We have been analyzing the function and localization of tobacco prolyl hydroxylase (PH) in tobacco cells. This type II membrane protein has a basic amino acid cluster in its N-terminal cytosolic region. We have shown that a mutation that contain the exchange of all the basic amino acids to neutral amino acids prevent the export from the ER. We introduced one or two amino acid substitutions in this region of a GFP-fused PH and these proteins were expressed in tobacco BY-2 cells. We found that two basic amino acids separated with one amino acid was sufficient for Golgi localization. We next analyzed the distances of basic amino acids and found that basic residues separated with 0, 1 or 2 amino acids were sufficient for the Golgi localization. Using this information we will discuss possible localizations of other type II membrane proteins.

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We have produced antibodies against various membrane proteins in tobacco BY-2 cells to recognize uncharacterized organelle in the secretory system in plant cells. Possible membrane proteins were chosen using the EST information of tobacco and homologies of the EST encoding proteins with yeast and mammalian's protein markers for secretory organelles. Using recombinant proteins made from the EST sequences or synthetic peptides based on the sequence of proteins encoded by the ESTs, we made rabbit polyclonal antibodies purified by affinity chromatography. Using these antibodies, we analyzed localizations of these possible markers using density-gradient separated membrane fractions from BY-2 cells followed by immunoblotting. We also employed immunocytochemical methods at LM and EM level to localize the antigen in situ. Based on these analyses, we found that some proteins will be localized in organelles that had not characterized at the morphological level.

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ER body is a compartment that is derived from endoplasmic reticulum in Arabidopsis thaliana. They are distributed in the epidermal cells of whole seedlings, while rosset leaves have no ER bodies. However, ER bodies are induced in rosset leaves by wounded stress and/or treatment with methyl jasmonate. This indicates that the ER body plays a role in the defense system of plants (Matsushima et al.,2003). Fluorescent ER bodies were observed in transgenic plants of Arabidopsis expressing GFP fused with an ER retention signal (GFP-h). To know the mechanism of ER body formation, we raised several kinds of morphological ER body mutants by EMS treatment of the epidermal cells of cotyledons from GFP-h seeds. We will report the morphological features of these mutants obtained through observations by conforcal laser scan microscope and transmission electron microscope. Based on these, we would like to discuss the mechanism of the formation of ER bodies.

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Rice endosperm tissues have storage proteins, which are accumulated in two types of protein bodies (PBs). Prolamin is deposited in PB-I, glutelin and globulin are stored in PB-II. To clarify the transport pathway for storage proteins, we studied the electron microscopic analysis of rice developing endosperm tissues.
We found novel vesicles derived from rough endoplasmic reticulum (rER). The structures of novel vesicles were different from that of the ER-derived PB- I and the Golgi-derived dense vesicles. They had an electron-dense core of PB-II type storage proteins surrounded by an electron translucent layer. Immunocytochemical analysis revealed that the novel vesicles had glutelin and globulin in the electro dense core. In addition, BiP, an ER-resident molecular chaperone, was localized in the novel vesicles, but also in PB-II. These results suggest that the novel vesicles mediate transport of glutelin and globulin directly from the ER to PB-II in rice endosperm.

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Autophagy is an intracellular process for vacuolar degradation of cytoplasmic components. A recent genome-wide search revealed significant conservation among autophagy genes (ATGs) in yeasts and plants. It has not been proved, however, that Arabidopsis ATG genes are required for plant autophagy. To evaluate this requirement, we examined the ubiquitination-like Atg8 lipidation system. We generated transgenic Arabidopsis expressing a GFP-ATG8 fusion protein and established a system monitoring autophagy in whole plants. In wild-type plants, GFP-ATG8s were observed as ring shapes in the cytoplasm and were delivered to vacuolar lumens under nitrogen-starved conditions. By contrast, in T-DNA-insertion mutants of the ATGs (atgs), autophagosomes were not observed and the GFP-ATG8s were not delivered to the vacuole. The autophagy defective mutants exhibited early senescence phenotype and a reduction in the growth rate of roots. The physiological roles of plant autophagy will be discussed.

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Clathrin has been suggested to be involved in removal of an excessive membrane in cell plate formation. In this study, we analyzed the role of clathrin in the formation of cell plate using tobacco cultured cell, BY-2. Immunocytochemical study using an antibody against clathrin revealed that this protein was distributed in cell plate. To further analysis the role of clathrin in cell plate formation, C-terminus of clathrin heavy chain, in which light chain binding domain and trimeraization domain are contained, was expressed in BY-2 cells. When the cell cycle of the transformant was syncronized, about half of the cell became multinucleate, and morphology and organization of cell plate and microtuble bundles in phragmoplast were aberrant.This result suggested that clathrin is involved in the formation of cell plate.

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The non-linear differential equation that describes the coupling between water transport and solute transport, that is dependent on the standing osmosis in narrow long canal systems, was proposed in plants (Katou and Furumoto 1986). In the previous work, we solved the equation analytically and obtained static characteristics of water absorption, proved the contribution of cell wall to water absorption by providing the apoplast canal system surrounding xylem vessels, and estimated roughly the structure of the apoplast canal (Mizuno & Mizuno 2002). We also solved analytically the non-linear partial differential equations that described the system response to a change of solute influx and obtained dynamic characteristics of water absorption to solute absorption (Mizuno & Mizuno 2003).
In the present work, we solved the simultaneous equations which described static and dynamic characteristics of water absorption respectively, and obtained more detailed estimation of the structure of the apoplast canal.

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We isolated 6 different cDNAs (VHA-c1 to VHA-c6) coding for the proteolipid subunit of Acetabularia acetabulum (A. a) V-ATPase. By heterologous expression in yeast, the 6 translation products were revealed to function as Vma3p, but not as Vma11p. By using specific antibodies, the two isoforms (VHA-c2/VHA-c4) were detected in a microsomal fraction prepared from stalks of A. a cells before cap formation (Adult). In whorl of hairs of Adult cells, VHA-c2 was detected. In caps of A. a cells after cap formation (Reproductive), VHA-c1, -c2 and -c4 were detected. V-PPase was detected in stalks, whorl of hairs and caps of Reproductive cells. In the case of stalks and whorl of hairs of Reproductive cells, similar results were obtained as Adult cells. VHA-c1, -c2 and -c4 were immunohistochemically revealed to be localized in vacuolar membrane of caps.

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Bacteria and plants have some K⁺ transporters and K⁺ channels, which play important role in the adaptation of cells to hyperosmotic conditions. The ktrB gene isolated from Synechocystis PCC6803 was similar to Na⁺-K⁺ transporters (HKT/Ktr/Trk). Although wild-type of Synechocystis had K⁺ transport, the mutant disrupted ktrB gene did not show K⁺ transport. Moreover the mutant could not grow in the high salinity medium. Three Synechocystis genes, ktrA, ktrB and ktrE, were all required for K⁺ uptake. When ktrABE was expressed in K⁺ uptake deficient E.coli LB2003, this strain could grow under low K⁺ condition, and was detected Na⁺-dependent K⁺ uptake. Measurement of KtrABE transport in Synechocystis showed enhancement of K⁺ uptake in response to high osmolality challenge when Na⁺ was present in the medium, and that KtrABE prevented hyperosmotically-induced K⁺ loss from the cells. These data show that KtrABE functions as an essential mechanism in the adjustment to hyperosmolality stress.

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There are several types of K⁺ channel in organisms and they play various roles in cells. In order to investigate physiological meanings of K⁺ channel in rice, we studied expression and function of putative outwardly rectifying K⁺ channel gene cloned from 2 varieties of rice, Hitomebore and Hidekomochi, Japonica cultivar. Phylogenetic analysis of plant K⁺ channels supported ROK in the outwardly rectifying groups. Molecular size of ROK expressed in yeast cells is about 33kD by gel shift analysis. By using real time PCR detection system, specific expression analysis of ROK in anthers revealed higher in the stage at 10 days than 5 days before the heading. Stage of anther at 10 days before the heading is sensitive to cool temperature in summer.
Study of fusion protein of ROK-GFP revealed expression change in the organella in suspension-cultured cells of Arabidopsis thaliana, which supported ROK is outwardly rectifying K⁺ across the plasmamembranes.

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The reddish-purple buds of the wild-type Ipomoea nil change into blue open-flowers. The color change is caused by vacuolar alkalization in the epidermal cells of the flowers. Although InNHX1 for Na⁺/H⁺ antiporter was thought to be a major gene responsible for the vacuolar alkalization, a mutant deficient in InNHX1 still shows partial increase the vacuolar pH and its reddish-purple buds become purple open flowers. We identified a novel gene for Na⁺/H⁺ antiporter, InNHX2, the deduced amino acid sequence of which showed 71% identity to that of InNHX1. Spatial and temporal expression of these genes and their response to NaCl treatment indicate that InNHX1 appears to be evolved specifically for blue coloration during flower-openning whereas InNHX2 is likely to perform dual functions: promote partial vacuolar alkalization in the petals and to confer salt tolerance on the plant. We will discuss the roles of InNHX1 and InNHX2 for blue flower coloration.

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Petals color of morning glory, Ipomoea tricolor cv. Heavenly blue, changes from reddish-purple to blue during blooming. The color change is caused by an unusual vacuolar pH increase from 6.6 to 7.7 of the adaxicial and abaxicial colored cells. To clarify the alkalization of epidermal vacuoles we focused on vacuolar membrane proteins. We isolated colored protoplasts from buds and full-opened petals and purified vacuolar membranes. The membranes contained V-ATPase, V-PPase and NHX1, which were immunochemically detected, with relatively high transport activity. NHX1 could be detected only in the vacuolar membranes of full-opened petals. These results suggest that the vacuolar pH increase of morning glory petals is due to an active transport of Na⁺ and/or K⁺ from cytosol into vacuoles through a sodium driven NXH1, which is supported by V-PPase and V-ATPase. This systematic ion transport brought alkalization of vacuolar pH to resulted sky-blue flower petals.

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We have reported that a rice vacuolar Na⁺/H⁺ antiporter, OsNHX1, play important roles in the tolerance of rice to salt and iron-deficiency stresses. We examined the expression pattern of OsNHX1 using transgenic rice carrying β-glucuronidase (GUS) gene under the control of the OsNHX1 promoter region. GUS activity was detected mainly in central cylinder and the bottom of lateral roots in rice roots, and in guard cells and vascular bundle in leaves. We isolated two rice osnhx1 mutants from Tos17 insertional mutants. No significant differences were observed in the growth between wild-type and mutant cells and plants under salt and osmotic stresses. In addition, we found no significant differences in the main elements between wild-type and mutant cells, and in Na⁺ and K⁺ contents between wild-type and mutant plants. We will show the expression of other NHX-type genes in osnhx1 mutants and discuss the physiological roles of OsNHX1 in rice.

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Cation/H⁺ exchanger (CAX) is an active transporter, which transports divalent cations using a H+ gradient. Previously, we cloned CAX from rice, OsCAX1a, and demonstrated its Ca²⁺/H⁺ exchange activity in yeast. The present study aims to elucidate the physiological function of OsCAX1a. First, we examined the response of OsCAX1a to several ions at mRNA and protein levels. Levels of mRNA and protein of OsCAX1a were increased by Ca²⁺. Second, subcellular localization was determined by expressing GFP-OsCAX1a. Green fluorescence was observed in vacuolar membranes in transiently expressed onion cells. OsCAX1a was immunochemically detected in vacuolar membranes by subcellular fractionation. Third, promoter-GUS analysis revealed that OsCAX1a was expressed in vascular bundles, stomal cells, trichomes, steles, flowers, embryo, and aleurone layers. These results suggest that OsCAX1a transports excess Ca²⁺ into vacuoles in rice cells, which suffer high Ca²⁺ concentrations, and is involved in Ca²⁺ homeostasis in various tissues.

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Characean internodal cells generate receptor potential in response to mechanical stimuli. The mechano-peception was studied in characeaean internodal cells. In this study, cells were stimulated by compressing them for 10 s with an acrylic stick. The responses of internodal cells to the compression was as follows. (1) The cell generated receptor potential at the both moments of compression and decompression. (2) The ΔE_m was significantly larger at the decompression than at the compression. Aequrorin studies revealed that the increase in [Ca²⁺]_c (Δ[Ca²⁺]_c) took place at both moments. The amplitude of Δ[Ca²⁺]_c was smaller at the compression than at the decompression, as was the case in ΔE_m. We discussed the ionic mechanism of mechano-perception under long term compression in relation with mechanosensitive activation of Ca²⁺ channels at the plasma membrane.

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Effects of rare earth elements (atomic numbers, 39, 57-60, 62-71; Y, La, Ce, Pr, Nd, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm, Yb and Lu) and Al added as chloride salts, on Ca²⁺ influx induced by two inducers of Ca²⁺ influx via distinct calcium channels, namely hypoosmotic shock and salicylic acid, were examined in a suspension-cultured transgenic cell line of BY-2 tobacco (Nicotiana tabacum) cells expressing a Ca²⁺-sensitive luminescent protein, aequorin. Most metals showed inhibitory effect against Ca²⁺ influx. Especially Nd, Sm, Eu, Gd and Tb showed the most robust inhibitory action. Interestingly, selective inhibition of the salicylate-induced Ca²⁺ influx by Pr at a low dose (ca. 0.1 mM) and by Al at high doses (5 - 10 mM) was observed. Lastly, the effects of above putative inhibitors were examined in the cell lines with modified TPC1 type channel expression.

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In the last JSPP meeting, we have shown that the Arabidopsis AtMID1A gene product has a Ca²⁺-permeable, stretch-activated channel activity. The data have suggested that AtMid1A acts as a sensor of mechanical stimuli and participates in generating a Ca²⁺ signal. We discovered AtMID1B, the homolog of AtMID1A in the Arabidopsis genome. Both had 73% amino acid identity and a similar hydropathy profile. Therefore, AtMid1B is expected to have a function similar to AtMid1A. Growth of the atmid1a/b double mutant delayed for 1-2 days under usual conditions. In addition, the atmid1a/b double mutant exhibited a severe growth defect when grown on media containing high concentrations of Mg²⁺, but this phenotype was alleviated by supplementing CaCl₂ to the media. Mg²⁺ is known to be a natural blocker for Ca²⁺ channels. Therefore, our findings suggest that the loss of AtMID1A and AtMID1B decreased Ca²⁺ influx and thereby caused Ca²⁺ deficiency in Arabidopsis plants.

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Aluminum (Al) ion is a major factor limiting plant growth in acid soils. Some wheat cultivars show higher Al tolerance that is associated with the Al-dependent efflux of malate from root apices. We previously cloned a wheat ALMT1 gene which is an Al-activated malate transporter. Heterologous expression of ALMT1 in cultured tobacco cells conferred an Al-activated malate efflux and increased Al tolerance, indicating that ALMT1 is Al-tolerance gene. Here, to examine whether wheat ALMT1 confers Al tolerance in other intact plants, we produced transgenic barley and tobacco plants overexpressing ALMT1. Transgenic barley roots exhibited Al-activated malate efflux and enhanced tolerance to Al in both hydroponic culture and acid soil. Transgenic tobacco roots also exhibited increased Al tolerance in hydroponic culture. These findings indicate that ALMT1 confers Al tolerance in both monocot and dicot plants, and also is acid-soil tolerance gene.

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Aluminum(Al) ion inhibits root growth and main constraint to crop growth in acid soils. Al tolerance in wheat and rye has been attributed to their ability to exude organic acids from roots to chelate and detoxify Al. We have recentry isolated wheat ALMT1 gene which encodes Al-activated malate transporter and is Al tolerant gene in wheat. In this study, we detected ALMT1 homologue in rye. Time-dependent enhancement of the ALMT1 homologue expression was correlated with the enhanced efflux of citrate and malate during Al treatment. The structure and functions of ALMT1 homologue(s) will be discussed.

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Plants evolved two strategies for the acquisition of Fe. Graminaceous monocots release phytosiderophores (PS), that chelate Fe(III) in rhizosphere. PS solubilize Fe(III)-compounds by chelation, and the Fe(III)-PS are taken up through a specific transport system in the root cell membrane. The maize yellow stripe1 (YS1) is the Fe(III)-PS transporter (Curie et al.2001). Searching the Rice genome based on the sequence similarity to the ZmYS1 gene, we identified 18 members of putative rice YS1-like (OsYSL) genes. To understand possible functions of these OsYSL (1-18) proteins, we analyzed the mRNA expression level of each gene, in the roots and shoots of Fe-sufficient (+Fe) and Fe-deficient (-Fe) rice plants by Northern Blot Analysis. OsYSL2 was expressed only in Fe-deficient shoots. We use Laser Microdisection to separate tissues (stele, cortex and epidermis) and examine more detailed expression patern of OsYSL (1-18). To investigate subcellular localization of these OsYSL (1-18), we made GFP-fusion constructs.

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Aquaporins play important roles in plant water transport. According to the former reports, Arabidopsis and maize have more than 30 different aquaporins, however, there have been little previous works on all rice aquaporins. We identified 32 different aquaporin genes from the rice genomic database. According to phylogenetic analysis, they were classified into four major subgroups; 11 plasmamembrane (PIPs), 10 tonoplast (TIPs), nine NOD26-like (NIPs) and two small basic intrinsic proteins (SIPs). Semi-quantitative RT-PCR analysis revealed the differences in expression patterns of each aquaporin gene; some genes were expressed in two or more organs, while others were mostly in one organ. In addition, some aquaporin gene expressions changed during developmental stages and in day/night cycle. Chilling stress decreased most aquaporin gene expressions, however, the variation of decrease was different among aquaporins. Further analysis is in progress to investigate water permeability of each rice aquaporin.

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A. thaliana has 35 aquaporin species. They are subdivided into four families, PIP, TIP, NIP, and SIP. In this study, we analyzed SIPs which are not well known. When SIPs fused with green fluorescent protein were transiently expressed in protoplasts of A. thaliana cultured cells, green fluorescence was observed specifically in the ER. Furthermore, SIPs were immunochemically detected in the ER fraction after sucrose density gradient centrifugation. Then SIPs were expressed in yeast and the water permeability of membranes from the yeast transformants was assayed by stopped-flow spectrophotometry. Three SIPs had little water channel activity. The SIP promoter region of 2 kb has been fused to a GUS reporter gene and expressed in A. thaliana. The GUS activity was detected in a tissue-specific manner. Thus, SIPs may function as slow water channel or the transporter for other small molecules at the ER membrane and maintain the ER homeostasis.

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Aquaporins are involved in rapid water transport across biological membranes. A. thaliana has 35 aquaporin species. In this study, we examined physiological changes in protein levels of eight species of plasma membrane aquaporins (PIP), two species of vacuolar membrane aquaporins (TIP) for understanding physiological meanings of each aquaporin isoform. Also, we analyzed their organ- and cell-growth-phase specific accumulation and responses to environmental stresses in Arabidopsis whole plants and suspension-cultured cells by using isoform-specific antibodies. In suspension-cultured cells, the protein levels of aquaporins remarkably increased after cultured for 2 weeks. The amounts of aquaporins in suspension cells were less than 10% of that in stems of whole plants. Furthermore, we determined changes in the aquaporin levels in response to osmotic (PEG) and salt (NaCl) stresses. The levels of PIP2-type and TIP1-type aquaporins increased more than twice under salt stress, while PIP1-type aquaporins kept constant levels.

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In order to develop biological monitoring system as indicator to genotoxicity in plants, we investigated the use of DNA-damage responsive promoter from Arabidopsis. A 0.7 kb fragment of the 5'-upstream region of AtRAD51 genomic DNA was fused to the modified firefly luciferase (Fluc+) reporter gene and introduced into Arabidopsis, tobacco and BY-2 cells. To test the AtRAD51 promoter induction in response to DNA damage, we tested treatment with a radiomimetic agent, bleomycin, which produces DNA strand breaks. Treatments at various concentrations of bleomycin resulted in the induction of AtRAD51 promoter in a dose-dependent manner in transgenic plants and BY-2 cells. No detectable induction of the AtRAD51 promoter activity was observed with xenobiotic stresses including treatment with heavy metals, plant hormones and defense gene inducers.

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We investigated the tissue specific expression and DNA damage-responsiveness of AtRAD51 gene in Arabidopsis to uncover the mechanisms involved in regulated expression of the DNA damage-responsive gene. Transgenic Arabidopsis plants harboring AtRAD51 promoter-β-glucuronidase (GUS) were used to study the detailed expression pattern of the AtRAD51 gene. A histochemical GUS assay of bleomycin-treated plants showed that the AtRAD51 promoter in young tissues is actively expressed particularly in meristematic cells of the root and shoot apex of seedlings. In mature plants, the activity of the AtRAD51 promoter is mainly expressed in flower bud, petal and stigma. Reporter gene expression pattern similar to bleomycin-treated plants was also observed in UV-treated plants. In the absence of genotoxic stress, only very low GUS activities were detected in these organs. These results suggest that the tissue-specific expression levels of AtRAD51 gene promoter are also controlled by the DNA damage-responsive regulation.

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We previously found that higher cyclobutane pyrimidine dimer (CPD) photorepair activity was observed in nucleus-rich fraction, but not in chloroplast-rich fraction. In these experiments, each nucleus- and chloroplast-rich fraction obtained from leaves by sucrose density gradient centrifugation was used. This study was aimed to reconfirm the above results and to study whether CPD photorepair ability is involved in mitochondria. We measured UVB-induced CPDs and their photorepair and darkrepair in each organellar genome in intact leaves by Real-time PCR. rbcS, cab and phr (nuclear genome-encoded genes), cox3, cob and orf288 (mitochondria genome-encoded genes) and atpB and rbcL (chloroplast genome-encoded genes) were targeted for analysis. DNA lesions were found to be induced in all of these genes by UV-B. On the other hand, photorepair was observed in nucleus and mitochondria, but not in chloroplast. Little or no excision repair was observed in all of organellars.

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Rice cultivars (Oryza sativa) vary widely in their sensitivities to UVB. Many UVB-resistant cultivars belong to Japanese lowland group and boro ecotype. Among Japanese lowland and indica cultivars, Sasanishiki was most resistant to UVB followed by Norin 1 and Surjamkhi (indica) in this order. Cyclobutane pyrimidine dimer (CPD) photorepair activity of Sasanishiki was greatest, while Surjamkhi was lowest. That of Norin 1 was intermediate. There were variations at positions 126 and 296 in amino acid sequences of CPD photolyases of these three cultivars. We suggested that the difference in the UVB sensitivity in rice might be caused by structural alteration of CPD photolyase due to variation in amino acid sequence. We report here correlation between UVB sensitivity, activity and amino acid sequence of CPD photolyase in wild rice (O. rufipogon from Asia, O. meridionalis from Australia, and O. barthii from Africa).

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The most major prevalent lesions produced by ultraviolet (UV) are cyclobutane (CPD) and pyrimidine (6-4) pyrimidone dimmers. These photoproducts impair DNA replication and transcription processes and induce mutation. In plants, these photoproducts are mainly restored with photolyase and NER. Moreover, it has been thought that these lesions which are not restored by photolyase and NER should be restored by homologous recombination (HR).
In the present study, we have analyzed intrachromosomal homologous recombination (ICHR) frequency in AtRAD1 or AtRAD2 knock-out mutant of Arabidopsis . With the result, AtRAD1 KO (uvh1 ) suppress ICHR, on the other hand AtRAD2 KO (uvh3 ) enhance ICHR. We have crossed photolyase deficient mutant of Arabidopsis (uvr2-1 ) with uvh1 or uvh3 to produce double KO mutant, uvr2-1 / uvh1 or uvr2-1 / uvh3 , respectively. We are currently analyzing ICHR frequency in these double KO mutants.

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Cyclobutane pyrimidine dimers (CPDs) constitute a major portion of UVB-induced DNA lesions. Cucumber plants adapt to the solar radiation by expressing CPD photolyase, which catalyzes rapid restoration of CPDs, synchronously with the UVB content of sunlight. Such an expressional pattern is attributable to light-dependent transcriptional activation of the CPD photolyase gene (CsPHR). The purpose of this research was to elucidate the molecular mechanisms leading to the light-driven transcriptional activation. Irradiation of monochromatic light revealed that UVB with wavelengths around 310 nm maximally induces the transcription of CsPHR. To identify the cis-acting elements responsible for the light-driven transcriptional activation, we established transgenic Arabidopsis lines expressing reporter genes (the β-glucuronidase gene [GUS] and the luciferase gene [LUC]) under the control of the CsPHR promoter. Histochemical localization of the GUS activity as well as the LUC activity under the control of the CsPHR-promoter derivatives will be presented.

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The ultraviolet B insensitive 4 (uvi4) mutant is one of the four UV-B resistant mutants that have been previously isolated. The fresh weight of uvi4 plants grown under supplemental UV-B light was more than twice of that of the wild type. No significant difference was found in the UV-B absorbing compounds extracted in acidified methanol. The induction and reduction in level of cyclobutane pyrimidine dimers was not significantly different between uvi4 and wild type. We found that the leaf cells of uvi4 undergoes one more round of endoreduplication as compared to those of wild type. This enhanced ploidy level may contribute the higher tolerance of uvi4 mutant. The uvi4 is mapped on the bottom of chromosome 2 and the positional cloning is ongoing.

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