Roots of plant alter their growth direction to escape obstacles that lie in their paths. This response has been analyzed with a simple method using inclined the agar plate. On the surface of the vertical agar plates, the roots of seedling of Arabidopsis thaliana grow straight to gravity direction. When the plate was inclined at 45° to gravity direction, the roots exhibit a wavy growth pattern. We screened six mutants, wav1-wav6, showing abnormal wavy growth pattern¹⁾.
wav2 mutant exhibits shorter wavelength pattern in wavy growth phenotype of root than wild type. The angle of the wave was two times and the pitch of wave was half to that of wild type. We revealed that WAV2 gene encodes a putative membrane protein by map-based cloning. We are analyzing the gene-expression and the function of WAV2 protein.
1) Okada & Shimura (1990) Science vol.250, pp. 274-276

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Effects of salts at low concentrations on growth were studied from the aspects of controlling factors for cell growth. When germinated lettuce (Lactuca sativa cv. Grand rapids) seeds were incubated in NaCl and KCl solutions for 4 days in the dark, each salt at concentrations lower than 50 mM stimulated hypocotyl elongation. Kinetic studies revealed that both salts (20 mM) stimulated elongation at similar extent until 4 days, then growth rate of NaCl-treated seedlings decreased as compared with KCl-treated ones. Osmotic pressure of cell sap increased in NaCl and KCl treatments, resulting in an increased-delta-osmotic pressure. Extensibilities of hypocotyl cell wall also increased, the effect being much larger in KCl treatment. These suggest that NaCl and KCl at low concentrations stimulate hypocotyl growth by affecting not only osmotic properties but also cell wall properties in etiolated lettuce seedlings, although effectiveness on cell wall properties is different from kind of salts.

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Cyanamide has been utilized as a synthetic agrochemical. Recently, cyanamide was isolated and identified as a natural plant growth inhibitor of hairy vetch (Vicia villosa Roth), a legume. Cyanamide may also play an important role in vetch-associated disease of cattle as a toxic substance. In this study, we developed the advantageous method to identify cyanamide, and reported the distribution of cyanamide in some leguminous plants, such as Vicia, Astragalus and Trifolium. It became clear that Vicia contained large amount of cyanamide but other genus hardly contain cyanamide.

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Hairy vetch (Vicia villosa Roth) a leguminous plant mainly utilized as forage, green manure or cover crop in many countries. It has been reported that this plant effectively suppresses weed growth. Recently, natural occurrence of cyanamide has been confirmed and isolated from hairy vetch and are reported to be involved in allelopathic phenomenon. Many plant species including hairy vetch has been reported for their allelopathic potentiality, however, detail studies related to the function and mechanism of allelopathic substances on plant growth has not yet been carried out. As to our first approach, here we investigated the effects of cyanamide on the germination and growth of Arabidopsis and hairy vetch. We found that different concentration of cyanamide completely inhibited seed germination of Arabidopsis, while in hairy vetch it was not. Detail studies on the interaction mechanisms are underway and will be discussed.

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T-2 toxin is one of the trichothecene mycotoxin family synthesized by various Fusarium species. Although trichothecenes are thought to be virulence factors in wheat head scab, their effects in plants are barely understood. We previously found that dwarfism was observed when Arabidopsis were grown on the medium containing T-2 toxin. We monitored global gene expression in T-2 toxin-treated Arabidopsis plants using an Affymetrix GeneChip system. This revealed that T-2 toxin-inducible genes contain many defence genes. It is likely that dwarfism is caused by continuous defence response to T-2 toxin. Furthermore, we found a gene (At2g03760), which is homologous to the BNST3 gene encoding steroid sulfotransferase in B.napus, is also upregulated in T-2 toxin-treated plants. It has been reported that BNST3 specifically inactivated brassinosteroid. In addition, GA4 gene is downregulated in T-2 toxin-treated plants. These two genes might play some roles for T-2 toxin-induced dwarfism in Arabidopsis plants.

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Nitrate reductase (NR) is a central enzyme in nitrogen metabolism in plants. However, NR protein is small amounts in plants, which makes recombinant expression an attractive approach for studying its biochemistry.
NR CcR+ fragemnt (43 kDa fragment containing FAD, Fe-heme and Hinge-1 including regulatory Ser residue) was expressed in yeast (Pichia pastoris). CcR+ fragment was expressed only 1 % of CcR- (41 kDa fragment without the Ser residue ) fragment. We modified nucleotide sequence in the front of ATG codon in the cDNA fragment coding CcR+. This modified nucleotide sequence was same as that of the cDNA fragment coding CcR-. This CcR+ fragment was expressed about 59 mg/L Pichia culture. Next, we modified full length NRcDNA in the same manner. Active NR protein was expressed about 0.7 mg/L culture and this amount of NR protein was ten times higher than that of unmodified one.

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In higher plants, expression of nitrate transporter(s) (NRT), nitrate reductase (NR), and nitrite reductase (NiR) is regulated both positively and negatively at the transcription level; Gln or its metabolite(s) downregulates transcription, whereas nitrate and/or nitrite upregulate transcription. In this study, we cloned the NR cDNAs from the moss P. patens and partially characterized them. P. patens was found to express two closely related species of NR mRNA when grown with nitrate. The deduced NR proteins are 80% identical to each other and 56% and 47% identical to the NR proteins from Arabidopsis thaliana and Chlamydomonas reinhartii, respectively. Expression of the NR genes is activated by nitrate and inhibited by ammonium. These results are essentially the same as those reported for higher plant systems, showing that P. patens can be used as a model plant for the studies on the mechanism of regulation of the nitrate assimilation genes.

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Nitrate-independent activity of nitrate reductase (NR) is generally found in legume root nodules. Therefore, effects of nitrate on the levels of plant NR activity and mRNA were investigated in root nodules of Lotus japonicus. NR activity and mRNA were enhanced by the addition of nitrate in roots and root nodules. They were also detected in root nodules without nitrate while they were undetectable in roots. Southern blot analysis indicated that NR was a single gene. These results indicate that inducible-type NR could be expressed in root nodules without nitrate. The activation state of the nitrate-independent activity of NR was as high as that of nitrate-induced activity of NR. mRNA expressed independently of nitrate in root nodules was localized in the infected region of root nodules. Thus the expression could be related to specific structure and environment of root nodules.

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Nitrate reductase (NR), a key enzyme of nitrate metabolism, is regulated at transcription and post-translation in response to environment at light conditions. Inactivation of NR occurs by phosphorylation followed by the binding with 14-3-3 proteins. We studied Ca²⁺ independent NR kinase from Brassica campesis . Immediately after transferring plants to darkness , we observed an increase of NR kinase activity. This NR kinase activity was inhibited by 10 mM glucose 6-phosphate (G6P) and Fructose 6-phosphate (F6P) approximately 40 %. NR kinase was fractionated by Resource Q chromatography. The major peak of NR kinase activity from dark treated leaves was much higher than those from light treated leaves. NR kinase in the major peak is inhibited by 10 mM G6P and F6P approximately 40 %, 20 % respectively . Taken together with these findings, results obtained suggest that G6P and F6P may be responsible for regulation of NR kinase activity.

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Nitrite reductase (NiR), an enzyme containing a single siroheme and an [4Fe-4S] cluster, catalyzes the six-electron reduction of nitrite to ammonia in cyanobacteria and higher plants. Ferredoxin is the typical electron donor for NiR, and donates six electrons by an intermolecular transfer to the [4Fe-4S] cluster through the siroheme active-site. Plectonema boryanum NiR (FL-NiR) contains a redox-active ferredoxin-domain at the C-terminal region of its polypeptide. FL-NiR is homologous to many known assimilatory nitrite reductases. The P. boryanum NiR gene has been cloned from the genomic DNA, and over-expressed in Escherichia coli. A truncated FL-NiR containing only the NiR domain, or only the ferredoxin domain, and an authentic Synechocystis sp. 6803 NiR were also expressed. Physiological and enzymatic properties of these authentic NiRs and the truncated versions will be presented.

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Primary ammonium assimilation into glutamine and glutamate is carried out by the GS/GOGAT cycle. In Arabidopsis, GS and GOGAT are coded by 9 isoforms gene families (GS1;Gln1;1-1;5, GS2; Gln2, Fd-GOGAT; Glu1, Glu2, NADH-GOGAT; Glt1).
In the present study, we identified the molecular species of GS and GOGAT responsible for assimilation of NH₄⁺ in roots. Real-time PCR analysis showed that levels of Gln1;2 and Glt1 mRNA were specifically increased following supply of NH₄⁺. In addition, these genes mainly expressed in epidermis or cortex cells that play important roles in absorption of NH₄⁺. These results strongly suggest that most of the absorbed NH₄⁺ can be assimilated to glutamine and glutamate by the NH₄⁺ inducible Gln1;2/Glt1 system at the surface of Arabidopsis root.

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Cytosolic glutamine synthethase (GS1) in rice (Oryza sativa L.) is probably important in the export of glutamine from senescing leaves. To prove this function of GS1, mutants lacking GS1 expression have been screened and characterized.
GS1 mutants (osgs1) were isolated from the Tos17-insertion lines of rice (distributed from the Rice Genome Project by Hirochika and Miyao). It is confirmed that Tos17 was inserted into the exon 8 of GS1 gene. This mutant contained undetectable amount of GS1 protein and its activity. GS1 protein is accumulated in companion cells in wild-type rice, but not in the mutant. Mutants were flowering but the seeds obtained were mostly sterile. Other four GS1-knock out lines were independently isolated by a PCR screening. The lines inserted Tos17 into either the exon 8 or exon 10 showed the same phenotype, i.e. growth retardation, dwarf, sterile, etc., as osgs1 mutant.

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Glutamate decarboxylase (GAD) is an enzyme that catalyzes the conversion of glutamate to γ-aminobutyric acid (GABA). We previously reported that rice has two distinct isoforms of GAD (OsGAD1 and OsGAD2). The former carries a C-terminal calmodulin-binding domain that is common in dicotyledonous plants, but the latter does not. In order to know regulatory mechanism of these rice GAD activities, we introduced these wild-type GAD genes and mutated genes lacking the region encoding the C-terminal peptide, respectively, into rice calli via agrobacteria. Transformed callus lines were used to determine content of GABA. Unexpectedly, overexpression of mutated OsGAD2 was 100~200 times higher than that of wild-type calli. Plants regenerated showed the same level of accumulation of GABA in all the tissues examined as in calli. Besides, the plants displayed aberrant phenotypes such as pale color, dwarf and curled leaf. These results suggest that C-terminal portion of OsGAD2 plays as an autoinhibitory domain.

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Regulation by Ca²⁺ of protein dephosphorylation in plants is not well understood, since calmodulin-dependent protein phosphatase (PP2B) conserved in both animals and yeasts has not been characterized in plants. Screening of the cDNA library of the moss Physcomitrella patens by using ³⁵S-labeled calmodulin identified PCaMPP gene encoding a protein similar to protein phosphatase 2C. Bacterially expressed recombinant GST-PCaMPP bound calmodulin-Sepharose. The protein exhibited phosphatase activity to phosphorylated myelin basic protein and the activity was dramatically increased by Mn²⁺. Furthermore, the activity was insensitive to okadaic acid. PCaMPP lost both calmodulin-binding ability and phosphatase activity by deletion of the C-terminal region containing basic amphiphilic amino acid sequence. A synthetic peptide with this sequence bound calmodulin in a Ca²⁺-dependent manner. A gene similar to PCaMPP was found in Arabidopsis genome suggesting that it is a protein phosphatase conserved in land plants.

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14-3-3 proteins play a pivotal role in intracellular localization of transcription factor RSG involving synthesis of gibberellins. We characterized protein kinase activity in tobacco which phosphorylates Ser-114 of RSG involved in its association with 14-3-3 proteins usingin-gel kinase assay. GST-fusion proteins of wild and S114A of RSG were prepared by E.coli cells. Ser-114 of RSG was phosphorylated by protein kinases at 50-60 kDa in the tobacco microsomal fraction in Ca²⁺-dependent manner. Thus, it was suggested that CDPKs phosphorylate Ser-114 of RSG. Tobacco CDPK1 cDNA was isolated from tobacco cDNA library and GST-fused CDPK1 was prepared. CDPK1 preferentially phosphorylates Ser-114 of RSG in vitro. Pull-down assay demonstrated that in vitro phosphorylated RSG binds to 14-3-3 proteins and that CDPK1 interacts with 14-3-3 proteins by itself. These results suggest that CDPK regulates interaction between RSG and 14-3-3 protens via phosphorylation of Ser-114, leading to modulation of intracellular localization of RSG.

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We have obtained 28K full-length cDNA sequence data from the rice full-length cDNA project and homology search was made against NCBI GenBank data. Comparative analysis of calcium ion transporter protein revealed that, the cell-to-cell calcium signal transduction systems(the voltage dependent calcium channel, Ins3 receptor and ryanodine receptor etc.) have severely changed between animal and plant. On the other hand, signal transduction systems at the internal of the individual cells (animal Ligand-gated channel protein, Ca2+ATPase, Ion-exchanger membrane proteins) are well conserved between plant and animal. Comparative analyses of calcium ion binding and/or controlling signal transduction protein were also carried out. It revealed that, plant developed directly calcium ion interacting system more than animal. There are many species of plant specific calcium ion binding protein, which has calcium ion binding domain (EF-hand) plus CAMK II type kinase domain (CDPK) or phosphates domain (PP2B) or Ca2+ /phospholipid binding domain.

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A proteinaceous elicitor from a pathogenic fungus, cryptogein, induced programmed cell death (PCD) in suspension-cultured tobacco (BY-2) cells. Changes in cell shape, cytoplasmic condensation as well as swelling and collapse of the vacuoles were observed during the PCD. Various plasma membrane ion fluxes such as Ca²⁺ influx, Cl^- efflux and H⁺ influx were induced within a few minutes after elicitation, suggesting that ion channel cascade play important roles in regulation of PCD. To elucidate the molecular mechanisms for the [Ca²⁺]_cyt changes, effects of various specific inhibitors were tested. Elicitor-induced production of reactive oxygen species (ROS) was shown to be regulated by ion fluxes including Ca²⁺ influx. Possible mechanisms and significance of ion fluxes and ROS production during the PCD will be discussed.

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We cloned a cDNA encoding a receptor-like protein kinase, PnLRK1 ((Populus nigra var. italica leucine-rich repeat receptor kinase 1), from a shoot cDNA library of lombardy poplar. The cDNA is about 2.7 kbp in length and contains an open reading frame encoding the PnLRK1 protein of 856 amino acids. The N-terminus of PnLRK1 has a potential signal peptide. This is followed by a putative extracellular domain consisting of 14 leucine-rich repeats (LRRs). The C-terminal catalytic domain of PnLRK1 has twelve subdomains, which are conserved in the protein kinase family. A putative hydrophobic transmembrane domain is localized between the LRRs domain and the catalytic domain. The PnLRK1 gene was expressed in leaves, stems and apical buds of lombardy poplar; trace amounts of the mRNA were detectable in roots.

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In Brassica self-incompatibility, the self/non-self recognition between pollen and stigma is controlled by the multiple alleles at a single locus, S. The S locus contain three highly polymorphic genes, S-locus protein 11 (SP11), S-receptor kinase (SRK) and S-locus glycoprotein (SLG) genes. To date, we revealed that SP11 on the pollen coat acts as a ligand for its cognate SRK, and that SRK and an SLG-like 60 kDa protein (60K) together form a receptor complex for SP11 on the stigma membrane. However, the precise molecular structure of 60K is still unknown. Here we show that SRK/SLG expressed in yeast or COS-7 cells did not exhibit SP11 binding activity. We also show that 60K affinity-purified from stigmatic membrane exhibited slightly larger molecular size compared with stigmatic soluble form of SLG. We are now analyzing the molecular structure of 60K.

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Many flowering plants have evolved self-incompatibility systems to prevent inbreeding. In Brassica, self/non-self recognition is controlled by the multiallelic gene complex (S-haplotypes) at the S-locus, which contains both determinants of the male (SP11: S-locus protein 11) and the female (SRK: S-receptor kinase). SP11 has been shown to activate its cognate SRK to trigger the signaling cascade that results in the rejection of self-pollen. However, the components working in this signaling cascade is still entirely unknown.
In this study, we searched for the stigmatic proteins that were phosphorylated after pollination by using in vivo ³²P-labelling system followed by 2D-PAGE analysis. We found one protein that was specifically phosphorylated after self-pollination, and two that were specifically phosphorylated after cross-pollination. The former was shown to be phosphorylated within 30 min after self-pollination.

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When a compatible pollen grain adheres to a papilla cell of a stigma in Brassicaceae, the pollen grain is hydrated and germinates the pollen tube. The pollen tube penetrates into the papilla cell wall and then enters the style. After these sequential steps, cross-fertilization can be achieved. In animal and plant cells, the oscillations of cytosolic Ca²⁺ occur in response to many kinds of extracellular stimuli. During pollination process, it is speculated that the oscillations of cytosolic Ca²⁺ occur in response to some stimuli from the pistil to regulate the pollen tube growth. In this study, we examined the distribution of Ca in the pollen grain and the papilla cell during pollination process using an EDX system fitted with the VP-SEM. Furthermore, we examined the Ca²⁺ dynamics during pollination process using Arabidopsis thaliana, into which the gene of a Ca²⁺ sensor protein, yellow cameleon, was transformed.

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Prant heterotrimeric G proteins have been proposed to be involved in the transduction of several extracellular signals such as plant hormones, elicitor and light. In the plant G proteins, the precise intracellular localization of the subunits have not yet been analyzed. In the present work, we analyzed the localization of the subunits of the rice G protein.
1. Western blot analysis showed that the α, β, γ1, and γ2 subunits were localized in rice plasma membrane fractions.
2. Membrane-proteins solubilized with cholate were fractionated by gel filtration (Superdex 200 PC3.2/30, SMART System, Pharmacia). Two fractions of the G protein were obtained; one seemed to be a heterotrimer(s) (αβγ1 and/or αβγ2), and the other, a βγ dimmer(s) (βγ1 and/or βγ2). We propose that, in rice plants, a large amount of βγ dimmer is present in the plasma membrane in a form without the α subunit.

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GH3 gene was originally found as a rapid-auxin inducible gene in Glycine max. In the Arabidopsis genome, there are 20 members of GH3 gene family, and the expression of the several genes is regulated by auxin and/or light. Recently it has reported that several GH3s showed adenylation activities to phytohormons such as IAA, JA and SA.
We isolated a novel short hypocotyl mutant from approximately 50,000 of activation tagging lines, named dwarf in light 1-like 1-D (dwl1-D). dwl1-D is dominant and has short primary root, epinastic leaves and shows dwarf phenotype in adult stage and loss of apical dominance. We cloned DWL1 and found that this gene was a new member of GH3 gene family. DWL1 was induced by applied exogenous auxin. 35S::DWL1 lines represented phenotype of dwl1-D.

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Previous experiments have established that the MYC-like sequence CATGTG plays an important role for induction of dehydration-dependent expression of the ERD1 gene that encodes a putative protein similar to the ATP binding subunit of the Clp protease. Three cDNA clones encoding NAC proteins that bind to the 63-bp fragment of the ERD1 promoter containing the CATGTG motif were isolated using yeast one-hybrid system. The cDNA clones were named NAC2, NAC3 and NAC4. The three encoded proteins formed a subgroup of the NAC transcription factor. All three proteins bound specifically to the CATGTG motif either in vitro or in vivo, and activated the transcription of the GUS reporter gene driven by the 63-bp fragment in Arabidopsis protoplasts. Expression of the three genes was induced mainly by drought, high salinity stress and ABA treatment, but not by low temperature. Using yeast one-hybrid system we determined the complete NAC recognition sequence (NACRS).

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Coccolithophorids are unicellular marine algae that produce CaCO₃ crystals on the cell surface and therefore very important for fixing large amount of carbon by both photosynthesis and calcification. We previously found that nanomolar level of selenium is essential for growth of coccolithophorids. In this study, we investigated on the utilization mechanism of selenium by a coccolithophorid, Emiliania huxleyi, by means of kinetic analysis of Se-uptake using ⁷⁵Se-radiolabelled selenite.
The intracellular concentration of Se increased 2400-fold where the initial concentration of Se in the medium was 3.2-nM (seawater-level). 50% and 20% of Se incorporated by cells were fixed into low-molecular-mass-compounds and proteins, respectively. Kinetic analyses of ⁷⁵Se-uptake in experiments with inhibitors for energy production revealed that Se-concentrating mechanism involves two steps: namely, rapid uptake of selenite by the combination of active and passive transport systems and subsequent synthesis and accumulation of Se-containing organic compounds, such as amino acid derivatives and proteins.

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Rice is a typical Si-accumulating plant and a specific uptake system for silicic acid in rice roots has been suggested. We characterized this specific system in rice roots. Si uptake increased linearly with time, but pre-treatment with Si did not affect the Si uptake, suggesting that the uptake system for Si constantly exists in rice roots. Kinetic study indicated that Si uptake is mediated by a kind of proteinaceous transporter. The Km was estimated to be 0.32 mM, suggesting that the transporter has a low affinity for silicic acid. Mercury chloride, phloretin significantly inhibited the Si uptake, but DIDS hardly affect the Si uptake. Si uptake was also not affected by the presence of 10 times more of boric acid. Taken together, the Si uptake by rice roots is a transporter-mediated process and this transporter contains Cys residues but not Lys residues.

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Genes for high-affinity nitrate transporter (NRT2) has been isolated and well characterized in many higher plants. However, biochemical properties of NRT2 proteins are not well investigated. To obtain a tool for biochemical analysis of NRT2, we have developed an antibody against C-terminus region of NRT2 protein, which is expressed in E. coli. In this study, we investigate the relationship between the expression patterns of NRT2 protein and nitrate flux during the nitrate induction in barley roots.
Barley seedlings grown hydroponically in N-free medium were supplied with nitrate and plasma membranes (PM) and other membranes were isolated from root with two-phase partitioning method. The immunoblot analysis of membrane proteins clearly indicated that NRT2 proteins located in PM of barley roots. There was no correlation between the accumulation patterns of NRT2 protein and nitrate flux during the nitrate induction suggesting that NRT2 are post-translationally regulated in barley roots.

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Blue light, absorbed by phototropins, activates the plasma membrane H⁺-ATPase via phosphorylation of C-terminus and concomitant binding of 14-3-3 protein to the phosphorylated C-terminus in guard-cells. In this study, we identified the binding proteins to the C-terminus of H⁺-ATPase, by two-hybrid system using yeast-cells. The C-terminus of VHA1 (position 846-951) used as a bait, and cDNA library was constructed from guard-cells of Vicia faba. Eight clones were isolated as positive from 4.2 x 10⁷ clones. Sequence analysis showed that 5 clones were encoded unknown proteins, and that 3 clones were encoded vf14-3-3a and vf14-3-3d, which are the 14-3-3 proteins in Vicia faba. Binding affinity of vf14-3-3a to C-terminus is 4 times higher than that of vf14-3-3d. Analysis of amino acid substitution of C-terminal end showed that both vf14-3-3 required T₉₅₀ for binding. This result confirmed the previous results that 14-3-3 protein binds to Yphospho-T₉₅₀V in the C-terminal end of H⁺-ATPase.

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In a fructose-containing medium in which rye root-microsomal membrane vesicles had reached the equilibrium of uptake of fructose, the presence of both Mg²⁺ and ATP caused the efflux of fructose from the vesicles. Because the fructose-containing medium did not contain any readily permeant anions, it is unlikely that proton pump-dependent, ΔpH-driven proton/monosaccharide cotransport was the cause of the efflux of fructose. Among nucleotides examined, ATP caused the largest efflux of fructose. The efflux of fructose dependent on Mg²⁺ and ATP was quite insensitive to a protonophore, CCCP, while it was largely inhibited by vanadate, which inhibits an ATP-binding cassette transporter (glutathione-conjugate pump) of higher plants. It was shown that the Michaelis-Menten constant (Km) of the efflux was 0.4 mM.

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We have found that elicitor and electric stimulus (30 V, 30 sec) induce the calcium influx, which increase chitinase mRNA levels in cultured rice cells (Oryza sativa L. japonica cv. Nipponbare). Some calcium channel blockers inhibited the increase of intracellular calcium concentration and chitinase gene expression. In this study we aimed at cloning and functional analysis of rice calcium channel gene.
A novel Oryza sativa calcium channel cDNA clone (OsCC1, 2791 bp) was isolated using RT-PCR derived from a Arabidopsis thaliana calcium channel (AtTPC1) sequence. The cDNA clone had a 2274 bp insert containing a single open reading frame of 758 amino acids and showed about 70 % homology to AtTPC1. Yeast mutant cells of calcium channel, were transformed with OsCC1 cDNA, and calcium uptake in yeast cells was measured. OsCC1 rescued the calcium uptake activity of the mutant. I suggested that OsCC1 has a function of calcium channel.

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Aequorin is widely utilized for analyzing calcium signaling in cells. We constructed chimeric proteins (GFP-AQ) consisted of GFP (S65T) and aequorin with various length linker peptides, according to Baubet et al (Proc Natl Acad Sci U S A.; 97(13):7260-5). Using GFP-AQ, calcium fluxes are converted to strong fluorescence of GFP without exitation light.
In this study, we characterized Arabidopsis Ca²⁺ permeable channel AtTPC1 using GFP-AQ in cch1 mutant yeast cells. Glucose addition to the sugar-starved yeast cells expressing AtTPC1 caused a transient increase of cytosolic free Ca²⁺ concentration ([Ca²⁺]_cyt). This glucose-induced [Ca²⁺]_cyt increase was clearly inhibited by BAPTA, an extracellular Ca²⁺ chelator and La³⁺, a Ca²⁺ channel blocker. This [Ca²⁺]_cyt increase was also specifically inhibited by K⁺, but not by other mono- and divalent cations. Moreover, the expression of AtTPC1 accelerated ⁸⁶Rb⁺ uptake into the cells, indicating that AtTPC1 is a Ca²⁺ and K⁺ selective cation channel.

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Calcium plays an essential role as an intracellular second messenger in plants. Rapid increases in [Ca²⁺]_cyt are mediated by calcium channels located on the plasma membrane and endomembranes. In animal cells, it is well known that the α-subunit of L-type Ca²⁺ channel is composed of 4 domains, each consisting of 6 transmembrane segments and 1 pore loop segment. Recently we reported about a novel plant Ca²⁺ channel, the A. thaliana AtTPC1, composed of 2 domains. From searching the last available release of the DNA databases, AtTPC1 homologues appear to be well conserved in planta.
AtTPC1 is supposed to function as a dimer and to form an ion channel with 4 domains. A hydrophobic region, between the 2 domains contains 2 EF-hand motifs (EF-1, 2) and possesses weak similarity with the consensus sequence for tetramerization from the animal K⁺-channels. Mutation D335A in EF-1 increased efficiency of Ca²⁺ uptake in yeast cells.

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When Characese cytoplasm is rapidly hydrated, Ca²⁺ is released from some intracellular stores. This is explained as a Ca²⁺ channel activation at the store membrane in a mechanosensitive manner. It may be expected that mechanosensitive Ca²⁺ channels function not only at the membrane of the stores but also other membrane system such as plasma membrane.
According to Shimmen, mechanical stimuli to an intact cell induces receptor potential which reflects activation of Cl^- channels at the plasma membrane. We studied possible contribution of mechanosensitive Ca²⁺ channels on the receptor potential.
Removal of Ca²⁺ in the bathing medium reversibly inhibits the receptor potential. Mechanical stimuli caused not only receptor potential but also increases in cytoplasmic level ofCa²⁺, and both of those amplitudes are dependent on that of mechanical stimulus. These results strongly suggest that mechanical stimulus is first received by mechanosensitive Ca²⁺ channels at the plasma membrane.

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By taking advantage of Characeae, I have studied the mechanism of electrical perception of *eath message Specimens composed of two internodal cells were prepared. When one cell (victim cell) was killed, another cell (receptor cell) generated depolarizing responses. A possibility was suggested that the depolarization was induced by loss of the turgor pressure of the victim cell. To examine this possibility, I decreased the turgor pressure of the victim cell by adding sorbitol to the bathing medium of the victim cell. Upon decrease in the turgor pressure of the victim cell, transient depolarization was induced by the receptor cell. However, duration of the depolarization induced by the osmotic stimulation was much shorter than that induced by killing the victim cell (Shimmen (2001) Plant Cell Physiol 42: 366-373). In the present study, the mechanism of membrane depolarization induced by osmotic stimulation was analyzed.

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Because of the viscoelastic property of the cell wall and of the osmotic pressure difference between in- and out-side the plasmalemma, the tension of Characean cell wall changes as a function of the water flow across the membrane. By giving a constant sinusoidal mechanical vibration (10Hz) to the internode, and by studing the absorption rate of the vibration, the relative velocity of water flow can be estimated. Then the water conductivity, Lp, of the membrane can be figured out (with a time resolution of 0.1sec). It was found that there was no significant difference in Lp between the efflux and the influx. This confirmed that the membrane itself was not a rectifier for the water flow (non-intrinsic).

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Full-length cDNAs are essential for the correct annotation of genomic sequences and for the functional analysis of genes and their products. Using the biotinylated CAP trapper method, we constructed full-length cDNA libraries from Arabidopsis plants and isolated 155,144 RIKEN Arabidopsis full-length (RAFL) cDNA clones. They were clustered into 14,668 nonredund ant cDNA groups, about 60% of predicted genes1). We have determined full-length cDNA sequences of 12,189 RAFL cDNA clones as of Nov. 29, 2002. We have also used the RAFL cDNAs for the microarray analysis of expression profiles of Arabidopsis genes under drought, cold and high-salinity-stresses2),3),4). In this meeting, overview of our RAFL cDNA project will be presented.
1) Seki et al. (2002) Science 296:141-145. 2) Seki et al. (2001) Plant Cell 13:61-72. 3) Seki et al. (2002) Plant J. 31:279-292. 4) Seki et al. (2002) Funct. Integ. Genomics 2:282-291.

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Soil salinity is one of the severe abiotic stresses for plant growth and agriculture. Recently, several salt overly sensitive (sos) mutants have been isolated in Arabidopsis. In this study, the expression profiles of sos2-1 and sos3-1 mutants were analyzed by cDNA microarray.
We regarded the genes with expression ratios (high-salinity/unstressed) greater than three times that of lambda control template DNA fragment in at least 1 time-course point as high-salinity-stress-inducible genes. We detected 347, 157, and 125 genes as high-salinity-inducible genes in wild type, sos2-1, and sos3-1 mutants, respectively. Among them, expression of DREB2A, RD29A and RD17 were high-salinity-stress-inducible in sos2-1 and sos3-1 mutants as well as in wild type. This result indicates that SOS pathway is independent of DREB/CBF pathway. The expression of sos1, a transcription factor, and some unknown protein genes were not high-salinity-stress-inducible in sos2-1 and sos3-1 mutants, suggesting that these genes are regulated by SOS pathway.

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Plants respond and adapt to drought stress to survive under stress conditions. Several genes that respond to drought at the transcriptional level have been described,*but there are few reports on genes involved in the recovery process from dehydration. Analysis of*rehydration-inducible genes should help not only to understand the molecular mechanisms of stress responses of higher plants, but also to improve the stress tolerance of crops by gene manipulation. We used a full-length cDNA microarray with ca. 7000 Arabidopsis full-length cDNAs and identified 154 rehydration-inducible genes. Among them, 59 genes contained the ACTCAT sequence involved in proline- and hypoosmolarity-inducible gene expression in their promoter regions. Venn diagram analysis showed relationship of the rehydration-inducible genes to proline-inducible genes and water-treatment-inducible genes. Functional analysis of rehydration-inducible genes and rehydration-repressed genes revealed their functions not only in the release from a stressed status but also recovery for growth of plants.

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Recently, the halophytic plant species, Thellungiella halophila, was discovered in high salinity coastal area in east China. T. halophila is thought to be a good model plant for the analysis of high salinity tolerance because it is closely related to Arabidopsis; such as similar morphology, small size, short life cycle, ability to self-pollinate and high seed number. There are great advantages in T. halophila for genetic and molecular analysis of salt stress tolerance. Furthermore, T. halophila genes share >90% nucleotide identity with Arabidopsis genes, and it can be transformed in planta according to Arabidopsis protocol.
T. halophila is quite salt tolerant and can grow in 500 mM NaCl medium. To analyze molecular mechanism of salt tolerance in T. halophila, we analyzed expression profiles of salt-inducible genes in T. halophilaby using Arabidopsis cDNA microarray to compare the profile with those of Arabidopsis. We also analyzed accumulation of osmoprotectants in T. halophila.

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The polyamines (PAs) - putrescine (Put), spermidine (Spd), spermine (Spm) are polycationic cellular molecules, ubiquitous in living organisms. Several studies have indicated that PAs are implicated in several cellular processes. However the specific physiological roles of PAs under water stresses in plant are not well understood. In whole genome of Arabidopsis, there were 10 genes supposed to be involved in PA biosynthesis and their expression patterns under stress were separated into stress-inducible, constitutive, and stress-repressible. In these genes, we focused arginine decarboxylase (ADC) that related to Put biosynthesis. Put functions as not only PAs but also precursor for next PAs. We report tissue specific expression and cellular localization of ADC and phenotypic analysis of adc2 Ds-tagged mutant in abiotic stress response.

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It is considered that some osmolites are accumulated in plants to acquire tolerance to environmental stresses. Proline accumulation is observed in many plants, and it plays a role as an osmoprotectant. We isolated a barley proline transporter (HvProT) gene from salt-stressed roots and identified its function and substrate specificity. To study contributions of HvProT to stress tolerance, we introduced HvProT gene in Arabidopsis and evaluated its tolerance under stress conditions and high concentration of proline conditions. As a result, enhancing of stress tolerance was clearly observed in transgenic plants. Especially, effects on initial growth stage were significantly. On the other hand, it was shown that high concentration of L-proline inhibited growth of transgenic plants. Similarly, transgenic plants showed more sensitive to D-proline than control plants. These results indicated regulation of proline concentration is quite important for acquisition of stress tolerance and suppression of plant growth.

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For the waterlogging-stress in plants, there are few studies in green leaves although many studies on anaerobic condition in roots have been done. The fern Ceratopteris richardii has trends to have broad sterile fronds in high-humid condition and narrow fertile fronds in dry condition. The 17-kDa protein is abundant in the sterile fronds, and no similar feature was found in homology searching.
In present work, we further studied the 17-kDa protein by the western blotting analysis. Two bands corresponding to the precursor and the mature proteins were clearly appeared in broad sterile fronds of Ceratopteris richardii, but not in roots and petioles. The immunochemical signal was not detected in narrow fertile fronds. No band was appeared in the leaves of Arabidopsis thaliana. This suggests that the 17-kDa protein may be a novel protein which is induced in fern sterile fronds by a high-water condition.

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We isolated a lily aquaporin gene, AqpL1, from lily petal using RT-PCR. Phylogenetic analysis showed that AqpL1 was a member of PIP1 subfamily. AqpL1 gene could be properly expressed by in vitro translation and showed a 26 kDa monomer band revealed by autoradiograph. Injection of AqpL1 cRNA into Xenopus oocytes resulted in an increase of the osmotic water permeability (Pf= 0.72●10-3 cm/s) for about 2-3 times compared to that (Pf=0.24●10-3 cm/s) of the cells injected with water. Overexpression of AqpL1 in tobacco could improve the content of aquaporin in the cells revealed by Western blotting, and subsequently resulted in a great increase of the osmotic water permeability of leaf protoplasts ( 0.7●10-2 cm/s vs 4.3●10-2 cm/s ) and water conductivity of leaf cells (8.3% vs 23.2%). The results of Northern hybridization indicated AqpL1 expressed in whole plant but it strongly expressed in young petal and remarkably decreased with flower age.

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We have studied on desiccation tolerance in a terrestrial Cyanobacterium, Nostoc commune, and got the following results. 1) Structures of PSI and PSII reaction center (RC) complexes and phycobilisomes change so that absorbed light energy is converted to heat. 2) PSI and PSII activities are inactivated in dryness, but small amounts of water are enough for their recovery. 3) PSII RC remains active in dryness in desiccation sensitive cyanobacteria.
Here, we will report changes in photosynthetic systems during desiccation in various mosses grown under different water conditions. 1) The cause of inactivation of PSII is supposed to be due to dissipation of light energy absorbed by PSII. Quenching of PSI fluorescence was not observed. 2) The PSII activity was less sensitive to the decline of the water potential in the cell than in N. commune. 3) The PSI and II RC remained active in dryness in desiccation-sensitive species.

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Roots display positive hydrotropism in response to a moisture gradient, which regulates the directional growth by interactions with other tropisms such as gravitropism. To dissect the molecular mechanism underlying hydrotropism in seedling roots, we established a method for screening Arabidopsis mutants defective in root hydrotropism, and we have successfully obtained 12 mutants of which root hydrotropism is reduced to various extents. Among them, roots of a mutant named rhy1 (root hydrotropism 1) were found to be unique because they showed ahydrotropic response although their growth rate was normal, and they normally displayed gravitropism, phototropism and waving response. Roots of other rhy mutants (rhy2, rhy3, rhy4) showed the reduced hydrotropism together with either a reduced gravitropism or a reduced waving response. Currently, we are attempting to clarify factor(s) responsible for hydrotropism by identifying the mutated genes in the rhy
mutants.

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We present water movement in a plant using a positron emitting nuclide, ¹⁵O. Because of the extremely short half-life of ¹⁵O, water uptake measurement could be performed only for 20 min. However, after an hour, the next experiment was able to perform using the same plant. A soybean or wheat grown after 3 or 4 weeks in water culture was used. After ¹⁵O-water was supplied from the root, an imaging plate (IP) was exposed to the plant periodically. In another way, the BGO detectors were set at the stem and the gamma-rays emitted from the nuclide were counted. Through these two kinds of the measurement, using an IP and BGO detector, we found that the water uptake activity of the plant was drastically reduced under high humidity and dark condition and there was a correlation between the uptake amount of water and light intensity.

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Aluminum (Al) ion binds to root apices and inhibits cell elongation, whereas cell expansion depends on water potential. We investigated whether Al causes water stress in suspension-cultured tobacco cells. After treatment without (control) or with Al (50 μM) in a simple medium containing calcium and sucrose for 18 h, cells exhibited decreases in water content (70% of control) and growth capability (30% of control). Osmotic pressure in Al-treated cells was less than that in control cells (80% of control). When water permeability was compared in the protoplasts prepared from Al-treated and control cells, Al-treated cells exhibited higher efflux rates in hypertonic solution and lower influx rates in hypotonic solution than control cells. These results suggests that Al causes a decrease in osmotic pressure in cells, which leads to the repression of water uptake, the decrease in water content and finally the inhibition of cell expansion.

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Tazawa and Okazaki (1997) measured transpiration of water from the radish hypocotyl segment by measuring a decrease in the weight and reported that transpiration of water occurred linearly with time. However, it seems to be curious, because transpiration of water should increase the concentration of the cell sap and this should decrease the transpiration rate. Thus, transpiration of water from the Chara internodal cell and evaporation of water from distilled water, 300 mM mannitol, 500 mM mannitol and 278 mM KCl solutions were measured at 25 C under controlled relative humidities. The results showed that transpiration of water from the cell and evaporation from the solutions were linear with time and the evaporation rates from distilled water, 300 mM mannitol, 500 mM mannitol and 278 mM KCl solutions were almost equal to each other.

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We investigated β-glucuronosyltransferase (GlcAT) involved in the synthesis of radish arabinogalactan-proteins (AGPs) whose basic carbohydrate structure comprises a β-(1→3)-galactan, branched with consecutive (1→6)-linked β-Gal residues, to which GlcA residues are attached through β-(1→6)-linkages.
The reaction was carried out in a mixture composed of a microsome fraction prepared from 6-d-old radish roots, UDP-[¹⁴C]GlcA, and β-(1→3)-galactan as an acceptor at 25ºC. The GlcA transfer occurred maximally at pH 6.0 in the presence of 30 mM Mn²⁺ and 0.75% triton X-100. Digestion of rdiolabeled product with exo-β-(1→3)-galactanase released GlcA-Gal and GlcA-Gal-Gal as the main products, which were further degraded into GlcA by the treatment with β-glucuronidase [1] purified from Aspergillus niger. GlcA transfer for mature root AGP was insignificant but increased by prior enzymatic digestion with α-L-arabinosidase and endo-β-(1→6)-galactanase [2].
[1] Carbohydr. Res., 333 (2001) 27-39
[2] Carbohydr. Res., in press

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The opening of stomata, mediated by K⁺ accumulation in guard cells, is induced by blue light (BL). BL activates the plasma membrane H⁺-pump in guard cells, and provides driving force for K⁺ uptake. In this study, we present evidence that the pump is the plasma membrane H⁺-ATPase, and that the H⁺-ATPase is activated by phosphorylation of C-terminus in Vicia guard-cell protoplasts. Furthermore, we found that BL induces phosphorylation of Thr950 in the C-terminus of Vicia H⁺-ATPase isoform1 (VHA1), and that binding of 14-3-3 protein to this site is indispensable to the activation of H⁺-ATPase. Analysis of BL-responses in stomatal guard cells using Arabidopsis mutants revealed that phot1 and phot2, BL-receptors for phototropism and chloroplast relocation, act redundantly as BL-receptors mediating stomatal opeing. These findings demonstrated that BL, absorbed by phots, activates the plasma membrane H⁺-ATPase via phosphorylation of the C-terminus with concomitant binding of 14-3-3 protein in stomatal guard-cells.

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