RSG is a bZIP transcriptional activator, which regulates shoot growth by controlling GA level. Suppression of RSG resulted in severe dwarfism. We have demonstrated 1) RSG interacts with 14-3-3 proteins, which are broadly conserved regulatory factors in eukaryotes, through phosphorylated serine-114(PS114) in RSG; 2) Binding with 14-3-3 inhibits the transport of RSG to nucleus; 3) RSG is not statically sequestered in the cytoplasm by 14-3-3, but dynamically shuttling between nucleus and cytoplasm; 4) endogenous level of GA regulates function of RSG by control of intracellular localization; 5) GA promotes nuclear export of RSG by 14-3-3 binding of RSG through phosphorylation of S114 of RSG. We identified CDPK as an RSG kinase that specifically phosphorylates S114 of RSG. Then, commitment of CDPK to the GA-induced cytoplasmic migration of RSG was examined. Furthermore, we examined the effects of over-expressing of CDPK on the feedback regulation of GA biosynthetic genes.

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Recently, we isolated a soluble GA receptor, GID1, from rice. Under the condition of presence of GA, GID1/GA complex interacts with SLR1, a repressor in GA signaling, which results in the degradation of SLR1 and the following GA responses. Here, we report the results of the domain analysis of GID1 for GA binding and SLR1 interaction.
GA binding analyses using mutated recombinant GID1 proteins revealed that the central and C-terminal regions of GID1 are important for the binding. Because GID1 resembles the hormone sensitive lipase (HSL) and the amino acids which corresponding to "catalytic triad" of HSL are present in these regions, we inferred that the conformation of the HSL catalytic active center is conserved in GID1 and is necessary for its GA-binding. We also determined the GID1-interacting domain of SLR1 using yeast-two-hybrid assay. The DELLA and TVHYNP domains of SLR1 were essential for the interaction with GID1.

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DELLA proteins, which are a subgroup of GRAS proteins, are key factors of gibberellin (GA) signaling. Our previous studies revealed that, in rice cells, DELLA protein SLR1 functions as a repressor for GA action and is degraded GA-dependently by ubiquitin-proteasome system through the SCF^GID2 function. However, it is unclear how the GA signal is transduced to SLR1 from bioactive GA molecular. Recently, we succeeded to identify a GA receptor, which has a similar primary structure to that of hormone sensitive lipase and is localized in nucleus. Interestingly, the yeast two-hybrid assay revealed that the interaction between GID1 and SLR1 occurs as a GA dependent manner. Thus we speculate that the complex composed by GID1, SLR1, and GA may recruit an F-box protein, GID2, to initiate the degradation of SLR1. To evaluate this, we have performed some biochemical experiments such as yeast three hybrid assay and pull-down assay in vitro.

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SPINDLY (SPY) encodes an o-GlcNAc transferase, which has been considered to be a negative regulator in gibberellin (GA) signaling by modification and regulation of DELLA proteins through unknown mechanism. We have studied GA signaling using GA related mutants of rice. In the absence of GA, a rice DELLA protein, SLR1, represses GA actions. When a soluble receptor, GID1, binds GA, GID1 interacts with SLR1, resulting in the degradation of SLR1 through the SCF^GID2 proteasome pathway. In order to understand the function of SPY in the GA signal pathway, we produced transgenic rice plants, in which SPY expression is reduced, in the gid1 and gid2 mutant backgrounds. Transgenic lines showed taller phenotype than the control plants not only in the background of gid1 but also in gid2. These results suggest that SPY functions to activate the suppression activity of SLR1 rather than to regulate SLR1 degradation though GID2 function.

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We previously reported a novel Arabidopsis auxin-related mutant, aar1 (antiauxin resistant 1) that shows specific resistance to 2,4-D, yet nevertheless shows a wild type response to IAA and NAA in root elongation assay. The aar1 mutant was initially identified during a process of screening mutants in Arabidopsis thaliana against an antiauxin, p-chlorophenoxyisobutyric acid (PCIB). 2,4-D uptake and metabolism experiments did not show any difference between WT and the aar1-1 plants, implying the mutation alters the pathway for 2,4-D response. Molecular analyses revealed that a lack of a novel gene encodes small acidic protein 1 (SMAP1) confers the most of the the aar1 phenotype. The results suggest that 2,4-D/IAA response pathway in planta is at least in part different.

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Loss-of-function mutants of NPH4/ARF7 are insensitive to auxin and have defects in hypocotyl tropism, hook formation and differential growth of leaf. Especially nph4-103 shows hyponastic cotyledons and leaves. We carried out screening of suppressor mutants of nph4-103 by checking M2 seeds derived from 10,000 EMS-treated seeds of nph4-103. We first isolated mutant seedlings with epinastic cotyledons and rosette leaves. Then, we selected seedlings with almost normal gravitropism in hypocotyl in the M3 generation. Finally, we succeeded in isolating a recessive mutant named suppressor of nph4 1 (snp1). The mutant line displayed serrated and epinastic leaves and suppressed nph4 defects in hypocotyl gravitropism. However, it did not rescue phototropic defects of nph4. Genetic mapping showed snp1 mutation in the lower arm of chromosome 1. We are carrying out fine mapping of snp1 to identify its molecular nature.

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Heat shock proteins (HSPs) are responsible for the refolding of denatured proteins or the folding of newly synthesized proteins. Among these HSPs, HSP90 is the most important HSP that involved in the regulation of various cellular signaling molecules in animals and yeast. HSP90 not only activates signaling molecules but also inactivate them in response to various stimuli. In plant, HSP90 seems to be involved in various cellular events, however the detail is obscure. We investigated the effect of specific inhibitors for HSP90 in Arabidopsis. We use several transgenic Arabidopis that express beta-glucronidase (GUS) genes in response to various stimuli. We found that inhibitors of HSP90 affected the expression of GUS gene in these transgenic Arabidopsis. The data indicates that HSP90 is involved in several cellular signaling. This also suggests that inhibitors of HSP90 illustrate unrevealed regulatory mechanisms on cellular signaling in Arabidopsis.

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Results of recent studies on Arabidopsis thaliana, the His-Asp phosphorelay signaling plays an essential role in the primary responses of plants to cytokinin. The phosphorelay circuitry consists of His-kinases (AHKs), phosphotransfer factors (AHPs), and transcription factors (type-B ARRs). Clarification of the downstream signaling network must await further extensive examination, which should include a comprehensive genetics of 11 type-B ARR genes. Here we isolated a set of mutant alleles, including arr1, arr10, arr12, arr13 and arr21. Plants carrying each one of these mutant alleles were characterized with reference to the inhibition of root elongation and the stimulation of callus development by cytokinin. The results suggested that the ARR1, ARR10, and ARR12 genes are more critically implicated in these cytoinin-responses. Then, an arr10/arr12 double mutant was selectively characterized deeper, by also employing a DNA microarray analysis. The results will be discussed with regard to the His-Asp phophorelay-mediated cytokinin-signaling network.

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Cytokinin is a plant hormone regulating plant morphogenesis through cell differentiation and proliferation. Early cytokinin signaling in Arabidopsis cells occurs through His-Asp phosphorelay from the cyrokinin receptors to the transcription-factor-type response regulators (type-B ARRs). ARR1 directly transactivats many cytokinin primary responsive genes (CPRGs). Each promoter region of CPRGs has many in vitro ARR1 recognition sequences ((A/G)GAT(C/T), ARR1-seq). Here, we tested contribution of each ARR1-seq preceding CPRGs on transactiation by ARR1 in vivo.
A type-A ARR, ARR6 is a CPRG diriectly transactivated by ARR1, is preceded by a 750-bp promoter region containing 5 copies of ARR1-seq. We constructed mutant ARR6 promoters which contains a base substitution within recognition sequences and then fused to the luciferase gene. Each of them was introduced into onion epidermal cells together with ARR1ΔDDK, generating constitutively active truncated ARR1 protein. As a result, we found that each ARR1-seq differently contributes to transactivation by ARR1 in vivo.

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Brz (brassinazole) was synthesized as the first specific inhibitor of brassinosteroid biosynthesis. Target of Brz220 is the cytochrome P450 enzyme encoded by DWF4. In order to analyze in detail the mechanisms of brassinosteroid signal transduction, we screened for mutants that showed longer hypocotyls than wild type when grown with Brz220 in the dark, and designated bil mutants (Brz-insensitive-long hypocotyl). We identified a semidominant mutant, bil5, from fast neutron-treated lines. Hypocotyl elongation of these plants on Brz medium was at least twice that of the wild type. Adult bil5 plants showed pale green, thin stems, thin leaves and shortened stem length. The pale-green leaves of bil5 were in contrast to the dark-green leaves of brassinosteroid-deficient mutants, and chloroplast gene expression was lower in bil5 than in wild type. We also identified a pale green mutant bpg1 (Brz-insensitive-pale green1) that could not be recovered to high green by Brz in the light.

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Brassinosteroids are steroid hormones that play an essential role in plant growth and development. Brz (brassinazole) is the first specific inhibitor for brassinosteroid biosynthesis. Target of Brz220 is the cytochrome P450 enzyme encoded by DWF4. In the light, plants treated with Brz220 display dwarfism with the dark-green leaves. In the dark, Brz causes deetiolation phenotype with short hypocotyls and open cotyledons, similar to those of BR-deficient mutants. We tried to screen hypocotyl-elongation mutants in the dark with Brz, which can be anticipated as brassinosteroid signaling-accelerated mutants. As a result, we identified Brz insensitive mutant bil1 from EMS-mutation lines, and bil5 from fast neutron-mutation lines. In order to clarify in detail the mechanisms of brassinosteroid signal transduction, we are trying to screen new bil mutants from Arabidopsis activation-tag lines.

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Photosystem I (PSI) cyclic electron transport is essential for efficient photosynthesis. However, the route taken by electrons is still unclear. Recently, it was found that the cytochrome b₆f complex associates with the FNR and contains an novel heme, which is not conserved in the cytochrome bc₁ complex. Based on the findings, it was suggested that the cytochrome b₆f complex was related with the PGR5-dependent cyclic pathway.
We evaluated whether the PGR5-depenedent pathway is through the cytochrome b₆f complex or not by using an Arabidopsis mutant, pgr1(proton gradient regulation). In pgr1, Q-cycle activity is hypersensitive to acidification of the thylakoid lumen because of an amino acid alternation in the Rieske subunit of the cytochrome b₆f complex. Neither in vivo nor in vitro assays showed that the defect in Q-cycle activity affected the PGR5-dependent pathway. It is suggested that Fd donates electrons directly to PQ in PGR5-dependent pathway.

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Cyclic electron transport through photosystem (PS) I has been believe to work in photosynthesis by supply of extra ATP. We investigated contribution of cyclic electron transport in C3 photosynthesis using pgr5, an Arabidopsis thaliana mutant deficient in the ferredoxin-dependent cyclic activity. For comparison of CO₂ assimilation between wild type (WT) and pgr5, we used low-light grown plants showed no difference in growth. Under light-limiting conditions or CO₂-limiting conditions, net CO₂ assimilation in pgr5 was the same as in the WT. Although at higher irradiance under non CO₂-limiting conditions where energy production by cyclic and non-cyclic electron transport limit photosynthesis, net CO₂ assimilation was 14% lower in pgr5 than in the WT, inactivation of PSI occurred in pgr5. We propose two possible functions of the PGR5-dependent pathway: 1) supply of ATP, and 2) lowering of the electron pressure at the accepter side of PSI.

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Light energy is excessive at low temperature due to lowered activity of CO₂ fixation. Higher plants have multiple mechanisms to adapt low temperature, including accumulation of pigments, carotenoids and anthocyanins, and modification of LHCII composition. In order to characterize the molecular bases of the adaptation to excessive light energy under low temperature conditions, Arabidopsis thaliana plants were treated with continuous low-temperature or fluctuating-temperature conditions. There was no significant different in maximum activity of photosystem II (Fv/Fm). However, the fluctuation of temperature modified the light-intensity-dependence of NPQ. Based on this information, mutans defective in the regulation of photosynthetic electron flow under low temperature were screened using chlorophyll fluorescence imaging. The mutants defective in PS I cyclic electron transport pathway were also characterized.

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We tested the hypothesis that the plants grown under high light-intensity (HL-plants) had a large activity of cyclic electron flow around PSI (CEF-PSI), compared to the plants grown under low light-intensity (LL-plants). To evaluate the activity of CEF-PSI, the relationships among photosynthesis rate, quantum yields of both PSII and PSI, and Chl fluorescence parameters were simultaneously analyzed in intact leaves of tobacco plants. Tobacco plants were grown under the different light-intensities (150 and 1,100 μmol photons m^-2 s^-1) and the different amount of nutrient supply. HL-plants showed a larger value of non-photochemical quenching (NPQ) of Chl fluorescence at the limited activity of photosynthetic linear electron flow (LEF), compared to LL-plants. Furthermore, HL-plants had a larger activity of CEF-PSI than LL-plants had. These results implied that HL-plants dissipated the excess photon-energy against photosynthesis through NPQ by enhancing the ability of CEF-PSI to induce acidification in lumen side of thylakoid membranes.

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Plant regulates metabolism in response to changing photoenvironments, which is an important physiological processes for optimal growth. Recent research proposed that the redox state of PQ functioned in quantitative and qualitative regulation of cell metabolisms as signals. To reveal its physiological function, we produced a transplastomic tobacco plant, which overexpresses Fd in chloroplast, hereafter called Fd-tobacco. F₀ of Fd-tobacco, a parameter of chlorophyll fluorescence, was strongly increased, indicating that the stimulated reduction of PQ in Fd-tobacco, compared to wild-type. Fd-tobacco did show no difference of both CO₂-assimulation and the content of Rubisco from wild-type. Further, Fd-tobacco did show no difference of total APX and chloroplast APX activities from wild-type. However, Cu/Zn-SOD and Fe-SOD activities in Fd-tobacco were reduced, compared to wild-type. Now, we analyze proteins functioning in photochemical systems to elucidate the redox network regulated by Fd.

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To understand molecular mechanisms of drought/strong light stress-tolerance in wild watermelon, changes in the protein composition in the leaves under the stress were comprehensively analyzed by proteomic approach. Approximately 900 protein spots were detected by two-dimensional gel electrophoresis analysis, 82 and 29 spots of which were up- and down-regulated, respectively by the stress. Mass spectrometry and database analysis revealed that the up-regulated proteins included heat shock, photosynthesis, antioxidative defense, protein degradation/synthesis, membrane traffic and signaling proteins. Interestingly, several Rieske proteins of chloroplast cyt b₆f complex newly appeared under the stress. By contrast, spot intensity for FNR was markedly decreased by the stress. These observations suggest that dissipation of excess light energy is regulated by a dynamic change in the protein composition of the photosynthetic apparatus, thereby enabling the survival of the plants under drought/strong light stresses.

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Excess light energy absorbed in the leaves must be dissipated safely to prevent photoinhibition, but molecular mechanisms for the energy dissipation are poorly understood. In this study, proteomics and western blot analyses of wild watermelon leaves revealed that the amount of the ε subunit of chloroplast ATP synthase decreased markedly in drought/excess light stress, whereas that of the β subunit did not change in this condition. Comparison of electron transport activities determined in the presence and absence of an uncoupler indicated that thylakoids prepared from stressed leaves were uncoupled, and addition of the recombinant ε subunit significantly restored coupling state of the thylakoids. These observations strongly suggest that the leakage of protons from thylakoid lumen to stroma is induced in excess light conditions. This proton leakage may avoid hyper-accumulation of protons in the lumen, enabling the continuous cyclic electron flow around PSI and energy dissipation under excess light conditions.

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Oxidative stress inhibits the repair of photosystem II (PSII), especially the de novo synthesis of proteins, but does not damage PSII. To investigate the mechanism of the inhibition of protein synthesis by oxidative stress, we constructed an in vitro translation system from cyanobacterium Synechocystis sp. strain PCC 6803 and analyzed the synthesis of the D1 protein under oxidative stress. Cell extracts including thylakoid membranes were incubated with psbA2 mRNA, ¹⁴C-labeled Leu, and several compounds required for the translational reaction. The synthesis of the D1 protein was detected in light. This translation system did not require DTT, which is indispensable for in vitro translation systems from wheat germ and tobacco chloroplast, indicating that some reducing compounds accumulated in cyanobacterial extracts. Hydrogen peroxide inhibited the synthesis of the D1 protein, but the inhibition was recovered by addition of catalase, suggesting that the oxidative stress-induced inhibition of translation might be reversible.

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Response of photosystem II (PSII) to high temperature in a cell-suspension culture of soybean (Glycine max [L.] Merr.) was studied. High temperatures inactivated PSII in two distinct manners. Inactivation of PSII by moderately high temperatures, such as 41^oC, was recovered by transfer of cells to 25^oC. The recovery of PSII required light but not the synthesis of proteins de novo. By contrast, temperatures higher than 45^oC inactivated PSII irreversibly. The thermal stability of PSII was enhanced when the growth temperature was shifted from 25^oC to 35^oC. This acclimative response was reflected in isolated thylakoid membranes; thylakoid membranes from cells that had been grown at 35^oC exhibited greater thermal stability of PSII than those from cells grown at 25^oC. These observations suggest that factors responsible for the enhancement of the thermal stability of PSII might be associated with the isolated thylakoid membranes.

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Photosystem II (PSII) is particularly sensitive to high temperature. When photosynthetic organisms are grown at moderately high temperatures, the thermal stability of PSII is increased. This acclimative response is observed in cyanobacteria and higher plants. We studied the molecular mechanisms responsible for the acclimation using purified PSII complexes from a mutant of Synechocystis sp. PCC 6803, which expresses a His-tagged CP47 protein. The thermal stability of the purified PSII complexes was increased by addition of the lumenal proteins of thylakoid membranes isolated from cells that had acclimated to a high temperature (38^oC). The lumenal proteins from non-acclimated cells were less effective in increasing thermal stability. These results suggested that proteins responsible for the enhancement of the thermal stability of PSII were involved in the lumenal proteins from the acclimated cells. A MALDI-TOF/MS analysis of the lumenal proteins associated with the PSII complexes identified several proteins, including hypothetical ones.

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Standard (1 mM N) and high (4 mM N) levels of N were supplied to rice plants at the early, middle and late stages of senescence of the eighth leaves of rice. The levels of rbcS and rbcL mRNAs and the amount of Rubisco synthesized were temporarily increased after each N treatments and were greater in the 4 mM-N treatment. The ratios of the amount of Rubisco synthesized to the levels of rbcS and rbcL mRNAs decreased with the progress of leaf senescence. The amount of Rubisco synthesized in the eighth leaf blade largely increased by the removal treatment of other leaves and all tillers at the late stage of leaf senescence. These results suggest that, although senescent leaves have an ability to synthesize Rubisco in response to N supply, the translational efficiencies decrease with the progress of leaf senescence.

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DNA microarray analysis in Synechococcus elongatus showed that under CO₂-limited conditions, sigD, nblA, hliA, and the genes required for the CO₂-concentrating mechanism were activated, whereas the genes related to ribosome, nitrogen assimilation, PSI, phycobilisome, and ATP-synthetase were repressed as previously reported in Synechocystis sp. Deficiency of CmpR, a LysR-family transcription factor, caused not only decline of the expression level of the cmpABCD operon for an ABC bicarbonate transporter but also de-repression of the ndhF3 operon, encoding a low CO₂-inducible CO₂ uptake system, under high CO₂ conditions. In Synechocystis sp., expression of the ndhF3 operon is negatively regulated by NdhR, a homolog of CmpR. Since S. elongatus lacks NdhR, it appears that Synechococcus CmpR functions both as CmpR and NdhR. Surprisingly, the expression of the nitrate assimilation genes was activated under high-CO₂ conditions in the cmpR mutant of Synechococcus.

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The psychrophilic phytoplankton perform photosynthesis under environments of low temperature and very weak light. In this study, we analyzed the photosynthetic property of psychrophilic phytoplankton and ice algae collected in Franklin Bay, Canadian Arctic, in late May, 2004, by using a pulse amplitude modulation fluorometer. These values were systematically compared with those obtained from mesophilic marine diatoms grown under various irradiances in laboratory. The highly shade-adapted features of ice algae and phytoplankton were disclosed through this comparative analysis. It was also found that the non-photochemical quenching was much higher in psychrophilic samples than that in mesophilic diatoms grown under moderate irradiance. Our comparative analysis in mesophilic diatoms also indicated that the acclimation strategies to growth irradiances were variable between species.

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In plant, hemes are distributed to various organelles. In addition, tetrapyrroles act as plastid-derived signals that regulate nuclear photosynthetic genes. However, mechanism of distribution of tetrapyrroles into organelles is poorly understood. In higher plants, tetrapyrroles are presumably distributed with tetrapyrrole binding proteins (TBP). Here, to clarify this mechanism, we characterized genes of Arabidopsis, which exhibited homology to animal p22HBP, a tetrapyrrole binding protein.
In Arabidopsis, 6 genes were identified as p22HBP homologs. When the gene with highest homology (At1g17100) was fused to GFP and expressed in onion, GFP fluorescence was detected in cytoplasm. Increase in absorption around 400 nm was observed, when heme was added to the recombinant protein with Kd 0.4 µM. The protein exhibited the affinity to protoporphyrin IX (Kd 0.4 µM). Reconstitution with apo-peroxidase showed that the protein binds heme reversibly. These results suggest that At1g17100 is cytosolic TBP, which carries tetrapyrroles in higher plants.

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Atmospheric NOx, which includes nitric oxide and nitrogen dioxide, is usually regarded to act as N source or to exert detrimental effects on plants. We found that Nicotiana plumbaginifolia cultured in air with ¹⁵N-labelled NOx (100 to 200 ppb) increased the biomass yield, element contents, total free amino acids and crude proteins¹⁾. Furthermore, Isotopic mass spectrometry has shown that the nitrogen derived from NOx made only a negligible contribution in the total nitrogen of plants. We are currently investigating the effects of atmospheric NOx on Arabidopsis plant. Our findings indicated that the atmospheric NOx vitalizes plants.
1) M. Takahashi et al. New Phytologist 168: 149-154 (2005).

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We recently reported that plants ubiquitously form previously unidentified nitrogen (UN) compounds from the airborne nitrogen dioxide (NO₂) and the soil nitrate, leading us to postulate that plants possess not-yet-unveiled metabolisms of inorganic nitrogen. Among the examples of such UN compounds is a group of S-nitrosylated forms of biological thiols called S-nitrosothiol (RSNO), possibly mediating plant NO signaling and reactive nitrogen stress. When exposed to NO₂ or treated with nitrate, Arabidopsis plants significantly accumulated soluble RSNO of high-molecular nature. Over- and down-expression in Arabidopsis of S-nitrosoglutathione reductase (GSNOR), the only reported plant metabolic enzyme for RSNO, revealed the central role of this enzyme in RSNO metabolism. Moreover, GSNOR overexpression improved the ability of transgenic Arabidopsis to uptake and assimilate NO₂ and nitrate, suggesting the possible crosstalk between RSNO metabolism and inorganic nitrogen assimilation.

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Approximately one-third of nitrogen of nitrogen dioxide (NO₂) or nitrate taken up into plants was found to be converted to a previously unknown organic nitrogen (designated UN) that was not recoverable by the Kjeldahl method^1,2). We isolated UN-rich fractions by using the chromatography from the extract of Arabidopsis leaves fumigated with ¹⁵N-labelled nitrogen dioxide. Their structures were analyzed by the EA/MS, LC/MS and NMR. A UN-bearing compound was found to be 1, 2, 3-thiadiazole and 1, 2, 3-thiadiazoline derivatives³⁾. This was the first report on the presence of thiadiazole rings in biological systems. Thiadiazole-based pesticides such as BTH [benzo(1,2,3)thiadiazole-7-carbothioic acid] and tiadinil (3'-chloro-4,4'-dimethyl-1,2,3-thiadiazole-5-carboxanilide, V-get) reportedly function as a vital signal to induce SAR (systemic acquired resistance) against rice blast fungus.
1) H. Morikawa et al., Planta 219:14-22 (2004).
2) H. Morikawa et al., Z. Naturforsch.,60c: 265-271 (2005).
3) K. Miyawaki et al., Org.Biomol.Chem.,2:2870-2873 (2004).

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Nitrite is generated in cytoplasm as an intermediate metabolite in the nitrate assimilation of plant and converted into ammonia in chloroplast (plastid). Rapid nitrite loading into chloroplast is essential for effeicent nitrate assimilation as well for removal of toxic nitrite ions. We cloned previously unknown nitrite transporter gene (CsNitr1) from cucumber leaves. CsNitr1 encodes CsNitr1-L with 11 transmembrane regions and one central hydrophillic loop and there is its isoform (CsNitr1-S) with N-terminal one transmembrane region missing in greening cucumber seedlings. In this study, induction and mechanism of expression of two isoforms of CsNitr1 was examined and their functions were elucidated from the subcellular localization of CsNitr1-L and CsNitr1-S that were fused with sGFP at their C-terminus. Outside-acidic pH gradient-driven transport function of CsNitr1-L is shown by the nitrite uptake by the intact chloroplasts isolated from leaves of tobacco transformed with CaMV35S::CsNitr1-L-sGFP.

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The nitrite transporter (CsNitr1-L) was cloned from cucumber. CsNitr1-L is localized in chloroplast envelopes. CsNitr1-L has an isoform (CsNitr1-S) having a core sequence overlapping with CsNitr1-L. CsNitr1-S is an efflux type nitrite transporter. L type may load nitrite into the chloroplast during nitrate assimilation. The CsNitr1-S is located in the plasma membrane and is anticipated to transport nitrite out of the cell. To better understand the function of the CsNitr1-S, transgenic tobacco lines overexpressing and suppressing the nitrite transporter were made by inserting the CsNitr1-S in the sense and antisense directions using Agrobacterium tumefaciens. The genetic and phenotypic traits of the transgenic plants were examined. The CsNitr1-S has a vital role in the germination stage of tobacco seeds. Other results have indicated some drought tolerance and early flowering of antisense plants. The relatedness of the nitrite transporter function with such phenotypes will be discussed.

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We analyzed the time course relationships between the expressions of three genes, encoding high-affinity nitrate transporter (NRT2.1, NRT2.2) and an important activator for NRT2 transport activity in plants (NAR2.1), and the amounts of nitrate uptake by rice seedlings. The expressions of NRT2.1 and NAR2.1 were induced by nitrate and delayed by ammonium pretreatment. During the duration of nitrate treatment, transcripts of both genes were decreased by ammonium pretreatment. These phenomena were also found in nitrate reductase gene expression, suggesting that the expressions of three genes are coordinately controlled by similar regulation mechanisms. On the other hand, NRT2.2 was transcribed by ammonium. The induction of nitrate uptake was synchronized with the changes of NRT2.1 and NAR2.1 expression. These results indicate that NRT2.1 and NAR2.1 play an important role in the nitrate uptake in rice roots.

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In rice, expression of NADH-GOGAT and OsAMT1;1-2 was up-regulated by glutamine (Gln), respectively. This suggests that the occurrence of Gln-sensing and -signaling systems in rice. To clarify the molecular mechanism of the Gln-sensing and -signaling systems, expression of rice ACT domain repeat proteins (OsACRs), as a candidate for Gln sensor, has been analyzed. OsACRs are composed of four ACT putative amino acid binding domains, which are similar to bacterial Gln sensor GlnD. OsACR3 could bind with Gln-Sepharose, and could interact with a chaperon, OsHSP18.0-CII in yeast two-hybrid system. In this annual meeting, we report the binding capacity of OsACR3 with native Gln by using Isothermal Titration Calorimetry. We also describe the interaction between OsACR3 and OsHSP18.0-CII proteins in vivo.

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NADH-glutamate synthase (NADH-GOGAT) reaction requires in
2-oxoglutarate(2-OG) as a carbon skeleton. We have suggested that mitochondrial NAD-isocitrate dehydrogenase (IDH) is a candidate for providing 2-OG to NADH-GOGAT reaction in NH4+ assimilation in roots of rice seedlings. In contrast, the origin of 2-OG in young leaf blades and spikelets is not well understood.
We have focused on three 2-OG-producing enzymes: four distinct isozymes of NADP-isocitrate dehydrogenase (ICDHs), IDH and glutamate dehydrogenase (GDH).
Immunoblot analysis showed that the change in contents of ICDH, IDH and GDH proteins were similar to thsese of NADH-GOGAT in leaf blades at the different leaf positions. The protein for ICDH, IDH and GDH accumulated in spikelets at the early stage of ripening. However, NADH-GOGAT protein content peaked at the middle stage of ripening. Thus, metabolic route of 2-OG generation is still unclear. We are now preparing RNAi-mediated OsICDH1-knockdown rice to obtain more imformation on this important event.

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To understand an individual role of three GS1 isoenzymes (GS1;1, GS1;2 and GS1;3) in rice (Oryza sativa L.) spikelets, expressions of these genes were determined during ripening. Localization of these gene products in situ were also performed. OsGS1;1 and OsGS1;2 were expressed in leaf blades, leaf sheath, roots and spikelets, while OsGS1;3 was expressed only in spikelets. OsGS1;1 mRNA was accumulated mainly at the early stage of ripening. In contrast, OsGS1;3 was the major gene product at the late stage. Transgenic rice expressing GUS under the control of corresponding GS1 promoter showed that all three genes were active in dorsal vascular bundles, nucellar epidermis and nucellar projection. In addition, OsGS1;3 was also expressed in small vascular bundles. Physiological function of each GS1 in the spikelets will be discussed.

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Rice (Oryza sativa L.) plants possess three homologous but distinct genes for cytosolic glutamine synthetase (GS1): these are OsGS1;1, OsGS1;2 and OsGS1;2. We characterized knockout mutants caused by insertion of endogenous retrotransposon Tos17 into OsGS1;1. This mutant contained undetectable amount of GS1 protein and its activity in the leaf blades.
Mutants showed severe retadation in growth rate under NH₄⁺-containing hydroponic solution. Glutamine pool in the leaf blades of the mutants was approximately 60% lower than that of the wild-type. Microarray analysis successfully identified several genes, either up- or down-regulated, in the mutants.
The results indicate that GS1;1 is important for normal growth of rice and other two GS1 isoenzymes as well as GS2 were not able to compensate the function of GS1;1.

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N-acetylglutamate kinase (NAGK) is an enzyme of the arginine biosynthesis pathway. It is activated by the PII protein in cyanobacteria and shown to interact with PII in arabidopsis and rice. We identified the NAGK gene of Physcomitrella patens, PpNAGK1, in the genome sequence database. The predicted protein is 55% identical (90% similarity) with A. thliana NAGK. Similar to NAGK of other plants, PpNAGK1 was shown to be located in chloroplasts. The PpNAGK1 mRNA level was not affected by the cellular nitrogen status but was high in the light and low in the dark. These results suggested that the NAGK activity is regulated by light and/or photosynthetic products. Since the highly conserved Ser residue in the T-loop of PII, which is essential for interaction with NAGK in cyanobacteria, is replaced with Thr in the moss PII protein, we are examining whether PII interacts with NAGK in the moss.

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Glutamate glyoxylate aminotransferase (GGAT) catalyzes the reaction of glutamate and glyoxylate to 2-oxoglutarate and glycine. GGAT plays an essential role in the photorespiratory transamination in the peroxisome. We previously identified a photorespiratory GGAT gene (GGAT1) by analyzing a knockout line of the alanine 2-oxoglutarate aminotransferase (AOAT) -like gene (Igarashi et al. Plant J 33, 975- 2003).
To investigate the function of the GGAT1 gene in amino acid and related metabolisms, we obtained transgenic Arabidopsis plants over-expressing GGAT1. The transgenic plants had markedly higher free serine and glycine levels. The serine and glycine content clearly correlated with the level of GGAT1 mRNA expression and with the GGAT activity.
Furthermore metabolic profiling by GC-MS analysis indicated that GGAT1 over-expression plants accumulate many kinds of metabolite including unidentified peaks. These results suggest that the existence of the new metabolic pathway related photorespiration.

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Nitrogen utilization in higher plants is regulated by many genes at several steps. Toward identification regulatory genes involved in NH₄⁺ uptake, assimilation and subsequent metabolism, chromosome segment substituted lines (CSSLs) developed between Koshihikari (japonica rice) and Kasalath (indica rice) were employed to detect QTLs of root elongation under various concentrations of NH₄⁺. Germinated seedlings were hydroponically grown in an outdoor-growth chamber supplied with 5, 50 or 500 μM NH₄⁺ for a week, and then maximum length of roots were determined. A probability of 0.01 (t-test) was used as the threshold for the detection of QTL. The root length of Kasalath was longer than that of Koshihikari under these NH₄⁺ applications. Many QTLs were detected for root elongation under various concentrations of NH₄⁺ on all chromosomes, except chromosome 3 and 10. In particular, Kasalath allele on QTL of chromosome 6 greatly promoted the root elongation. High-resolution mapping is now in progress.

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It was reported that the administered sinapate was incorporated into syringyl lignin of Robinia pseudoacacia and that 4CL isoenzyme in R. pseudoacacia utilized sinapate unlike other plant 4CLs. These findings are indicative of an alternative route of syringyl lignin biosynthesis where sinapyl alcohol is biosynthesized passing through sinapate and sinapoyl CoA. We therefore examined enzyme activities involved in lignification in the developing xylem of R pseudoacacia. Protein extract from the developing xylem of R pseudoacacia exhibited methylation activities toward 5-hydroxyferulate and 5-hydroxyconiferyl aldehyde when these substrates were mixed. In addition, 5-hydroxylation activity with ferulate was not completely abolished by the presence of coniferyl aldehyde in the reaction. These results suggest the presence of biosynthetic route of syringyl lignin via sinapate in R pseudoacacia and that the syringyl lignin biosynthetic pathway differs among angiosperm species.

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Lithospermum erythrorhizon produce and highly accumulate a red naphtoquinone pigment shikonin in the root epidermis. A key regulatory enzyme of shikonin biosynthesis is p-hydroxybenzoate(PHB):geranyltransferase (LePGT), which belongs to membrane-bound prenyltransferase family involved in CoQ biosynthesis. LePGT shows strict substrate specificity for geranyl diphosphate (GPP) unlikely to other members of this enzyme family. LePGT composed of 307 amino acids has 9 transmembrane domains, which was functionally expressed in yeast, but the protein yield was very low. In this study we have expressed LePGT in Baculovirus system for the purpose of X-ray crystallographic analysis. The recombinant LePGT expressed in Sf9 have shown much higher specific activity and high protein yield than those of in yeast, which has been solubilized retaining the enzymatic function. We have further established the purification protocol using His-tag attached LePGT.

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Catharanthus roseus cell suspension cultures are capable of converting curcumin applied exogenously to a series of glucosides with drastically enhancing water solubility. A cDNA clone encoding an enzyme catalyzing the transfer of glucose from UDP-glucose to phenolic hydroxyl groups of curcumin (UCGT), was already isolated. Another enzyme, UDP-glucose: curcumin glucoside 1,6-glucosyltransferase (UCGGT), which catalyzes the transfer of glucose to a sugar moiety of curcumin glucoside, has not been characterized so far. In this study, we partially purified UCGGT from cultured C.roseus cells by ammonium sulfate precipitation, anion exchange-, hydrophobic interaction-, hydroxyapatite-, affinity dye-ligand-, and gel-filtration chromatography. The UCGGT activity was separated from the UCGT activity, and purified 250-fold in specific activity. Partially purified UCGGT catalyzed glucose transfer to a sugar moiety of curcumin glucoside to produce its gentiobioside. The enzyme also exhibited the glucose transfer activity to flavonol glucosides.

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Cultured Coptis japonica cells produce a large amount of anti-microbial isoquinoline alkaloid, berberine. Here, we report the isolation and partial characterization of bHLH transcriptional factor in the regulation of berberine biosynthesis.
After the sequence determination of about 5000 EST isolated from high alkaloid producing cells, we analyzed the regulatory function of some EST clone as the transcriptional regulator using RNAi. Among 27 candidates, dsRNA of clone#48 induced a marked reduction of transcripts of 6OMT and all other biosynthetic genes in berberine biosynthesis, but the transcript levels of actin, GAPDH, Chorismate mutase were not affected. Since this clone had conserved bHLH motif, we named it CjbHLH1. Our results suggest that CjbHLH1 would be a key regulator of berberine biosynthesis. To confirm this idea, we constructed a constitutive expression vector and transformed some alkaloid producing plant species. Preliminary characterization of transformants will be discussed.

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Coenzyme Q (CoQ), an electron transfer molecule in the respiratory chain and a lipid-soluble antioxidant, is present in almost all organisms. CoQ is composed of a benzoquinone moiety and an isoprenoid side chain. The length of side chain varies depending on the organism, and is defined by the nature of the polyprenyl diphosphate synthase. In Arabidopsis, ER-localization of polyprenyl diphosphate synthase was reported. By producing the decaprenyl diphosphate synthase (DdsA, from Gluconobacter suboxydans) at the mitochondria, we successfully changed the length of side chain of CoQ in rice. The production of endogenous CoQ9 was almost completely replaced with that of CoQ10. Expression of DdsA at the mitochondria increased accumulation of total CoQ amount in seeds. These results show that the synthesis of polyprenyl diphosphate was far more effective in CoQ biosynthesis at the mitochondria than at the ER, and demonstrated the effectiveness of foreign enzyme expression at the non-native site.

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Plants have two independent routes for the biosynthesis of isoprene units; the cytosolic mevalonate (MVA) pathway and the methylerythritol phosphate (MEP) pathway in the plastid. Recent studies have shown that a fraction of common isoprenoid precursors is exchanged between the cytosol and the plastid. To identify regulators of isoprenoid biosynthesis in plants, we screened Arabidopsis mutants that were resistant to fosmidomycin (FSM), an inhibitor for 1-deoxy-D-xylulose 5-phosphate reductoisomerase (DXR) of the MEP pathway, during seedling development. Genetic analysis of an isolated mutant revealed that a loss-of-function mutation in the PRL1 gene, which encodes a regulatory WD-repeat protein, conferred the FSM-resistant phenotype. Our results from immunoblot analysis and feeding experiments with ¹³C-labeled precursors suggest that upregulation of the MEP pathway, but not an enhanced influx of MVA-derived precursor(s), is responsible for the FSM-resistant phenotype of the prl1 mutant.

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Sterols are essential components in eukaryotes. In animals and fungi, lanosterol is an intermediate of sterol biosynthesis. In contrast, in higher plants, only cycloartenol has been recognized as the biosynthetic intermediate. Oxidosqualene cyclases (OSCs) called lanosterol synthase (LAS) and cycloartenol synthase (CAS) are responsible for the respective reactions. We recently found that the OSC7 gene of a model legume Lotus japonicus encodes a LAS. In addition, the phylogenetic relationship between LAS and CAS in eukaryotes suggested that CAS is the more primitive OSC. The two amino acid residues that were reported to be essential for CAS were introduced into OSC7 by site-directed mutagenesis. The OSC7-N478H-V482I double mutant produced cycloartenol, together with larger amount of parkeol. It was thus suggested that OSC7 has been optimized for LAS activity after it was diverged from CAS. The optimization implies some physiological importance of lanosterol synthesis in plants.

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Petal 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 of the epidermal colored cells, which is arise from an active transport of Na⁺ and/or K⁺ through NHX1. During flower opening process the cell volume of epidermal layers become three to four times larger than those of buds. This elongation growth may be caused by water transport into vacuoles responding to increasing of osmotic pressure due to transport of cations into vacuoles. To clarify the mechanism we analyzed the enlargement, the osmotic pressure and ion concentration of the colored cells during flower opening process. The major cation in the colored protoplast was K⁺ and the concentration of Na⁺ was low. Major counter anion in the cells was Cl^-. The development of NHX1 and ion transport will be discussed.

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The seismonastic movement of Mimosa plant is triggered by a sudden change in turgor pressure in the motor cells of the pulvinus. Aquaporin plays a role in the rapid water transport through a plasma membrane according to the change in such an osmotic pressure. We focused our analysis on the roles of aquaporins during the seismonastic movement. In the present study, we isolated nine cDNA clones of Mimosa aquaporin and we measured the water membrane permeability (Pf) using a Xenopus oocyte expression system. MpPIP1;1 exhibited a low Pf value, whereas MpPIP2s exhibited high Pf values in an isoform-dependent manner. In addition, a significant increase in Pf value was observed when MpPIP1:1 and MpPIP2;1 cRNAs were coexpressed in oocytes. We will elucidate whether this acceleration of water permeability occurs by coexpression of MpPIP1;1 and other MpPIP2 isoforms and how this regulatory mechanism contributes to the seismonastic movement.

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K⁺ uptake system by KtrB is essential for the adaptation to hyperosmotic change and other types of transport proteins also seem to be involved in the osmoregulation in cyanobacterium Synechocystis sp. PCC 6803. Aquaporins comprise a family of water-transporting membrane protein. The Synechocystis genome has a single copy of the gene encoding putative aquaporin, aqpZ (Shapiguzov et al. 2005). We have characterized the transport activity of AqpZ in Xenopus laevis oocytes. The oocytes expressing AqpZ showed an efficient water channel activity. The AqpZ-mediated water permeability was not inhibited by HgCl₂. The activity was also not influenced by phosphorylation or different external pH. Synechocystis AqpZ-null mutant grew better than that of wild-type strain under the hyperosmotic stress. This suggested that mutation of the aqpZ gene led to prevent from water loss from the cells in the hyperosmotic conditions.

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Characean internodal cells generate receptor potential (ΔE_m) at the moment of both compression and decompression ((ΔE_m)_S and (ΔE_m)_E, respectively) in long-lasting stimulation. In this report, to verify our hypothesis that the ΔE_m depends on the degree of membrane deformation, the ΔE_m in internodal cells treated with a potent water channel inhibitor, HgCl₂, was investigated.
After the whole-cell treatment with HgCl₂, a long-lasting stimulus induced relatively small membrane deformation and ΔE_m, compared with those of intact cells. On the other hand, the cell, in which only the stimulated portion had been treated with HgCl₂, presented deformation with similar amplitudes of untreated cells but small ΔE_m as is the case in the whole-cell, suggesting a possibility that the generation of the ΔE_m may be linked to water movement especially across the plasma membrane of the stimulated portion.

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KAT1 encoding a K⁺_in channel is expressed primarily in Arabidopsis thaliana guard cell and KAT1 contains six-membrane spanning regions. Several putative phosphorylated residues exist in the cytosolic region of the channel. It has been indicated that the activity and the gating of KAT1 channel is modulated by phosphorylation, such as cAMP-dependent protein kinase or ABA-responsive protein kinase mediating phosphorylation, and the evidence for KAT1 phosphorylation in planta has been provided by biochemical approaches. Thus phosphorylation could be considered as a switch that allows the channel to respond to their usual stimuli. In this study, we performed two-electrode voltage clamp analysis of KAT1 channel activity under the condition where protein kinase was activated. Recordings of KAT1 currents were performed in Xenopus laevis oocytes. Time course of changes in current were obtained in oocytes expressing wild type KAT1 and the mutants.

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