RuBisCO is a key enzyme that catalyzes the addition of CO2 to ribulose-1,5-bisphosphate (RuBP) and produces two molecules of 3-phosphoglycerate (3-PGA). This enzyme is localized in the stroma of plastids and consists of two octamers of large subunit (encoded by the rbcL gene) and small subunit (encoded by the rbcS gene).
Cyanidioschyzon merolae, a unicellular rhodophytes contains an operon that is constituted of rbcL, rbcS and cbbX in a tandem array. The cbbX genes are not found in the chlorophytes, glaucophytes and cyanobacteria but are detected in some kinds of proteobacteria. Phylogenetic tree inferred from RbcLs indicates that RbcL of rhodophytes are more closely related to those of β-proteobacteria than those of non-rhodopyte plants or cyanobacteria. Interestingly, a cbbX homologue is also encoded in the cell nucleus of C. merolae. In this study, we performed functional analysis of the plastid and nuclear CbbX proteins and discussed the relationship between two CbbXs.

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In the protein-coding genes of plants, core promoter elements such as TATA-box and initiator (Inr) are thought to be the cis-determinants of transcription start sites (TSS). However, many of the nuclear-encoded PSI genes have neither TATA-box nor Inr. Furthermore, these genes have multiple TSS distributed over tens of nucleotides. Therefore it is likely that some alternative to the classical core promoter elements should determine the TSS of PSI genes. In order to reveal the possible cis-elements that deterimine the TSS of PSI genes, we made a series of chimeric promoter constructs and introduced them into Arabidopsis. The analysis of the transgenic plants suggests that a novel cis-determinant for TSS should be located downstream of TSS. We would like to show further characterization of this novel element in this talk.

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Low-affinity sulfate transporter SULTR2;1 has been considered to function in sulfate translocation from roots to shoots because of its predominant localization in vascular tissues. The mRNA level of SULTR2;1 is highly up-regulated responding to sulfate starvation (-S) in roots but decreased in shoots. In this study, we identified the -S-responsive region of SULTR2;1 and found that -S-responsive expression of SULTR2;1 in roots requires the 3'non-transcribed region. In transgenic plants containing the SULTR2;1-5'region::GFP::SULTR2;1-3'region fusion construct, GFP accumulation was induced by -S not only in vascular tissues but in cortex. Physiological relevance of this 3'region was verified in plants containing T-DNA insertions in SULTR2;1-3'region (tKO). The -S response of SULTR2;1 was completely abolished in roots while unaffected in shoots by tKO. In addition, both the sulfate uptake and translocation were decreased in tKO under -S condition, suggesting that SULTR2;1 contributes to both processes through its induction driven by the 3'region in roots.

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SRD2 is an Arabidopsis gene encoding snRNA transcription activator. Phenotypic analysis of a temperature-sensitive mutant of this gene srd2-1 showed that SRD2-dependent snRNA accumulation is required for various aspects of development, including tissue culture responses such as hypocotyl dedifferentiation and shoot regeneration.
A reporter (SRD2::GUS), consisting of the -1,908 to +152 region (+1 = translation initiation site) of SRD2 and the GUS structural gene, was expressed strongly at the shoot apex, root apex, and root stele but not in the hypocotyl. When hypocotyl segments were cultured to form callus, dedifferentiation-associated expression of SRD2::GUS was induced in the stele and non-tissue-specific expression, possibly due to wounding, was observed near the cut ends. Deletion analysis of the regulatory region of SRD2::GUS suggested that cis elements important for spatial expression in planta and dedifferentiation-associated expression lie within the -1,241/+152 region while wound-responsive elements may be located in the region upstream from -1,214.

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We have identified a High-Light inducible NAC transcription factor (HLN) in Arabidopsis. The plant-specific NAC transcription factors consist of an N-terminal conserved NAC-domain required for DNA binding and a C-terminal activation domain. Interestingly, HLN also contained a transmembrane motif (TM) in the C-terminal region. Moreover, the expression of HLN-target genes was not up-regulated under normal conditions in the HLN-overexpressing Arabidopsis, suggesting that HLN might be expressed as a membraneassociated, dormant form, and then translocated to nucleus to activate its target genes under stressful conditions. Therefore, we investigated the subcellular localization of GFP fused full-length HLN and truncated form of HLN without TM motif (HLNΔTM). GFP-HLN was localized in the cytosol. In contrast, GFP-HLNΔTM was localized in the nucleus. These finding suggest that HLN is translocated to nucleus by the release from the membrane under stressful conditions and then functions as a transcriptional activator.

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Previously, we reported that the expression of a gene for soybean early light-induced protein (ELIP) was strongly induced by blue/UV-A light, and two cis-elements in the ELIP promoter, a GT-1-box-like sequence (GTGTGGACAA) and a G-box-like element (CCACGTGA), were necessary and sufficient for the blue/UV-A light-dependent expression of the gene. Here, we report a transcription factor that binds to these cis-elements. We determined the transcription start site in the ELIP promoter as C located 222 bp upstream of the start codon by primer-extension analysis. Also, we cloned a cDNA for soybean transcription factor GmGT-1 by PCR and expressed it in E.coli. The purified recombinant GmGT-1 tightly bound to the short sequence containing both GT-1 box and G box in the ELIP promoter, suggesting that GmGT-1 is involved in the blue/UV-A light-dependent expression of ELIP gene.

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Arabidopsis transcription factors DREB1s/CBFs specifically interact with a cis-acting element DRE/CRT/LTRE involved in the expression of low-temperature-, drought- and high-salinity-inducible genes. Expressions of the DREB1 genes is induced rapidly by low temperature stress. We analyzed transcriptional regulation of the DREB1 genes under low temperature condition.
Using a GUS reporter gene fused to the DREB1C promoter, we identified a 67-bp region that contains cis-acting elements and isolated a cDNA clone encoding a protein that binds to this 67-bp region by using the yeast one-hybrid screening. The cDNA encodes a bHLH-type transcription factor named bHLH072. We found that the bHLH072 has an active phytochrome binding (APB) motif in the C-terminal region and functions as a transcriptional repressor in Arabidopsis protoplasts. We are analyzing function of the bHLH072 in the expression of the DREB1 genes under low temperature stress condition using transgenic plants and T-DNA insertion mutants.

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Arabidopsis genome contains about 2000 genes that encode the transcription factors, and about 10% of them have been suggested to be transcriptional repressors because they share the common EAR-motif that act as a repression domain. This indicates that repressors might have important roles in the regulation of gene expression as well as transcriptional activators in plants. In this study, we report novel family of repressors, which possess a novel repression domain different from known EAR-motif. When the short peptide containing the novel repression domain was fused to the transcriptional factor CUC2 and AGAMOUS, the resultant transgenic plants had similar phenotype of the loss of function of these genes. The data suggest that the novel repression domain function as a potent repression domain as in EAR-motif. Because the novel domain is found in various transcription factors and in various plant species, it may play an important role in transcriptional regulation.

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With the availability of rice genomic sequence, full length cDNA sequence (35,187 clones), many ESTs and expression profiling using 22K oligoarray system, we focused our holistic approach to study the plant specific transcription factors- the NAC (NAM, ATAF and CUC). In rice genome, NAC has been predicted in 124 loci and 112 loci had FL-cDNA or EST evidence. 24 NAC genes were mapped as tandem duplicated genes and 16 pairs of NAC genes were assigned to TIGR segmental duplication blocks. Based on the phylogeny, NAC proteins have been classified into two groups (Group I and Group II). All the characterized NAC proteins fell into Group I. Group II included many of the predicted NAC proteins from tandem duplicated genes. Phylogeny of NAC genes revealed tight grouping of stress regulated NAC members. Expression profiling with 22K oligoarray system showed forty six NAC genes are differentially regulated under several stress conditions.

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We have analyzed transcription factor genes in organisms (A.thaliana (2465), O.sativa(2326),N.crassa(445),H.sapiens(2699), C.elegans(998), D.melanogaster(1395), S.cerevisiae(322) and E.coli(271) )to detect the specificities of transcription regulation in organism. To better describe and understand the differences we have focused on features of their DNA-binding structures. Among these organisms, the zinc-coordinating factor genes are especially divergent in animals. In contrast, plants are more divergent in genes containing the other DNA-binding domains (the basic domain with bZIP and HLH; HTH with HD, Myb and NAC; the beta-scaffold factor with MADS and CCAAT factors; and plant-specific domains including AP2/EREBP and B3). Analyses of alternative splicing events and gene duplication by chromosomal mapping indicated different tactics of the increase of the protein species in plants (C2H2, WRKY, bHLH, MYB, NAC, B3 =segmental duplication; MADS, homeodomain=tandem duplication; Ring finger, AP2/EREBP=Alternative splicing).

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We previously reported that the ERF-associated Amphiphilic Repression (EAR) motif, which is conserved in the C-terminal region of a number of transcriptional repressors in plants, is essential for the activity of transcriptional repression. Surprisingly, an amphiphilic motif composed of only six amino acids (XLxLXL) is sufficient to confer the strong repression activity on a transcription factor when fused. However, the mechanism of the repression mediated by the EAR-motif is still unknown. Because of the short peptide, the EAR-motif as itself is unlikely to have enzymatic activity. Therefore, we predict that the EAR-motif may interact with other factor(s), such as co-repressor, to repress the transcription of the target genes. In order to isolate such factor(s) that interact with the EAR-motif, we carried out the screening using Yeast two-hybrid system. In this report we discuss the possible function of factors that we isolated and the mechanism of the EAR motif-dependent transcriptional repression.

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Lipid peroxide-derived aldehydes produced in plants under various environmental stresses are potentially cytotoxic. Production of aldehydes inside chloroplasts can inhibit photosynthesis, in particular, at the Calvin cycle. Because the reactivity of aldehydes depends on their structure, it is important to determine the chemical species occurring in chloroplasts and their contents. In this study, we analyzed profile of aldehydes in spinach chloroplasts. LC/MS analysis showed that chloroplasts without stress treatments contained the following aldehydes at 10-600 μM; (i) C6-aldehydes (3Z)-hexenal, (2E)-hexenal and n-hexanal, (ii) α,β-unsaturated aldehydes such as crotonaldehyde and 4-hydroxy-2E-hexenal, (iii) short chain aldehydes formaldehyde, propionaldehyde and acetaldehyde and (iv) supposedly several oxo-fatty acids. Because these aldehydes were mainly distributed to thylakoids, their concentrations can reach mM levels in the membrane. In model experiments, it was found that formaldehyde, propionaldehyde, crotonaldehyde, butyraldehyde, (3Z)-hexenal, 4-hydroxy-2E-nonenal are produced from linolenic acid via its oxidation by singlet oxygen.

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Bioconvection of motile cells of Chlamydomonas reinhardtii was observed directly by a custom-made horizontal microscope. Upon illumination from above, the cells accumulated at the surface of culture medium, and then distinct paths were formed through which cells were rapidly dropping. The dropping cells were aggregated. This observation suggests that intercellular interaction is important in the bioconvection in this alga. This is in clear contrast with previous studies of fluid dynamics that were based on the assumption that each cell is a linearly moving ellipsoid having negligible intercellular interactions.

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There seems to be two important factors for trapping and/or nursing the cyanobacterial symbiont cells in the thalli of Peltigera polydactylon. One is the nitrogen nutrition probably produced by the host fungi. The cyanobacteria isolated from P. polydactylon contain heterocyst cells endowed with nitrogen fixation especially under the nitrate deficiency. However, the typical spectra derived from the heterocyst, which lacks functional photosystem II, was not observed in the thalli of P. polydactylon. Another candidate is carbon dioxide or bicarbonate probably supplied with fungi cells. During the strong light illumination, oxygen evolution activity of the isolated cyanobacteria decreased. With incubation of bicarbonate, however, the activity of the isolated cyanobacteria did not decrease, which was similar to the activities of the thalli of P. polydactylon. Protection from the photoinhibition of photosynthetic apparatus by the strong light was observed in the thalli of P. polydactylon.

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Photosystem II (PSII) is particularly sensitive to high temperature. However, the thermal stability of PSII is enhanced upon the acclimation of photosynthetic organisms to moderately high temperatures. In the present study, we studied the molecular mechanisms of the thermal acclimation of PSII in Synechocystis sp. PCC 6803. We found that the enhanced thermal stability of PSII was retained in the isolated PSII complex. Proteome analysis suggested several candidate proteins in the PSII complex with enhanced thermal stability. Screening of their knockout mutants revealed a protein responsible for the thermal stability. This protein was assigned to FabI, which is a key enzyme in fatty acid synthesis. Incubation of cells with a specific inhibitor for this enzyme prevented thermal acclimation completely. These observations suggest that synthesis de novo of lipids around the PSII complex is required for the thermal acclimation of PSII.

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In the plant respiratory chain, the ATP-uncoupling cyanide-resistant pathway catalyzed by the alternative oxidase (AOX), is supposed to prevent ROS production and maintain a carbon/nitrogen (C/N) ratio.
The aim of this study is to clarify roles of AOX in low temperature. We used Arabidopsis thaliana WT and aox1a knock-out transgenic treated at 4C. In WT, AOX protein amounts and cyanide-resistant respiration rates increased in 4C, but total respiratory rates were higher in aox1a than those in WT. In aox1a, an ATP-uncoupling NADH dehydrogenase, NDB2, was significantly induced. A malondialdehyde amount, an index of oxidative damage, in aox1a was significantly lower than that in WT. These results suggested that a lack of AOX did not induce ROS production and damages, and was complemented by other proteins. However, the C/N ratio in aox1a was significantly higher than that of WT, which suggesting that AOX maintain the C/N ratio under low temperature.

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Any visible injury can not be observed when the whole rice seedlings (Akitakomachi) are exposed for a week to chilling conditions at around 10°C. We, however, reported the severe injury in leaves that often led to leaf death, after the chilling only for a few days, only when the roots were not chilled, being preceded by a severe inhibition of photosynthetic electron transport. Based on a hypothesis that an excess water supply from roots is involved in the cause of this high-root-temperature (HRT)-induced chilling injury, we explore the relationship between the chilling injury and the nutrient solution. The results showed that it was not water but nitrate ion to affect the vulnerability to HRT-chilling stress. The current report also suggests that the HRT-chilling destroys some component between photosystem II and I without the aid of light, causing over-reduction of photosystem II in the light, thereby the visible injury is induced.

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Photosynthesis is one of the most important determinants of crop productivity. To identify photosynthesis-related genes of rice, we generated Rice-Arabidopsis FOX lines that rice full-length cDNAs were over-expressed in Arabidopsis. We have used imaging of chlorophyll fluorescence to screen photosynthesis-related mutants. We generated transformants overexpressing each cDNA isolated from candidates, resulting that thirteen re-transformed lines showed the same phenotype as the original lines. Candidates could be classified into five categories depending on the chlorophyll fluorescence parameters. We analyzed gene expression profile of three lines showing similar photosynthetic characteristics using DNA microarray. We found commonly regulated genes among three candidates, suggesting that candidates have shared influences on gene expression. We analyzed the metabolites of candidate lines, resulting that each category showed similar metabolite profile. This research is supported by the Special coordination funds for Promoting Science and Technology entitled Rapid identification of useful traits using rice full-length cDNAs.

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We examined the effects of temperature and [CO₂] on Rubisco activation, ΦPSI, ΦPSII, and NPQ in rice including the transgenic plants with increased or decreased Rubisco. The Rubisco activation was higher at low temperature, declined with increasing temperature, and decreased to 60% above 42˚C. TheΦPSI/ΦPSII and NPQ were also higher at low temperature, and declined with temperature. NPQ increased again above 42˚C. The Rubisco activation was higher at low [CO₂] and declined at elevated [CO₂]. The ΦPSI/ ΦPSII and NPQ were also higher at low [CO₂] and declined with elevating [CO₂]. Whereas rbcS-antisense rice showed a high activation of Rubisco and high ΦPSI/ΦPSII and NPQ, rbcS-sense rice had a lower activation of Rubisco. Thus, under the conditions of low temperature, low [CO₂] and low Rubisco contents, high activation state of Rubisco is always associated with high ΔpH generated from the cyclic electron flow around PSI.

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We examined the differences in the effects of temperature on photosynthesis and plant growth between rice and wheat. The optimal temperatures of photosynthesis were 30 to 35C in rice and 25 to 30C in wheat, respectively. These were not affected by growth temperatures except rice grown at 19/16C and wheat grown at 13/10C, which showed a relatively higher rate of photosynthesis at lower temperature. Whereas NAR in rice decreased at low temperature (19/16C), NAR in wheat decreased at high temperature (37/31C). These declines in NAR led to decreases in the relative growth rates (RGR) in both species. A species-dependent correlation was found between NAR and RGR in each species. The results indicate that photosynthesis in rice is more adaptive for high temperature than in wheat and that difference in temperature responses of NAR between two species mainly leads to a difference in growth rate.

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High night-temperature often leads to a decrease in grain yield of rice. We examined the effects of night temperature on yield of a large-grain rice cultivar, Akita 63. Plants were grown under two different night-temperatures for 45 days after flowering, 27˚C/27˚C (day/night) as high night-temperature (HNT) and 27˚C/22˚C (day/night) as the control. The dry weights of brown rice and filled spikelets were decreased by 8% and 9% in the HNT plants, respectively. Total dry-weights of the whole-plant at 45 days after flowering showed no significant difference between the treatments. Ratio of panicle dry weight to total biomass in the HNT plants was higher than in the control. This suggested that decrease in the grain yield in the HNT plants is due to changes in biomass allocation at the level of the whole-plant.

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In CAM plants, intercellular CO₂ concentration (C_i) increases during daytime. This is due to CO₂ released by malate decarboxylation. In leaves of Kalanchoe daigremontiana, the rates of CO₂ uptake and malate accumulation differ between the adaxial and abaxial parts (Winter, 1987, 1989). Then, C_i would differ considerably within leaf. We measured C_i for each surface and the photosynthetic products in successive three horizontal layers cut from leaf discs. In daytime, C_i was greater in the side that showed a greater CO₂ uptake rate at night. Moreover, in some occasions, the two surfaces showed uptake and efflux of CO₂. Therefore, C_i was not constant inside the leaf, implying a large CO₂ resistance to diffusion. We are now examining localization of Rubisco, PEPCase and chlorophyll to understand vertical heterogeneity in CAM photosynthesis.

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Most plants change their leaf characteristics, depending on growth temperatures. Recent studies showed acclimation of leaves is affected not only by their local environment but also by the environment of lower mature leaves. Araya et al. (2007) suggested that the photosynthesis of the young leaves is affected by the redox state of the mature leaves. Thus, it is possible that temperature acclimation of young leaves is also affected by the temperature of mature leaves. In this study, we examined effects of temperature of a mature leaf on development of young leaves in Helianthus annuus. When only the mature leaf was treated with air at 16 ºC, the palisade layer of the young leaves became thicker, and photosynthetic rates of the young leaves were higher than those of the control plants. These results suggested that the temperature of the mature leaves affects both morphologic and photosynthetic properties of the young leaves.

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Malate excretion is major aluminum (Al) tolerant mechanism in Arabidopsis and which is mainly regulated by AtALMT1 gene expression and AtALMT1 protein activation. Malate release and gene expression are sharply induced by Al within a couple of hours. Once induced gene expression, AtALMT1 protein would be activated by Al. All these responses are nearly specific to Al because other ion treatments (e.g. proton, heavy metals and Al-mimic trivalent cations) do not induce malate release and activate the protein. A set of pharmaceutical characterization indicates that both gene expression and protein activation involve a typical reversible protein phosphorylation. Promoter GUS reporter assay showed that Al inducible gene expression is adapted to protect the root apex. In addition, one natural Al-hyper sensitive accession, Wa-0, involves nonsense mutation in AtALMT1. These results indicate that AtALMT1 system is specialized for Al tolerant mechanism, which is adapted to minimize negative impact, namely carbon loss.

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Acid soils are one of the major factors to limit crop production. We have isolated a single recessive mutant showing H⁺-hypersensitivity (designated as stop1; Sensitive TO Proton rhizotoxicity) from Arabidopsis thaliana EMS/M₂ population. The root growth of stop1 mutant was suppressed under low pH conditions. Positional mapping analysis and transgenic complementation test revealed that stop1 has a missense mutation at the zinc-finger domain of predicted Cys2/His2 type zinc-finger protein, designated as STOP1. Moreover, stop1 mutant shows hypersensitivity to Al³⁺ treatment. The stop1 mutant lacks the induction of AtALMT1 gene encoding a malate transporter, and malate exudation by Al³⁺ treatment does not take place in stop1. The AtALMT1 knockout mutant shows hypersensitive to Al³⁺, whereas the stop1 mutant shows hypersensitive to both Al³⁺ and low pH. These results suggest that STOP1 is a key gene that affects Al³⁺ tolerance and low pH tolerance.

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Rice is known as an Al-resistant species, but the mechanisms involved have not been understood. We have isolated a rice Al-resistant gene (Als1), which encodes a putative ABC transporter containing ATP binding domain only. Here, we report a gene (Als3) which also encodes an ABC transporter, but containing transmembrane domain only. A transient assay with fluorescent fusion proteins showed that Als3 was localized at the plasma membrane, while Als1 alone was present as particles in the cytoplasm. However, when Als1 was co-expressed with Als3, Als1 was trafficked to the plasma membrane. A yeast two hybrid assay also showed that Als1 interacted with Als3. Both Als1 and Als3 were mainly expressed in the roots and their expression level was enhanced by Al. These results suggest that like bacterial-type ABC transporter, Als1 and Als3 are respectively translated and then function as an ABC transporter complex, which is required for high Al resistance.

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We recently identified an Al-tolerant gene (HvAACT1) from barley, which encodes an Al-activated citrate transporter. Here, we report the closest homolog of this gene in rice (OsFRDL1). We compared the Al-induced citrate secretion between the wild-type rice and Tos-17 insertion lines. However, there was no difference in the citrate secretion between these lines. When these lines were grown in a nutrient solution with low Fe concentration, chlorosis on the new leaves was observed only in the knocknot lines. The concentration of Fe and citrate in the xylem sap was much lower and Fe precipitation in the root stele was observed in the knockout line. The protein encoded by this gene showed transport activity for citrate. This gene was expressed in the roots and the expression level was not affected by Fe nutrition. These results suggest that OsFRDL1 is a citrate transporter, which is necessary for Fe translocation to the shoot.

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Aluminum ion (Al³⁺) promote the root growth inhibition in acidic soils. Nevertheless, a variety of plants have a capability to grow in acidic soils by excluding and/or detoxifying the Al³⁺. By the exposure to Al³⁺, a sort of organic acids such as malate or citrate are secreted from the root tips, and the organic acids chelate Al³⁺ and detoxify. Although these organic acids are synthesized from sugars which obtained by photosynthesis, the impacts of Al³⁺ to the photosynthesis and the sugar translocation are still obscure. In the present study, we monitored the sugar translocation toward roots and photosynthetic activity in the presence of Al³⁺, and revealed that both photosynthetic activity and amount of sugars accumulated in roots are promoted. Interestingly, sugars are specifically accumulated in Al³⁺-treated roots. These results suggest that plant has a capacity to optimize their growth and development in acidic soils.

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In cultured tobacco cells in calcium (Ca)-sucrose medium, Al causes cell elongation inhibition and other symptoms [reactive oxygen species production, callose secretion, cell death]. In this study, the mechanism of elongation inhibition and the relationship between these symptoms were examined. Cell elongation was estimated by measuring fresh weight (FW) of cell culture. After 3 to 6-h of the treatment time, the increases in FW, osmolality and soluble sugar content were observed in control cells, but not in Al-treated cells. The ¹⁴C-sucrose uptake was inhibited in Al-treated cells by 40% of the control level. When tobacco cells were incubated in sucrose-free Ca medium, the increases in FW, osmolality and soluble sugar were inhibited, indicating that sucrose uptake is necessary for water uptake and elongation. However, other Al symptoms were not induced. Taken together, we conclude that Al inhibits sucrose uptake, which leads to cell elongation inhibition, but not other Al symptoms.

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To better understand plant Se toxicity and resistance mechanisms, we compared the physiological and molecular responses of two Arabidopsis accessions, Col-0 and Ws-2, to selenite treatment. Measurement of root length demonstrated a clear difference between selenite-resistant Col-0 and selenite-sensitive Ws-2. Macroarray analysis showed more pronounced selenite-induced increases in mRNA levels of ethylene or jasmonic acid (JA) biosynthesis and -inducible genes in Col-0 than in Ws-2. Indeed, Col-0 exhibited higher levels of ethylene and JA. The selenite-sensitive phenotype of Ws-2 was attenuated by treatment with ethylene precursor or MeJA. Conversely, the selenite resistance of Col-0 was reduced in mutants impaired in ethylene- or JA-biosynthesis or signaling. Furthermore, the generation of reactive oxygen species (ROS) by selenite was higher in Col-0 than in Ws-2. Together these results indicate that JA and ethylene play important roles in Se resistance in Arabidopsis.

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Glutathione (GSH) is involved in many aspects of plant growth and development including redox control, storage and transport of reduced sulfur, and response to biotic and abiotic stresses. The transport and compartmentalization of GSH is essential to perform all these functions. We have cloned an Iron (Fe) deficiency regulated GSH transporter (IGT) from rice. IGT is a putative member of oligopeptide transporters (OPT) family, and was identified through microarray analysis as its expression was highly upregulated in response to Fe-deficiency. IGT showed high homology i.e. 82% homology to BjGT1 and 80% homology to AtOPT3. Electrophysiological measurements using Xenopus leavis oocytes showed that IGT is a functional GSH transporter. Northern blot analysis and IGT promoter-GUS analysis confirmed that its expression is induced in response to Fe-deficiency in root as well as in shoot tissue. These results suggested that IGT plays a critical role in mitigating Fe-deficiency induced stress in rice.

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A transgenic European pear (#32) overexpressing apple spermidine synthase (SPDS) and wild type were subjected to the stress for CdCl₂, PbCl₂, ZnCl₂ or their combination. Based on the shoot growth, #32 was much better than wild type. SPDS expression and Spd titer in #32 were higher than those in wild type. Glutathione was significantly depleted in line #32 with stress, but not so much in wild type. Activities of glutathione reductase/superoxide dismutase and malondialdehyde content were changed under stress toward a more favorable direction for survival in #32: these changes were closely related to the Spd titer. Accumulation of heavy metals tended to be less in #32 than that in wild type except for ZnCl₂ stress, while calcium content showed the reverse trend. Therefore, Spd levels are implicated in enhanced heavy metal tolerance possibly via the antioxidant property of Spd per se as well as by exerting an antioxidant system.

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Pteris vittata is an arsenic (As) hyperaccumulating fern, which is a candidate for phytoremediation of As-contaminated soils. As mostly occurs as arsenate in soils and was reduced to arsenite in plant tissues by arsenate reductase (AR). It has been shown that the detoxification of As in plant is achieved by binding this arsenite with thiols, such as γ-glutamylcysteine (γEC), glutathione, and phytochelatin (PC). Since γEC is a precursor of glutathione and phytochelatin, γEC synthetase (γECS) is considered to be a key enzyme in detoxification of As in plant. In the present study, therefore, As detoxification mechanisms in P. vittata was investigated with emphasis on γECS.
A full-length cDNA sequence encoding γECS has been isolated from P. vittata. Effects of As concentration and species (arsenate or arsenite) in medium on gene expression of PC synthase and AR, which had been cloned before, were analyzed in addition to of γECS.

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It was believed that adaptation responses to phosphate-deficiency were controlled by two mechanisms; systemic long-distance signal transduction and local sensing mechanism for phosphate status. This study was aimed to clarify how these mechanisms regulated expression of OsPI1, a phosphate-deficiency responsible gene of rice. Rice plants were grown hydroponically under phosphate deficient (-P) and sufficient (+P) conditions. Their roots were split and transferred into -P (-P roots) and +P solutions (+P roots). Plants were continuously harvested until 5 d after root splitting. OsPI1 expression in -P roots transferred from +P were significantly higher than in +P roots. OsPI1 expression in -P roots remained high until 2 d after transfer from -P and strongly repressed thereafter. However, ANOVA showed that repressed expression level was still higher than in +P roots. Therefore, it was indicated that OsPI1 was controlled by not only long-distance signal transduction but also local sensing mechanism under -P conditions.

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Plant develops several strategies to overcome low phosphorus condition. In this study, we focus on rice, Arabidopsis thaliana, and Lotus japonicus as the model plant of Gramineae, Brasicaceae, and Leguminosae, respectively. Plants were grown hydroponically and subjected to with or without phosphorus deficient treatment during their juvenile stage. Transcriptome analysis was performed on shoot and root of each plant, and subjected to pathway analysis by using KaPPA-View. mRNA expression of several genes related to phosphorus metabolism in plant were further analyzed by using real time PCR. In all the three species, acid phophatases, nucleases, and those genes related glycolysis and starch synthesis pathway were upregulated by phosphorus deficiency. Clear difference was observed in TCA cycle or glyoxylate cycle among three species, and the most remarkable change was observed in secondary metabolite synthetic pathway. We are going to present the quantitative data of several flavonoids in the plant tissue also.

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Saponins are suger-modified triterpene derivatives. In oats, the saponin was implicated as determinants of the resistance to the root-infecting fungus Gaeumannomyces graminis. We were not able to detect the saponins and the intermediates in rice using LC/MS and GC/MS. Our final goal is that the anti-microbial saponin is available for the rice. Therefore, to be clear this secondary metabolite biosynthesis pathway in rice, we are performing molecular analyses. First, we focused one of the pivotal genes, OxidoSqualene Cyclases (OsOSCs), on the pathway, and performed gene expression and evolution analyses. Here, we report that we found eleven OsOSC homologues in the rice genome using the DNA sequence data from oats and rice. Five out of eleven were likely to pseudogenes from the genome structures and the data of gene expression analyses. Other five genes out of eleven were expressed. We are going to measure their enzymatic activity in next step.

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A monoterpene-indole alkaloid, camptothecin (CPT), is produced in hairy roots but not in dedifferentiated cell-suspension culture of Ophiorriza pumila . Using these systems, the genes specifically expressed in CPT-producing hairy roots were profiled. Among them, full length cDNA, OpERF, encoding a transcription factor witch belongs AP2/ERF family, and OpCYP1-4 encoding P450 family proteins were isolated. To clarify the function of these hairy root specific genes, the expression of OpERF and OpCYP1-4 was modified by RNAi technique and the effects on the expression of biosynthesis genes and on the CPT accumulation were studied.

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Vitamin E in the latex of Hevea brasiliensis act as an antioxidant of natural rubber products. H. brasiliensis contains two groups of isomers of vitamin E, tocopherols and tocotrienols. Tocopherols are abundant in mature leaves, while tocotrienols are in the latex. However, their biosynthesis pathways and related genes are unknown in H. brasiliensis. In this research, we obtained ten candidate genes of vitamin E biosynthesis from our EST database and confirmed their gene expression pattern in various tissues using real-time PCR. We successfully obtain homogentisate geranylgeranyltransferase (HGGT) from dicotyledonous plant for the first time. Our results suggest that homogentisate geranylgeranyltransferase (HGGT) and homogentisate phytyltransferase (HPT) control each tocopherol and tocotrienol biosynthesis.

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Lignans' various useful physiological effects on humans have been attracting attention. For example, lignans have antioxidant and anti-cancer effects. However, it was quite difficult to establish a comprehensive analytical system for lignans as various analogs and metabolic intermediates exist in plants; some in extremelyt small amount. In this study, we established a high-sensitive microanalytical analytical method for lignans and phenylpropanoids using capillary LC-ESI-MS. Multiple reaction monitoring (MRM) was adopted for the analysis. Limit of detections were several fmol. Using this analytical method, we investigated correlations between light conditions that are a critical factor for plant metabolism, and lignans profile in leaves of a lignan-rich plants, Forsythia suspensa.
As a result, ten lignan related compounds were detected in leaves of F. suspensa. Additionally, the amount of five lignans was increased by coutinuous blue light irradiation.

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Latex of a succulent shrub Euphorbia tirucalli includes triterpens, sterol, or other secondary metabolites, and can be processed into biofuel, or medicinal and insecticidal compounds. To isolate a homologue gene for a mevalonate pathway key enzyme, squalene synthase, we performed RT- and genomic PCRs to reveal putative ORF encodes 411 residues of amino-acid sequence, in which 380-residues of N-terminal region exhibits high homology with those from other organisms, while 31-residues of C-terminal hydrophobic region was less homologous. The cDNA fragment which encodes the N-terminal 380-residue sequence was subcloned into a bacterial expression vector, pET32b, and the resultant vector was overexpressed in E. coli BL21(DE3). Using soluble fraction of the bacterial proteins, FPP and NADPH, in vitro squalene synthase activity was being examined by GC-MS. This work was partly performed as one of the technology development projects of the "Green Biotechnology Program" in NEDO.

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Coumarins consist of 2H-1-benzopyran-2-one core structure and are ubiquitously found in the plant kingdom. They are originated from the general phenylpropanoid pathway via ortho-hydroxylation of cinnamates, trans-cis isomerization of the side chain and lactonization. We have already reported that the feruloyl-CoA 6'-hydroxylase of Arabidopsis thaliana (AtF6'H1) is involved in ortho-hydroxylation of ferulate unit, which is the key step of scopoletin biosynthesis. There is less information about the steps in the lactone formation of coumarins following AtF6'H1 catalysis. In this study, we analyzed formation of scopoletin after AtF6'H1 reaction. We found that formation of scopoletin non-enzymatically proceeds in the mixture.

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Coumarin is the main component of sweet aroma of cherry flower (Cerasus lannesiana) and is utilized for the Japanese sweets. Coumarin is biosynthesized via ortho(2'-)hydroxylation of trans-cinnamic acid, while an ortho-hydroxylase (ClC2'H) has not yet been identified from C. lannesiana. We have already identified feruloyl-CoA ortho(6')-hydroxylase from Arabidopsis thaliana, which is a key in scopoletin biosynthesis. Because both ClC2'H and AtF6'H1 catalyze ortho-hydroxylation of cinnamate, it is possible that ClC2'H is a homolog of AtF6'H1. The leaves of C. lannesiana leaves contain the very low level of coumarin but accumulate the β-glucoside of 2'-hydroxycinnamic acid as the precursor of coumarin. After wounding, the glucoside is hydrolyzed by a β-glucosidase, and the released 2'-hydroxycinnamic acid is spontaneously lactonized to coumarin. In this study, we have performed screening of ClC2'H and β-glucosidase from C. lannesiana.

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We have identified that feruloyl-CoA ortho-(6'-) hydroxylase (AtF6'H1) catalyzes the key step of scopoletin biosynthesis in Arabidopsis thaliana. AtF6'H1 exhibits the high substrate specificity for feruloyl-CoA. Arabidopsis mainly accumulates scopoletin. These results indicate that the substrate specificity of the ortho-hydroxylases in the respective plants seems to determine the accumulation patterns of the coumarins.
Many AtF6'H1 homologs are found in the EST database of the plants. Of these plants, sweet potato and tobacco accumulate both scopoletin and umbelliferone. It is, therefore, assumed that the AtF6'H1 homologs from sweet potato and tobacco are involved in biosynthesis of the respective coumarins. These homologs may also exhibit the substrate specificities different from that of AtF6'H1. In order to investigate the substrate specificity of these homologs, we performed cloning and functional analysis of these AtF6'H1 homologs from sweet potato and tobacco. The total sequences of the homologs were obtained by RT-PCR, 3'- and 5'-RACE methods.

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Preparing 1-O-hydroxycinnamoyl-β-D-glucoses (HCA-Glc) as an acyl donor is prerequisite to studying HCA-Glc-dependent acyltransferase to anthocyanins. Four HCA-Glcs (sinapoyl-, feruloyl-, caffeoyl-, and 4-coumaroyl-glucose) were synthesized using a recombinant protein for Gomphrena globosa sinapate glucosyltransferase coupled with a UDP-glucose recycling system using Arabidopsis thaliana sucrose synthase. The substrate preference for HCA-Glc-dependent acyltransferase activity was examined in a protein extract prepared from anthocyanin-synthesizing cultured cells of Daucus carota and Glehnia littoralis. The sinapoyl moiety in D. carota and the feruloyl-moiety in G. littoralis were modified to be identical to the anthocyanin molecule of in vivo aglycone and sugar moieties. The protein extracts from both D. carota and G. littoralis cultured cells showed higher activity for feruloyl-Glc than for sinapoyl-Glc. These results suggested that these species have different specificity for acyl donor molecules in vivo rather than indicating a substrate preference of acyltransferase enzymes in vitro.

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Two types of red pigment, anthocyanins and betacyanins, never occur together in the same plants. Although anthocyanins are widely distributed in higher plants, betacyanins are characteristic constituents of most species of Caryophyllales.
We have cloned two cDNAs of DOPA dioxygenase (DOD) which is known as a betalain biosynthetic enzyme from red cell cultures of Phytolacca americana (PaDOD1, PaDOD2). PaDOD1 and PaDOD2 showed approximately 80% homology to each other. Expression patterns of PaDOD1 and PaDOD2 in cell cultures and in various organs of Phytolacca plants were investigated by semi-quantitative RT-PCR. In Phytolacca cell cultures and wild plants, the transcripts of PaDODs were found not only in betalain-containing cells but also in green or white cells. Moreover, we have isolated promoter regions of these PaDODs by inverse PCR. Further analyses of regulatory system and function of two PaDODs in Phytolacca plants are in progress.

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Multiple copies of flavonoid biosynthetic genes with different patterns of expression of tissue or cells and induction following environmental stimuli were identified in Legume. We are trying to analyze regulatory networks controling flavonoid biosynthesis in Legume. Sequence homology searches in model legume Lotus japonicus genome were performed to gain the information about transcription regulators involved in flavonoid biosynthesis. We identified gene duplication in the transcription factors regulating proanthocyanidin biosynthesis. Three copies of a homologue of Arabidopsis TRANSPARENT TESTA2 (TT2), which is an MYB transcription factor that regulates proanthocyanidin biosynthesis, were present in the Lotus japonicus genome. The organ specificity and stress responsiveness differed among the three LjTT2s. Moreover, it was shown that three LjTT2s could function homologously to AtTT2 in transient expression experiments. Further analysis using yeast two-hybrid and transgenic plants to examine the protein interaction properties of LjTT2s and contribution to proanthocyanidin biosynthesis in vivo are in progress.

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PEPC catalyzes the irreversible carboxylation of PEP to yield oxaloacetate and Pi. Although it has long been considered to be cytosolic, we recently found PEPC targeted to the chloroplast in rice (Osppc4). Genes encoding the chloroplastic isoform have been identified only in the genus Oryza, namely, in cultivated and wild rice species.
Osppc4 was expressed in all organs tested, exhibiting high expression in leaf blade and leaf sheath. Its expression was confined to green parenchymal cells in these organs. In contrast, it was expressed in vascular bundles in addition to green parenchymal cells in spikelet. Suppression of Osppc4 expression by RNAi technique led to 30-35% decrease of total PEPC activity of leaf blade, while the activity of spikelet remained almost unaffected. The suppression did not affect the photosynthetic rate but slightly inhibited growth during vegetative stage, while it significantly retarded grain filling and reduced grain yield.

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PEPC, PPDK, NADP-MDH, and NADP-malic enzyme (ME) were overproduced in the mesophyll cell of rice plants. Overproduction of PEPC alone stimulates respiration in the light and slightly reduces the carboxylation efficiency, thereby suppressing the photosynthetic CO₂ assimilation. The stimulated respiration remained unaffected by overproduction of all the four enzymes, but the carboxylation efficiency was restored. The apparent CO₂ assimilation rate was also restored to reach levels comparable to or slightly higher than that of non-transformants. The extent of the restoration, however, was more marked at higher CO₂ concentrations, indicating that overproduction of the four enzymes did not act to concentrate CO₂ inside the chloroplast. The δ¹³C value was unaffected by overproduction of any of four enzymes but slightly increased by overproduction of all the four. Since a similar increase was observed in other transformants overproducing PEPC and ME, it likely reflected some metabolic path constituted of foreign PEPC and ME.

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PEPC involved in C₄ photosynthesis is subject to an allosteric inhibition by malate. The inhibitor-sensitivity is high at pH7, while very low at pH8. There is no such pH-modulation in E.coli enzyme (EcPEPC). Previous our study had revealed that the flexible loop I (631-651 in maize C₄-form PEPC, ZmPEPC) takes two states in association with allosteric activation and inhibition. In the present study a recombinant ZmPEPC was crystallized and X-ray analyzed. The loop I was revealed to be stabilized through interaction with the plant-specific loop II (124-140) at pH7, while it is mobile at pH 8. Furthermore, protonated H653 in the loop I was speculated to play a key role in this stabilization. The kinetic studies with many mutant enzymes as given below essentially supported the speculation and more detailed mechanisms: ZmPEPC (H653N, H653D, H653K, H653R, D133A/E134A, E140A/D142A, G650A/T652A) and EcPEPC (A590G/A592T, A592G/A594T, here H593 corresponds to H653 of ZmPEPC).

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E. vivipara has a unique nature that expresses C₄ characteristics under terrestrial conditions and C₃ characteristics under submerged aquatic conditions. Both characteristics are interconvertible to each other in response to environmental changes. Since not only the expression profile of the enzymes but also the leaf morphology (Kranz anatomy) change in association with this conversion. E. vivipara is a suitable plant to investigate the genes indispensable for the functional C₄ photosynthesis. During the course of our study on the regulatory mechanisms for gene expression and enzyme activity of PEPC for C₄ photosynthesis, we noticed that the previously published method for PEPC extraction from the culms of E. vivipara was quite inefficient when grown in the glasshouse. Here we report that the use of skim milk as an additive to the extraction buffer was 50 to 100-fold more efficient than the previous one, and discuss possible usefulness of this method.

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