Reactive oxygen species (ROS) inhibit the synthesis de novo of proteins that are required for the repair of photosystem II (PSII). To alter the sensitivity of the repair system to ROS, we constructed mutants of the cyanobacterium Synechococcus sp. PCC 7942 that overexpressed ROS-scavenging enzymes. Overexpression of catalase from Vibrio rumoiensis increased the tolerance of the photoautotrophic growth of cells to H₂O₂. In this mutant, the PSII activity became more tolerant to strong light in the presence of H₂O₂ than that in wild type. In vivo labeling of proteins revealed that the synthesis of the D1 protein de novo was enhanced under the oxidative conditions. Overexpression of superoxide dismutase from Synechocystis sp. PCC 6803 increased the tolerance of the photoautotrophic growth of cells to the superoxide radical generated by the presence of methyl viologen.

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Lichens, characterized by having photobionts, can live even in the drought environment. Photobiont cyanobacteria in thalli of Peltigera polydactylon harvested in Akita was studied morphologically especially about their distribution, showing the typical cyanobacterial absorption and emission spectra. During the drought stress condition, inactivation of PSII measured by PAM system was observed in intact thalli, but the PSII fluorescence increased and recovered after re-watered. Using pico-second time resolved spectrophotometer, the PSII fluorescence decayed much faster and excitation energy transfer within PSII was still observed in the dried thalli. Lifetime of PSII increased within 1 min after water-absorption. The gene encoding alpha-subunit of nitrogenase (nifD) was cloned from cyanobacterial symbionts isolated from two locations in Akita (Mt Takao and Lake Tazawa). The deduced NifD sequences were 97.8% identical to each other between two isolates, which were much more similar to the genes from Anabaena than those from Nostoc.

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Rice cultivar, Akenohoshi produces heavier dry matter than those of Nipponbare. The main causal factor is the relatively smaller decrease in the rate of photosynthesis during the ripening stage in Akenohoshi. Cytokinin levels in xylem sap are high in Akenohoshi. However, the mechanisms in the suppression of the decrease in photosynthetic rate are not clear. The effect of exogenous cytokinin on the rate of photosynthesis and its causal factors were investigated in Nipponbare with or without cytokinin (BA).
The decrease in leaf photosynthetic rates was smaller in BA-treated plants than those of control plants. The carboxylation efficiency was higher in BA-treated plants. The decreases in levels of Rubisco and rbcL and rbcS mRNAs were smaller in BA-treated plants. These results indicate that the higher rate of photosynthesis with BA-treated plants is caused by the higher carboxylation efficiency due to the higher levels of Rubisco brought by higher rbcL and rbcS transcripts.

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In C₄ plants, mesophyll (M) chloroplasts are randomly distributed in the cells, but bundle sheath (BS) chloroplasts are located in a centripetal or centrifugal position. We previously found that the intracellular arrangement of BS chloroplasts is acquired during cell maturation and can be re-established after disturbance by centrifugation. The intracellular positioning is dependent on actomyosin system and cytosolic protein synthesis but not tubulin or light. In this report, we investigated whether the intracellular positions of M and BS chloroplasts are affected by environmental stresses. When mature leaves of finger millet (Eleusine coracana) were exposed to high-intensity light, M chloroplasts showed light-avoidance movement but BS chloroplasts didn't. The orientation movement of M chloroplasts was also observed under the drought-stress condition. We also observed the similar phenomena in maize. These findings suggest that M and BS cells are also differentiated in regulation system of intracellular chloroplast positioning in response to environmental changes.

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The plant nuclear genome has acquired many genes from the cyanobacterial endosymbiont, but most of these genes have not been identified. Gclust program finds out proteins encoded by these genes and conserved among many organisms. We selected proteins acquired from cyanobacterial endosymbiont. Consequently, 73 protein groups were identified and these groups contained 56 unidentified proteins of Arabidopsis. We designated these proteins as Chloroplast Proteins of Endosymbiont Origin (CPRENDO). The SALK T-DNA insertion lines for the genes encoding protein in CPRENDO (cpr mutant) were systematically characterized. Some of The cpr mutants showed high NPQ phenotypes by the analysis of the saturation pulse method. This suggested that these mutants including ycf65 and ycf20 have abnormal thermal dissipation system. The ycf65 and ycf20 mutants had lower Fv/Fm ratios and showed cotyledon-specific pale green phenotype and variegated-leaf phenotype, respectively. The photosynthetic properties of the mutants are being analyzed.

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Lichens are photoautotrophical symbionts containing algae. It has been suggested that lichens convert absorbed light energy into thermal energy under the drought conditions. We investigated 20 pieces of lichens to clarify the excitation energy transfer and electron transfer processes under the drought conditions. We classified lichens into 3 groups based on the drying-responses of fluorescence spectra at 77 K. Fluorescence lifetime analysis further indicated that the light energy absorbed by photosystem II is rapidly quenched in the two groups in the dry conditions. The remaining one group was shown to protect photosystem II by decreasing the antenna size in the dry conditions. The delayed fluorescence emitted from photosystem II at 50-100 ns indicated the electron transfer from P680 to pheophytin is still active. We also measured the delayed fluorescence in the millisecond domain and the thermoluminescence in the dry conditions. We will discuss the energy dissipation mechanisms from these results.

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In mammals, the electron-transfer flavoprotein (ETF) is a heterodimeric protein composed of two subunits, α and β, which is responsible for the oxidation of at least nine mitochondrial matrix flavoprotein dehydrogenases. Electrons accepted by ETF are further transferred to the main respiratory chain via the electron transfer flavoprotein ubiquinone oxidereductase (ETFQO). There are the unique homologues of two subunits of ETF in Arabidopsis genome. Isolation and characterization of two independent T-DNA insertional Arabidopsis mutants of the ETFβ revealed early death phenotype compared to wild type during extended darkness. Furthermore the etfb mutants demonstrated a significant accumulation of several amino acids, isovaleryl-CoA, and phytanoyl-CoA during dark-induced carbohydrate deprivation. These phenotypic characters of etfb mutants are a phenocopy of those that we observed previously in Arabidopsis etfqo mutants, suggesting functional association between ETF and ETFQO in Arabidopsis, and confirming the essential roles of the ETF/ETFQO electron transfer complex during dark-induced carbohydrate deprivation.

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Plant mitochondrial respiratory chain has not only the cytochrome pathway, which is common to animals, but the alternative oxidase (AOX). AOX is uncoupled with proton extrusion. Thus, AOX apparently wastes the energy, but can dissipate the excess reductants effectively. In the plant cells, substantial reductants are consumed by nitrate reduction. This suggests both AOX and nitrate reduction can be regarded as the reductant-consuming systems. This study clarified any interaction between the nitrate reduction and AOX. Thus, we examined whether the activity of nitrate reduction could influence the AOX activity. Nitrate reduction was inhibited by tungstate, which is the inhibitor of Nitrate reducase (NR). The extent of decrease in NR activity was positively correlated with that of increase in AOX capacity. This result indicated that the excess reductants accompanied by decrement in nitrate reduction could be consumed by AOX. We also examined mechanism of interaction in detail.

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In the plant respiratory chain, there are two major uncoupling pathways, the alternative oxidase (AOX) and uncoupling protein (UCP). These are supposed to prevent production of ROS and maintain the TCA cycle activity.
Arabidopsis thaliana has AOX1a, 1b, 1c, 1d, 2 and UCP1-6. We used rosettes in plants transferred from 25^oCto 10^oC. The expressions of AOX1a and UCP5 increased after 3h. The cyanide-resistant respiratory rate and AOX protein amounts increased after the transfer. The amount of malondialdehyde, an index of oxidative damage, decreased with the AOX protein increase. AOX and UCP would function together for depletion of reducing equivalents at 10^oC. These expression patterns are different from those at 4^oC reported by Borecky et al. (2006). Thus, we questioned whether regulatory systems differently control AOX and UCP depending on the intensity of cold stress. We compared expression patterns of the respiratory chain components between 10^oC and 4^oC.

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Although assimilation of inorganic nitrogen is a critical biochemical process for plant growth, the underlying regulatory mechanism has remained to be resolved. Here, we investigated a transcription factor (TF) that contributes to expression of nitrogen assimilation genes in the primitive red alga Cyanidioschyzon merolae.
DNA microarray and Northern blot analyses showed that expression of MYB-1 encoding an R2R3-type MYB TF was significantly increased under nitrogen deprivation condition (-N). Accumulation of MYB-1 protein and its nuclear localization were also observed in response to the -N. Furthermore, chromatin immunoprecipitation analysis with anti-MYB-1 antibody and gel-mobility shift assay using C. merolae crude extract containing nuclear proteins demonstrated that MYB-1 specifically binds to promoter regions of nitrogen assimilation genes, NRT, NIR, and GS under the -N. These results clearly suggested that MYB-1 controls nitrogen assimilation-related gene expression as a positive regulator in response to nitrogen status.

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The unicellular red alga Cyanidioschyzon merolae 10D has simple cell structure, which contains one nucleus, one mitochondrion and one plastid. C. merolae is considered to be a model organism to analyze the basic construction of eukaryotic cells, and it is indispensable to develop transformation techniques to facilitate functional genomics.
Previously we demonstrated that a lesion in the URA5.3 gene was repaired by the introduction of wild type URA5.3 gene in a homologous recombination dependent manner. In this study, we constructed a URA5.3 gene in which the URA3 portion was substituted by the URA3 of Galdieria sulphuraria, a related red alga, which prevents the recombinational transformation. Using plasmids containing this marker gene, plasmid constructs were stably retained in C. merolae in some cases, which may indicate the plasmid DNA replication in C. merolae cells.

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Arabidopsis thaliana possesses three types of hemoglobin (Hb) genes: class I, class II, and truncated Hb (trHb). AtGLB3 is a homologe of trHb that are widely distributed in lower organisms such as bacteria and unicellular eukaryotes. The function of AtGLB3 in vivo has remained unknown. The T-DNA insertion mutant ΔGLB3 lacks the functional gene of the trHb. We have found that ΔGLB3 cannot germinate above 32ºC where the wild type shows normal germination. The mutant is extremely sensitive to high temperature stress during germination. The germination was partially restored by adding carboxy-PTIO, a nitric oxide (NO) scavenger, chlorogenic acid or bovine serum Hb. Furthermore, the volatile organic compound isoprene also restored the germination capability of ΔGLB3 at high temperature. This strongly suggests that the failure of ΔGLB3 to germinate at high temperature is caused by NO scavenging activity and physiological function of ΔGLB3 have been first reported in this paper.

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Dof1 is a plant-specific transcription factor which had been showed to improve nitrogen assimilation in transgenic Arabidopsis. To investigate if the transcription factor is widely functions in all plants, the cDNA of adof1 from Arabidopsis was overexpressed in tobacco leaves under the control of tomato rbcS-3C promoter. The assay for pyruvate kinase (PK) and phosphoenolpyruvate carboxylase (PEPC) indicated the overexpression of adof1 led to a 1.5-5 fold increase in PK activity and 1.5-3.2 fold increase in PEPC activity, respectively, in transgenic tobacco. When grown under lower (1 mM) and moderate (5 mM) nitrogen conditions, the transgenic tobacco displayed better growth as compared with the control. The height of the transgenic lines was 1.3-1.7 fold of the control while their fresh weight was 1.1-1.3 fold of the control. No significant differences were observed in the growth of transgenic lines and the control when they were grown under higher nitrogen conditions.

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Recent studies of nitric oxide (NO) in plants have suggested crucial roles of NO in diverse physiological processes, although the pathway of NO production in plants has not been established yet. When rice plants were grown with nitrate, NO emission from leaves was low in air under the dark, but stimulated by light. NO emission in dark strongly increased upon flushing with nitrogen. When rice plants were grown with ammonium, NO emission from leaves was very low in any conditions. In cultured cells grown with nitrate, addition of nitrite to the medium resulted in a rapid NO emission, and that was stimulated in nitrogen. NO emission from the cells grown with amino acids was also enhanced after addition of nitrite in nitrogen, but the steady state level was lower than that of the cells grown with nitrate. Possible pathway of NO production in rice leaves and cells will be discussed.

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Polyamines (PAs) are aliphatic amines with low molecular weight that can be found throughout most living organisms. They are associated with a wide spectrum of cellular activities, such as growth, morphogenesis, or environmental stress response. In spite of the presence of PAs at considerable concentration, their physiological roles in plants yet remain as a subject to be explored. Here we present evidence showing that PAs are involved in rapid root abscission of the aquatic fern Azolla pinnata. The PAs spermine and spermidine induced rapid root abscission. Interestingly, spermine-NONOate, an nitric oxide (NO) conjugated form of spermine, showed much faster root abscission than that induced by the parent compound spermine. Because nitrite, an oxidized form of NO, also exhibited a similar root abscission, we consider it plausible that PAs may induce the root abscission of Azolla through the production of reactive nitrogen species in vivo.

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Transfer of ammonium-grown cells of Synechococcus elongatus to nitrogen-free medium increased expression levels of -80 genes involved in nitrogen acquisition and metabolism. In addition, two genes encoding sigma factors of RNA polymerase (syc0015 and syc0953) and a gene encoding a response regulator (syc1148) were upregulated by nitrogen depletion, with 2.5-, 11.5-, and 7.6-fold increase in the transcript levels, respectively, and found to contain an NtcA-binding site in their promoters. The upregulation of syc1148 was abolished in an NtcA deletion mutant, whereas significant upregulation of syc0015 and syc0953 was observed even in the mutant upon nitrogen depletion, indicating involvement of an NtcA-independent as well as an NtcA-dependent mechanism in gene activation. In a mutant deficient in syc0953, the extent of upregulation of nblA, a representative of NtcA-activated genes, was reduced to a level similar to that in the NtcA mutant, indicating direct and indirect regulation of nblA by NtcA.

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Nitric oxide has been thought to play a key role on signal transduction involved in many important physiologic functions. To elucidate the molecular mechanisms for NO signaling in higher plants, we made two-types of the transgenic Arabidopsis excessively producing NO. One (cdeiNOS) constitutively overexpressed NOS gene derived from Deinococcus radiodurans, and the other (ideiNOS) transiently overexpressed deiNOS gene with the ethanol-inducible promoter derived from Aspergillus nidulans. We previously showed that the expression levels of many genes already reported as NO response genes in some articles are almost same as that of WT using microarray analysis in cdeiNOS. To analyze NO early response genes, we analyzed the expression pattern of genes in ideiNOS using microarray analysis. We observed remarkable differences in the expression levels of 1731 genes (up-regulated 887,down-regulated 844) between no-treatment and ethanol-induced ideiNOS. We are analyzing genes which exhibited similar expression profiles between cdeiNOS and ideiNOS.

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As reported previously, about one-third of inorganic nitrogen, such as N of nitrogen dioxide and nitrate, taken up into plant body is converted to Kjeldahl-unrecoverable, hitherto unknown organic nitrogen (UN). UN bearing compounds include those in which a nitrogen atom is linked to an oxygen atom or atoms, e.g. nitro, nitroso, and oxime groups, or to a second nitrogen atom, e.g. azo compounds and hydrazines (Morikawa et al., 2004). There is an alternative pathway in the N metabolism to form the UN-bearing compounds. Some UN-bearing compounds, if not all, may be involved as a signal molecule in the plant vitalization effect of atmospheric nitrogen oxides (Takahashi et al., 2005). This communication summarizes what have been done and what are to be done in the study on the novel aspect of N metabolism.
Morikawa, H. et al. Planta.219: 14-22 (2004).
Takahashi, M. et al. New Phytol. 168: 149-154 (2005).

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The growth of plant is influenced by temperature. In this study, we aimed to clarify the effect of night temperature on biomass production. Plants were grown under three different night-temperatures(17°C, 22°C and 27°C) for 63 days. The day temperature was 27°C. The biomass of plants grown at high night-temperature was the greatest. Net photosynthesis at the level of the whole plant was the highest in the high night-temperature treatment. RGR in the high night-temperature treatment was also the highest during d21-42. In plants grown at high night-temperature on d42, the biomass and N allocation into leaf blades at the level of the whole plant tended to increase. Thus, the greater biomass at high night-temperature was caused by higher RGR during the early stage of the growth as well as by greater biomass and N allocation into leaf blades.

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Among six BOR1 paralogs present in Arabidopsis thaliana genome, we studied roles of BOR6 and BOR7 in pollen development.
Transcripts of BOR6 and BOR7 were detected in flowers. Transgenic A.thaliana lines expressing GUS under the control of each gene promoter exhibited specific GUS staining in pollen and pollen tubes, suggesting that BOR6 and BOR7 are expressed in pollens.
Expression of BOR6 decreased B concentration of yeast cells, suggesting that BOR6 is a functional efflux B transporter, similar to the case of BOR1.
GFP fusion proteins of BOR6 and BOR7 are located at the tip of pollen tubes.
Experiment of pollen tube elongation in vitro under limited boron supply showed that length of pollen tubes of bor6-2/bor7-2, double T-DNA insertion mutant were shorter than those of the wild type.
These results suggest that BOR6 and BOR7 are B transporters important for pollen elongation under low boron supply.

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MicroRNAs (miRNAs) are small, noncoding RNAs that can play important roles in eukaryotes by mRNA degradation, and translation repression. To investigate whether miRNAs regulate gene expressions in the flowering process of woody plants, we analyzed small RNAs expressed at flowering stage of Eucalyptus. First we isolated RNAs from flower buds of Eucalyptus, and then separated small RNAs by polyacrylamide gel electrophoresis. Twenty-two bases of the small RNAs were sequenced by MPSS method, and about 120,000 sequence signitures were determined. Next the sequences that have homology with rRNA, tRNA, snRNA, scRNA were removed from the 120,000 sequences, and then genome sequences including the small RNAs were sereached and about 30,000 sequences were taken. Finaly we identified about 500 miRNA candidates by prediction of secondary structures of the 30,000 sequences. Some miRNA candisates have sequence conservation in Arabidopsis or Populus, but unexpectedly many new miRNA candidates were found in Eucalyptus.

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Pollen coat is a surface structure of Arabidopsis pollen grains, which consists mainly of proteins and lipids. These components are synthesized in tapetal cells and are deposited on the pollen surface after the tapetal cells are ruptured. Pollen coat is important for pollen fertility. The EXTRACELLULAR LIPASE4 (EXL4) and EXL6, which belong to the GDSL lipases, are abundant proteins in Arabidopsis pollen coat. To reveal their function, we carried out several experiments. Both EXL4 and EXL6 were strongly expressed in the flower buds of stage 10 to middle 12, where the lipid components of pollen coat were synthesized actively. The plants that carry both a T-DNA insertion in EXL6 and an RNAi construct for EXL4 showed reduced fertility. Thus it is proposed that both EXL4 and EXL6 are expressed in the tapetum and have important roles to create lipid component such as free fatty acids enriched in the pollen coats.

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Polypyrimidine tract-binding protein (PTB) belongs to a member of heterogeneous nuclear ribonucleoproteins(hnRNPs). In animals, PTB proteins are known to play multiple roles in splicing, intracellular sorting and nucleocytoplasmic transport of mRNA, and be involved in differentiation and development. However, function of PTB in plants reminds open. RT-PCR analyses for two Arabidopsis PTB genes (AtPTB1 and 2) indicated ubiquitous expression in vegetative and reproductive organs in Arabidopsis plants. Expression profiles using promoter::GUS system suggested that expressions of AtPTB1 and 2 are induced in mature pollen grains, papillae and placentas but not in developing embryos. The null mutants of atptb1 and atptb2, were selected, however, growth and development of these mutants were normal when they were compared to those of wild type plants. Double mutant (ptb1/ptb2) is under preparation.

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Plant reproductive development is more sensitive to various environmental stresses than vegetative growth. Especially, high-temperature stress causes abortive male reproductive development in lots of plant species. Here, we report the mechanism of high-temperature injury during anther early development in barley plants. Under high-temperature conditions, cell proliferation arrest, abnormalities of the mitochondria, nuclear membrane, and rough endoplasmic reticulum and premature degradation of tapetum cells and premature progression to meiotic were specifically observed in anther cells. To monitor the entire gene expressions during high-temperature injury, we performed DNA microarray analyses. Certain genes involved in histone, DNA replication initiation, mitochondria and ribosomal proteins were specifically repressed in the injured panicles. Series of genes, meiosis specific gene and anther specific LTP, was prematurely up-regulated at the earlier stage under the high-temperature conditions, suggesting that high temperature causes premature progression of anther early development programs, accompanied by comprehensive alterations in transcription.

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PnTFL1, an ortholog of TERMINAL FLOWER 1 (TFL1) in Lombardy poplar (Populus nigra L. var. italica Koehne) was cloned to evaluate its function in poplar. RT-PCR studies showed that the gene was mainly expressed in roots, stems, apical buds and lateral buds. The transgenic Arabidopsis that over-expressed the gene for PnTFL1 decelerated flowering as well as TFL1 over-expressing Arabidopsis. To determine that PnTFL1 acts as TFL1 in lombardy poplar, we constructed binary vectors generating RNAs capable of duplex formation of sequence specific for the PnTFL1 cDNA under control of the cauliflower mosaic virus 35S promoter. The resultant plasmids were introduced into a female clone of Lombardy poplar. Some transgenic lines showed early flowering phenotype that flowered within five months. The most severe transgenic line flowered within five weeks after vegetative propagation by cutting in phytotron, and it also showed late growth, light-green leaves and abnormal leaf shape.

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The Arabidopsis PROTODERMAL FACTOR2 (PDF2) gene is required for epidermal differentiation durning plant morphogenesis. The overexpressed plants of the PDF2 gene by the control of the CaMV 35S promoter were found to delay flowering. To understand the molecular basis of a genetic network of flowering control, we analyzed the PDF2 overexpression plants. The PDF2 gene encodes the HD-ZIP START transcription factor. To identify the essential domains responsible for the late-flowering phenotype of the PDF2 overexpression plants, chimeric genes consisting of deleted fragments of the PDF2 cDNA expressed by the CaMV 35S promoter were transformed into wild-type plants. Furthermore, we investigated whether PDF2 interacts with the floral pathway integrators in the yeast two-hybrid system. To isolate mutants that suppress the effect of overexpression of PDF2, we screened an EMS-mutagenized population of 35S::PDF2 transgenic plants for early-flowering mutants. Here, these results will be presented.

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To investigate the flowering mechanism of apple, the expression of AFL1 and 2 genes, which are orthologues of the LEAFY gene from Arabidopsis, are analyzed. The respective 3' noncoding region from each AFL gene was used as a specific probe on in situ hybridization (ISH). The ISH of 10 μm thick sections of floral meristems with AFL1 or 2 probe in 2005 summer to 2006 summer showed the staining of tunica and leaf primodia, respectively. There were no differences between each stained tissues with the probes, even in developing inflorescences. The apple cultivars have different period for each inflorescence development. The AFL1 and AFL2 ISH staining also showed common tissues in spite of morphological changes. These results suggested that both AFL1 and AFL2 played important role in the flowering of apple tree.

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The maize INDETERMINATE1 gene, ID1, regulates the transition to flowering in Maize. ID1 encodes a protein including four zinc fingers and defines a gene family (IDD family) unique to plants. Among IDD families, a domain of about 200 amino acid residues including zinc fingers (ID domain) is highly conserved. Our previous biochemical studies showed that some IDD families bind to 11 bp of specific sequence via ID domain. The result suggests that IDD families are transcription factors.
Genome database analysis revealed that there are sixteen, fourteen, sixteen of IDD family genes in maize, rice and Arabidopsis, respectively. However, functions of all family genes except for ID1 of maize remain to be elucidated.
To clarify the functions of IDD family genes in Arabidopsis we analyzed expression profile of these family genes and also analyzed transgenic plants overexpressing these genes or RNAi plants that the expression of these genes are suppressed.

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LKP2 (LOV kelch repeat 2), which belongs to ADO/FKF/LKP/ZTL family protein in Arabidopsis, possesses an LOV, an F-box and a Kelch repeat domain. LKP2 is postulated to regulate the degradation of certain regulatory proteins that control circadian rhythm, photomorphogenesis or flowering time, through ubiquitin-proteasome system. We isolated LIF1 (LKP2 interacting factor 1) through a yeast 2-hybrid system using LKP2 as a bait. In this study, we will show our experimental results on LIF1 gene expression, intracellular localization of LIF1 protein, and phenotypic analysis of LIF1 transgenic plants, and discuss possible LIF1 function. This work was supported by a grant from the Program for Promotion of Basic Research Activities for Innovative Bioscience (PROBRAIN).

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Reproductive organ development in plants is regulated by endogenous and exogenous factors. At 25C with a 14-h photoperiod in a phytotron, male strobili of Cryptomeria japonica showed delayed development. Here, the effects of changing temperatures on the morphogenesis of male cones were examined. Two year-old saplings were treated with GA₃ in mid-July to induce male strobilus formation and these plants were grown in a phytotron at 30/25C (day/night) with a natural photoperiod. At the 7th week after GA₃ treatment, some saplings were transferred to a lower temperature (20/15C), and some of them were subsequently transferred to the field at the 13th week. Histological observation showed that the delayed development of male strobili under the high temperature treatment was recovered in the lower-temperature environment. Pollen production was normal in those saplings transferred to the field. The pattern of gene expression in the male cones under different temperatures will also be presented.

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Circadian clock generates internal 24-h rhythm in plants, allowed them to predict changes of environment like daily and seasonal changes. Resent studies suggest CARCADIAN CLOCK ASSOCIATED 1(CCA1)/ LATE ELONGATED HYPOCOTYL(LHY), PSEUDO RESPONSE REGULATOR9/7/5, TIMIG OF CAB EXPRESSION 1(TOC1, also called PRR1), and GIGANTEA(GI) regulate each other in transcription, and form interlocked feedback loop in Arabidopsis.
Photoperiodic flowering induction is most important physiology regulated by clock, and arrows many plant to flower at their favorite seasons. Flowering is induced when CONSTANS(CO) gene expression coincides with the exposure of plants to light, in that condition, CO protein is highly stabilized. However, how circadian clocks induce CO gene expression at dusk is unknown.
By comparative genetics of cca1/lhy, prr7/5, and gi, we show circadian clocks induce CO at dusk via at least two pathways(PRR7/5-CO, and CCA1/LHY-GI-CO). And more, we discuss molecular mechanisms of these genes for the strict phase regulation of CO.

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Proteasome-mediated protein degradation pathway is implicated in all kinds biological responses in eukaryotes. In mammals and Drosophila, proteasome pathway is shown to play important role at the core of circadian clock. It is also known that the pathway is involved in plant circadian clock processes. However, the underlying molecular mechanisms of these processes in plants are still poorly understood. In this study, we show that an F-box protein ZEITLUPE (ZTL) plays a role in targeting PSEUDO-RESPONSE REGULATOR 5 (PRR5) to proteasome-dependent degradation in Arabidopsis thaliana. Characterization of PRR5 protein levels and stability in WT, ztl mutants and ZTL-overexpression plants in various circadian conditions demonstrate that ZTL is required for proper degradation of PRR5. We also provide evidence for genetic and physical interaction of ZTL with PRR5.

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It has totally remained a mystery about the evolution and origin of circadian clocks in plants, because the studies on the molecular mechanism of the plant clock have so far mostly been limited to Arabidopsis thaliana. To obtain some hints about this mystery, we started to study the circadian clock using the moss Physcomitrella patens. Previously, we reported that clock-controlled genes of this moss showed rhythmic expression in continuous dark, while they were completely arrhythmic in continuous light (LL). This is contrasting to the cases of higher plants, which show robust circadian rhythms of gene expression in LL. We report here the results of recent analyses on a Physcomitrella homolog of the Arabidopsis clock gene PCL1, and phylogenetic relationships between the clock gene homologs in plants.

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In plants, the studies on the circadian clock have been so far concentrated on Arabidopsis thaliana, while there are only few studies on the mechanisms of circadian clocks in other plants. Therefore, it is unknown how similar or diverged are the molecular mechanisms of the circadian clocks in plants. Moreover, in plants the evolution and origin are completely a mystery. For these reasons, we started to study the circadian clock at the molecular level using the moss Physcomitrella patens, which diverged from the higher plants before hundreds million years. In A. thaliana, CCA1/LHY, TOC1(PRR1), PCL1 (LUX)have been so far isolated as clock genes. We found some homologs of these genes in P. patens. We report here results of expression analyses using a bioluminescence reporter and those of functional analyses by gene targeting of PpCCA1a and PpCCA1b, CCA1 homolog-genes in P. patens.

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The Pex protein in cyanobacterium Synechococcus elongatus PCC 7942 fine-tunes the period and the phase of the circadian clock. We carried out X-ray crystal structure analysis of Pex. We prepared the Pex lacking the 14 residues and obtained good crystal of the protein. Then we determined the crystal structure at 1.8 angstrom in resolution. As a result, we revealed that Pex had a winged-helix structure, which was a typical DNA binding motif. By DNA gel shit assay, binding of Pex to a DNA fragment of kaiA promoter region was confirmed. While the arginine residue at 106th in the protein was estimated as an important portion to contact DNA, its substitution to alanine diminished the binding activity. We also established the Synechococcus cell constitutively expressing the mutant Pex, and examined the clock by bioluminescence monitoring. Then, no activity of the period lengthening was observed.

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In Arabidopsis thaliana, results from recent studies have begun shed light on the molecular mechanisms underlying clock-controlled circadian rhythms. In this context, we recently realized that each member of the small PRR family, including TOC1/PRR1, plays important roles close to the central oscillator (or within the input and/or output pathways). However, their individual molecular functions of PRRs have not yet been fully clarified. To this end, here we set up all possible transgenic lines each over-expressing constitutively a given one of the PRR genes (designated as PRR1-ox, PRR3-ox, PRR5-ox, PRR7-ox, and PRR9-ox), and their phenotypes were compared at once with each other, with special reference to "free-running circadian rhythm", "red light response during early photomorphogenesis", and "photoperiodic flowering time". Furthermore, we attempted to characterize transgenic plants mis-expressing a set of truncated form of PRR5 and PRR7. Taken these results together, we will discuss about the structure-functions of these PRR clock-components.

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In Arabidopsis thaliana, results form recent studies have begun shed light on the molecular mechanisms underlying clock-controlled circadian rhythms. In this context, we recently realized that each member of the small PRR family, including TOC1/PRR1, plays important roles close to the central oscillator. In addition to the clock-associated transcription factors (such as CCA1 and LHY), there must be many as yet unidentified transcription factors, which play important roles close to the input and/or output pathways. To address this issue, here, we focus out attention on a large family of B-box-type Zn-finger (B/Zn) transcription factors, which enclosed the CONSTANS sub-family members, some of which are implicated in the clock-controlled photoperiodic flowering time. In this study, we uncovered and characterized a number of clock-controlled novel B/Zn family genes, for which extensive genetic studies were carried out, in the hope of clarifying their circadian-associated roles in A. thaliana.

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Woody plants in boreal zone respond to short daylength in fall and induce an initial phase of cold acclimation. To accurately measure the daylength, plants have an endogenous mechanism called as "circadian clock". In Arabidopsis, it is thought that a central oscillator of the circadian clock consists of LHY/CCA1 and TOC1/PRR1, which form a transcriptional feedback loop. To elucidate involvement of the circadian clock system in the phase of cold acclimation in boreal woody plants, we determined full-length cDNA sequences, circadian expressions and genomic structures of LHY/CCA1- and TOC1/PRR1-like genes in a model tree, Populus nigra, and subsequently conducted phylogenetic analyses of these genes. We found that, in leaves, two LHY/CCA1-like genes were expressed in the morning phase and the TOC1-like gene was expressed in the evening phase, which is similar to those in Arabidopsis. Furthermore, our results suggest that two LHY/CCA1-like genes were paralogs of each other.

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AtC401 is an Arabidopsis homolog of Pharbitis nil C401 (PnC401) that relates the photoperiodic induction of flowering and shows a circadian oscillation at the transcriptional level. We previously reported that the AtC401 minimal promoter does not required the canonical TATA box but required the 5' UTR sequence for circadian regulation, and suggested that the novel transcription machinery regulates the circadian transcription of AtC401. This minimal promoter contains two candidates of cis-elements; GATA motifs and CCA1 binding like motifs. Then, we constructed the promoter fragments mutated these elements, and fused a firefly luciferase reporter gene (luc+). The reporter analysis using the transgenic plants showed the CCA1 binding like motifs is required for circadian regulation. Moreover, we confirmed the binding of recombinant CCA1 protein to the motif by electrophoretic mobility shift assay. These results suggest that these CCA1 binding like motifs and CCA1 protein are required for circadian regulation of AtC401.

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Grain hardness of wheat is determined by the Hardness (Ha)-locus region, which contains three similar genes; puroindoline-a (Pina), puroindoline-b (Pinb) and GSP-1. Although Krishnamurthy and Giroux (2001) showed that expression of Pina or Pinb in transgenic rice seeds reduced rice grain hardness, functional relationships of the three genes have not yet been clarified.
In this study, we produced the transgenic rice plants harboring large genomic fragments of the wheat Ha-locus region containing Pina and GSP-1 genes by Agrobacterium-mediated transformation (Nakano et al. 2005). We confirmed homozygosity of the T-DNAs in the four independent T₂ lines by using fluorescence in situ hybridization (FISH) and DNA gel blot analyses. RT-PCR and Western blot analyses using the T₃ seeds showed that Pina and GSP-1 were expressed in endosperms of the transgenic rice plants. We are now trying to examine functional contribution to grain hardness in the transgenic rice plants.

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Carrot embryogenic cells (EC) release 4-hydroxybenzyl alcohol (4HBA), an inhibitory factor of somatic embryogenesis in carrot. Thus, high cell density culture of EC causes to failure of somatic embryo formation by 4HBA released from EC. In order to know how 4HBA acts as the inhibitor of somatic embryogenesis in carrot, 2,4-dichloroacetic acid-treated hypocotyl explants were placed on the solidified medium containing 4HBA or its related compounds and somatic embryogenesis was examined. Although 4HBA did not show inhibitory effects of somatic embryogenesis and its related compound vanillyl alcohol (VA) showed stimulatory effect. Histological and molecular biological analyses suggested that 4HBA and VA have an inhibitory effect on the early process of somatic embryogenesis, but stimulatory effect on the late process. The mode of action of the inhibitory and stimulatory effects of 4HBA and VA on somatic embryogenesis in carrot will be discussed.

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Previously, we isolated a rice TPR-containing nuclear protein, IABP4, that is specifically bound to particular rice importin α protein whose expression is down-regulated by light. In an effort to elucidate the function of IABP4, first we tried to examine the phenotype of null mutation in its Arabidopsis homolog, At2g06210 (vip6/elf8). T-DNA insertional-mutagenized lines were obtained from the Salk Institute or Kazusa DNA Research Institute. Though we repeated PCR-based screening for the null mutant seedlings that have T-DNA insertion into both copies of the gene, only seedlings of wild or heterozygous genotypes were found, and not those with homozygous genotype. Therefore, we examined seed development in immature siliques. On average, 40% of seeds on heterozygous plants were aborted, whereas 10% of seeds on wild-type plants were aborted. These results suggest that homozygous knockout mutation of this gene might have caused embryonic lethality.

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FUS3 is one of the key transcription factors controlling seed maturation in Arabidopsis. Microarray analysis comparing the transcriptomes of developing seeds beteween the fus3 mutant and the wild type revealed altered expression of the various genes functioning in the synthesis, metabolism, transport and signal transduction of plant hormones such as auxin, gibberellins, abscisic acid and cytokinins, as well as those thought to be regulated by these plant hormones. For example, the expression of the auxin-induced genes, such as PIN7, PIN3, SUR2 and GH3 family members, were increased in the immature seeds of fus3 compared to wild type. Conversely, the expressions of some of the IAA/AUX genes were decreased in the mutant. However, no large differences in the IAA levels in the developing seeds were found between of the mutant and wild type. We will discuss about the functioning of FUS3 in seed maturation from the view point of hormonal regulation.

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Arabidopsis FUS3 is one of the key transcription factors that regulate seed storage protein (SSP) genes expression. Previously, we proposed that the regulation of FUS3-dependent SSP expression involves an indirect mechanism requiring the ABA- and FUS3-dependent synthesis of intermediate regulatory factor(s). Here, we further conducted molecular analysis of FUS3-dependent SSP expression. By in vivo footprinting analysis of the CRC SSP promoter, we identified two ABREs and one ABRE-like element as potential cis elements for the proposed intermediate regulatory factor(s). Northern analysis revealed that the expression of bZIP67, one of the ABRE-binding factors, is regulated in an ABA- and FUS3-dependent manner. Over expression of bZIP67 was found to enhance FUS3-dependent CRC expression. Furthermore, specific in vitro interactions between bZIP67 and the two ABREs in the CRC promoter was confirmed. These results suggested that bZIP67 is one of the intermediate regulatory factors for FUS3-dependent SSP expression.

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In higher plants, alcoholic fermentation is important to survive under hypoxic conditions. Alcohol dehydrogenase (ADH), which is responsible for alcoholic fermentation, is required to supply NAD⁺ to glycolytic pathway. We characterized the reduced adh activity (rad) mutant, whose coleoptile elongation is repressed under submergence. It was found that the reduction of ADH activity was caused by decrease in the amount of ADH1 protein as a result of point mutation in the Adh1 gene.
In this study, we analyzed difference in gene expression between the rad mutant and the wild type during coleoptile elongation. Thus, the coleoptiles were isolated by use of Laser Microdissection (LM). Thereafter, the rice 22K oligo-DNA microarray was performed using RNA extracted from the LM-isolated tissues. We selected 315 up-regulated genes and 153 down-regulated genes in the rad mutant (p-value<0.05). Now, functional analyses of some interesting genes are under way.

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A rice proteinase, REP-1 degrades seed storage proteins, and expression of the gene, Rep1 is induced by gibberellin (GA). The promoter region contains GA response element (GARE) to which GAMyb bind and a cis-element including CAACTC core sequence designated CARE, and these elements are required for GA response of Rep1. CARE interacts with a transcription factors containing a Myb motif in the C-terminal region, its gene designated CTMyb1. The differences of the transcription start sites of CTMyb1 result in two polypeptides, designated CTMyb1L (521 amino acid residues) and CTMyb1S (409 amino acid residues). The transient expression of CTMyb1L had no effect on activity of Rep1 promoter, but CTMyb1S activated Rep1 promoter with OsGAMyb synergistically. CTMyb1S interacted with rice prolamin box binding factor (RPBF) known to interact with OsGAMyb. In addition, RPBF activated Rep1 promoter. These results suggest that Rep1 expression is involved in complex consisting of CTMyb1S, RPBF and OsGAMyb.

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Expression analysis of β subunits of OSK in germinating rice seeds using semi-quantitative RT-PCR techniques revealed high expression of all the four β subunits during seed germination. Analysis of OSK gene promoters fused to GUS gene exhibited very high expression of GUS in the scutellum during germination. An attempt to purify the OSK complex from seeds during the germination stage resulted in isolation of a complex with SNF1-specific kinase activity and molecular weight similar to the proposed hetero-trimeric complex. However, visualization of phosphorylation pattern of the purified complex revealed that in addition to OSK auto-phosphorylation, additional proteins were phosphorylated. These could be substrates of OSK in the germinating seeds. Furthermore, suppression of OSK1, OSK3 and OSK4 during germination, using chemical-inducible RNAi resulted in delayed germination. The results of RNA-interference experiments also suggest that each species of OSK could have distinct function during seed germination.

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The seed germination has been shown to require ROS. However, little is known about the molecular mechanism how ROS promote seed germination. Here, we report that ROS promotes seed germination by increasing the expression of GA biosynthesis genes. O{SUB}2{/SUB}{SUP}-{/SUP} was immediately generated following seed imbibition. DNA microarray analysis and seed germination test showed GA biosynthesis genes were upregulated by H{SUB}2{/SUB}O{SUB}2{/SUB} treatment that promoted seed germination. The effect of H{SUB}2{/SUB}O{SUB}2{/SUB} treatment was observed only at an early period of seed germination. DPI slightly inhibited generation of O{SUB}2{/SUB}{SUP}-{/SUP} with decrease in the expression level of GA biosynthesis genes. Unexpectedly, a mutant of NADPH oxidase show that increased generation of O{SUB}2{/SUB}{SUP}-{/SUP} promoted with seed germination and expression of GA biosynthesis genes. These results suggest that during seed germination O{SUB}2{/SUB}{SUP}-{/SUP} or H{SUB}2{/SUB}O{SUB}2{/SUB} is an early signal inducing GA biosynthesis genes, and that NADPH oxidase is a component of O{SUB}2{/SUB}{SUP}-{/SUP} generation following seed imbibiiton.

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It has been reported that mature dry seeds contain RNAs stored during maturation and protein synthesis in the early stage of seed germination occurs utilizing such long-lived RNAs. However, the mechanism for keeping the activities of long-lived RNAs and the induction system of protein synthesis after imbibition remains to be elucidated. There is a possibility that RNA binding proteins in dry seeds play important roles in stabilization of long-lived RNAs and/or regulation of protein synthesis. It is well known that RNA binding proteins could be purified selectively by single stranded DNA (ssDNA)-affinity column chromatography. Therefore, we carried out proteome analysis of proteins in the ssDNA binding fraction from dry seeds of rice. It was found that RNA binding proteins such as glycine rich RNA binding protein and KH domain protein were detected in the fraction. In addition, the KH domain containing protein was disappeared until 24 hr after imbibition.

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msi1 mutant was identified as one of the mutants which seeds autonomously develop without fertilization. Nuclear division normally progress in endosperm of msi1 gametophyto and endosperm become to polyploid cell. However cellularization of polyploid endosperm do not complete. MSI1 repress gene transcription as one of the component of polycomb protein complex.
msi1 homozygote plant is lethal at early embryogenesis. On the other hand, the others one of the component of polycomb group complex, medea's mutant is viable. Thus MSI1 might have another function except component of polycomb group complex. Here we aim at finding new role of MSI1 protein in plant development by genetic and cell biological analysis.

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