Salicylic acid (SA) is an important signal molecule in plant disease resistance. Two pathways for the biosynthesis of SA are proposed: the isochorismate pathway and the benzoic acid (BA) pathway. In the former, ICS1 gene encoding isochorismate synthase has been identified. However, genes involved in the latter pathway are not known. To study the BA pathway for SA biosynthesis, mutant screening was performed. The growth of wild-type is inhibited in the presence of SA. On the other hand, BA has no such inhibitory effect. Based on this property, we isolated benzoic acid hypersensitive (bah) 1 mutant that exhibit an inhibited growth in the presence of BA. bah1 grew normally in the absence of BA. The transcript of SA-inducible PR1 gene accumulated in bah1 in the presence of BA. These results raise a possibility that the accumulation of SA occurs in bah1 by application of BA.

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Rapid accumulation of reactive oxygen intermediates (e.g., H₂O₂) is a striking early event in the hypersensitive response including programmed cell death. Signalling pathways leading to these responses, however, remaine to be dissolved. To elucudate the signaling pathways, eleven rice lesion mimic mutants spl (spl1~11) with spontaneous cell death on their leaves, were investigated using suspension-cultured cell. Three of the mutants (spl2, spl7, spl11) were found to accumulate higher amount of H₂O₂ than the wild type, when treated with elicitor, indicating that these mutations are involved in accumulation of H₂O₂. Calyculin A (CA), an inhibitor of protein phosphatase, enhanced production of H₂O₂ in spl7 but not in the wild type, spl2 and spl11. Moreover, CA-enhanced H₂O₂ production in spl7 was reduced by addition of a Ca²⁺ chelator EGTA. These results imply that spl7 participates in regulation of H₂O₂ production at a protein dephosphorylate- and Ca²⁺ - dependent step.

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Arbuscular mycorrhizal (AM) fungi are ancient symbionts on plant roots. AM fungi uptake phosphate (Pi) through extraradical mycelium that extends beyond rhizosphere and supplies Pi to plants. In return, plants supply photosynthates to AM fungi. An AM-specific Pi transporter gene of Lotus japonicus, a model legume, was identified and its knockdown transformants were prepared. Under Pi-limiting conditions, the transformants showed reduction of Pi uptake via AM roots. The transformants also exhibited reduced numbers of fungal arbuscules and vesicles, and an increased nuumber of plant root cells showing hypersensitive response-like symptom. Thus, sufficient Pi uptake through the AM-specific phosphate transporter is necessary for establishment of normal symbiosis including development of fungal structures and suppression of plant defense response.

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Acidophilic algae, Chlamydomonas acidophila DVB 238 and Chlamydomonas acidophila KT-1, had very strong heavy metal tolerance in comparison with other neutrophilic algae. To explore the possibility for use of phytoremediation, two acidophilic algae were investigated the accumulation of metal (Cd and Ni) and the protein synthesis under various metal concentration.
C. acidophila DVB 238 showed higher accumulation (13.7 μg g Cd and 2.59 μg g Ni per 10⁸ cells) than C. acidophila KT-1. Moreover Cd treatment did not induce an increase of protein in C. acidophila KT-1 but C. acidophila DVB 238. Therefore these two strains seemed to have different system for accumulating Cd. So we detected changes in the soluble protein synthesis pattern by two-dimensional polyacrylamide gel electrophoresis for C. acidophila DVB 238 cells grown under different concentrations of Cd and Ni. Some of characteristic spots were analyzed by protein sequencer and MALDI-TOF Mass.

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To investigate the toxic mechanisms of boron, we identified Arabidopsis thaliana genes that confer boric acid tolerance to yeast. Among the identified genes, several putative splicing factors were included. To examine the possible effects of boric acid on the splicing, we performed RT-PCR to detect accumulation of splicing intermediate depending on the boron supply. We found that the splicing of RPL7B was inhibited by high levels of boric acid application. RPL7B is a ribosomal protein and a paralog RPL7A is present in the yeast genome. It was known that disruption of both RPL7A and RPL7B is lethal. Yeast cells with disrupted RPL7A were sensitive to high B whereas those with disrupted RPL7B was not, suggesting that inhibition of splicing of RPL7B is critical in the RPL7A mutant background. The present study is the first demonstration of a molecular mechanism of boron toxicity.

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To date, >32,000 cDNA clones have been collected and sequenced by the Rice-Full-Length-cDNA Project. Mapping of the cDNAs to rice genome sequence (Pseudomolecule ver 2 from TIGR) indicated that 1094 clones may be transcribed from the different regions of rice genome. Among them, 681 clones may be produced by the trans-splicing event, and we attempted to detect the corresponding transcripts by RT-PCR. A fragment from one of these clones, AK100704, suggested to be generated from two transcripts, which were derived from different chromosomes. They were joined differently from that shown in the previously reported cDNA. The RNA fragment with the same structure was also detected in several tissues such as leaves, stems, roots, and panicles. The corresponding transcriptional origins are located in the chromosome 1 and the chromosome 5, respectively, and no other sequence was found. Therefore, these data suggest that the trans-splicing event may involved creating this transcript.

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In red-leaf lettuce, although the leaf is usually dark reddish-brown, if it grows by hydroponics, the anthocyanin content will decrease and it will become the shortage of coloring. In our experiments, irradiation of blue and/or UV-B light at night, cooling of hydroponic solution, and restriction of phosphorus fertilization had effects in the improvement of coloring. Accumulation of anthocyanin occurred in the immature leaf and the mature leaf was not able to obtain sufficient coloring. Then, the cloning of genes which encode biosynthesis of anthocyanin was carried out, and expression analysis was performed. The transcript level of LDOX increased in the immature leaf by which coloring was promoted. On the other hand, the transcript level of LDOX was low on the cooling conditions of hydroponics culture solution, or the restriction conditions of phosphorus fertilization. These results suggest that the transcript level of LDOX is variously controlled according to environmental factors.

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Plant roots perceive various environmental stimuli such as water,gravity and touch.Recently,the bending response of roots towards higher concentrations of oxygen (oxytropism) was found in Pea plants.To elucidate the molecular mechanism of oxygen sensing in plants,we developed a new simple method of measuring the response of roots to hypoxia in Arabidopsis thaliana.
Seedlings were grown on GM medium for 6 days under continuous light in petri dishes.Following 6 days growth,an oxygen-remover was added to expose the seedlings to a low concentration of oxygen for 2 days.A 90% reduction in oxygen induced ectopic formation of elongated root hairs at root tips.
The role of ethylene in root hair formation is well documented.Therefore we compared hypoxia-induced root hairs with the root hairs of plants treated with ACC. Moreover,we assessed the effects of the ethylene synthase inhibitor AVG on hypoxia induced root hair formation.

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We have investigated transcriptome and metabolome of Arabidopsis under sulfur deficiency stress to elucidate a holistic response of sulfur deficiency. In our studies, the genes involved in jasmonates(JAs) biosynthesis and those induced by JAs treatment were up-regulated. These results suggest that there is a relationship between sulfur deficiency response and JAs signaling. Arabidopsis mutants which produce less JAs were grown under sulfur deficient condition and their transcriptome and metabolome were analyzed. Involvement of JAs signaling in response to sulfur deficiency is discussed.

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Graminacious plants secrete mugineic acid family phytosiderophores (MAs) from their roots to solubilize the external insoluble iron and absorb the chelated iron. The MAs secretion from root increases under Fe-deficient condition and followed the circadian rhythm. The particular vesicles derived from rER were observed to accumulate at the cell periphery near the cell membrane in Fe-deficient barley roots before sunrise. Immunocytochemical analysis on nicotianamine synthase (NAS), nicotianamine aminotransferase (NAAT), and IDS3, enzymes in MAs biosynthesis pathway, revealed that these enzymes were increased in Fe-deficient root epidermis. The subcellular localization of NAS, NAAT, and IDS3 were determined to be on the membrane, inside, and in the vicinity of the vesicle, respectively. It is suggested that deoxymugineic acid could be synthesized in the vesicle, and then DMA could be transported to the cytoplasm to convert to MAs. The polar vesicle transport may regulate the diurnal rhythm of MAs secretion from barley root.

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The ozone-sensitive and ozone-tolerant tobacco varieties Bel-W3 and Bel-B have been widely used as bioindicator of ozone. However, the mechanisms for the difference in sensitiveness to ozone in above two varieties are not fully understood. Ozone is readily converted other members of reactive oxygen species (ROS) such as superoxide anion. Here, cell suspension cultures were prepared from Bel-B and Bel-W3 plants and propagated for the study of generation and metabolism of ROS. When both cell lines were treated with salicylate (1 mM) and AlCl₃ (1 mM), 5-fold greater superoxide generation was observed in Bel-W3 cells compared to Bel-B cells. The in vivo catalase activity was measured by monitoring the H₂O₂-dependent O₂ evolution using a Clark-type oxygen electrode. The in vivo catalase activity in Bel-B cells was ca. 2-fold greater than that in Bel-W3 cells, and apparent V_max in Bel-B cells was slightly greater than that in Bel-W3 cells.

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It is already known that hypocotyl elongation of Arabidopsis thaliana is controlled by gibberellins (GA) and light through phytocrome (red/far red) and cryptochrome (blue light) response. However, effects of other monochromatic lights, especially yellow light, are still unclear. In this study, Arabidopsis thaliana seedlings were cultured on media added GA and/or uniconazole under different monochromatic lights or dark condition. On the medium added only GA, hypocotyl growth was strongly suppressed by blue light compared with other lights. Hypocotyl growth under yellow light was almost equal that under dark. However, yellow light was most promotive on hypocotyl elongation among tested monochromatic lights, on the media added both of GA and uniconazole. These results suggested that the responsiveness of hypocotyl to GA changed depending on light quality. Now, using a mutant lacking of GA synthesis enzyme, the responsiveness to GA is going to examine under different monochromatic lights.

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Plant mitogen-activated protein kinases (MAPKs) have been involved in a multitude of biotic and abiotic stress responses, hormone responses, and regulation of cell division. Some MAPK cascades are involved in wound/JA signaling pathway. One of them, Arabidopsis MPK4 (AtMPK4) is required for JA-responsive gene expression, and AtMPK4 is activated by various stress responses in Arabidopsis. However, in our knowledge, there is no evidence about function of this MAPK in other plant species. In this study, to assess whether the MPK4 is involved in wound/JA signaling pathway in other plant species, we isolated tobacco MPK4 gene (NtMPK4) and generated the NtMPK4-silenced tobacco plant by RNA interference (RNAi). Using the transgenic plant, we show that NtMPK4 has an important role in wound response as well as WIPK and SIPK, and also is involved in ozone response by regulating a JA signaling pathway.

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Plants develop sun and shade leaves depending on light environment. Although information of differences in physiological traits and leaf anatomies between sun and shade leaves has been accumulated, analyses of their developmental processes were rarely carried out. Recently, it was reported in several plants that development of new leaf is affected by light environment of mature leaves but not their own one. These facts indicate long-distance signaling from mature leaves to leaf primordia. We hypothesized that photosynthates might be the signal. To test this hypothesis, we anatomically analyzed Arabidopsis thaliana grown on various sucrose-concentration medium under 60 μmol m^-2 s^-1, under which plants grown on soil develop thin leaves with one-cell layered palisade tissue. With the increase of sucrose concentration, the leaves developed thick lamina and two-cell-layered palisade tissue. These results support our hypothesis. Analyses of some mutants with defects in photo- or sugar- sensing will also be introduced.

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Lesion mimic mutants develop spontaneous cell death without pathogen attack. To understand the molecular mechanism of cell death in lesion mimic mutants, we isolated len1 and lin2 mutants. The len1 mutant develops lesions on its leaves under short-day conditions. LEN1 was identified to encode a chloroplast chaperonin 60b (Cpn60b), a homologue of bacterial GroL. However, we do not know the mechanism of the lesion initiation of len1 plants. The lin2 mutant develops lesion formation on leaves and siliques in a developmentally regulated and light-dependent manner. LIN2 encodes coproporphyrinogen III oxidase, a key enzyme in the biosynthetic pathway of chlorophyll and heme, a tetrapyrrole pathway, in Arabidopsis. The len1lin2 plants showed reduced lesion formation than len1 plants under short-day conditions. This would suggest that LIN2 would be necessary to execute lesion formation in len1 plants.

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Temperature is a primary factor for seed germination.To classify genes expressed in imbibed Arabidoopsis seeds and to find genes related to germination inhibition at high temperature, we adopted 22k Arabidopsis2 oligo-microarray from Agilent. We picked 9,138 genes that showed significant signal change more than 2 fold by imbibition. We found that the number of genes and the degree of change increased with imbibition time at 22^oC, and the number and the change were relatively small at 34^oC. The patterns of gene expression were clustered into 4 groups; 6h at 22^oC, 6h at 34^oC, more than 12h at 22^oC and more than 12h at 34^oC. There was no visible germination by 24h at 22^oC, and rupture of seed coat was observed after 36h. These results suggest that temperature determined the pattern of gene expression until 6h, and induced the gene expression related to germination induction or inhibition after 12h.

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Storage proteins in cotyledon of legume plants are degraded by proteases expressed during seed germination and are mobilized to the embryonic axis for its growth. In aleuron layers of rice or barley, expression of proteases is induced by gibberellin (GA). We examined whether GA regulate expression of a protease, EP-C1 in germinated cotyledons of common bean. Expression of EP-C1 was repressed by GA biosynthesis inhibitors, suggesting that GA regulates expression of EP-C1 in germinated cotyledons. Because a DELLA protein, RGL2 is known to be involved in GA-regulated germination of Arabidopsis seeds, we examined whether RGL2 regulated EP-C1 expression. The promoter activity of EP-C1 was repressed by over-expression of RGL2, suggesting that DELLA proteins regulate EP-C1 expression in germinated cotyledons.

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Gibberellins induce gene-expression for hydrolytic enzymes in germinated cereal aleurones. Products of GA-primary genes, such as GAMYB, act as key molecules leading to transcription-activation of these genes. To dissect true mechanisms of GA-mediated gene-expression, it would be critical to understand how GA-primary genes themselves are regulated. Previous analyses indicated that 5'-upstream portion from GA-primary genes could not confer GA-responses. Several regulatory domains were located throughout gene-region. The most notable finding was enhancer-activities present in a large 1st intron. In Arabidopsis, intron-enhancers have been identified in floral-control-genes, AG and FLC, in which large intron should not only contribute to proper gene-expression, but also associate with gene-silencing by covalent modifications of DNA and histone. These evidences allow us to suggest that primary event caused by GA-action might be recruitment of primary genes from silent chromatin region. Based on this assumption, we have been investigating DNA-methylation-status of GA-primary genes, using bisulfite sequence method.

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FUS3, a key transcription factor controlling seed maturation, regulates the expression of seed specific genes including the SSP genes. Microarray analysis using transgenic plants carrying an inducible FUS3 transgene revealed that FUS3 regulated genes are categorized into several groups with respect to induction kinetics and ABA-dependencies, suggesting distinct mechanisms of FUS3 action. Members of the SSP gene families (the Napin and Cruciferin families) belonged to different groups. The FUS3 induction of CRC (a Cruciferin member) took much longer periods than that of At2S3 (a Napin member), indicating that the FUS3 regulation of CRC required intermediate regulatory factors whose synthesis was regulated by FUS3 and ABA. The microarray analysis identified eighteen FUS3-induced transcription factors. Cotransfection experiments revealed that the bZIP67 and bZIP12 among them were able to activate the CRC promoter. Thus, these bZIP factors are the strong candidates for the intermediate factors that function in the FUS3 regulation of CRC.

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In seed germination, expression of many genes is induced, and various transcription factors play important roles in this process. However, it is hard to detect the transcription factors in proteome analysis, because the content of these proteins in cells is known to be extremely low. To identify differentially displayed transcription factors of rice seeds during germination, 2D-PAGE analysis was carried out after prefractionation of DNA binding proteins by DNA affinity column chromatography. It was found that (i) three major DNA binding proteins (42kDa, 40kDa and 38kDa ) decreased rapidly after absorbing water in seeds, and (ii) these three proteins existed in halfseeds without embryo. Moreover, the significant decrease of three proteins in DNA binding fraction was observed when the embryoless half seeds were soaked in water. These results suggest that any factor from embryo is not required for decrease of these three proteins.

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Photoperiods regulate expression of various genes involved in circadian rhythm, photosynthesis and flowering. In Pharbitis nil, the exposure to a single dark period of 16 h can induce flowering. In order to identify novel genes involved in flowering induction, we have isolated two genes induced by a 14 h dark treatment from P. nil using a cDNA subtraction. These genes are highly homologous to ubiquitin 1,2 and unknown gene in Arabidopsis. The expression of both genes in cotyledons was induced by dark treatment of 12-16 h. Accumulation of these transcripts was reduced by a brief exposure to white light at the 8 h of the dark period (night break treatment) or by exposure to far-red light at the end of the light period (end-of-day far-red treatment). Possible involvement of these genes in flowering induction will be discussed.

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The most important phase change in plants is the transition from vegetative to reproductive growth. A MADS-domain gene, AGL24, was identified as an important promoter of flowering in Arabidopsis. To gain insights into the expression mechanism of AGL24 we have analyzed the AGL24 promoter using a series of AGL24:: GUS reporter genes. There deletion studies revealed cis-elements to lie between nt -850 and nt -353, and in the 2nd intron. The AGL24 2nd intron contains the consensus CArG box sequence, the MADS-box protein binding element. We also found that MADS-box proteins, AP1, SOC1 and SVP bind to this CArG box in vitro by gel retardation experiments. These results suggest that the transcription of AGL24 is directly controlled by AP1, SOC1 and SVP.

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Arabidopsis AGL24 encodes a MADS-box transcription factor, and it promotes flowering. A second MADS-box gene SVP, which is phylogenetically very close to AGL24, represses flowering. In this study we focused on the target genes of AGL24 and SVP to understand the mechanisms of their transcriptional regulation. To identify their downstream genes microarray experiments were performed using shoot apices of agl24 and svp mutants and transgenic plants constitutively overexpressing AGL24 or SVP. Results indicated that 56 genes in AGL24-overexpressing plants, 171 in SVP-overexpressing plants, 7 in agl24, and 3 in svp were up- or down-regulated at least 2-fold compared to wild type. Most of them were not previously identified as having roles in flowering except for SOC1, a flowering pathway integrator. SOC1 expression increased in AGL24-overexpressing plants and svp, but decreased in SVP-overexpressing plants. These results suggest that AGL24 and SVP may antagonistically control flowering partly through the regulation of SOC1.

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Arabidopsis MADS-box genes, AGL24 and SVP are phylogenetically very close, while AGL24 promotes flowering, SVP represses it. Both genes encode transcription factors which consist of MADS-box, I, K, and C domains. To investigate the mode of action of AGL24 and SVP proteins, we tried to identify the domains responsible for their functional specificities. We generated and analyzed transgenic plants constitutively overexpressing chimeric AGL24 and SVP genes. As a result, plants carrying the construct which contains the SVP MADS-box and the AGL24 I, K, and C flowered earlier than wild type. In contrast, plants with the construct which contains the AGL24 MADS-box and SVP I, K, and C showed a late flowering phenotype. These results indicates that I, K, and C domains are important for the regulation of flowering, suggesting that differences in the formation of higher-order complexes between AGL24 and SVP may determine their functional specificities.

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Ubiquitin-mediated protein degradation pathway is known to be involved in varied regulatory processes of plant development. F-box protein is a component of SCF complex (Skp1, Cullin, F-box) that functions as an ubiquitin ligase(E3). At least, 568 F-box genes are present in Arabidopsis genome, while others such as Skp1 and Cullin consist of small family, (Kuroda et al. Plant Cell Physiol.43: 1073-1085(2002)), suggesting that the F-box protein plays a central role in specific recognition to the target protein that is to be degraded.
We isolated a late flowering mutant, F-box related to late flowering 1 (fbl1), from Arabidopsis F-box antisense line. FT transcription was decreased in the fbl1 background. In the 35S::FT fbl1 double mutant, the 35S::FT suppressed the late flowering phenotype of fbl1. From the lines of evidence, it is likely that the fbl1 mutation is not caused by autonomous promotion pathway but by long-day period promotion pathway.

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To investigate the flowering mechanism of apple, the expression of AFL1 and AFL2 genes, which are orthologues of the LEAFY gene from Arabidopsis, are analyzed. These 5' upstream region (about 2.5kb length, respectively) were linked to GUS gene, and transduced to Arabidopsis. The transformed Arabidopsis with AFL2:GUS showed GUS expression in young flower buds, but AFL1:GUS not. Both transformed plants showed heavy stains at stipules of rosset and cauline leaves. These results suggested the AFL2 gene correlate to flower development. Next, the apple JM2 (M. prunifolia x M. 9), which was selected as dwarf root stock in this department of apple research, was used for transformation. We found the JM2 had much higher transfomation efficiency than otheer cultivars. Five hundred seventeen pieces of leaves from JM2 were transformed with AFL2:GUS, and 17 lines with GUS staining and Kanamycin resistance were obtained.

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We previously indicated that epigenetical regulation is involved in the photoperiodic flowering in Perilla frutescens var. crispa whose flowering state lasts long as the low temperature effect lasts long in vernalization. Accordingly, we investigated a possible correlation between the character of permanent flowering state and the ability to flower by DNA demethylation.
Four plant species were photoperiodicaly induced to flower and then transferred to under non-inductive condition. Pharbitis nil and Lemna paucicostata reverted to vegetative growth, whereas Silene armeria and Xanthium strumarium did not. Treatment with 5-azacytidine caused flowering in S. armeria, but not in X. strumarium. The results suggest that the permanent flowering state is not necessarily correlated with the flower-induction by DNA demethylation. The mechanism of flowering in X. strumarium may differ from that in P. furutescens and S. armeria. This paradox will be discussed by analyzing the level of DNA methylation in rDNA intergenic spacer region.

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Several environmental stress induces seeds sterility in a lot of plant species via injury to their reproductive development. However these molecular mechanisms are not well understood. We study the high-temperature injury of barley microsporogenesis at the molecular level. Barley is a useful experimental plant for investigation reproductive development, because its development is well synchronized. High-temperature stress (30^oC day/25^oC night) during early development of anther cell layers causes complete male sterility. In this presentation, we would like to show the result of differences of entire protein expressions between high-temperature injured panicles and control panicles. We found several proteins fluctuated by high temperature stress via proteomics analysis including 2D SDS-PAGE and peptide mass finger printing wiht MALDI-TOF MS.

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High-temperature stress (30^oC /25^oC) for 5 days during early development and differentiation of anther cell layers induced male sterility in barley plants. Tapetum and pollen mother cells completely failed to differentiate and "no pollen grain" phenotype was revealed at anthesis. Several transcripts including histone and glycine-rich RNA-binding protein genes drastically increased just before development and differentiation of anther cell layers. In contrast, under the high-temperature condition, the significant induction did not occur and also normally abundant mRNAs, such as 60S ribosomal protein L27a and glyoxalase I, were reduced. We found a strong correlation between the failure of transcriptional re-activation following down-shift to optimal temperature and the sterility. Hyper-phosphorylation of the CTD ser-5 of RNA polymerase II largest subunit occurred under high-temperature condition. These results suggest that the early development and differentiation of barley anthers is sensitive to high-temperature stress due to inhibition of general transcription.

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Meristem keeps supplying cells to organs while maintaining its defined structure. tebichee (teb) mutant of Arabidopsis is defective in meristem structure and shows abnormal organ shape. TEB gene encodes a homolog of MUS308 of Drosohila, which is involved in repair and response to DNA damage. In teb plants, expression levels of genes inducible by DNA doublestrand-breaks were higher than the wild type. In addition more cells in meristems of teb expressed cyclinB1::GUS than those in the wild type, suggesting that defect in cell cycle progression leads to meristem disorganization in teb. Double mutant of teb and atr-2, which has a mutation in ATR involved in DNA replication checkpoint, showed enhanced phenotypes of teb, suggesting that TEB is involved in progression of DNA replication and/or checkpoint mechanism. Further functional analysis of TEB will reveal a relationship between the DNA replication or cell cycle regulation and the structure of meristems and organs.

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A soluble ABC protein gene, GCN20-3, was isolated by gene trap mutagenesis in Arabidopsis. The gene was expressed in root tips, and involved in root development. An Arabidopsis GCN1 gene whose product was interacted with the ABC protein was also expressed in root tips, and shoot meristems, flowers. Two genes are orthologs of yeast GCN20, GCN1 genes that function in amino acid starvation conditions. In yeast, other GCN2, eIF2α genes were concerned with this system. To gain more insight into this system in plants, promoter regions of the Arabidopsis GCN2 gene or eIF2α gene were fused to a GUS gene, and then the genes were introduced into Arabidopsis plants. The GCN2 gene and one of the eIF2α gene (AteIF2α_II) were expressed in root tips, shoot meristems, and flowers similar to the GCN1 gene expression. These results suggested that plant GCN proteins play important role in development.

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Typical angiosperm B-class MADS box genes are expressed in petals and stamens, but their expressions in other organs have also been reported. Up to now, the gymnosperm homologs are reportedly expressed exclusively in male cones. However, detailed analysis has not been carried out to determine the expression of the genes in other organs. Earlier we showed that the two B-like genes in Cryptomeria japonica have a male-cone-specific expression pattern as demonstrated by Northern blot analyses. In this study, we performed the RT-PCR analysis to determine whether they are expressed in organs other than the male cones. Our results indicate that these genes could be detected in leaves of 1-year-old saplings and field-grown trees, in leaves that associated with the early stages of strobilus development, and in female cones till the seeds were produced. These results indicate that the conifer B-like genes may have additional roles to play in plant development.

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One of the previously identified meiosis-associated genes from Lilium longiflorum, designated LlM532, has shown to contain a nuclear localization signal and sigma-70 like RNA binding motif, and has homology with yeast Rpf2, a pre-rRNA processing factor of ribosome biogenesis. Immunostaining and protein gel blot analysis indicated that the LlM532 gene product is a nuclear/nucleolar protein highly expressed during early stages of microsporogenesis. Nuclear targeting and subnuclear localization of the LlM532 protein was confirmed by expressing the GFP::LlM532 fusion protein in onion epidermal cells. We have also isolated an Arabidopsis homolog, designated AtM532. The similarity between LlM532 and AtM532 is high and the intracellular localization of GFP fusion protein indicated that the AtM532 protein is targeted to the nucleolus. The AtM532 gene exists as a single copy in the genome and the mRNA is expressed at high levels in reproductive organs and root.

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RDD1 was identified as one of genes exhibited daily expression in constant darkness in leaf blades of rice (Oryza sativa L.). This gene has a similarity to a transcription factor gene, Dof. RT-PCR analysis showed that rhythmic expression of two photosynthesis-related genes, Cab1R and rbcS, was altered in both sense (S) and antisense cDNA-overexpressing (AS) transgenic rice plants. Change in accumulation patterns of the both transcripts was different between the transgenic plants under long days and those under short days. Furthermore, the presence of antisense RNA for endogenous RDD1 was detected, suggesting that it may have a role in regulation of the RDD1 expression. The AS plants were shorter and showed later heading than wild-type and the S plants. These results suggested that RDD1 seems to be one of the transcription factors to regulate expression of photosynthesis-related genes and involved in the control of growth and heading date.

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The central circadian oscillator, kaiABC genes, has been identified in cyanobacterium Synechococcus elongatus PCC 7942. It is proposed that the transcriptional feedback loop involving kaiABC provides a circadian oscillation to each cyanobacterial gene expression. We found these oscillations are able to be classified by two types of amplitude shapes, which are a high amplitude-type and a low amplitude-type. The former expression pattern shows restrict "stop and go" on the transcriptional level during the circadian times. On the other hand, the latter keeps a certain amount of gene expression all the day through, showing a weak circadian oscillation. However, there is no specific cis-element on their promoter region to determine the amplitude shapes. Our micro array data suggested that these oscillation patterns are raised by different mechanisms, and additional experiments provided a new model for circadian output cascade.

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Cyanobacteria are the simplest organisms known to have a circadian clock. A random promoter-trap experiment in Synechococcus elongatus sp. PCC 7942 demonstrated circadian rhythms in the activities of almost all gene promoters. It seems plausible that the cyanobacterial clock system regulates basic transcriptional machinery or a process closely linked to it. Here we show physiological significance of circadian gene expression by using high-density DNA microarrays (Affymetrix Gene Chip) of S. elongatus 7942. Gene expressions of a whole genome were analyzed every 4 hours during 52 hours of continuous culture. It will be discussed that circadian gene expression is involved in controlling physiological processes in the cyanobacterium.

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A variety of circadian rhythms have been observed in angiosperms. In contrast, only a very few examples have only been reported in relatively primitive plants: gymnosperms, ferns and bryophytes. We started the study of circadian rhythms and its molecular basis using the moss Physcomitrella patens, to which gene-targeting techniques are applicable. From P. patens, we have so far identified several clock-controlled genes (ccgs), all of which exhibited damping oscillation in continuous darkness while showing arrhythmic expression profiles in continuous light. The latter feature is contrasting to ccgs in angiosperms, which generally show rhythmic expression in continuous light. We show here results of recent analyses on some ccgs such as Lhcb genes and on moss homologs of Arabidopsis "clock gene" CCA1.

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Plants must properly respond to daily light/dark cycles and changes in seasonal day-lengths. In such processes, a circadian clock plays crucial role. In the model higher plant Arabidopsis thaliana, it has been proposed that the central oscillator is composed of the Myb-related transcription factors CCA1 (LHY) and the pseudo response regulator PRR1/TOC1. The PRR1 clock component is a member of small family including PRR9, PRR7, PRR5, and PRR3. We already suggested that these PRR members all together play roles close to the circadian clock. To gain further insight into the clock-associated functions of PRRs, here we characterized a series of double prr mutants, such as prr5/prr7, prr5/prr9, prr7/prr9, with reference to the circadian-associated phenotypes. The results suggested that these PRRs play clock-associated roles coordinately and/or complementarily. These results will be discussed in the context of the current molecular model of the plant circadian clock.

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In the model higher plant Arabidopsis thaliana, a number of circadian clock-associated protein components have recently been identified. Among them, a small family of ARABIDOPSIS PSEUDO-RESPONSE REGULATOR (APRRs) < is interesting because the most probable clock component TIMINING OF CAB EXPRESSION 1 (TOC1) belongs to this family. Here we dissected the regulatory cis-elements of the light-induced and/or circadian-controlled APRR9 promoter, by employing not only a mutant plant carrying a T-DNA insertion in the APRR9 promoter, but also a series of APRR9-promoter::LUC (luciferase) reporters that were introduced into an Arabidopsis cultured cell line (T87). we provided several lines of evidence for that the APRR9 promoter contains at least two distinctive and separable regulatory cis-elements: namely, "L element" responsible for the light-induced expression, which is followed by "R element" necessary for the fundamental rhythmic expression of APRR9. Furthermore, APRR1/TOC1 was implicated in the L-element-mediated light response of APRR9, directly or indirectly.

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Greening of etiolated seedlings is an important process of photomorphogenic responses. The chlorophyll synthesis under light must be finely controlled because certain intermediates of chlorophyll synthesis act as oxidative reactants, which might cause dangerous effects on plants. However, the regulatory mechanism underlying the chlorophyll synthesis during de-etiolation (or greening) is not fully understood. In this respect, it was recently reported that a phytochrome-interacting bHLH transcription factor, name PIF1, serves as a negative regulator of the chlorophyll synthesis in etiolated seedlings. PIF1 is interesting because this bHLH factor turned out to be identical to PIL5, which we previously identified as a factor that directly interacts with the Arabidopsis clock-component PRR1/TOC1 in yeast two-hybrid analyses. This fact led us to envisage that the clock-associated factors, such as PRRs and CCA1, might closely implicated in the control of the chlorophyll synthesis. Here we report several lines evidence that strongly support this intriguing hypothesis.

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Tomato accumulates a variety of secondary metabolites in its fruits. Therefore tomato is one of the best candidates for the Metabolome Project in plants. A whole genome sequencing, analysis of the full-length cDNAs and ESTs and screening of mutants are essential to step forward the Tomato Genome Project. Actually we are performing the EMS-mutagenesis of tomato (Micro-Tom) to generate 5,000 M1 plants. We will show our current status on the EMS-mutagenesis and screening of tomato mutants using these lines.

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Eucalyptus is planted worldwide for pulp and paper production with more than 10 million hectares throughout Asia, South America, southern Europe and Australia. The importance of this genus to industry has encouraged its genetics and the molecular biological researches. Consequently, Eucalyptus has become a key forestry species that has a potential for exploring genomic research. With the aim of understanding the genetic system carried by eucalypt, we launched on a large-scale genome analysis of Eucalyptus camaldulensis. Its genome size is estimated to be 650 Mbp. The Whole Genome Shotgun (WGS) method was applied to produce the Eucalyptus draft genome sequence. For the sequencing, another genomic library was constructed in BAC-vector in parallel with WGS. All clones (57,600 BACs) were subjected to end sequence analysis in order to establish the scaffold of WGS-contigs. We are going to report the current status of the E. camaldulensis genome project.

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Cyanobacteria are a diverse group of prokaryotes known also as blue-green algae. They are known to inhabit a variety of habitats, and show many responses to changes in milieu exterior. Recently, many cyanobacterial genome projects are ongoing, and we can use many genome sequences for informatics analysis. Thus, we performed the phylogenetic profiling analysis using the Pfam, which is a database of protein domains. We performed hmmpfam program for 14 cyanobacterial genomes and obtained about 2000 Pfam domains, then we used hierarchical clustering and selected the result on the viewpoints of biological characters and their habitats. We focus on signal transduction-related domains (GAF, PAS, HisKA and Response_reg, etc.). Interestingly, an approximately three-fold increase in the number of these domains were observed in freshwater cyanobacteria compared to seawater species. In addition, we predict that some domains of unknown function are relevant to nitrogen fixation and heterocyst formation.

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