We first set up on the experiment system with tobacco suspension cultured cells, of which Hypersensitive Response (HR) can be induced by the TMV avirulence factor p50 upon dexamethasone (DEX) treatment. This system makes it available to synchronously induce HR, by which sets of relevant genes are easily screened. A transcription profiling with an oligonucleotide array representing about 10,000 Nicotiana tabacum genes, identified over 400 genes that were up regulated at 10 h after DEX treatment. The numbers of down regulated genes were 300. Depending on these results, we classified genes into two major groups, one those directly controlled by the N factor, and the other those participating in general defense response. In addition, there is the signal transduction pathway that does not mediate genes. Therefore we performed a search of the factors that interacted with N factor by yeast two-hybrid method. As a result, three candidates were provided.

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WIZZ, a gene of which was immediate-early and transiently expressed upon mechanical wounding in tobacco, was previously shown to bind the W-box element. The promoter analysis indicated that the region between -155 and -97 contained cis-element. Yeast one-hybrid screening was conducted using the region as bait, resulting in an identification of an EREBP/AP2-type transcription factor, which belongs to DREB subfamily. DREB subfamily is known to bind to DRE element (GCCGNC). In fact, DRE element exists in the region between -155 and -97 of WIZZ promoter. Gel shift assay confirmed that DRE element was a binding site for the identified factor. Previous microarray analysis led to identification of ACC oxidase gene, which is related in ethylene biosynthesis, to be a candidate of target gene(s) for WIZZ. Promoter of ACC oxidase contains some W-box elements, and in vivo transient transactivation assay revealed WIZZ to be a suppressor for ACC oxidase gene expression.

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RIM1 (rice dwarf virus multiplication1), which is identified as a host factor involved in RDV multiplication, encoded a novel NAC transcription factor of rice. To know the function of the RIM1, we identified loss-of-function mutants from the Tos17 mutant panel. The rim1 mutations showed the phenotype of root growth inhibition, which was similar to the phenotype induced by jasmonic acid (JA) treatment in WT. Microarray analysis revealed mutant's expression profile is significantly correlated with the profile of JA-treated WT, indicating JA-responsible genes are constitutively expressed in the rim1 mutant. To know if the rim1 mutation affects endogenous JA accumulation or not, we measured the JA level under normal condition. The level of JA in the mutant is similar to that in WT, implying that the rim1 mutation is not involved in JA biosynthesis but in JA signaling. These results indicate RIM1 acts as a negative regulator in JA signaling pathway.

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In suspension-cultured rice cells, chitin elicitor treatment induces defense responses, such as production of pathogenesis-related proteins and phytoalexins. Although jasmonic acid (JA) has been suggested to be involved in some of these elicitor-induced defense responses, physiolosical functions of JA in rice defense responses are largely unknown. In this study, by microarray analysis, elicitor-induced genes in suspension-cultured cells were compared between normal-type rice and cpm2, a photomorphogenesis mutant that is thought to be JA-deficient, to investigate the roles of JA in the elicitor-induced defense responses. The results show the expression of 720 genes of 3384 elicitor-induced genes identified in the normal-type rice cells were suppressed in cpm2. The genes suppressed in cpm2 include those coding for phytoalexin biosynthesis enzymes, transcription factor involved in JA signaling, JA biosynthesis enzymes, and phospholipid-metabolizing enzymes. It was also shown that elicitor-induced phytoalexin production was suppressed in cpm2.

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Jasmonic acid (JA) is a plant hormone that acts as a secondary signal molecule in plant defense against pathogens. RERJ1 which encodes a bHLH transcription factor was isolated as a JA-early-responsive gene from suspension-cultured rice cells, and candidate target genes of RERJ1 were suveyed by microarray analysis of transformants overexpressing RERJ1. In the present study, we generated a transgenic rice (XS-line) in which RERJ1 expression could be induced by estradiol treatment. Expression of RERJ1 in the XS-line under 1 mM estradiol treatment was almost equal to that after 0.1 mM JA treatment. Now, we are investigating relationships between expression of RERJ1 and that of the candidate target gene by expression analysis and reporter-gene assay using XS-line in a time-dependent manner after RERJ1 expression. We also suggested by transient gene expression assays that RERJ1 functions as an transcriptional activator of target genes in vivo.

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The diterpene cyclase gene OsKS4 is involved in the biosynthesis of momilactones, major phytoalexins in rice. In suspension-cultured rice cells, transcriptional expression of OsKS4 and production of momilactones were strongly induced by treatment with a chitin elicitor. We identified a TGACG-sequence as an elicitor-responsive cis-element in the promoter of OsKS4 using reporter gene assays. This strongly suggests that a bZIP transcription factor regulates expression of OsKS4. We identified two elicitor-induced bZIP transcription factor genes OsTGA1 and OsbZIP21 using microarray followed by real-time PCR analysis, and found OsKS4 expression was robustly suppressed in a tos17 insertion mutant for OsTGA1 gene. In this mutant, production of momilactones was also suppressed. Additionaly, gel-mobility shift assays indicated that GST-OsTGA1 could specifically recognize and interact with the elicitor-responsive cis-element in the promoter of OsKS4. These results suggest that OsTGA1 participates in regulation of the elicitor-induced production of momilactones through controlling of OsKS4 expression.

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To examine mechanisms involved in HR-induced cell death, FOX hunting system was used to isolate gain-of-function mutants. The isolated overexpressor, which shows constitutive cell death on rosette-leaves and accelerated leaf senescence, is caused by over-expression of the DEAR1 (DREB and EAR motif protein) gene encoding transcriptional repressor that contains a the DREB domain and EAR motif.
Expression of DEAR1 was enhanced by pathogen infections. The overexpressor showed transcriptional promotion of PR genes and highly accumulation of endogenous SA. These results indicate that the SA-dependent defense pathway is activated in the overexpressor, leading to the HR-like cell death phenotype. Expression of DEAR1 is also induced by cold treatment. Transcriptional repression of the cold tolerance genes in the overexpressor results in reduction of cold tolerance. These results indicate that the DEAR1 is a key regulator for cross-talk between biotic (pathogen tolerance) and abiotic (cold tolerance) stress responses.

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Plants are constantly exposed to environmental stresses, including biotic and abiotic stresses, all of which give occasionally serious damages to their survival. To cope with these stresses, plants have evolved various functional proteins by fitness. This study focuses on protein evolution by analyzing the mechanism of localization change from nucleus to plastids. A wound- and pathogen-responsive bHLH protein, NtWIN4, was previously identified in chloroplasts of tobacco leaves. Since bHLH proteins are generally known to function as nuclear transcription factor, NtWIN4 was conceivably converted from nucleus-residence to plastid-specific protein during evolution, resulting in participation in hypersensitive response through chlorosis. Plastid-resident NtWIN4 was predicted to be synthesized from the in-frame second AUG. In fact, further analysis identified short mRNAs lacking the first AUG, from which plastid-type polypeptide was translated in vitro. Results suggested that the different size of mRNAs is one of factors that induce localization change of NtWIN4.

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The axial growth of plant organs changes under genetic and environmental influences, which can be observed as alternations in the spatial profiles of cell proliferation and elongation. We have analyzed temperature effects on root growth in Arabidopsis thaliana, using the kinematic method in combination with a mathematical model that incorporates basic relationships between cell proliferation and volume growth. Our preliminary analysis on root growth of seedlings cultured at 28°C, 22°C, and 16°C showed a remarkable decrease in the rates of volume growth and cell production at 16°C. We have extended a similar analysis to more temperature conditions, and utilized the CYCB1;1::GUS reporter line to evaluate temperature effects on cell proliferation independently of the kinematic data. Based on these results, we discuss the cellular basis of the temperature effects on root growth.

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It is important for root hairs to keep touching soil in order to uptake water and nutrients efficiently. In Arabidopsis grown on agar, root hairs become longer when the roots are apart from the agar surface.
In the timid (tmd) mutant, however, the root hairs became shorter in the air. The TMD gene encodes a GPI-anchored protein. When the cells expressing GFP:TMD fusion protein were plasmolyzed, GFP signal was observed in the outside of the plasma membrane at the root-hair tips as well as around the cell boundary. The TMD protein, which lacks C-terminal region and was expected to be secreted from the plasma membrane, was able to complement the tmd mutant. These results suggest that the TMD protein is released from plasma membrane in the root-hair tip, and regulates the root hair elongation in response to touching stimulus.

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Root system is responsible for acquisition of water from the soil. It is important for plants to avoid drought stress by controlling the orientation of root growth. Hydrotropism is the directed growth of roots in response to moisture gradients, but its mechanism remains to be clarified. We therefore isolated Arabidopsis mutants whose roots are impaired in hydrotropic response and named them mizu-kussei (miz). Here, we identified the mutated gene responsible for its phenotype by map-based cloning. MIZ1 was expressed in the columella cells of the root cap, suggesting that it could function in a perception or signaling pathway of hydrotropism. Interestingly, MIZ1 contained a domain conserved only in terrestrial plants. Thus, MIZ1 is probably a gene that played a role in adaptation of plants to terrestrial environment. This work was supported by the Program for Promotion of Basic Research Activities for Innovative Biosciences.

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The inflorescence stems of Arabidopsis sgr9 and sgr5 mutants exhibit weak gravitropic response. The SGR9 and SGR5 genes encode a RING finger protein and a putative transcription factor, respectively. Both genes function in the endodermis that is responsible for gravity sensing in shoot gravitropism.
The sgr5sgr9 double mutant loses the ability to respond gravi-stimulation. However, the endodermis and vacuoles in endodermal cells were normally formed in sgr5sgr9. Histological analysis displayed that almost all amyloplasts sediment to the direction of gravity in wild-type endodermal cells, while a few amyloplasts did not seidimented in considerable number of endodermal cells of sgr9. Interestingly, a lot of amyloplasts appear to float at the central part of endodermal cell of sgr5sgr9.
These results suggest that SGR9 is involved in sedimentation of amyloplasts in endodermal cells. In addition, SGR9 and SGR5 may be involved in distinct genetic pathway regulating shoot gravitropism.

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It is well known that there is a positive correlation between ploidy level and cell size. However, it is unknown how endoreduplication induces cell enlargement.
When bean plants grown in low intensity red light for 10 days (cell division in the primary leaves had completed) were transferred to bright white light, cells in the primary leaves rapidly enlarged (Van Volkenburgh and Cleland, 1979). This method was used in the present study with slight modification to induce rapid cell enlargement. After transfer to bright white light, nuclear DNA content in lower epidermal cells remained at the 2C level, whereas it increased to 4C in upper epidermal cells at 4 d after transfer. On the other hand, the size of lower epidermal cells increased by 60% in 5 d and remained unchanged thereafter, whereas upper epidermal cells continuously enlarged for 12d. This suggests that endoreduplication supports cell growth from 5 to 12d.

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Pondweed (Potamogeton distinctus A. Benn.), a monocot floating plant, forms overwintering buds, called turions, underground. Turions show extremely high tolerance to anoxia. Degradation of starch stored in cells and active sucrose metabolism are thought to be important in supplying sugars for sustenance of energy production under anoxia. Sucrose synthase (SuSy) is a key enzyme in sucrose metabolism, but its role on anoxia tolerance has not yet been clarified.
We isolated a SuSy gene (PdSUS1) up-regulated in pondweed under anoxia and introduced it into suspension-cultured tobacco BY-2 cells. Viability of BY-2 cells were evaluated by an Evans blue staining method. Sugar contents and invertase activity in transgenic cells were higher than those in wild type cells. Moreover, viability under anoxia was incerased in two cell lines overexpressed SuSy activity. These results suggest that SuSy plays an important role in controlling anoxia tolerance of plant cells.

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During the embryo maturation phase in Arabidopsis, cell division is arrested around 8 DAP. The fus3 mutants have been shown to fail to undergo such arrest. However, little is known about the molecular mechanisms underlying this process. We have been analyzing the downstream events regulated by FUS3 using transgenic plants, in which ectopic expression of FUS3 was induced artificially by estrogen. Ectopic expression of FUS3 during early seedling development resulted in the reduction of cell division activity and caused auxin-insensitivity. These results indicate that FUS3 negativelly regulates auxin-signal transduction, whereby cell division may be arrested during embryo maturation. We screened a mutagenized population of above transgenic plants for novel mutants defective in the FUS3-imposed growth arrest in seedling and isolated several lines that are impaired in embryonic growth arrest. Presentlly, we are investigating of alterations in auxin-signalling in the isolated mutants during the embryonic growth arrest phase.

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Makorin RING finger protein gene (MKRN) family encodes proteins with a characteristic array of zinc-finger motifs. We characterized the structure and expression of MKRN gene from pea and rice. Pea and rice MKRN cDNAs encoded putative *makorin* consisting of four C3H motifs, one Cys-His motif, and one RING motif. This feature clearly established that this gene in pea and rice as true MKRN orthologs. RT-PCR studies in pea and rice revealed that MKRN transcripts were present at very low levels even in dry seeds. Its expression was induced during imbibition and germination periods. A tissue-dependent expression pattern of MKRN was observed in root and shoot tissues in the later stages. In situ hybridization study, in rice, revealed that MKRN was expressed in young plumule, lateral root primordia, leaf primordia, leaves and roots. These findings suggest that MKRN plays an important role in germination and development in both pea and rice.

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Apyrase is a low substrate spesificity enzyme that hydrolize NTP and NDP. Apy1 and Apy2 are paralogues of apyrase in pea. In our study we found that APY1 is present more in the cytoskeletal fraction obtained from first internode of dark-grown pea. In order to find the role of apyrase in germination and differentiation, we analyzed the expression of apyrase using western blotting, RT-PCR and immunohistochemistry. The expression of APY1 appeared suddenly after 10 h of germination in all tissues studied and it reached a maximum in 62 h in root, stem and leaf tissues. Our reference genes (ACT and TUB) were induced during imbibition and germination but, we found that APY1 is the induced only during germination and not during imbibition. And in the later stages a tissue specific expression of Apy1 was observed. These results suggest that APY1 plays an important role in germination and differentiation in pea.

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Seed dormancy is one of important agronomic traits of rice as well as other cereal crop species, because it is associated with pre-harvest sprouting. Seed dormancy is a typical complex trait determined by a series of quantitative trait loci (QTLs). Previously, five QTLs have been detected for the seed dormancy of rice using Nipponbare (japonica)/Kasalath BILs. Here we demonstrate molecular cloning of a major QTL designated as Seed Dormancy 4 (Sdr4). High-resolution mapping enabled to define a genomic region of 8.7-kb as a candidate for Sdr4. Expression analysis showed that predicted gene 1 and 2 were expressed in the panicles. Introduction of 3.3-kb fragment encompassing the predicted gene 2 alone complemented Sdr4 function, indicating that Sdr4 is the predicted gene 2. An Arabidopsis T-DNA tagged line of Sdr4 homologue showed lower germination ratio, suggesting that function of Sdr4 or its homologues in seed dormancy/germination was conserved across monocots and dicots.

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We previously reported that the treatment with 5-azacytidine, a DNA demethylating reagent, induced flowering of Perilla frutescens under long-day condition (LD) and hypomethylation occurred in rDNA intergenic spacer region. These suggest the involvement of epigenetics in photoperiodic flowering. If it was the case, the demethylated genes may be remethylated and silenced in the next generation. Indeed, progeny of the 5-azacytidine-treated plants did not flower under LD and the rDNA intergenic spacer region had been remethylated. These results suggest the existence of DNA-remethylating system which controls the epigenetic state of flowering-related genes. Thus, it is possible that the flowering-related genes of P. frutescnens are demethylated in short-day condition (SD). We used AFLP technique to compare DNA of the cloned plants grown under LD or SD, and detected the SD-specific bands. This indicates that the level of DNA methylation changed under SD. Based on this result, we are cloning the SD-spacific genes.

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To investigate the flowering mechanism in boreal forest trees, we have isolated genes homologous to Arabidopsis flowering genes LEAFY, LGY1 and LGY2, from Larix gmelinii var. japonica, a dominant species in the boreal forest. The expression of both genes was higher in buds, which become floral primordia, than in male and female flowers. The LGY1 expression in the buds increased from May and decreased in September, while the LGY2 expression was constant. The expression of the Arabidopsis AGAMOUS-like gene isolated initiated from July, indicating that LGY1 is expression prior to the initiation of floral organ development. These results suggest that LGY1 is involved in the floral determination. We have been constructing the transgenic Arabidopsis plants harboring LGY1 or LGY2 driven by the cauliflower mosaic virus 35S promoter. We will present the results from the transgenic plants and discuss about possible functions of LGY1 and LGY2.

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In the leaf vein development, local distribution of auxin, which is dynamically changed during the leaf development, is known to be prerequisite for leaf-vascular formation. It is known that polar transport of auxin is necessary for the proper distribution of auxin and that polar localization of PIN proteins is required for the polar auxin transport. However, molecular mechanisms for regulated auxin distribution are still largely unknown. Toward the understanding the mechanisms of formation of leaf-venation pattern, we have identified NO VEIN (NOV) gene which is necessary for prepro/procambial cell formation in leaves. It turned out that NOV is required for formation of developmentally regulated auxin-distribution pattern both in the leaf and in the embryo. NOV encodes a protein with unknown function. Analyses of expression and subcellular localization of NOV, genetic interaction between NOV and GNOM, expression and subcellular localization of PIN proteins in nov mutants will be presented.

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In the leaves of many higher plants, the tissues on the adaxial (upper) and abaxial (lower) sides are morphologically different, owing to genes specifically expressed on the respective sides. Adaxial-abaxial polarity pre-formed in leaf primordial will guide the specific expression. However the molecular mechanism of the process is not understood yet.
FILAMENTOUS FLOWER (FIL) is an abaxial specific gene and the abaxial side specific expression initiate in early stages of the leaf development. We isolated a mutant, #2.0-07-4, where the FIL-expressing region is expanded. In mature leaves of #2.0-07-4, the adaxial tissues resemble spongy tissues observed on the abaxial side in wild type leaves. These results suggest that the responsible gene has a role in restricting the expression of an abaxial specific gene in the proper region and keeping the adaxial tissues from abaxialization in wild type plants.

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In Arabidopsis thaliana, leaves have adaxial side (upper side) and abaxial side (lower side), which differ in shapes of cells and in the number of stomatas or trichomes. In order to examine genetic mechanism determining the adaxial/abaxial boundary in leaf primodia, we have isolated new mutants in which the expression pattern of the abaxial specific gene, FIL, is abnormal. One mutant, #1-63, formed abaxializing or adaxialing leaves. Expression domein of PHB, adaxial marker, and FIL were also changed. These results indicate that #1-63 was defective in the determination of adaxial-abaxial boundary site. We will report analyses of phenotypes and expression patterns of adaxial/abaxial makers in #1-63. Cloning of the gene is in progress.

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In plant lateral organs, including leaves, floral organs and flowers, tissues characteristic of different regions (i.e. adaxial, abaxial and lateral regions) are developed in the axis-dependent manner. In contrast to adaxial and abaxial regions, it is not well understood how lateral region is specified at the molecular level.
PRS was identified to be necessary to form the lateral-specific tissues, and specifically expressed in the region (Matsumoto and Okada, 2001).
For further investigation of factors involved in lateral specification, we examined deletion series of the PRS promoter to identify cis elements. A 140-bp region, named region A, is identified to be necessary for the lateral-specific expression. This region A was also sufficient for the expression in the early stages of lateral-organ development. Now we are screening for trans-acting factors binding to the region A using yeast one-hybrid system.

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Plant cells are surrounded with rigid cell walls and cannot move during development. Cell shape plays an important role for organ shaping and depends on the directions of cell elongation. However, the genetic regulation of anisotropic cell expansion is not well known.
We isolated an Arabidopsis mutant, itosugi (itg), showing defects in elongation of hypocotyls and roots. Cytological analyses of itg mutants revealed that longitudinal cell elongation was suppressed and radial cell expansion was promoted in hypocotyls and roots, suggesting that ITG is required for cell anisotropy.
The ITG gene encodes a plant-specific protein containing armadillo repeats and a C2 domain, which are involved in the protein-protein interaction and lipid binding, respectively. ITG:GFP fusion protein was observed in cytoplasm and around cell periphery. To clarify molecular function of ITG protein, we are analyzing phenotypes of 35S:ITG:GFP transgenic plants.

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The ASYMMETRIC LEAVES2 (AS2) and ASYMMETRIC LEAVES1 (AS1) genes of Arabidopsis thaliana are required for symmetrical and flat lamina expansion. AS2 encodes a plant specific protein that contains AS2/LOB domain, and AS1 encodes a myb (SANT) domain protein. AS2 and AS1 are thought to act as a transcriptional regulator of certain genes including class 1 KNOX genes. To identify the downstream target of AS2, microarray analysis of mRNA from shoot apices was performed. Several candidate genes to be downstream target were identified, and ETTIN(ETT)/ARF3 was selected for the genes whose expression levels were increased in as1 and as2 but decreased in pAS1::AS2 plants. ETT specifies abaxal organ polarity, and known to be a target of TAS3 ta-siRNA. The altered expression levels of ETT were confirmed with real-time PCR. We report how AS2 regulates the ETT expression in the process of leaf development.

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In the Japanese morning glory, there are various mutants not only in color or pattern of petal, but also in morphology of lateral organs. A classic morphological mutant, delicate (dl) has narrow leaves and separated flowers. We succeeded to isolate the responsible gene DL using STD (simplified transposon display) method. The DL encodes the ortholog of FILAMENTOUS FLOWER (FIL) in Arabidopsis and GLAMINIFOLIA (GRAM) in Antirrhinum. These genes are a member of YABBY family transcriptional factor expressing in the abaxial side of lateral organs and specify the abaxial identity. The collaborative expressions of adaxial/abaxial-specific transcriptional factors are known to be important for normal lamina growth, suggesting that a partial loss-of-abaxial identity in dl mutant causes narrow laminae.

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S-adenosylmethionine (SAM) synthesized from methionine serves as a common substrate for the biosynthesis of ethylene and polyamine. To clarify the relationship of ethylene and polyamine metabolism during senescence of Hibiscus syriacus L. flowers, ethylene production and changes in the contents of ACC, ACC-conjugate, and polyamines in the petal were examined. Ethylene production greatly increased just around the beginning of petal in-rolling and paralleled an increase of ACC-conjugate in the petal. Spermine decreased with the petal senescence in contrast to the sharp rise in ethylene production. AVG, which inhibits ACC synthesis from SAM, extended the flower longevity and maintained a high spermine content in the petal. In contrast, MGBG, which inhibits SAM decarboxylation, promoted ethylene production and shortened the flower longevity. These results suggest that a competition for SAM between ethylene and polyamine biosynthesis plays a key role in determining the flower longevity in H. syriacus L.

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The primitive red alga, Cyanidioschyzon, has only a set of organelles, such as nucleus, mitochondrion and chloroplast in the cell. The cell cycle of this organism coordinates with division cycle of chloroplast and mitochondrion. However, mechanisms for the regulation of this coordinated cell cycle have not been elucidated. Here, using synchronized cultivation technique, we investigated regulatory mechanisms for organelle DNA replication and that to coordinate organelle cell cycle with that of the nucleus.
We identified that replication of the chloroplast genome and mitochondrial genome starts before that of the nuclear genome by real-time PCR method and BrdU incorporation. We also identified that any inhibitor effect. These results suggest that proliferation cycle of each organelle in Cyanidioschyzon is regulated independently and that unknown mechanisms may involve to coordinate organelle DNA replication and division in Cyanidioschyzon.

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Regulation of organelles gene expression in optimal timing is crucial for the modulation of cellular metabolisms in eukaryotic plant cells. A primitive red alga, Cyanidioschyzon, containing only one nucleus, one mitochondrion, and one plastid in the cell and those genome sequences are already determined, is a good model for the analysis of regulatory mechanisms of organelle gene expression. We designed and used the DNA microarray system which covers the whole ORFs in chloroplast and mitochondrion to analyze the regulatory mechanisms of the organelle gene expression. As a result of clustering analysis of the DNA microarray data, we found that putative transcriptional units in these organelles and also could predict the functions of a part of conserved hypothetical chloroplast genes (ycf genes). Further analysis will be discussed.

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Chloroplasts have their own genome and gene expression machineries derived from endosymbiosis of ancestral cyanobacteria. During subsequent evolution, chloroplasts lost their autonomy and most of regulatory function became under control of the nucleus. In this work, we used the primitive, unicellular red alga Cyanidioschyzon merolae. This alga shows ancestral characteristics on many aspects including chloroplast genome or transcription regulation. Therefore, some chloroplast functions in C. merolae can be partly regulated more autonomously than those in higher plants.
Here we performed run-on transcription assay using isolated chloroplasts to understand light-dependent transcription regulation in C.merolae chloroplasts. Transcription of every chloroplast gene was induced by light treatment of dark-adapted cells. On the other hand, transcription activity of ycf27 and psbD was specifically activated when isolated chloroplasts were shifted to the light after prepared from dark-grown cells. This result suggests that transcription of particular chloroplast genes is regulated by chloroplast-specific mechanisms independent of the nucleus.

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Maturation of D1 protein in photosystem II completes after proteolytic processing of carboxyl-terminal extension of precursor protein. Since carboxyl-terminus of mature D1 protein is required for the assembly of 4Mn-1Ca cluster and integration of oxygen evolving complex, the processing is important step for photosynthetic water oxidation. In addition, the processing is speculated to be involved in stabilization of D1 protein; although its functional role has not been fully resolved. D1 protein is well conserved among a wide variety of organisms. However, the absence of C-terminal extension has been reported in some photosynthetic protists of secondary symbiotic origins. The present study is focusing on Dinoflagellate which inherited its plastid from red algae (haptophyte). We found multiple genes coding D1 protein (psbA) in a strain. These psbA genes were characterized from the view points of gene structure and deduced C-terminal sequence of D1 protein.

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KaiC phosphorylation oscillation is the pacemaker of the cyanobacterial circadian clock. We recently demonstrated a self-sustainable robust circadian oscillation of KaiC phosphorylation by reconstituting KaiA, KaiB and KaiC proteins with ATP in vitro. We show here that the temperature-compensated ATPase activity of KaiC oscillates in a circadian manner in vitro. KaiC showed very low ATPase activity (16 ATP day^-1), which was stimulated and suppressed by KaiA and KaiB, respectively. The activities of KaiC mutant variants were directly proportional to their cycle frequencies, indicating that the ATPase activity defines the circadian oscillation period. The KaiC ATPase activity is the most elementary mechanism underlying circadian periodicity in cyanobacteria.

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The Lemna genus in duckweeds includes two species that show photoperiodic flowering responses of a long-day type (L. gibba) and a short-day type (L. paucicostata). We tried to reveal the molecular basis for their timing systems. We isolated a number of Lemna homologues of circadian clock-related genes of Arabidopsis. We then tried establishing the methods for analysis of functions of those homologues in the Lemna plants. To monitor the circadian rhythm, we developed a bioluminescent reporter system in which the luciferase gene under a circadian promoter was introduced by using a particle bombardment method. Then, this semi-transient bioluminescence monitoring system was combined with overexpression/RNAi procedures to manipulate the expression levels of clock-related homologues. This enabled us to try various combinations of effectors/promoter-reporters for an approach to the gene network underlying the circadian system. Physiological functions of Lemna clock-related homologues and the comparison with those in Arabidopsis will be discussed.

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Hd6 encodes a casein kinaseII (CK II) α subunit and suppresses flowering under long-days in rice, a short-day plant. Since CKII has been shown to be a key component of the circadian clocks in several model organisms, we analyzed free-running rhythms of Cab1R::luc and diurnal mRNA rhythms of circadian clock-related genes, such as OsLHY, OsPRR1, OsGI and Hd1 using nonfunctional, functional and over- expressing lines of Hd6, but could not detect any significant differences of those expression patterns among them. In contrast, Hd3a and RFT gene expression were repressed in both Hd6 functional and Hd6ox lines. We further revealed that Hd6 suppress flowering only with Hd1 functional alleles. Therefore, we performed a phosphorylation assay by rHd6 after co-immunoprecipitation with FLAG - Hd1 protein, which was transiently expressed in rice Oc-cell. The result suggests Hd6 may phosphorylate a member in Hd1 complex, but not Hd1 itself in rice.

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In Arabidopsis thaliana, it is currently believed that the members of a small family of clock component PSUEDO-RESPONSE REGULATOR(PRR) proteins including TOC1 (PRR1) coordinately play crucial roles within the clock. Among them, the PRR9 gene is particuarly unique in that its expression is circadian-controlled and also rapidly induced by light at the level of transcription. Here we first dissected the regulatory cis-elements of the light-induced and/or circadian-controlled PRR9 promoter by employing a PRR9::LUC reporter. Nonetheless, these results did tell us nothing about the abundance of PRR9 protein product it self in plant. We then established a transgenic line expressing PRR9-TAP-tag polypeptides, by demonstrating that the occurrence of PRR9 polypeptides in plants is indeed diurnally oscillated. In addition, PRR9 polypeptides are accumulated rapidly in response to light. Interestingly, PRR9 polypeptides appear to be subjected to a proteasome-mediated programmed-degradation in the dark.

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The model plant Arabidopsis has provided valuable information about genes and genetic interactions involved in the functions of circadian clock. We have reported that a family of Pseudo-Response Regulators (PRRs) plays important role for the Arabidopsis clock. Other representative components of Arabidopsis clock are CCA1/LHY, ZTL, ELF3/4, GI and LUX. Based on these, the completion of genome sequencing of several other higher plants, such as rice, now make it possible to verify the universal molecular mechanism underlying the circadian clock among higher plants. Here, we carried out comparative analyses as to such clock components between rice and Arabidopsis, and identified and characterized rice clock-associated genes (e.g. OsPRRs, OsCCA1, OsZTL1, and OsLUX). These results suggest that most of the Arabidopsis major clock-associated components are highly conserved also in rice.

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It is obvious that many types of transcriptional factors play roles downstream of the red and far-red photoreceptors, phyA/phyB, which are important for various photo-signal transduction pathways. In this respect in Arabidopsis thaliana, the best characterized are a small family of phytochrome-interacting bHLH transcription factors, which are collectively referred to as the PIF/PIL family, including the founding member PIF3. They play roles in various photo-responses, such as germination, greening, hypocotyl-elongation, and/or shade avoidance. Such phytochrome-dependent photo-responses are highly conserved in many higher plants, and therefore, it is highly probable that another model plant rice also has such PIF/PIL-type of transcription factors. To address this issue, here, we compiled a whole list of rice PIF/PIL-type transcription factors (designated as OsPIL11 to OsPIL16), by characterizing mainly their light-responsive expression profiles. When these OsPIL genes were expressed in A. thaliana, the resulting transgenic plants commonly displayed light-response-associated phenotypes, suggesting their common roles.

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Central clock genes, kaiABC, are encoded on a genome of cyanobacterium Synechococcus elongatus PCC 7942. These function as central autonomous oscillatory proteins. Additionally, it was proposed that the transcriptional feedback loop involving kaiABC provides a global circadian expression. As a fact, we have never found non-oscillatory genes from cyanobacteria using luxAB reporter system. In this study, we approach the mechanism for global circadian gene expressions in cyanobacteria. A microarray analysis exhibited that only 30% genes had a possible oscillation in the mRNA accumulation level. These genes showed negative feedback regulation against excess KaiC condition, but the others did not. Thus, the global oscillation was not generated in transcriptional level, suggesting it is conferred in post-transcriptional level. In further molecular analysis, an interaction between translation elongation factor and Kai complex was observed in subjective night. According to these facts, we will propose new model for circadian output cascade in cyanobacteria.

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Male reproductive development in rice is highly organized and very sensitive to various environmental stresses including high temperature. Heat treatment at the young microspore stage induces pollen sterility. To investigate stress responsive mechanisms in rice male reproductive development, RNA was extracted from anthers at the early microspore stage, with or without heat treatment, and subjected to microarray analysis. We found that expression of several tapetum-specific genes was drastically reduced just after two days heat treatment. About 150 genes, which are expressed at high levels specifically in the mature anther under normal conditions, were identified as elevated expression just after four days heat treatment. However, expression of these genes was lower in the mature anther of heat-treated rice plants. These results suggest that heat treatment damaged function of tapetum and caused disruption of organized gene expression.

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A rice DNA transposon nDart is a non-autonomous element that was found from a mutable virecent line and its excision activity is controlled by an active autonomous element, aDart. A map-based cloning indicated that aDart is identical to an inactive element iDart1-27 (inactive Dart1-27) residing on chromosome 6 in Nipponbare that is probably silenced by epigenetic regulation. To test this hypothesis, cloned iDart1-27 was grown in E. coli in order to release its methylation state in Nipponbare and introduced into plants together with nDart. The results clearly demonstrated that the cloned iDart1-27 element is capable to act as aDart and to excise the nDart element. We can thus conclude that iDart1-27 in Nipponbare is an epigenetically silenced autonomous element.

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Considerable attention has recently been given to gene tagging in rice as a functional genomic tool for elucidating the function of rice genes since the complete sequencing of rice genome had been achieved. We are characterizing a novel DNA transposon system, Dart, and are trying to assess whether the Dart system is useful for gene tagging. While some of the nonautonomous elements nDart in the genome can be transposed in pyl-v line containing an active autonomous element aDart, both nDart and iDart (epigenetically silenced aDart-like elements) remain inactive in pyl-stb and Nipponbare. Some of these dormant nDart and iDart elements are activated by 5-azacitidine (5-azaC) treatment and their transposase gene appear to be expressed. We also found that DNA methylation of the epigenetically silenced dormant aDart becomes hypomethylated after the 5-azaC treatment. These results will be discussed with regard to developing a new gene tagging system using the Dart elements.

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Helitrons are eukaryotic rolling circle replication transposons. Computer-predicted autonomous Helitrons encode a putative transposase, Rep/Hel-TPase, containing a nuclease/ligase domain for the replication initiation protein and a DNA helicase domain. Plant Helitrons carry an additional gene, RPA-TPase, which is related to RPA70. Although Helitrons are found in diverse genomes, neither an autonomous element nor a transposition event has been reported.
A spontaneous pearly-s mutant of Ipomoea tricolor, exhibiting white flowers and isolated in approximately 1940, has an novel Helitron, named Hel-It1, integrated into DFR-B. Hel-It1-related elements are scattered in the Ipomoea genome, and only a fraction of the pearly-s plants was found to carry Hel-It1 at another insertion site. Hel-It1 carries the two putative transposase genes, Rep/Hel-TPase and RPA-TPase, which contain a nonsense and a frameshift mutation, respectively. The wild-type RPA-TPase transcripts are expressed in the pearly-s mutant. We will discuss mobility of Hel-It1 and a putative autonomous element.

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CTP:PHOSPHORYLETHANOLAMINE CYTIDYLYLTRANSFERASE 1 (PECT1) is a gene encoding a rate-limiting enzyme of phosphatidylethanolamine biosynthesis in Arabidopsis. A null mutation, pect1-6, caused embryo abortion before the octant stage. However, reciprocal crosses revealed that pect1-6 caused no significant gametophytic defect. pect1-4/pect1-6 transheterozygotic plants were created by crossing pect1-6 heterozygotic plants with pect1-4 homozygotic plants, which showed moderate level of PECT activity. The transheterozygotic plants, whose PECT activity was decreased by 81 % when compared to that of the wild type, displayed severe dwarfism and low fertility. In these plants, anthers produced no or reduced volumes of pollen, and many ovules remained unfertilized, because of no or abnormal embryo sac development. Therefore, sufficient PECT activity is required for development of normal gametophytes in floral organs. In addition to these defects, partial lethality of pect1-4/pect1-6 embryos decreased seed production in the siliques of pect1-4/pect1-6 transheterozygotic plants.

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BiP is an Hsp70 in the endoplasmic reticulum (ER). Arabidopsis thaliana has three BiP genes, AtBiP1, AtBiP2 and AtBiP3. AtBiP1 and AtBiP2 are ubiquitously expressed under normal growth condition, while AtBiP3 is expressesed only under the ER stress conditions.
Single deletions of each AtBiP gene did not show any obvious phenotype. On the other hand, atbip1 atbip2 double mutant was not obtained, indicating essential and redundant functions of these two AtBiP genes. To investigate functions of AtBiP1 and AtBiP2 in male and female gametophyte, we performed reciprocal cross experiments of atbip1/+ atbip2/atbip2 mutant and found that atbip1 atbip2 double mutation was lethal in the female gametophytes, but not in the male gametophytes. Embryos generated by fertilization of atbip1 atbip2 female gametophyte aborted in development at the early stage of embryogenesis. Interestingly, the wild type AtBiP1 gene from the male gametophytes did not complemt this embryonic lethality.

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The sexuality of homosporous fern gametophytes is generally controlled by antheridiogen, a pheromone that promotes maleness. In this work, we examined whether photomorphogenically active light affects the antheridiogen-induced male development in the gametophytes of Ceratopteris richardii. It was found that the gametophytes of her1 mutant, which are insensitive to Ceratopteris antheridiogen (A_Ce), developed into male when they were grown under blue light in the presence of A_Ce. In the wild-type gametophytes, however, the blue light-irradiation did not affect the sensitivity to A_Ce, probably due to the saturation of A_Ce-sensitivity in the dark. Indeed, in the gametophytes of C. thalictroides, which is other Ceratopteris species and showed low antheridiogen-sensitivity in the dark, the blue light-irradiation increased the male development. Thus, we think that another A_Ce-signal transduction pathway that is activated by blue light latently exists in the gametophytes of C. richardii.

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K034 that is an asexual mutant of the dioecious plant, Silene latifolia has asexual and female-like flowers in the ratio of 9:1. The asexual flower lacks a mature gynoecium and stamens while the female-like flower has an imperfect but fertile gynoecium to bear seeds. K034 is presumed to derive from male since the fourth whorl is smaller than that of a female flower. R025 has been obtained by heavy-ion beam accelerated by RIKEN Ring Cyclotron. R025 has bisexual flowers on most branches and normal male flowers on a few branches. Its genetic stability has been examining with individuals propagated by selfing and cutting. The bisexual flower has mature stamens like a wild-type male and a mature gynoecium fused five carpels like a wild-type female. K034 has the Y chromosome carrying two deletions in gynoecium-suppressing and stamen-promoting functional regions. We are now examining some deletion on the Y chromosome of R025.

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Some nuclear tRNA genes from Eucarya and Archaea contain introns. Pre-tRNA introns are cleaved by the successive reaction of three enzymes: an endonuclease (Sen), an tRNA ligase (tRL) and a 2'-phosphotransferase (Pt). In plants, only two kinds of nuclear tRNA genes for tRNA^Tyr and tRNA^Met are interrupted by introns. Using a GFP reporter fused with genes encoding the subunits of plant Sen (Sen1 and Sen2), tRL and Pt from rice or Arabidopsis, the subcellular location of these splicing proteins was examined. Transient expression of these fusion genes in the onion cells or bean guard cells showed that both Sen proteins are mainly located in the nucleus, but mitochondrial location was also observed. Surprisingly, the same assay revealed that tRL and Pt mainly located in the chloroplast (plastid). These results suggest the possibilities not only of cytosolic tRNA splicing in plants but also of unknown function(s) of splicing enzymes in chloroplast.

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J-domain containing molecular chaperones (J proteins) are functional partners for Hsp70. Budding yeast has three J proteins in the ER: Scj1p and Jem1p functioning in the ER quality control, and Sec63p functioning in protein translocation across the ER membrane as partners for BiP, an Hsp70 in the ER. We found that Arabidopsis has homologs of these yeast ER J proteins, which we designated AtScj1A, AtScj1B, AtJem1, AtSec63A and AtSec63B. Analyses of deletion mutants of these J protein genes showed that only AtSEC63A is essential for the Arabidopsis growth. Although deletion mutants of each of the other ER J protein genes were viable, we found induction of the AtSCJ1A and the AtJEM1 genes in the atscj1b mutant and induction of the AtSCJ1A gene in the atjem1 mutant, suggesting the functional redundancy between these J proteins. We also found that the atscj1b mutants became sterile when they grew at high temperature.

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Previously, we isolated a novel rice protein, IABP4, that is specifically bound to particular rice importinα protein whose expression is down-regulated by light. IABP4 consists of a long N-terminal tetratricopeptide repeat (TPR) domain, the presence of which implies activity to form a protein complex, and a small C-terminal domain that contains three putative nuclear localization signals (NLSs). However, there has been no direct evidence that IABP4 is nuclear-localized. Therefore, we examined the intracellular localization of green fluorescent protein (GFP)-IABP4 fusions in onion epidermal cells, and only the GFP-fusion with the C-terminal fragment containing three putative NLSs was exclusively localized in the nucleus. By further experiments, we identified the one among three putative NLSs as the functional NLS. However, because the GFP-fusion with the entire IABP4 protein failed to localize exclusively in the nucleus, we suppose that the N-terminal domain is involved in the control of intracellular localization of IABP4.

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