To identify novel components of the ABA signaling pathway, we isolated ABA hypersensitive mutants of Arabidopsis using ABA analogs. An analog named #18 inhibits seed germination similarly to ABA.
The sensitivity to #18 differs among ecotypes; Col shows hypersensitivity to #18 when compared with Ler, while Col is less sensitive than Ler to ABA. We tried to identify the responsible gene for this difference of sensitivity. Map-based cloning is now in progress.
We previously demonstrated that two PP2C genes AHG1 and AHG3 are strongly expressed in seed, and ahg1ahg3 double mutant showed strong ABA hypersensitivity at germination. We are searching for targets of PP2C by comparing the amount of phosphoproteins extracted from ahg1ahg3 and Col seeds.

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Suaeda salsa is a main halophyte in saline soils of China with dimorphic seeds (brown and black seeds). Brown seeds show higher germination percentage than black seeds. The effect of salinity on gibberellins (GAs) and abscisic acid (ABA) during germination of S. salsa seeds was invested. ABA content in dry brown seeds was about 2.7 times of black seeds, and decreased quicker than black seeds after imbibition. NaCl stress slightly prevented decreasing of ABA content in germination seeds and fluridone alleviate seed germination under salinity stress. Bioactive GAs and their biosynthetic precursors were higher in brown seeds than black seeds in all treatmens. Deactivated GAs forms were higher in black seeds than brown seeds in all treatments. GA4 declined in response to salinity for both seeds in early stage of germination. GA4 is more active than GA1 in promote seed germination.

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Onion plants form bulbs in response to stimuli generated under long-day conditions. The stimuli seem to consist of bulbing and anti-bulbing hormones and the their ratio determines the commencement of bulb formation. However, the nature of these hormones still remains unknown. Simultaneous application of sucrose at a high concentration and an inhibitor for GA biosynthesis can induce bulb formation in vitro. Using the culture system as a bioassay method, we tried to isolate these hormones. Strong anti-bulbing activity was found in hexane-soluble fraction obtained from onion leaves and the active compounds were identified to be free linoleic and linolenic acids. On the other hand, bulb-inducing activity was found in water-soluble fraction. Purification of the active compound is now in progress.

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GAMYB was firstly identified as a transcriptional regulator of gibberellin (GA)-responsive genes in barley aleurone. Previously, we revealed that OsGAMYB-dependent CYP703A3 expression is essential for the GA-mediated exine formation in the rice. Our recent studies have also shown that the GID1/DELLA-mediated GA perception system appeared after the divergence of vascular plants. In this study, we tested whether the regulation of CYP703A expression by GAMYB is also conserved in Selaginella.
In situ hybridization showed that SmGAMYB and SmCYP703A were expressed in the tapetum. SmGAMYB could specifically interact with the SmCYP703A promoter region in vitro. Moreover, introduction of these genes from Selaginella rescued the male sterility of rice gamyb and cyp703a3 mutants. The treatment of GA biosynthetic inhibitor to Selaginella inhibited its normal exine formation. Taken together, these results suggest that the GA signaling for exine formation is conserved after the divergence of vascular plants.

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We have previously reported that a cytochrome P450 gene that encodes steviol synthase was identified in Arabidopsis. Transgenic Arabidopsis plants overexpressing this steviol synthase gene showed semi-dwarfism like mild gibberellin (GA)-deficient mutants. GA analysis indicated that the levels of non 13-hydroxy GAs including GA₄ were decreased, whereas those of 13-hydroxy GAs including GA₁ (which is less active in Arabidopsis) were increased in the transgenic plants. To identify rice ortholog of this gene from the same gene family (CYP714), we made transgenic Arabidopsis plants that overexpress each rice CYP714 gene whose function is unknown. We found that two of these overexpressor plants show semi-dwarfism. GA analysis indicated that the endogenous GA profiles of these plants were similar to those of the overexpressor of the Arabidopsis steviol synthase gene. These results suggest that rice has at least two cytochrome P450 genes that are involved in GA 13 hydroxylation.

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Genetic studies have revealed that brassinosteroids (BRs) are essential plant hormones for growth and development of seed plants. However, little is known about the occurrence and physiological roles of BRs in ferns and moss. While the biosynthesis genes of sterols are highly conserved in the plant kingdom, down-stream genes responsible for BR biosynthesis are not likely to be present in the moss, Physcomitrella patens. Furthermore, no homolog of BRI1, a BR receptor, is found in the moss genome. In order to know whether P. patens produces BRs, we analyzed endogenous BRs using GC-MS. As the result, neither castasterone nor brassinolide that are biological active forms in seed plants were identified, but some of the precursors of these BRs such as 6-dexoxocastasterone were identified in P. patens. We discuss the biosynthesis of BRs in the moss in terms of presumptive BR biosynthesis genes.

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Brassinosteroids are steroid hormones of plants that regulate plant growth and development. While the physiological function of brassinosteroids in the angiosperm Arabidopsis thaliana has been well studied, that in other plants has not. In this study, we report the physiological function of brassinosteroids in the moss Physcomitrella patens.
When P. patens was grown on media with brassinosteroid biosynthesis inhibitors, such as brassinazole or spironolactone, the number of gametophores was decreased and the gametophores developed short rhizoids. Further, we found the Arabidopsis DWF7 homolog in P. patens (PpDWF7) and constructed PpDWF7 disruptants by homologous recombination. Phenotypes of the disruptants were very similar to wild type grown on media containing brassinosteroid biosynthesis inhibitors. These results suggest that brassinosteroids function in P. patens and control gametophore formation and rhizoid development.

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Among vascular plants, seed plants (Angiosperms and Gymnosperms) have been well studied for the endogenous brassinosteroids. However, any concrete evidence for the occurrence of brassinosteroids in ferns, seedless vascular plants, has not been obtained. In this study we analyzed some ferns for the presence of brassinosteroids by using GC-MS. The ferns examined were Equisetum arvence (vegetative shoot and reproductive shoot, Thelypteris decursive-pinnata (leaf), Pteridium aquilinum (leaf), Osmunda japonica Thunb. (leaf). As the results, brassinosteroids were identified from all the tissues examined. As biologically-active brassinosteroids, castasterone but not brassinolide was identified. The biosynthetic precursors of castasterone were also identified, being the same as those in seed plants. The endogenous brassinosteroid profiles of the respective ferns will be discussed by comparing with those in Arabidopsis.

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For the purpose of isolating brassinosteroid(BR)-induced genes of rice, we monitored the transcriptomic responce of brd1, a mutant that has a defective BR biosynthetic gene OsBR6ox, to BL treatment. Among the up-regulated genes, we present detailed analyses of OsBU3 and OsBU17, both encoding putative bHLH proteins.
Rice overexpressing OsBU3 (OsBU3:OX) showed severe bending of the lamina joint. In contrast, RNAi plants designed to repress both OsBU3 and its paralogues displayed erect leaves. In addition, OsBU3:OX plants were resistant to brassinazole, an inhibitor of BR biosynthesis, suggesting that OsBU3 is not involved in BR biosynthesis but BR signaling. Analysis of promoter-GUS plants demonstrated that OsBU3 is specifically expressed in the lamina joint. Further, OsBU3:OX plants showed large grain and reduced fertility.
In contrast, OsBU17:OX showed erect leaves but did not show dwarfism. OsBU17 may be mainly involved in bending of the lamina joint.

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Brassinosteroids (BRs) are inactivated by cytochrome P450 monooxygenases in plants. It is known that Arabidopsis CYP734A1 and tomato CYP734A7 inactivate BRs by C26-hydroxylation of castasterone and brassinolide. Rice has four CYP734A homologues (CYP734A2/A4/A5/A6). Transgenic rice plants overexpressing these CYP734As, except for CYP734A5, showed a severe dwarf phenotype, and their endogenous BR levels were decreased. In order to investigate BR inactivation mechanism in rice, we have prepared the recombinant CYP734As expressed using a baculovirus insect cells system, and performed the enzyme assays with various BR-biosynthetic intermediates as a substrate. Rice CYP734As showed wide substrate specificities for BRs and exhibited high activity for early intermediates. Furthermore, rice CYP734As was found to catalyze three-step oxidations to form the carboxylated-BRs. These results indicate that BR inactivation mechanism in rice is different from those in Arabidopsis and tomato.

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It has been established that the jasmonic acid (JA) is involved in the plant resistance to insect and pathogen attacks as well as anther dehiscence and senescence. However, little is known about the mechanism how the genes of JA biosynthetic pathway are induced by the attacks or wounding. We have found that the expression of Arabidopsis DEFECTIVE IN ANTHER DEHISCENCE1 (DAD1) gene, which encodes a lipase responsible for the JA biosynthesis in stamen filaments at the anther dehiscence, is immediately induced in rosette leaves when the leaves are wounded. Recently, it was revealed that the DAD1 is also involved in the wound induction of JA. These findings strongly motivated us to determine the cis-regulatory element of DAD1 wound induction. The promoter-GUS analyses revealed that the element is located in a 40-bp region at the 3' downstream of DAD1 gene.

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Jasmonic acid (JA) is a plany hormone that has multiple important roles, such as anther dehiscence at anthesis and resistance to pathogen infection and insect wounding. We have identified Arabidopsis DAD1,which encodes a lipase catalyzing the initial step of JA biosynthesis. Arabidopsis has six homologs of DAD1, namely DAD1-LIKE LIPASE1(DAL1) to DAL6,which encode lipses with transit peptide at their N-terminals. Our studies with multiple mutants showed that DALs also required for JA biosynthesis. To reveal the functional differences among them, we analyzed their expressions with transformants containing promoter-GUS genes. To date, we have examined the expressions of DAL1,2,5,and 6. These four genes were induced by wounding in rosette leaves, but in different cells each other. DAL2 was expressed in stamen filaments as similar to DAD1, while DAL1 and 6 were expressed in other floral organs. DALs might have individual functions in JA biosynthesis.

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Cellulose biosynthesis inhibition (CBI) causes changes in root morphology and in the expression of genes. Recent studies revealed that CBI induces change in the amount of plant hormones. We have isolated a mutant of Arabidopsis (css1) that showed altered sensitivity to 2,6-dichlorobenzonitrile that inhibits cellulose biosynthesis. The css1 mutant also showed altered fundamental metabolism due to the defect in the mitochondrial function. The growth of this mutant was highly sensitive to sugar concentration of the medium (PCP 47:772-83(2006)). We tested ABA sensitivity of this mutant, since ABA strongly interacts with the growth regulatory effect of sugar. Our result showed that the post-germination growth of css1 mutant was more sensitive to ABA than that of wild type. We also determined mRNA levels of several ABA-related genes. The results indicated that not only post-germination growth but also mRNA levels of the genes were affected by css1 mutation.

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Ovule produces female gametes, egg cell and central cell in flowering plant angiosperms. In the other side, anther produces male gamete, pollen. Two sperm cells in pollen migrate to fertilize egg cell and endosperm in the process termed double fertilization. Fertilized egg cell and central cell differentiates to embryo and endosperm to form seed , respectively.
These reproductive developmental processes, in which many kind of differentiated cell join, are sensitive to environmental stresses. However, we do not know why these processes are so sensitive to environmental stresses.
To identify hormonal signaling pathways involved in these phenomena, we searched for mutants show sensitivity to high temperature stress at plant reproductive development in A.thaliala.
Consequently we identified three lines that are sensitive to high temperature at flowering stage.In this meeting, we will discuss about how these mutants get weak/strong to stress and report further search for stress sensitive/tolerant mutants.

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We have elucidated that AtMPK4 was involved in cytokinesis in Arabidopsis. The ANQ/AtMKK6 MAPKK activated AtMPK4 specifically in vitro. And, we investigated the activity of AtMPK4 in the anq mutant using immnocomplex kinase assay with anti-AtMPK4 antibody, and that activity was reduced. Furthermore, it was observed that multinucleate cells with incomplete cell plates in the root and the leaf in the atmpk4 mutant. These results suggest that AtMPK4 regulates the growth of cell plate positively downstream of ANQ. But, the atmpk4-2 mutant was not lethal, we postulated that other MAPK also might regulate cytokinesis. To investigate this possibility, we observed the phenotype of atmpk11 mutant with atmpk4-2 mutation.

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Many G2/M phase-specific genes in plants contain mitosis-specific activator (MSA) elements, which act as G2/M phase-specific enhancer and bind with R1R2R3-Myb transcription factors. Here, we examined the genome-wide effects of overexpression of NtmybA2, one of the R1R2R3-Myb transcription factors in tobacco. Microarray analysis was performed with stably transformed BY2 cell lines that ectopically express NtmybA2 or its hyperactive form lacking the negative regulation domain. This analysis revealed that overexpression of the truncated hyperactive form of NtmybA2, but not its full-length from, preferentially up-regulates many G2/M phase-specific genes in BY2 cells. We determined the promoter sequences of several such up-regulated genes, showing that all of them contain MSA-like motifs in their promoters. We propose that transcription of many G2/M phase-specific genes in tobacco is commonly and positively regulated by NtmybA2, in most cases through its direct binding to the MSA elements.

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TONSOKU (TSK) of Arabidopsis thaliana is responsible for mutants defective in both shoot and root apical meristems, and it encodes a protein of unknown function⁽¹⁾. TSK is expressed in the S phase of the cell cycle⁽²⁾, and tsk mutants show enhanced expression of DNA damage-responsible genes and increased number of cells arrested at G2/M ⁽³⁾. DNA damage activates ATR and ATM checkpoint kinases and regulates the cell cycle. Phenotypes of tsk such as meristem disorders and enhanced expression of DNA damage-responsible genes, but not expression of heterochromatic TSI, were suppressed by atr. ATR-dependent cell-cycle arrest might be important in causing meristem disorder in tsk. We compared gene expression patterns in Col-0, tsk and tsk atr to understand the mechanism responsible for the meristem disorder in tsk. ⁽¹⁾Plant J. 38:673 (2004), ⁽²⁾Plant Cell Physiol. 46:736 (2005), ⁽³⁾Plant Cell 18:879 (2006).

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The cell-cycle checkpoint monitors the progression of the cell cycle, detects a problem such as DNA damage, and arrests the cycle until the problem is solved. We previously identified the AtRAD26 gene being involved in the checkpoint in Arabidopsis. The AtRAD26-disrupted plant was sensitive to DNA damaging agents or cell-cycle inhibitors, which is reminiscent of the phenotype of AtATR-disrupted plant. The AtRAD26 protein contained two strokes of SQ sequences, potential target sites of PIKK including ATR and ATM.
To clarify whether the AtRAD26 is regulated by PIKK, we used the bacterially-expressed N-terminal region of AtRAD26 as a substrate for in vitro phosphorylation assay. It incorporated ³²P when incubated with [γ-³²P]ATP and the nuclear extract from wild-type plants. By contrast, the ³²P-incorporation was reduced when incubated with the nuclear extract from AtATR-disrupted plants. This suggests that the AtRAD26 is phosphorylated by (a) checkpoint-related kinase(s) including AtATR.

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Plant cell cycle is tightly controlled by various environmental stresses including elicitors from pathogens. Ubiquitin-mediated proteolysis plays crucial roles in cell cycle regulation, and Anaphase-Promoting Complex/Cyclosome (APC/C) , a multi-subunit E3 ubiquitin ligase, regulates cell cycle in animals and yeasts. Though homologs of all vertebrate APC/C subunits have been computationally identified in Arabidopsis thaliana, functional analyses of APC/C and its subunits are limited in plants. We have been characterizing AtAPC5, APC/C subunit 5 from Arabidopsis. A null atapc5 allele could only be maintained as a heterozygote. Genetic analysis showed that the mutant had female gametophytic defect. Confocal laser scanning microscopic observation of the ovules revealed that the female gametophytes arrested at the one-nucleus stage. We also identified a weak atapc5 allele and characterized its phenotype in planta. We will report phenotypes of loss-of-function mutants as well as overexpressing lines of AtAPC5 and discuss its possible functions.

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Cyanidium caldarium grows at acidic pH but not at neutral pH. The intracellular pH of Cyanidium cells has been reported to be kept constant in the neutral pH region by pumping out the proton leaked into the cells according to steep pH gradient. It is not, however, clarified why Cyanidium cells can not grow at neutral pH. In this study, we found that Cyanidium cells survived even at pH 6 over one month, and large cells involving four endospores significantly increased when the cells were cultured at pH 6. This indicates that the cells cultured at pH 6 grow until formation of endospore but can not divide. Furthermore, we found two extracellular proteins at apparent molecular masses of about 34 kDa and 29 kDa in the culture medium at pH 6 but not at acidic pH. Relationship between these extracellular proteins and cell division will be discussed.

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The stages of tapetum development are characterized by the accumulation of substantial intracellular lipid deposits. Much of the pollen coat material, which is essential to pollen-stigma recognition and pollen germination, derives from tapetal lipid bodies found in two types of organelles: tapetosomes and elaioplasts. Tapetosomes are single-membrane-enclosed organelles containing relatively large lipid-rich structures; elaioplasts originate from plastids by the gradual accumulation of osmiophilic droplets. On the other hand, male gametophyte also contains various and characteristic lipid-rich organelles. We examined the ultrastructural analysis of anthers by chemical-fixation and cryo-fixation using a high pressure freezing method. We found some organelles showed poor electron density on cryo-fixation, while they contained osmiophilic materials on chemical-fixation. Furthermore, we observed the process of exocytosis and fuse of particles. We report novel structures of organelle in tapetum and male gametophyte and process of deposition of pollen coat.

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The Arabidopsis genome is predicted to encode about 20 proteins homologous to the yeast or mammal peroxisome biogenesis factors (peroxin), but their functions remain unclear. In this study, we generated transgenic plants that were suppressed each peroxin gene expression using RNAi method and analyzed whether these homologues actually function for peroxisome biogenesis or not. The transgenic plants that stably expressed GFP-PTS1 were generated as a parent plant for RNAi plant, and we therefore could observe peroxisomes by GFP fluorescent. In the results, we could classified two groups of Arabidopsis peroxin, one group is that GFP is localized in the cytosol and the other defect on peroxisome morphology. Interestingly, PEX10 knock-down plant phenotype is different from wild type plant whereas other peroxin knock-down plants have similar phenotype of wild type plant. Then, we analyzed comprehensive gene expression in PEX10 knock-down plant and discuss PEX10 specific function.

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Almost proteins in peroxisomes are synthesized in the cytosol and transported via PTS1 or PTS2 pathways. Whereas PTS1 proteins are synthesized as mature sizes, PTS2 proteins are done as large precursors with N-terminal presequences. We focused on AtDeg15 that is a homologue of E.coli DegQ protease, which is a candidate for processing protease of presequences. We analyzed an Arabidopsis mutant of T-DNA insertion in AtDeg15. Peroxisomes of deg15 had normal import machinery for PTS1 and PTS2 proteins, but accumulated precursors for PTS2 proteins. Complementation studies of the mutant showed that AtDeg15 is essential for PTS2 processing. β-oxidation activity of deg15 was lower than that of WT and an enzyme activity of recombinant precursor for PMDH was also lower than that of mature PMDH. These results indicated that AtDeg15 is involved in maturation of PTS2 proteins and the processing seems to be necessary for assembly of peroxisomal functions.

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In higher plants, peroxisomes are known to differentiate into several classes. Glyoxysomes that are abundant in cotyledons, contain enzymes of β-oxidation and glyoxylate cycle to degrade storage lipid for postgerminative growth of oilseed plants. Leaf peroxisomes that are found in green leaves, function together with chloroplasts and mitochondria in phororespiration. Malate dehydrogenase (MDH) is an enzyme to catalayze the interconversion of malate and oxaloacetate. Two isoforms of peroxisomal MDH (PMDH) have been identified, the glyoxysome-type isoform, PMDH1 (At2g22780) which is involved in glyoylate cycle and in β-oxidation, and the leaf peroxisome-type isoform, PMDH2 (At5g09660) which is induced by light but those function are not elucidated. We investigated pmdh2 and pmdh1pmdh2 mutants of Arabidopsis to elucidate physiological function of PMDH2. In this study, we report the phenotypes of these mutants that are grown under high light condition and discuss the function of PMDH2 in leaves.

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Most of proteins functioning in organelle are encoded by nuclear genes. It is accepted that these genes were translocated from the endsymbiotic bacteria and acquired organelle targeting sequence. However the acquiring process is unknown.
We previously propose a hypothesis that the genes inserted to nuclear genome by horizontal transfer tend to acquire additional sequence to 5' terminal, lead to the acquisition of cryptic organelle targeting sequences.
In this presentation, we reported that N-terminal additional sequences by horizontal transfer to nuclear genome tend to be disordered region. This bias of disordered region was common to N-terminal additional sequences and organelle targeting sequences.
Although disordered regions were unstructured, intrinsic protein disorder is considered to play an important role in protein-protein interaction. Thus horizontal transfer to nuclear genome frequently donates flexible N-terminal sequence to transfer gene. It is expected that a parts of these flexible N-terminal sequence were used as organelle targeting sequences.

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We have identified 38 albino or pale-green (apg ) A. thaliana mutants in a collection of 11000 Ac/Ds transposon-insertion mutant lines. One of these apg mutants, apg13 shows an albino phenotype and has a Ds insertion in the MurE gene. We found that the expression of genes that depend on a multi-subunit plastid-encoded RNA polymerase was decreased, suggesting that A. thaliana MurE is functionally divergent from the bacterial MurE. In this study, we discovered that corresponding gene to apg17 is an Arabidopsis homolog of bacterial gene for PG sacculus maintenance. Accumulation of the apg17 mRNA in the wild-type plants was detected in leaf, stem, imflorescence and flower, but not in root. GFP-fusion protein analysis suggested that Apg17 localizes in chloroplasts. apg17 mutant showed variegated or pale-green leaf phenotype, and chlorophyll content was decreased to 70% to that in wild type leaf. Promoter analysis and morphological studies are now in progress.

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A mutation in CRL of Arabidopsis thaliana causes a severe defect in plastid division. Besides the inhibition of plastid division, the mutation causes distortion of cell division planes and abnormality in cell differentiation (Plant J., 38, 448-459, 2004). Our previous data indicated that CRL protein localized in the outer envelope membrane of plastids. We also have shown that the crl mutants contained the cells without detectable plastids. In this study, we have attempted to identify a CRL protein complex in chloroplast envelope membrane. CRL protein was solubilized by 1% n-dodecyl-β-D-maltoside (DDM) or 1% digitonin. The DDM-solubilized CRL complex was subjected to 2D gel electrophoresis with blue-native PAGE and SDS-PAGE, and was blotted on a PVDF membrane. Detection of CRL protein by an anti-CRL polyclonal antibody revealed that CRL was included in a protein complex with apparent molecular mass of 640 kDa.

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In plant cells, mechanisms for regulation among organelles and nuclear DNA replication have not been elucidated. Using quantitative PCR analysis, we have already presented that organelle DNA replication (ODR) regulate nuclear DNA replication (NDR) and the tetrapyrrole intermediates protoporphyrin IX or Mg-protoporphyrin IX acts as a signaling molecule to regulate the initiation of NDR. It indicates that the novel function and the importance of organelle to regulate the progression of nuclear DNA replication via retrograde signal. Here we monitored ODR and NDR by directly counting intensity of the DAPI stained DNA contents by fluorescence microscopy. And we measured intracellular accumulation of ProtoIX and Mg-ProtoIX through the G1 and S phases. As a result, transient accumulation of Mg-ProtoIX, which is correlating with the ODR occurrence, was observed.

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Centromeres are universally conserved functional units in eukaryotic linear chromosomes. However, little is known about the structure and dynamics of the centromere in lower eukaryotic plant cells. Previously, our group reported that dynamic centromere reconstitution during the cell cycle in a primitive red alga, Cyanidioschyzon merolae, using anti-CENP-A antibody (Maruyama et al., 2007). In this work, we determined centromere regions of all chromosomes in C. merolae by ChIP-on-Chip method using anti-CENP-A antibody and whole-genome tiling array with high probe density. Averaged length of centromere regions was estimated about 2 kbp suggesting that the centromere of C. merolae is categorized into regional type centromere, although they did not have any highly repeating DNA sequences which are found in several higher eukaryotic species.

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The unicellular red alga Cyanidioschyzon merolae 10D shows simple cell structure, containing one nucleus, one mitochondrion and one plastid. In order to use this organism as a model cell, it is indispensable to develop transformation techniques to facilitate functional genomics.
URA5.3 gene of C. merolae is the fusion gene of orotate phosphoribosyltransferase (URA5) and orotidine-5'-phosphate decarboxylase (URA3). To use it as a selection marker, we previously constructed a fusion gene of URA5.3, whose URA3 region was substituted to that of Galdieria sulphuraria, other unicellular red alga. Adjacent regions of CfxQ gene, a regulator of RubisCO, were connected to the marker to construct a plasmid for disruption of CfxQ gene. Uracil prototroph strains were obtained by transformation using the linearized plasmid. PCR analyses showed the marker was inserted into genome of the transformants by the single-crossover.

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Mitochondrial-nucleus (nucleoid) is defined as a DNA-protein complex of mitochondria. Mitochondrial DNA molecules are packed into highly condensed structure, while the genome function must be conducted properly in the structure. However, mechanism of organization of mitochondrial DNA was almost unknown. When mitochondrial-nuclei isolated from BY-2 cultured tobacco cells were treated with micrococcal nuclease (MNase) and their DNA was subjected to agarose-gel electrophoresis, DNA ladder (minimum size was ~75 bp) was observed, suggesting that nucleosome-like structure was present in mitochondrial-nucleus. The DNA ladder did not observed when isolated mitochondrial-nuclei were pretreated with proteinase K or NaCl (≥ 1 M) before treatment with MNase, suggesting that mitochondrial DNA was protected by the proteins that bound DNA through electrostatic interaction. Further experiments, such as electromicroscopic observation of the fine structures of isolated and MNase-treated mitochondrial-nucleus, are now on progress.

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Plastid is a typical organelle in plants, showing a spectrum of differentiation according to plant development. It is known that the plastid contains its own genome in the form of nucleoids, comprising a circular DNA of 120-150 kbp depending on plant species, and various proteins including DNA polymerase and RNA polymerases. We propose a new approach for isolation of plastid nucleoids. The PEND protein is a DNA-binding protein and a DNA-binding domain called cbZIP is present at its N-terminus. In transformants of Arabidopsis thaliana cbZIP-GFP fusion proteins were localized in the nucleoids of plastids. Plastid nucleoids were isolated from various tissues using GFP antibody, and determined proteins of plastid nucleoids by peptide mass fingerprint.

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In plants and algae, organellar genomes are replicated by POPs(plant organellar DNA polymerases). In a previous annual meeting, we reported that POP purified from the cells of rhodophyte Cyanidioschyzon showed high processivity and severely inhibited by phosphonoacetate. POPs are widely conserved in eukaryotes except metazoa and fungi. A ciliate Tetrahymena has a single POP gene. Tetrahymena does not have a chloroplast, but it is thought to be a plant in a wide sense (Plantae) (Nozaki, 2003). There are a few studies about mitochondrial DNA polymerase in Tetrahymena, but detailed analysis was not performed. The purpose of this study is purification of POP from Tetrahymena cells and comparison of the enzymatic activities of POP between Tetrahymena and Cyanidioschyzon. Immunoblotting showed that Tetrahymena POP was localized to mitochondria. Phosphonoacetate strongly inhibited the DNA synthesis activity of Tetrahymena mitochondria. These results suggested that inhibition by phosphonoacetate is a common property of POPs.

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NOG1 is a P-loop GTPase that belongs to the Obg-Hflx super family and is conserved among archaea and eukaryotes. In yeast, and mouse, NOG1 has been shown to be essential for cell viability and functionally linked to 60S ribosomal maturation. Arabidopsis thaliana contains two NOG1 genes (NOG1-1 and NOG1-2), but only NOG1-1 is constitutively expressed in all developmental stages of plant. The NOG1-1 was accumulated in nucleolus and nucleoplasm. Fractionation analysis suggested that the NOG1-1 was present in the preribosomal fractions. Moreover, FRAP analysis revealed that the distribution of NOG1-1 protein between nucleolus and nucleoplasm was sensitive to transcription and translation inhibitors, and carbon and nitrate starvation, suggesting that NOG1-1 plays an essential role in early and intermediate steps of ribosomal biogenesis. Furthermore, The NOG1-1 protein disappeared in pre-metaphase, and is rapidly re-accumulated in peripheral chromosomal region in early anaphase. NOG1-1may also play another important role in nucleolus dynamics.

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