NIMA-related kinases (NEKs) are a family of Ser/Thr protein kinases conserved in eukaryotes and implicated in cell cycle regulation. Arabidopsis NEK6 interacts with armadillo-repeat kinesins (ARKs) and modulates directional expansion of epidermal cells (Motose et al., 2008; Sakai et al., 2008). Here, we describe functional analysis of NEK family in Arabidopsis with special emphasis on NEK6. NEK6 was highly accumulated during cell division and cell expansion and concentrated in dot-like structures exhibiting dynamic movement along with cortical microtubules and phragmoplasts. The kinase assay and microtubule imaging indicated that NEK6 phosphorylates tubulins and regulates homogeneity of cortical microtubule array. NEK6 genetically interacted with microtubule-associated proteins; SPR2, GCP2/SPR3, and ARK kinesins. Especially, ARK kinesins were required for localization and activity of NEK6. The down-regulation of NEK4 compensated ibo1/nek6 mutant phenotype and the ibo1-3 mutation reduced interaction of NEK6 with NEK4. These results indicated that NEK6 controls microtubule organization and regulates anisotropic cell growth in concert with NEK4 and ARKs.

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In higher plants, metabolism of sugar (C) and nitrogen (N) is mutually regulated. To clarify the C/N regulator, we isolated novel C/N regulatory gene ATL31. The ATL31 belongs to ubiquitin ligase ATL family, sharing ATL6 with most similarity to the amino acid alignment. Plants overexpressing the ATL31 and ATL6 showed insensitivity to C/N stress conditions, whereas the single knockout mutants did the hypersensitivity. To investigate the detailed functions of both genes, we have generated double knockout mutants (atl31-1 atl6-1). The atl31-1 atl6-1 developed the etiolated true leaves and the elongated primary root length under normal growth conditions. Now we are going to try to figure out the characterization of atl31-1 atl6-1 in terms of these phenotypes. How both genes contribute to C/N response regulation in plants will be discussed.

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Plants sense balance of carbon (C) and nitrogen (N) metabolites to regulate metabolism and development, called C/N response. To clarify the mechanism of C/N response, we isolated a novel C/N response regulator ATL31, an ubiquitin ligase. Ubiqutin ligase recognizes specific proteins for degradation by ubiquitin-proteasome system. Previous studies demonstrated that the ATL31 is involved in C/N response to degrade the polyubiquitinated protein. To identify the target protein of ATL31, we screened interaction proteins by using immunoprecipitation and MS analysis. Characterization of the identified proteins will be reported.

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Perennial plants growing in temperate zone show annual growth cycle. In the autumn the newly formed buds enter endodormancy. After that, buds are released from endodormancy by a prolonged period of chilling during winter. This regulation of endodormancy allows perennial plants to survive in low temperate during winter. However, the molecular mechanism of endodormancy in perennial plants is not well understood. We isolated SVP-like gene of Prunus mume, named DORMANCY ASSOCIATED MADS-box6 (DAM6), which shows high expression during endodormancy. Seasonal expression pattern of DAM6 showed correlation with endodormancy. Moreover a prolonged period of chilling was required to decrease the expression of DAM6. Interestingly over-expressed DAM6 in poplar repressed bud growth under long-day condition. These results could indicate that DAM6 controls endodormancy in Prunus mume, suggesting the possible role of SVP homologs in regulating bud endodormancy in perennial plants.

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Leaf trichomes in Arabidopsis develop through several distinct cellular processes, such as patterning, differentiation and growth. Here we report a novel trichome mutation in Arabidopsis, which in contrast to previously identified mutants, increases trichome cell size without altering its overall patterning or branching. We show that the corresponding gene encodes a GT-2-LIKE1 (GTL1) protein, a member of the trihelix transcription factor family. GTL1 is present within the nucleus during the post-branching stages of trichome development and its loss of function leads to a ploidy increase only in trichomes that have completed branching. Our data further demonstrate that the gtl1 mutation modifies the expression of several cell cycle genes and partially rescues the ploidy defects in the cyclin-dependent kinase (CDK) inhibitor mutant siamese. In addition, studies of GTL1 and its closest orthologues in Arabidopsis provide further genetic evidence that these proteins are involved in the transcriptional regulation of plant cell growth beyond trichome development.

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In higher plants, organ sizes depend on its cell sizes and cell numbers. The correlation between ploidy, nuclear DNA content, and cell size has long been reported in Arabidopsis thaliana and in many other plant species. Our previous study showed that a plant-specific trihelix transcription factor, GT-2-LIKE1 (GTL1) functions as a negative regulator of ploidy-dependent cell growth in Arabidopsis trichomes. In this study we analyzed organ size phenotypes of GTL1 mutants. We are also setting up a new mutant screen to identify novel regulators of organ growth in Arabidopsis. Based on our progress in these studies, we will discuss how organ size is controlled in plant development.

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Cyanidium caldarium are unicellular primitive red alga that exists in many parts of the world in hot acidic habitats. C. caldarium grow in acidic environments (pH 1-3), but did not survive over pH 7. It would be expected to possess fundamentally different physiological controls in comparison with similar species restricted to neutral pH. In our previous study, we clarified that only one protein was secreted from C. caldarium at pH 6. Gene of this extracellular protein was cloned and only showed high homology with hypothetical protein of Cyanidioschyzon melorae in photosynthetic organism. Moreover, this protein showed relative high homology with lysily oxidase, which found in animal cells. It is known that lysil oxidase plays a role in morphogenesis of the extracellular matrix by oxidizing lysine residues in collagen. However, little is known about the function of extracellular protein from C. caldarium. In order to elucidate this problem, crosslinking reaction with various proteins by extracellular protein was performed. Several cross-linked products were obtained by this reaction. Possible implications of the results will be discussed.

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Thellungiella halophila a highly salt tolerant species with high genetic and morphological similarity to classical model plant Arabidopsis thaliana has begun to serve a model for salt tolerant plants. Seeds from salt cress showed a deep dormancy at maturity. The level of dormancy decreased during after-ripening and cold stratification. After-ripend seeds of salt cress failed to germinate in dark and strong light while best seed germination was obtained in weak white light at 22 C. Light enhanced the sensitivity to GA4 in dark-imbibed salt cress seeds and strong light inhibited biosynthesis of GA4. The content of ABA was not affected by strong light implying that ABA signals is not the mechanism in inhibiting seed from germination by strong light. Temperature also affected seed germination at constant 20 C and variable 15/25 C regime are the best germination temperature. The seeds are sensitive to salt in comparison to some other halophytes, and showed similar dose response as Arabidopsis seeds to salt. However, salt cress remained dormant under salinity stress without loosing viability, while most Arabidopsis seed suffer heavy mortality under 400 mM NaCl after ten days.

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Brassinosteroids (BR) affect a range of developmental characteristics, including hypocotyl and pollen tube elongation, flowering time and germination. Proteins involved in biosynthesis and in signaling have been identified through loss-of-function mutants, while enzymes of BR metabolism have been found by screening of overexpression lines.
Searching the Arabidopsis FOX collection for altered organ size, we identified PIZZA. The PIZZA overexpression line strongly resembles BR deficient plants. This phenotype could be rescued by supply of exogenous brassinolide and castasterone. The expression of PIZZA due to promotor-GUS reporter lines seems to be specific to root and floral tissues. Based on the complementation phenotype and sequence analysis, we suggest a role for PIZZA in BR metabolism rather than biosynthesis. As PIZZA knock out lines show no developmental phenotype, redundant enzymes are likely to exist. We are currently investigating the in vitro and in vivo function of the PIZZA protein, especially in comparison to the known P450 hydroxylases BAS1 and SOB7. Preliminary data from double mutant analysis also indicates a role of PIZZA in the inhibition of organ elongation.

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The transcriptional regulation system of auxin response indirectly controls the expression of auxin-responsive genes via auxin receptor TIR1/ABFs-dependent degradation of transcriptional repressor Aux/IAAs, which interact with auxin-responsive transcription factor ARFs. The functional analysis using dominant negative mutants demonstrated that Arabidopsis Aux/IAAs participate into the auxin-related organ formation system. In this study, we tried to clarify IAA8 function on organ formation system and identify IAA8-interacting ARFs. The estrogen-inducible IAA8 overexpressors showed reduced lateral root formation and abnormal gravitropism. In addition, yeast two hybrid assay revealed that IAA8 strongly interacts with ARF5, ARF7 and ARF19. Taken all, we suggested that phenotype changes caused by IAA8 overexpression would be derived from the functional suppression of ARFs interacting with IAA8. The analyzed data of dominant negative transgenic plants with IAA8 own promoter and IAA8 with the mutation on TIR1/ABFs recognition site and of protein-protein interaction between IAA8 and ARFs in plant cells using BiFC method are also reported.

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Several cytochromes P450 (P450s) are involved in biosynthesis and catabolism of phtohormones in flowering plants. Genome sequencing projects of Physcomitrella patents and Selaginella moellendorffii have revealed that some orthologous genes to phytohormone-related P450s from the flowering plants may be present in bryophyte and pteridophyte. We think that the comparative analysis of plant P450s conserved across the evolutionary stages can give us a clue to study the evolutionary aspects of phytohormone homeostasis. In this study, we performed functional analysis of P450s involved in abscisic acid (ABA) catabolism from P. patents and S. moellendorffii. While there are no CYP707A-family genes in the genome of P. patents, we have found five CYP707-family genes, CYP707A20, 707A43, 707B1, 707B2, and 707C1 from S. moellendorffii. The full-length ORFs of the P450 genes were isolated by RT-PCR, and the recombinant P450 proteins were expressed with a baculovirus-insect cell system in order to analyze their catalytic activities in vitro. CYP707A20 and 707A43 showed the ABA 8'-hydroxylase activity, while the other three P450s did not metabolize ABA at all.

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Gibberellins (GAs) are essential endogenous regulators of growth and developmental processes throughout the life cycle of higher plants. We are interested in genetic engineering of the biosynthesis of GAs to control the growth of lombardy poplar (Populus nigra). We isolated more than twenty genes for the putative GA oxygenase enzymes belonged to the 2-oxoglutarate dependent oxygenase superfamily in lombardy poplar. Phylogenetic analysis revealed that the genes fall into four clades: eight into the GA20ox clade, four into the GA3ox clade, seven into the C19GA2ox clade and six into the C20GA2ox clade. To explore the expression of the genes of GA biosynthesis in Lombardy poplar, we performed an analysis by real-time RT-PCR using primers specific for each gene. We have isolated transgenic Arabidopsis introduced the open reading frame of each gene under control of the cauliflower mosaic virus 35S promoter. The transgenic Arabidopsis that expressed the gene of the C19GA2ox clade or the C20GA2ox clade showed dwarf phenotypes.

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Jasmonates are known as plant hormones that are involved in stress responses. In most of the earlier studies, jasmonates levels and expression of their biosynthesis genes have been analyzed 15 minutes or later after stress treatment. Recently we found that accumulation of jasmonates occurred in much shorter time. Plants grown in sealed agar plates were corrected in two methods. When plants were frozen two minutes after opening the lid, concentration of jasmonic acid and JA-Ile were 52.2 and 9.7 ng/gDW, respectively. On the other hand, when plants were immediately frozen after opening the lid, concentrations of jasmonic acid and JA-Ile were 3.51 and 0.98 ng/gDW, respectively. In these conditions, abscisic acid and salicylic acid that are involved in stress response did not show significant differences. In addition to these hormones, auxin, gibberellin and cytokinin did not show significant differences.
Time-course change during this process and the effect of jasmonates accumulation on the levels of other hormones will be analyzed by using jasmonates biosynthesis/signaling mutants.

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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 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 at the 3'-region of the gene. By yeast one-hybrid screening, we identified two candidate transcription factors that bind to the region.

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Jasmonic acid (JA) is a plant hormone that modulates anther dehiscence, fruit ripening, and plant resistance to insects and pathogens. From the research of the Arabidopsis mutant defective in anther dehiscence1 (dad1), we found that the DAD1 protein is a chloroplastic phospholipase A1 that catalyzes the initial step of JA biosynthesis. The JA amount in young flower buds of the dad1 mutant was decreased to one fifth of the wild-type level, whereas comparable amounts of JA were detected in wounded rosette leaves of both dad1 mutant and wild type, indicating that additional lipolytic enzymes other than DAD1 must be present to participate in the wound induction of JA biosynthesis.
So we found six DAD1-like lipases (DAL1-6) including predicted transit peptides, and made dad1 dal1 dal2 dal3 dal4 dal6 sextuple mutant. We quantified the JA level in wounded rosette leaves and flower bud clusters of sextuple mutant plants, and found that they were considerably decreased as compared with those of wild-type plants. These results indicate that DAD1 and DALs are contributed to JA biosynthesis both in wounded leaves and in flower buds.

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Chloroplasts are differentiated from proplastids in response to light and/or hormonal signals. However, the overall mechanisms have not been well documented. We have previously shown that OsGLK1, a GARP-type transcriptional regulator, is a key factor regulating chloroplast development [Nakamura et al. (2009) PCP 50: 1933-1949]. We also observed that the transcript level of OsGLK1 increased synchronously with the greening of wild-type calli after transfer to regeneration medium containing a low level of auxin and high concentration of cytokinin. So we carefully examined the relationship between auxin/cytokinin concentrations and the expression of OsGLK1, and found that auxin strongly repressed and cytokinin promoted the expression of OsGLK1. The transcriptional control may change OsGLK1 activity, and consequently affect chloroplast development. To uncover the mechanisms of the hormonal regulation of OsGLK1 expression, we are currently investigating whether and which factor(s), possibly involved in the cytokinin signal transduction, regulate the expression of OsGLK1.

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Jasmonates(JAs) are plant signaling molecules, which have crucial roles in developmental processes and defense responses against wounding and pathogens. In JA signaling pathway, JAZ family proteins are the repressors of MYC2, a JA responsive transcription factor. After the degradation of JAZs by SCFCOI1, MYC2 is released and regulates the expression of jasmonates-responsive genes. Although MYC2 is well known to interact with JAZ proteins, other transcription factors, which interact with JAZ proteins, are still unknown. To identify transcriptional regulators in JA signaling, we performed cluster analysis of JA responsive genes. Based on the results, we focused on a bHLH transcription factor and designated the protein as INU1. Here, we tested interactions between INU1 and JAZ proteins using yeast two-hybrid system. A few JAZ proteins were found to interact with INU1. Moreover, INU1 formed heteromeric dimers with some other bHLH proteins including MYC2. In this presentation, we will show phenotype of inu1 and related single and double knockout mutants in Arabidopsis.

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Brassinosteroids (BRs) are plant steroid hormones that regulate plant growth and development. To identify detail molecular mechanism of brassinosteroid signaling, we tried to screen mutants from the Arabidopsis activation tagging lines by using Brz (brassinazole), which is the specific inhibitor of brassinosteroid biosynthesis. Brz causes deetioation and dwarf phenotypes that are similar to the phenotypes of BR-deficient mutants. To analyze BR signaling, we tried to screen Brz insensitive mutants bil (Brz-insensitive-long hypocotyl), which showed longer hypocotyls than the wild type with Brz in the dark. Here we identified new bil semidominant mutant bil3 from activation tagging line, which shows longer hypocotyl with Brz at the germination stage, and a slender dwarf phenotype related to excessive epinastic leaves and thin inflorescences at growth stage in the light. In the adult, compared with the wild strain, bil3 shows that the number of inflorescences is about 1.3 times, the number of branches is about 2.2 times, and the number of siliques is about 2.2 times.

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Saponins are secondary products in plants and their function in plant development is little known. We propose a hypothesis that chromosaponin I (CSI), a major saponin in leguminous plants, is required for the growth and development of plants under short days. This idea is based on two experimental results. (1) The flowering of low-CSI pea mutant, where CSI level is about 20% of wild type, is greatly delayed compared with wild type under short days. Furthermore, the growth of the mutant is reduced as well under short days. (2) We examined CSI content of leguminous plants (41 genera 51 species) and found CSI in 32 genera 41 species out of 34 genera 43 species of Papilinonoideae, but not detected in both Caesalpinioideae (4 genera 5 species) and Mimosoideae (3 genera 3 species) plants. Although almost all Papilinonoideae plants have CSI as a major saponin, CSI was not detected in 2 genera 2 species of Papilinonoideae, i.e. Tephrosia candida and Canavalia ensiformis, which are tropical plants. Since it is not required for tropical plants to adapt to short days, these plants might lose CSI. Based on these results we propose that CSI is required for plant development under short days.

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Plant hormones such as auxin and cytokinins function in various stages of vascular development, and play central roles for its regulation. In another case, CLE peptides act as cell-cell communication tools, and play crucial roles for plant morphogenesis to regulate cell differentiation. In particular, TDIF (CLE41/44), which is secreted from phloem and inhibits xylem differentiation, is necessary for strict regulation of vascular radial patterning. However, the roles of CLE peptides except for TDIF have not yet revealed during vascular development. To understand the function of Arabidopsis 26 CLE peptides in vascular development, we examined the effect of exogenously supplied peptides on vascular development in roots. As a result, we found some CLE peptides that inhibited protoxylem vessel formation. To elucidate the action mechanism of CLE peptides, we performed Gene-chip analysis. Based on these results, we discuss the relationship between CLE peptides and other phytohormones during vascular development.

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Root parasitic plants and arbuscular mycorrhizal fungi receive strigolactones as host recognition signals in the rhizosphere. In host plants, strigolactones play a key role in shoot branching. Genetic evidences have illustrated that strigolantones are derived from the carotenoid pathway in plants. However, the biosynthesis pathway of strigolactones has not yet been fully elucidated. Establishment of their biosynthesis pathway and subsequent studies on regulation of their biosynthesis will provide important clues to control their physiological functions. To better understand the biosynthesis of strigolactones in plants, we investigated the occurrence of strigolactones and regulation of their biosynthesis in plant cultured cell lines provided from RIKEN BioResource Center.

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Strigolactones (SLs) are plant secondary metabolites which function as host recognition signals for arbuscular mycorrhizal fungi and root parasitic plants, Striga and Orobanche. Recently, strigolactones have been identified as a novel class of plant hormones inhibiting shoots branching. However, the biosynthetic pathway of SLs in plants remains elusive. In this study, strigolactones produced by Physcomitrella patens subsp. patens, a bryophyte which is a basal lineage of land plants, were examined. Chloronemas of both wild type (WT) and the loss-of-function mutant of carotenoid cleavage dioxygenase 8 (Ppccd8) of P. patens were found to produce SLs including 7-oxoorobanchyl acetate, 7α-hydroxyorobanchyl acetate and orobanchyl acetate at similar levels. This result indicates that other genes are redundant with the CCD8 homolog in chloronema of P. patens.

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Tomato (Solanum lycopersicum) has been an excellent model system for analysis of plant mechanism, which cannot easily be studied in Arabidopsis or rice. To obtain information on the genetic mechanism of fruit setting and ripening, we induced mutations in the tomato cultivar 'Micro-Tom' by irradiation with accelerated heavy ions or gamma ray, and screened for associated phenotypes and tried to identify loss-of-function mutations in some genes that involve in the processes.
In an attempt to uncover the molecular mechanism of fruit setting and ripening, we designed an oligonucleotide-based microarray from whole set of tomato unigenes. The microarray consisted over 41,000 probe sets. Now we start to monitor the differences of gene expression level in the various stages of fruit ripening between mutagenized lines and wild type. We here report the expression profile of genes involving biosynthesis and signal transduction of phytohormones, and also will report the genes that co-express with these genes.

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Indole-acetic acid (IAA) plays central roles in plant growth and development and in responses to environmental factors, such as light and gravity. In this study, we employed emerald luciferase with C-terminal fusion of PEST sequence (Eluc-PEST) which improves its dynamic range of the signal. This short half-life luciferase enabled us to estimate AUX/IAA19 promoter activity in vivo. The luminescence of transgenic Arabidopsis was investigated to exogenous auxin. In the case of excised root segments, the arbitral maximum signal increases according to the auxin concentration, however, beyond 1-3 μM, the maximum signal intensity decreases. Interestingly, the time to reach maximum signal also prolonged according to the auxin concentration, but this trend is not observed in the mesophyll protoplast. We will discuss the kinetics of AUX/IAA19 promoter activity combined with the results of anti-auxin.

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Chloroplast development in cotyledons differs in a number of ways from that in true leaves, but the cotyledon-specific program of chloroplast biogenesis has not been clarified. The cyo1 mutant in Arabidopsis thaliana has albino cotyledons but normal green true leaves. Chloroplasts develop abnormally in cyo1 mutant plants grown in the light, but etioplasts are normal in mutants grown in the dark. CYO1 has a C4-type zinc finger domain similar to that of Escherichia coli DnaJ. Recombinant CYO1 accelerates disulfide bond reduction in the model substrate insulin and renatures RNase A, indicating that CYO1 has protein disulfide isomerase activity. In this study, we analyzed the kinetics of CYO1 in detail. The optimum temperature and pH of CYO1 were 40˚C and pH 6.7, respectively. Since the [S]-[V] plot was sigmoid, CYO1 should be an allosteric enzyme. To determine the amino acids important for CYO1 activity, we generated a series of 7 point mutations of cysteine residues in CYO1, and report these results.

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To evaluate effect of heavy-ion beam irradiation on mutation induction, we have examined the frequency of chlorophyll deficient mutants. Line 22-4Y was a virescent mutant isolated from M₂ populations irradiated with a C-ion beam (LET 22.5 keV/μm, 20 Gy). This mutant developed chrolotic leaves during the early seedling stages, on only first to fourth leaves, and produced mostly green leaves during the late stages. In addition, when the 22-4Y plants were grown at 25 degrees C, chlorophyll deficiency was observed, but the plants produced green leaves at 30 degrees C. The temperature dependent chrolosis was similar to that reported virescent mutants, virescent-1, 2 and 3.
Backcrosses and F₂ analyses indicated that 22-4Y was a single nuclear recessive mutant. Map based cloning identified that the mutation locus of 22-4Y was located at around 72 cM to 80 cM on chromosome 5. A classical phenotypic marker virescent-10 previously has been mapped in this region, but the responsible gene for the virescent phenotype is not isolated. We suggest that the mutation of 22-4Y caused by the same genes as virescent-10.

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The chloroplast is not only the site of photosynthesis but also performs biosynthesis of amino acids, lipid and phytohormones. Because of its endosymbiotic origin, chloroplasts proliferate by division. Recent studies in Arabidopsis mutants such as arc have revealed important molecular components involved in chloroplast division. Generally, mutants defective in chloroplast division machinery have enlarged chloroplasts. However, many of these mutants exhibit normal growth and the biological significance of chloroplast division remains unclear. To examine this question, we attempt to isolate rice mutants that have large chloroplasts. We screened an EMS-mutagenized M1 population of a Nipponbare line derived from tissue culture and showing coarse grain yield. Because chloroplasts were difficult to observe in intact rice leaves unlike Arabidopsis, protoplasts were prepared from third leaves of each plant. Overall, we screened 2,170 samples from 162 independent M1 lines by this method and isolated one line that had enlarged chloroplasts and was termed gic. This mutant so far grows normally and its phenotypic characterization will be presented.

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Photosynthetic proteins are constantly subjected to photooxidative damage. To minimize the damage, plants had developed PSII repair cycle as a quality-control system, in which the damage was targeted to D1 reaction center protein of PSII. In the PSII repair cycle, photodamaged D1 proteins are specifically degraded by an ATP-dependent metalloprotease FtsH ,which functions as a main factor in this cycle. In Arabidopsis, loss of the major chloroplast FtsHs involved in PSII repair cycle (FtsH2 and FtsH5) results in a typical leaf-variegated phenotype. In contrast to extensive Arabidopsis studies, mutants lacking FtsH are not reported in other species. To further investigate the effect of FtsH deficiency on leaf variegated and PSII repair cycle, we generated FtsH knockdown tobacco plants and characterized their properties. FtsH knockdown tobacco plants showed leaf variegation, although the development and pattern of leaf variegation is distinguishable from that in Arabidopsis variegated mutants such as var2. These transgenic plants also showed decreased PSII photochemical yield Fv/Fm upon high-light irradiation, supporting the important role of FtsH in PSII repair.

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The variegated phenotype in Arabidopsis var2 mutant is attributed to a defect of FtsH2, a metalloprotease localized in thylakoid membranes and critically involved in the quality control of photosystem II. We previously showed that the green sectors of var2 contain the same chloroplasts as wild-type except for accumulation of reactive oxygen species. The white sectors of var2 contain undifferentiated plastids with high accumulation of scavenging enzymes such as CSD2 and APX. To understand the physiological significance of variegated leaves, microarray analysis was performed using total RNA extracted separately from green and white sectors. We found that the gene expression profile of var2 green sectors was comparable to that of wild-type. On the other hand, we found that a significant number of genes were up- or down-regulated between var2 green and white sectors. Genes related to photosynthesis and chloroplasts were down-regulated, while genes related to stresses, especially oxidative stress, were up-regulated in white sectors. Among the up-regulated genes, we focused on CSD2, and based on these results, we propose a novel function for CSD2 as a copper buffer in white sectors.

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Vacuoles play many important roles in plant cells. In the root meristematic tissue, the presence of mature vacuoles is less evident, but they are always present as small provacuoles. It is known that provacuoles are produced by the trans Golgi network (TGN). When a cell begins to mature, the provacuoles fuse to produce large central vacuoles that are characteristic of most mature plant cells. We tried to prepare ultrathin sections of various plant samples by high-pressure freezing method and immunogold staining. In the root meristematic cells of Arabidopsis spp. and tobacco, vacuolar H+-pyrophosphatase (V-PPase) signals were detected in the TGN, tonoplast, and certain ring-like structures. These ring-like structures existed as flat and/or double-membrane structures and contained cellular components. Moreover, the structures fused with the central vacuoles. Therefore, these ring-like structures containing V-PPase may play an important role in vacuole formation and degradation of cellular components.

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The Obg/Era proteins of P-loop GTPase superclass is conserved in various organisms. Some of these proteins are critical regulators in many aspects of cellular processes. However, a genome-wide overview of these genes in plants is not available. Comprehensive genomic analyses of Arabidopsis thaliana identified 19 (in 9 subfamilies) of Obg/Era genes. Subcellular localization of Arabidopsis Obg/Era homologues revealed that archaea-derived proteins are mainly targeted to cytoplasm and nuclei (5). All other eubacteria-related proteins are targeted exclusively to mitochondria (2) or chloroplasts (8) and dual targeting proteins (3), suggesting their endosymbiotic origin. Phylogenetic analysis showed only four proteins that are derived from cyanobacteria by endosymbiotic gene transfer among 11 chloroplast-targeting proteins. Surprisingly, four other chloroplast-targeting GTPases do not show cyanobacterial or α proteobacterial affiliation. Instead, these homologues are closely related to other eubacteria, such as Bacteroides and Green nonsulfur bacteria, suggesting the important role of horizontal gene transfer in chloroplast- and mitochondria localized GTPase evolution.

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The peroxisome is one of the organelles that ubiquitous organelles in eukaryotic cells and metabolically diverse organelles with essential through life of the plant such as lipid metabolism, photorespiration, and biosynthesis of plant hormone. Higher plant peroxisomes undergo reversible functional transition depending on environmental, tissue and developmental stages. Some peroxisomal proteins are thought to be degraded in the transition, however, the machinery of the degradation system is unclear. In this study, we search for transition regulating factors that were related to the functional transition of the peroxisome by the proteomic analysis.
Glyoxysomes and peroxisomes in the transition stage of soybean cotyledons were isolated by iodixanol density gradient centrifugation. We performed peptide mass fingerprinting of each peroxisomal proteins with LTQ-Orbitrap. A quantitative change of the peroxisomal proteins in the transition stage was analyzed by iTRAQ. We will show dynamics of the peroxisomal transition from the analysis of these proteome and transcriptome.

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SNARE (soluble N -ethylmaleimide-sensitive factor attachment protein receptor) molecules play a pivotal role at the membrane fusion in membrane trafficking. Out of 65 SNARE proteins in Arabidopsis cell, SYP123 and SYP132, are localized to the plasma membrane of root hair, simultaneously. Although SYP123 is only expressed in root hair cells, and localized at the apical zone of the root hair, SYP132 is ubiquitously expressed in all plant cells so far examined. These differences in the localization pattern between SYP123 and SYP132 remind us of which these SNAREs function in distinct transport pathways to the plasma membrane in root hair cells. To clarify this possibility, we investigated the interaction among SNAREs and Myosin XI family proteins, and found that SYP132 interacts with myosin XI family, but SYP123 do not.

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Cell wall plays a key role in controlling the size and shape of the plant cell during plant development. Unlike the dicotyledonous cell wall, rice cell walls have less xyloglucan and pectin. The major glycan crosslinked with the cellulose microfibrils is arabinoxylan. However, the physiological role of such matrix polysaccharide is not well understood.
To understand the function of matrix polysaccharide in rice, we analyzed the xylanase, xylosidase, and galactanase over-expressed rice, using by the FOX (full-length cDNA overexpresser) gene hunting system, which places individual cDNA under the control of Zm Ubi-1 promoter. We found some significant phenotypes in the development and cell wall sugar composition.

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Liquid fuels derived from cellulosic biomass is one of the candidates of alternative to conventional fossil fuel source. However, cell walls, which consist of cellulose, hemicellulose, pectin and lignin, are locked in complex polymers. In particular, pectin might be a barrier for enzymatic saccaharification of cellulosic ethanol, because pectin degradation treatments are effective in ethanol production process. Compared with the pectin-abundant dicotylednous cell wall (40%), the cell wall of rice contains less pectin (10%). Therefore, pectin was thought to be less important for the growth and development in rice. To understand the function of pectin in the development of rice, we analyzed the polygalacturonase over-expressed rice driven by maize ubiqutin promoter. Overexpression of PG caused the reduction of pectin content and alteration of sugar composition. These results suggest that pectin might be important for the normal development and the property of cell wall in rice.

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In primary cell walls, cellulose microfibrils are crosslinked with cell wall matrix polymers. Their cross-linking glycans consist of linear chain and side chains. Although Ca2+ binding to backbone homogalacturonan has been known, the function of side chains are not well understood. To understand the function of the arabinose-containing side chains, we analyzed phenotype of RNAi suppression line of UDP-arabinopyranose mutase (UAM) gene. The abnormal phenotypes were observed in mature pollen grains and expanding leaves of the transformant. To clarifying the relationship between microsporogenesis and UAM expression, we analyzed expression pattern of UAM by qRT-PCR. In transformant leaves, the abnormal developments, such as earlier etiolation, were observed. And, abnormal cell wall compositions were also observed in the expanding leaves. These results suggest that UAM expression might be important for microsporogenesis and the maintenance of leaf development.

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Silicon (Si) is a beneficial element for plant. Several benefits of Si have been reported, such as enhancement of pest and pathogen resistance, drought and heavy metal tolerance, and improvement of agricultural crop quality and yield, in a wide variety of plant species. Plant species differ greatly in silicon accumulation, ranging from 0.1% to 10% in shoot dry weight. Rice (Oryza sativa) is a typical Si-accumulating plant and is able to accumulate Si to >10% of the shoot dry weight. In the shoot, it is known that Si exists as a polymerized form in motor cells or beneath the cuticle layer. On the other hand, it is suggested that there are silicon-containing high molecular weight compounds in cell wall. Therefore, our objective is to investigate the relationship between Si and cell wall matrix, especially its sugar composition. Sugar composition of cell wall fractions isolated from rice grown in hydroponics with or without 1.5 mM silicic acid were analyzed by gas-liquid chromatography. As a result, in the pectic fraction, amounts of some sugars were different between two Si conditions. Based on these results, the interaction between Si and cell wall matrix will be discussed.

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Boron (B) as borate cross-links pectic polysaccharides at rhamnogalacturonan II regions, thereby contributes to building the cell wall structure. We previously showed that B deprivation immediately induces Ca²⁺ influx and upregulation of stress-responsive genes in suspension-culture tobacco BY-2 cells. As a possible mechanism of such quick responses, we hypothesize that the cell wall with insufficient borate cross-linking cannot resist the turgor and allows the plasma membrane to stretch, thereby triggers Ca²⁺ influx through mechanosensitive ion channels.
Adding 0.4 M mannitol to reduce the membrane stretch suppressed the B deprivation-responsive gene expression. Higher dose Ca²⁺ (30 mM), which is expected to mitigate a structural disturbance of cell wall through enhanced cross-linking of polygalacturonate, also suppressed the upregulation of several genes. These results are consistent with our hypothesis that a weakened cell wall structure mediates the early responses of tobacco cells to B deprivation.

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We examined the effect of gibberellic acid (GA) on colchicine, microtubule-disrupting agent, in tissue culture of Arabidopsis. In shoot inducing medium, 5.6x10^-5 M colchicine inhibited greening of callus and shoot induction without inhibition of cell division. Almost cortical microtubules (cMT) of cells in these calli were disrupted by 5.6x10^-5 M colchicine. Treatment of 1.0x10^-4 M GA together with 5.6x10^-5 M colchicine diminished inhibition by colchicine of greening and increased the number of cells having intact cMT. The effect of GA is evident under condition of concentration of 1.0x10^-7 M. These results show that GA stabilized microtubules against microtubule-disrupting by colchicine in cells of callus.
Using this system, now we are trying to screen for colchicine-resistant line from activation-tagged mutants in order to identify genes related to stabilization of microtubules. We selected 6 candidates for colchicine-resistant mutants and 17 candidates for colchicine hyper-sensitive mutants from 10,000 lines.
In the future, we will investigate the relationship between stabilization of microtubules and plant growth by examining the function of causative gene for these mutants.

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In plant cells, microtubules are essential for cell elongation and mitosis, and have great influence on plant shape. It has been suggested that alignment of cortical microtubule bundles in expanding interphase cells determine the direction of cell elongation. However, how cortical microtubules are nucleated and become progressively organized has not been well understood. Our previous screening identified PROPYZAMIDE HYPERSENSITIVE1 (PHS1). PHS1 encodes a MAP kinase phosphatase which is preferentially expressed in elongating cells. When a phosphatase-inactive-form PHS1 was expressed in Arabidopsis plants, cells at the elongation zone swelled and root growth was strongly inhibited. In addition, cortical microtubule arrays are strongly depolymerized by transient expression of inactive-form PHS1. Therefore cortical microtubule arrays are possibly regulated by phosphorylation pathway. Thus we are now trying to understand the pathway in detail.

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Plant cells initiate nascent cortical microtubules from gamma- tubulin-containing complexes dispersed on existing microtubules as branching patterns. Newly formed microtubules are released from the nucleation sites, and then free microtubules are transported on the cell cortex by polymer treadmilling. By using live cell imaging, we found that the nucleation complexes labeled by fluorescently-tagged gamma complex protein 2 (GCP2) or GCP3 transiently associate with cell cortex in a microtubule-independent manner, and a fraction of the complexes immediately nucleate nascent microtubules upon association with previously established mother microtubules. Daughter microtubules are sometimes nucleated parallel to the mother microtubules, thereby generating instantaneous bundles. The GCP2/3-containing complexes are anchored at the basis of branching points until the Katanin-dependent activity severs daughter microtubules at their minus ends or their dynamic plus ends completely depolymerizes. We are analyzing spatiotemporal control of katanin protein that contributes to a microtubule release event.

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mRNA-ribonucleoproteins (RNPs) complexes mediate various mRNA metabolism, such as transcription, nucleus-cytoplasm transport, translation and mRNA degradation. Interestingly, RNPs complexes were concentrated in the fraction of microtubule-associated proteins, indicating the relationship between microtubule networks and mRNA metabolism. Because animal and yeast mRNA-RNPs complexes transport along microtubules, plant mRNA-RNPs may transport along microtubules.
We generated GFP-fused DCP2 that labeled one of mRNA-RNPs complexes, P-bodies, which function in mRNA degradation. Dual time-lapse observation using GFP-DCP2 and mcherry-tub6 revealed that P-bodies do not move along microtubules but step between microtubules and often stop on microtubules. The movements of P-bodies were inhibited by the treatments of actin filaments destabilizing agent, Lat B, suggesting that actin filaments deliver P-bodies between microtubules. We conclude that plant P-bodies move dependent on actin filaments and microtubules provide anchoring places.

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