Brassinosteroids (BRs) are essential plant hormones that control many aspects of plant growth and development. Our previous works revealed that the levels of endogenous BRs, especially those of typhasterol and castasterone, in rice seedling shoots, but not in the roots, were increased under continuous white or blue light condition as compared with continues dark condition. However, no such effects were observed under far-red and red light conditions. In this study, we analyzed transcript levels of BR biosynthesis genes using quantitative RT-PCR technique, in order to address the possible contribution of light quality to altering BR levels in rice seedlings shoots. As the result we found that the transcript levels of CYP85A1, encoding a protein that catalyzes C-6 oxidation step in BR biosynthesis, were up-regulated under white or blue light, but not under far-red or red light conditions. These results suggest that the increased levels of endogenous BRs in shoots of white or blue light grown rice seedlings should be a consequence of blue light-mediated up-regulation of CYP85A1 gene.

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We have already reported that chlorophyll levels in cucumber plants grown under low nitrogen conditions are preserved by abscisic acid (ABA). In this study, we investigated expression of chlorophyll biosynthesis and degradation genes in cucumber plants under low nitrogen conditions. In contrast to the reduction of expression of chlorophyll biosynthesis genes in older cucumber plants grown without ABA, ABA maintained relative high levels of transcripts until 20 days after treatment. The transcript levels of chlorophyll degradation genes in cucumber plants applied ABA were smaller than that in drug free plants. In addition, the transcript level of chlorophyll a/b binding protein reduced in cucumber plants grown under low nitrogen conditions in the absence of ABA compared to those applied ABA. Although the structure of thylakoid stack was disordered in drug free plants, it was well defined in plants in the presence of ABA under low nitrogen conditions. These results suggest that ABA promotes chlorophyll biosynthesis and suppresses chlorophyll degradation in cucumber plants grown under low nitrogen conditions. ABA also allows chlorophyll to be stable in thylakoid membrane.

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Cytokinins play crucial roles in diverse aspects of plant growth and development. In the biosynthesis pathway, nucleotide form is initially produced as a cytokinin precursor. It is expected that two pathways are involved in producing active form from the nucleotide: two-step activation pathway and direct activation pathway. The direct pathway mediates production of the active nucleobases from the nucleotides. LONELY GUY (LOG) was previously identified as a cytokinin-activating enzyme that works in the direct activation pathway in rice shoot meristems. However, it is not clear whether there is a functional differentiation between these two activating pathways. In Arabidopsis, we have identified seven LOG-like genes (AtLOG1-5, 7, 8), and analyzed the phenotypes of various combinations of the multiple knock-out mutants. Our results strongly suggested that the direct activation pathway plays a crucial role in cytokinin activation.

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We have developed a highly sensitive and high-throughput method for the simultaneous analysis of 43 molecular species of cytokinins, auxins, ABA, and gibberellins (GAs) using an automatic liquid handling system for solid phase extraction, UPLC-ESI-qMS/MS, and chemical derivatization with bromocholine. Our current method needs less than 100 mg (fresh weight) of plant tissues to determine phytohormone profiles and enables us to analyze simultaneously more than 180 plant samples. Application of this method to plant hormone profiling enabled us to draw organ-distribution maps of hormone species in rice and also to identify interactions among the 4 major hormones in the rice GA-signaling mutants, gid1, gid2, and slr. Combining the results of hormone profiling data with transcriptome data in the GA signaling mutants allows us to analyze relationships between changes in gene expression and hormone metabolism. We are now trying to identify key genes regulating hormone levels in rice using QTL analysis. We have measured hormone contents of Sasanishiki X Habataki BILs. The data will be presented.

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Agrobacterium tumefaciens infection induces tumor formation by integrating the T-DNA region of Ti-plasmid into the plant nuclear genome. Tumors are formed because the T-DNA encodes enzymes that synthesize auxin and cytokinin. We had demonstrated that a cytokinin biosynthesis enzyme isopentenyltransferase (IPT), Tmr, which is encoded by the T-DNA region, is targeted to and functions in plastids of infected plant. Tmr creates a metabolic bypass for direct synthesis of tZ-type cytokinin by using HMBDP. To reveal biological importance of the Tmr function for gall formation, we studied biochemical properties of Tmr with comparing to Tzs, which functions in the bacterial cell. We generated transgenic Arabidopsis overexpressing Tmr, Tzs, or transit peptide-fused Tzs (TP-Tzs) under the control of DEX-inducible promoter. DEX-treatment induced the accumulation of tZ-type cytokinin in Tmr-overexpressing Arabidopsis, whereas it induced that of both tZ and iP in Tzs and TP-Tzs-overexpressing plants. Tmr and Tzs show similar substrate specificity in vitro. Thus, it is strongly suggest that Tmr has a specialized function to predominantly use HMBDP for tZ biosynthesis in the host plant plastids.

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One of the plant hormones, ethylene, causes pleiotropic effects on plant growth. However, the effects of ethylene on cells are not fully elucidated. In the present study, the effect of ethylene on cucumber at the early stage of development was analyzed. Application of 0.35mM ethephon to cucumber plants when leaf blade of cotyledon is 2 cm, caused not only epinasty of cotyledons but also development of many small true leaves compared to the control plants. Application of ethephon (0.014mM, 0.07mM, 0.35mM) caused expansion of epidermal cells around trichomes and increased DNA content of the same cells on the surface of cotyledons. Toluidine blue O stained the expanded epidermal cells around trichomes. In many true leaves that are developed by the application of 0.35mM etephon, both leaf area and number of epidermal cells were smaller compared to the control plants. These results suggest that ethylene induces cell expansion, endreduplication, and accumulation of polyphenolic compounds in particular cells (epidermal cells around trichomes) at the tissue that finished cell division (cotyledons) whereas inhibits cell division at the shoot apical meristem (true leaves).

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MSG2/IAA19, an auxin-inducible Aux/IAA gene, functions in tropisms in hypocotyls and formation of lateral roots. IAA19 is expressed mainly in hypocotyls and the stele of roots in etiolated seedlings. In root tips it is expressed in root cap, lateral root cap, protoxylem and metaxylem. In this study, we mutagenized about 1000 IAA19 pro::GFP lines with EMS and carried out screening of mutants lines by examining GFP signal of each M2 lines, in order to identify regulatory factors for IAA19 expression. As a result, we isolated about 300 mutant lines in which the GFP signals were changed in various manners in etiolated seedlings. One of the isolated mutants, F10, exhibited reduced GFP signals, and showed defects in formation of root hair. Though F10 seedlings had few root hairs, F10 adult plants showed no different phenotype from wild type. Real-time PCR experiments showed that mRNA level of IAA19 was reduced to about 20% of wild type in F10. The F10 locus was mapped in chromosome 5. We would like to identify more mutant loci as well as the F10 locus by genetic mapping to study regulatory network of IAA19 expression.

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The Small Acidic Protein 1 (SMAP1) gene was identified as a regulator for 2,4-D response in Arabidopsis root. SMAP1 has the conserved phenylalanine and aspartate rich region (F/D region) in its C terminal. Previously, we reported that the F/D region is essential for the SMAP1 function and that SMAP1-GFP binds to COP9 signalosome (CSN). In this study, to further elucidate the interaction between SMAP1 and CSN, GST-tagged SMAP1 proteins were expressed in E. coli, purified, mixed with total proteins of aar1 that lacks SMAP1 gene, and examined by pull-down and western analysis. The GST-SMAP1 and GST-F/D proteins were pulled down with CSN but not GST-SMAP1ΔF/D, suggesting that the F/D region was required and sufficient for the interaction between SMAP1 and CSN.

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RecQ helicases are involved in the process of DNA replication, recombination and repair. Arabidopsis genome contains seven RecQ like genes. Recently, Hartung et al reported RecQl4A and RecQl4B knockout mutant in Arabidopsis. AtRecQl4A mutant showed enhanced frequency of homologous recombination compare with that of wild type. In contrast, AtRecQl4B mutant showed reduced frequency of HR. Furthermore, AtRecQl4A mutant showed sensitivity to DNA damaging agents, but AtRecQl4B mutant did not. In monocotyledon model plant, rice had been reported to have seven RecQ homologues. Interestingly, in contrast to Arabidopsis, rice has a single RecQl4-like gene. We cloned the full length OsRecQl4 cDNA, which is 3.5 kb in length and encodes an open reading frame of 1174 amino acid. It has a HRDC (Helicase and RNase D C-terminal) domain, which shows function of dissolution of double Holliday junctions (Wu L. et al. 2005). In addition, we characterized T-DNA and Tos17 insertion mutant of RecQl4 in rice.

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Endoreduplication involves a doubling of chromosomal DNA without corresponding cell division. In plants many cell types transit from the mitotic cycle to the endoreduplication cycle or endocycle, and this transition is often coupled with the initiation of cell expansion and differentiation. In spite of the importance of the cell cycle transition, it is still largely unknown how this process is developmentally regulated. We recently found that a SUMO E3 ligase, HIGH PLOIDY2 (HPY2), functions as a repressor of endocycle onset in Arabidopsis meristems. Loss of HPY2 results in a premature transition from the mitotic cycle to the endocycle, leading to severe dwarfism with defective meristems. HPY2 functions as a SUMO E3 ligase in vivo and in vitro through an SP-RING domain characteristic of MMS21-type SUMO E3 ligases. HPY2 is predominantly expressed in proliferating cells of root meristems and acts downstream of meristem patterning transcription factors PLETHORA1 (PLT1) and PLT2. These results establish that HPY2-mediated sumoylation modulates the cell cycle progression and meristem development in the auxin-PLT-dependent signaling pathway.

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Habituation is the least understood phenomenon in plant cell culture. To dissolve this, we compared the tobacco BY-2 cells with a habituated cell line of 2B-13 derived from the BY-2 cells. In fact, the culture filtrates from 2B-13 cells caused the induction of cell division in auxin-starved non-dividing BY-2 cells. The cell division factor (CDF) from the culture filtrates of 2B-13 cells has been ascribed to a glycoprotein, while those from BY-2 cells have been also identified a kind of glycoproteins, whose molecular masses are different from the CDF from 2B-13 cells. However, one difficulty was that the amounts of these CDFs were extremely low for biochemical characterization. In this communication, we tried to purify similar CDFs in the cell lysates from the BY-2 cells. As we have succeeded in purifying and characterizing the CDFs in this fraction, these outcomes will be discussed with the previously characterized CDFs and possibly deduce the role of these CDFs in the auxin-signaling pathway.

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Plastids and mitochondria have their own genomes descended from their bacterial ancestors. In C. merolae, dark incubation arrests cell cycle at the G1 phase, and the cell cycle synchronously initiates after light illumination. During the G1 to S phase transition, organelle DNA replication (ODR) precedes the nuclear DNA replication (NDR), and ODR is required for the NDR initiation. Thus, NDR is tightly coupled with ODR in C. merolae. As the molecular mechanism, we recently found that intracellular accumulation of Mg-Protoporphyrin IX, which is induced by ODR, activates CDKA and thus initiate NDR^{1, 2}. However, little information is available on the ODR control mechanism. We have recently made clear that CDK-type kinase other than CDKA is involved in the ODR initiation. In this meeting, we will present recent results on the characterization of this CDK enzyme.
¹ Kobayashi et al. Proc. Natl. Acad. Sci. U S A 106:803-807 (2009)
² Kanesaki et al. Plant Signal. Behav. in press (2009)

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We previously isolated Arabidopsis sog1-1 (suppressor of gamma response) as a checkpoint defective plants. SOG1 encodes a putative transcription factor belonging to the NAC protein family. Hundreds of genes are upregulated within 1.5 hr of IR treatment in wild-type Arabidopsis. Surprisingly, almost every gene was not induced in sog1-1 mutants, indicating that SOG1 is a master transcription factor that governs transcriptional response to IR. Although genes associated with cell-cycle progression are suppressed by IR in wild-type plants, this suppression was impaired in sog1-1 mutants. To understand the functional role of SOG1 in plant development, we examined its expression pattern using a SOG1-GUS fusion constructs. SOG1-GUS staining was observed in the apical meristems of shoots and roots, and in lateral root primordia, indicating that the function of SOG1 is important for actively dividing cells. These studies reveal a new aspect of the plant's damage checkpoint system, which is to direct the flow of information through a central regulatory transcription factor, SOG1, in response to genomic stress.

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Genome integrity is continuously threatened by external stress or by errors in DNA replication, thus a response to DNA damage is essential for survival and continuous growth of plants. To prevent DNA-damaged cells from proliferating, plants may provoke the DNA damage checkpoint, but its regulatory mechanisms remain unknown.
In plants, A- and B-type cyclin-dependent kinases (CDKA and CDKB) are assumed to mainly control the cell cycle progression. CDKB is further classified into two subtypes, CDKB1 and CDKB2. CDKA can complements the yeast cdc2/cdc28p mutant, while CDKB cannot, suggesting that CDKB is a plant-specific type of CDK.
Recently, we found that the protein degradation of CDKB2 is enhanced in response to DNA double-strand breaks (DSBs).We also found that DSBs trigger transition to the endocycle, leading to endoreduplication, in Arabidopsis. These results promoted us to speculate that CDKB2 degradation may be involved in switching from the mitotic cell cycle to the endocycle in response to DSBs. To answer this question, we are investigating the ploidy distribution of Arabidopsis cultured cells that are knocked-down for the expression of CDKB1 and/or CDKB2.

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During cell cycle progression, it is essential that DNA damage is repaired before cell division. However, in plants, such regulatory mechanisms remain unknown. Recentaly we found that CDKB2 is degraded in response to DNA double-strand breaks (DSBs), which also trigger transition from the mitotic cell cycle to the endocycle.
The first exon of CDKB2 encodes 75 amino acids, and contains the putative PEST motif in the region of amino acid No. 46-59 I made GUS-fusion constructs, which had mutations or deletions in the first exon and observed GUS expression in root tips. A deletion in the region of No. 46-75 suppressed the proteolysis of CDKB2 in response to DSBs. In contrust, mutations in the putative PEST motif did not affect the CDKB2 expression under genotoxic stress conditions. This suggests that the first exon contains regulatory element(s) that is important for CDKB2 degradation, but the PEST domain does not function in response to DSBs.
We are also testing the sensitivity to DSBs by using Arabidopsis mutants that have different levels of DNA ploidy. Based on these results, we shall discuss the physiological significance of endocycle induction in response to DSBs.

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Differential auxin distribution has been proposed to act as a morphogen to set up distinct zones for cell division, cell expansion, and cell differentiation. It is well known that cell differentiation is usually associated with transition from the mitotic cell cycle to the endocycle. This switch is assumed to be controlled by spatiotemporal regulation of cyclin-dependent kinase (CDK) activities, but its regulatory mechanisms remain unknown.
Plants have six types of CDK, among which CDKA and CDKB play a key role in cell cycle progression. We have recently found that a reduction in the CDKB2 level was well correlated with endocycle induction in response to genotoxic stress. Therefore, we assumed that quantitative regulation of CDKB2 may be also important in transition to the endocycle during plant development. When the CDKB2 reporter lines of Arabidopsis were treated with auxin antagonist, the CDKB2 level was downregulated by proteolysis earlier than by a reduction in the transcript level. Our recent report suggested that CDKB2 may be also controlled by protein stabilization, thus we are testing how the CDKB2 level is balanced in root tips to maintain the continuous root growth.

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We labeled DNA fibers of rice (Oryza sativa L. cv. Nipponbare) chromosomes with fluorescent marker and detected replication forks. The suspension cultured rice cells used in this work were subcultured every 7 days. We used cells at 5 days after subuculture because cell division was most active in this stage. First, cells were cultured with Bromodeoxyuridine (BrdU) to label nascent DNA. Next, nuclei were prepared by a simple procedure. Then, chromosomal DNA was extracted and extended on a microscopic slide. The BrdU labeled regions of DNA were detected using anti BrdU antibody and FITC-conjugated secondary antibody. As a result, we were able to observe replicating regions of rice chromosomal DNA. Moreover, we could measure the size of replicons. This is the first step for cloning and characterizing rice DNA replication origins.

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The posttranslational conjugation of small ubiquitin-related modifiers (SUMOs) to other proteins is involved in regulation of many processes in eukaryotic development; although its role in plant development is beginning to be dissected. Previously, we demonstrate that the siz1 mutant, impaired in a SUMO E3 ligase, showed dwarf-like shoot phenotype with accumulation of salicylic acid (SA) and the expression of nahG, a bacterial SA hydroxylase, in siz1 reduced SA level and suppressed dwarfism. Herein, we provide evidence that the SIZ1 controls cell division and elongation through regulation of SA level. siz1 plants exhibited a dwarf-like phenotype that is attributable to decreased leaf cell volume and number. Cell division and expansion defects were restored in siz1 nahG. Expression of XTH8 and XTH31, encoding xyloglucan endotransglycosylase/hydrolase, which are thought to facilitate leaf cell expansion, was down-regulated in siz1 leaves. But it was restored in siz1 nahG plants. These results indicate that SIZ1 regulates cell growth and plant development with regulation of SA accumulation. And XTH8 and XTH31 genes may be responsible for reduced leaf cell expansion.

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The peridinin-containing dinoflagellate is an algal group of Alveolata with plastids that originated from secondary endosymbiosis involving a red algal lineage. Plastomes of the peridinin-containing dinoflagellates are composed of a limited number of genes that are contained separately on small circular molecules, termed minicircles. We discovered multiple variants of the gene psbA in the minicircles of the dinoflagellate Alexandrium tamarense, which have persisted for almost three years in culture. Each variant, like the ordinary psbA, existed on distinct minicircles of similar size. These psbA variants retained all, or almost all, the coding sequence of the ordinary gene and all four were transcribed and edited after transcription, even though they could not encode the entire protein due to intervening or translocated sequences. Repeat elements were generally found in the relatively large non-coding region of these minicircles. The fact that these minicircles are transcribed, individually edited and maintained in the genome suggests that they are functionally important, although their precise roles remain unclear.

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It is widely accepted that plastids have evolved from free-living cyanobacteria. In the process of endosymbiosis, most of genes were relocated from ancestral plastids to the host nucleus. Most nuclear-encoded plastid proteins are cytosolically synthesized with N-terminal transit peptide (TP). It has been experimentally demonstrated that several TPs lack defined secondly structure. Such unstructured proteins are also known to be unstable in bacterial cells.
Here, we hypothesized that stabilization of unstructured proteins in eukaryotic cytoplasm would be prerequisite for acquisition of TP. To assess this hypothesis, we examined the fate of a TP of RbcS fused to GFP in E.coli. Addition of N-terminal stable tag allowed us to purify the fusion protein. After removal of the N-terminal tag by specific protease, the TP-GFP was rapidly degraded in the presence of E.coli cell extract. This degradation was inhibited by addition of o-phenanthroline, suggesting that metalloproteases mainly degrade the TP. Based on these results, it is assumed that such protease activity would be lost in eukaryotic cytoplasm before the acquisition of TP.

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Among several Plastid Specific Ribosomal Proteins (PSRPs), PSRP-1-like proteins are distributed in most bacteria. PSRP-1-like proteins in E coli, HPF and YfiA, were involved in dimer ribosome formation and translation inhibition.
We isolated a psrp-1 disruptant and an overexpression strain of Synechocystis sp. PCC 6803. Growth of either strain was similar to that of wild-type strain in liquid media at 30 degree or 40 degree, and in light-dark cycle or continuous light. In the competition culture between wild-type and psrp-1 disruptant in liquid media at 30 degree, psrp-1 disruptant gradually became dominant in the population, suggesting PSRP-1 moderately suppresses cell growth. On the other hand, growth of the psrp-1 disruptant was remarkably suppressed in the competition culture on solid media. These results suggest that PSRP-1 confer resistant to a kind of stress which may be related to growth condition on solid media. We then examined ribosome profiles of cell culture and found that dimer ribosomes were detected in the cells grown on solid media or grown at high temperature.

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Chloroplast contains multicopy of the genome, which form a complex with a number of proteins. This DNA-protein complex is called nucleoid. Earlier microscopic observations established that the localization of nucleoids changes during cell differentiation in land plants and that nucleoids are nearly equally inherited by daughter chloroplasts during chloroplast division. In this study, in order to understand the significance of morphological change of chloroplast nucleoids, we tried to identify chloroplast nucleoid proteins. We performed with mass spectrometry analysis and genetic analysis for identification of nucleoid protein. As a result, some factors are newly identified as the nucleoid protein. We discuss the role of them in the morphological change of nucleoids.

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When nuclei were isolated from Arabidopsis thaliana leaves in the presence of detergent, a DNA region visualized by Hoechst staining was maintained though the nuclear envelop was degraded. Isolated DNA regions were spindle-shaped, reflecting the shape of nuclei in vivo. We are examining factors involved in the maintenance of DNA regions. We asked a participation of nuclear lamina structures. A. thaliana lacking LITTLE NUCLEI 1(LINC1), one of the main lamina components, shows spherical nuclei. However, isolated demembranated nuclei of LINC1 knockout plant retained spherical DNA regions. This result suggests that LINC1 participates in the maintenance of shape of DNA region, but not in the maintenance of DNA region itself. Next, cytoskeleton inhibitor did not disperse the DNA region while α-amylase did. Assuming that possible targets of α-amylase were glycoproteins and/or carbohydrates, we treated demembranated nuclei with N-linked oligosaccharide hydrolyzing enzymes. However, the DNA region was maintained. Identification of targeted glycoproteins included in demembranated nuclei by other enzyme treatments and mass spectrometry are under investigation.

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In Arabidopsis thaliana, phosphatidylethanolamine (PE) biosynthesis occurs in the endoplasmic reticulum via the CDP-ethanolamine pathway (Mizoi et al., 2006). CTP:phosphorylethanolamine cytidylyltransferase (PECT1; EC 2.7.7.14), a rate-limiting enzyme in PE synthesis, is associated with the outer surface of mitochondria. Thus, the significance of mitochondrial localization of PECT1 remains to be evaluated. An N-terminal hydrophobic domain (PECT1N) was sufficient for PECT1 targeting to mitochondria, whereas the removal of PECT1N from PECT1-EYFP resulted in targeting of the protein ΔNPECT1-EYFP to the nucleus and the cytoplasm. ΔNPECT1-EYFP expression in pect1-6 mutants complemented the embryonic lethality, indicating that the mitochondrial localization of PECT1 is not necessarily required for embryogenesis of Arabidopsis. We will also report the evaluation of the chilling sensitivity, flowering, and pollen maturation in transΔNPECT1-EYFP pect1-6 plants.

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The regulation of mitochondrial morphology, intracellular distribution and inheritance are vital for plant growth and development. An evolutionarily conserved mitochondrial GTPase, MIRO1, has been previously shown to be required for embryogenesis in Arabidopsis thaliana (Yamaoka & Leaver, Plant Cell 2008), although the mechanism involved is still unclear. We created transgenic Arabidopsis plants of which mitochondria are visualized by GFP fluorescence in the egg cells and early-stage embryos. Two-photon laser scanning microscopy revealed that the miro1 mutant egg cell and embryos exhibit abnormally enlarged mitochondrial morphology, leading to the defective mitochondrial inheritance. Our findings suggest that MIRO1 plays a vital role for regulating mitochondrial morphology and inheritance during embryogenesis.

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Mitochondrial DNA molecules are packed into the 3-dimentionally organized structure, called mitochondrial-nucleoid. The function of mitochondrial genome must be conducted properly in this highly condensed structure. However, the mode of organization of mitochondrial DNA into nucleoid is largely unknown. When mitochondrial-nucleoids isolated from BY-2 cultured tobacco cells were treated with micrococcal nuclease (MNase) and their DNA was subjected to agarose-gel electrophoresis, DNA ladder was observed, suggesting that nucleosome-like repeated structure was present in the mitochondrial-nucleoid. MNase digests with minimum size (ca. 75 bp) purified through sucrose gradient centrifugal fractionation contained several kinds of proteins distinct from histones. This result suggests that the nucleosome-like repeated structures in mitochondrial-nucleoid are constructed by original proteins different from those present in cell-nuclear chromatin. Electromicroscopic observation of the fine structures of isolated and MNase-treated mitochondrial-nucleoids, is now on progress.

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The cell line named 1/20-B is tolerant to low boron condition (5 μM). In these cells, activity of pectin methylesterase (PME) and expression of its gene (paPME1), isolated from P. alba cells, were higher than that of 1/1-B cells which required enough boron (100 μM). However characteristic and transient increase of PME activity recorded in 2 days after subculture was only accompanied with low gene expression of paPME1. The participation of other genes encoding PME was deduced.
In this study, expression pattern of another PME cDNA (paPME2) isolated from 1/20-B cells in 2 days after subculture by RT-PCR was examined. A high homology was observed between paPME2 and PME2, isolated from hybrid poplar, rather than paPME1. It was expected that the expression of paPME2 regulated the high activity in 2 days after subculture. However expression pattern of paPME2 was similar to that of paPME1. This result indicated that there were post-transcriptional and/or post-translational regulations in PME activity.

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Cellulases are enzymes that digest celluloses, but some of them have been known to play essential roles in cellulose biosynthesis. How these cellulases work in the cellulose synthesis has not been clear. Gluconacetobacter xylinum, a cellulose-producing bacterium, has a cellulase that involves in the cellulose synthesis. We previously showed that cellulose-like fibrils produced by a cellulase-disruptant, strain F2-2, were highly twisted. Here, we characterized these cellulose-like fibrils. As the highly twisted cellulose-like fibrils were detected in the alkali-insoluble fraction of F2-2 culture by electron microscopy, this sample was analyzed by the ¹³C nuclear magnetic resonance (NMR) and by the Fourier transform infrared (FT-IR) spectrometer. The low-crystalline cellulose II was detectable in the alkali-insoluble fraction by these methods. However, cellulose fibrils produced by the cellulase-gene complemented strain of F2-2, F2-2 (pSA-CMCase/k), were shown to have the high-crystalline cellulose I. These data indicate that this cellulase facilitates cellulose biosynthesis by increasing the amount of crystalline cellulose in the fibrils.

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To generate a transgenic rice plant suitable for efficient bioethanol production by the use of a cellulase gene, we examined effects of overexpression of the cellulase gene on rice development. A rice cellulase cDNA driven by the maize ubiquitin promoter was introduced into the rice genome. The transgenic plants showed overexpression of the cellulase gene, and a high cellulase activity was detected in their protein extracts. The transgenic plants showed various physiological and morphological abnormalities such as leaf colour change, division of leaves and sterility. These results suggest that constitutive overexpression of cellulase can be used for improvement of rice plants, but induced expression of cellulase will be necessary to avoid unsuitable effects on rice development.

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We have isolated loss of function mutant of TRG1/XYL1 gene which encodes a cell wall hydrolyzing enzyme, α-xylosidase, as one of the high temperature resistant germination mutants of Arabidopsis. Over-expression of TRG1/XYL1 inhibited germination of the seeds at 28 ^oC, indicating that the gene has suppressive role on germination at supra-optimal temperature conditions. α-xylosidase removes xylose residue from the non-reducing end of xyloglucan oligosaccharides (XGOs), and it has been suggested to be a key step of further degradation of the oligosaccharides. HPLC and MALDI/TOFMS analyses revealed the over-accumulation of XGO molecules in developing fruits, leaves and seeds of trg1/xyl1 mutant. In addition to the germination phenotype, trg1/xyl1 had small flower organs, short and fat siliques, and the cells of the fruit wall showed disordered array and shape. The creep-extension analysis indicated that the elongating flower stem of trg1/xyl1 had lower elasticity and viscosity values than wild type. These results suggest that XGOs have enhancing role on cell wall loosening, and α-xylosidase plays a role to keep cell wall loosening state normal during cell elongation.

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We characterized peptidyl hydroxyproline O-galactosyltransferase (HGT), which is the initial enzyme in the arabinogalactan (AG) biosynthetic pathway. An in vitro assay of HGT activity was established using chemically synthesized fluorescent peptides as acceptor substrates and extracts from Arabidopsis thaliana T87 cells as a source of crude enzyme. The galactose residue transferred to the peptide could be detected by HPLC and MALDI-TOF-MS analyses. HGT required Mn²⁺ for maximal activity and consumed UDP-D-galactose as a sugar donor. HGT exhibited an optimal pH range of pH 7.0 to 8.0 and an optimal temperature of 35 degree. The favorable substrates for the activity seemed peptides containing two alternating imino acid residues including at least one acceptor hydroxyproline (HYP) residue, although a peptide with single HYP residue without any other imino acids also functioned as a substrate. The cellular localization of HGT activity is identical to those of ER markers, indicating that HGT is predominantly localized to the ER. This is the first characterization of HGT and the data provide evidence that AG biosynthesis occurs in the protein transport pathway.

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TBK5 is a higher plant-specific kinesin constantly expressed in tobacco BY-2 cells. On the basis of the finding that, in transiently transformed BY-2 protoplasts green fluorescent protein-tagged TBK5 (GFP-TBK5) proteins tend to form aggregates that function as microtubule-organizing centers (Goto and Asada, 2007), we hypothesize that TBK5 might be involved in nucleation of microtubules and/or the regulation of microtubule arrangement. To estimate the subcellular distribution of TBK5, we prepared stably transformed BY-2 cells that inducibly express GFP-TBK5 proteins, and examined the distribution of expressed GFP-TBK5 in interphase and mitotic cells. In interphase cells, GFP-TBK5 co-localized with peri-nuclei microtubules, microtubules in cytoplasmic strand, cortical and preprophase band microtubules. In mitotic cells, it co-localized with microtubules of mitotic spindle and phrogmoplast. Interestingly, the distribution of GFP-TBK5 along phragmoplast microtubules was not uniform, and biased toward their minus end. This pattern is similar to the reported distribution pattern of gamma-tubulin. We now carry out knock down experiments to test the above-mentioned hypothesis.

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Since co-alignments of microtubules with microfilaments are observed in various types of intracellular structures including premitotic cytoplasmic strands, preprphase bands and phragmoplasts, cross-talk between the two type of cytoskeletons might be important in premitotic nuclear positioning and cytokinesis. But, little is known about the molecules that link the two cytoskeletons. Recently, kinesins with a calponin-homology domain (KCHs) were identified as a subset of the kinesin-14 family in plants and some of them have an ability to associate with both microtubules and microfilaments. Here we report the molecular cloning of the cDNAs encoding the four KCHs, TBK1, TBK2, TBK9 and TBK11 in tobacco BY-2 cells. Co-expression of GFP-tagged KCHs and RFP-tubulin in BY-2 cells revealed that the TBK9-GFP partially decorated the microtubules in cytoplasmic strands and phragmoplasts. In contrast, the TBK11-GFP decorated the nuclear membrane. Co-localization studies of these KCHs with microfilaments are now in progress. Also, we are now engaging in inducible expression experiments of truncated KCHs or rigor mutants of KCHs that are expected to affect the function of endogenous KCHs.

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We examined the effects of 1-aminocyclopropane-1-carboxylic acid (ACC), the immediate precursor of ethylene, on growth, orientation of cortical microtubules, and the transcript levels of γ-tubulin complex (VaTUG and VaGCP3) and katanin (VaKTN1) genes in azuki bean (Vigna angularis Ohwi et Ohashi) epicotyls. ACC induced inhibition of elongation growth as well as stimulation of lateral expansion in epicotyls, the degree of which increased with increasing ACC concentration. The percentage of cells with transverse microtubules was decreased, while that with longitudinal microtubules was increased, by increasing the concentration of ACC. ACC increased transiently the transcript levels of VaTUG, VaGCP3 and VaKTN1. These results suggest that ethylene induces the branching of microtubules via up-regulation of γ-tubulin complex genes, and the separation of the newly synthesized microtubule branch via up-regulation of katanin gene. Repeat of these processes may induce the reorientation of cortical microtubules, which induces modification of growth anisotropy from elongation growth to lateral expansion growth in azuki bean epicotyls.

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During anaphase, the movement of sister chromosome is accompanied by spindle elongation. It has been unclear whether spindle elongation occurs during anaphase in meristematic cells of land plants because land plant cells have no centrosome. We have established the quantitative method of spindle elongation in acentrosomal spindle using tobacco BY-2 cells. In this study, we investigated the spindle elongation in another two land plant cells, Arabidopsis thaliana root tip cells and Physcomitrella patens protonemata cells. We used the cell lines expressing GFP-tubulin. We measured the spindle length by using live cell imaging technique, and found that the spindle of both cells elongated during anaphase. Next we calculated the spindle elongation rate and compared the results of Arabidopsis root tip cells and Physcomitrella protonemata cells with the result of BY-2 cells. We conclude that the spindles of these three plant cells elongate 1.2-1.3 times during anaphase. We will discuss the role of spindle elongation in highly proliferating plant cells.

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The cultured cells, GTH4, of Nicotiana interspecfic F₁ hybrid die after temperature shift from 37C to 26C. The burst of H₂O₂ have a pivotal role in this programmed cell death (PCD), because it was not detected in a mutant GTH4S which is no longer to die at 26C. However, when GTH4 was treated with O₂^- generating agents, eventually generating much H₂O₂, cell death was suppressed. To dissect this contradictory results, we examine a role of NO which reacts with O₂^- to form ONOO^-, in connection with ROS . Amounts of NO was increased in GTH4 after temperature shift, while no such increment was detected in GTH4S, suggesting that NO is involved in PCD. Through several pharmacological experiments which fluctuate NO or ROS amounts, following results were obtained; 1) Increase of NO inversely reduced the amounts of O₂^- and H₂O₂, and cell death rate as well. 2) Increase of O₂^- suppressed both NO evolution and cell death rate. 3) Simultaneous generation of NO and H₂O₂ induced cell death severer than H₂O₂. Taken together, these results indicate that synergetic effect of NO and H₂O₂, each of which was determined in balance of generation rates of NO and O₂^-, plays an important role in PCD of GTH4.

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Previously we reported cell-specific anthocyanin expression patterns that originated at stomatal complexes in the abaxial leaf epidermis. We showed that anthocyanin distribution patterns are a common phenomenon in higher plants. In addition, we have observed that expression patterns of peroxidase showed a similar pattern. These results suggested that anthocyanins work as antioxidants in vivo. In this study, we conducted pH fluorescence imaging studies to test for a similar pattern of stomatal complex-based distribution in cellular pH. For pH imaging, HPTS (8-Hydroxypyrene-1,3,6-Trisulfonate), a pH-dependent fluorescence reagent was used. Because of its high water solubility, non-toxicity, and high quantum yield, the reagent is suitable for living cells. In addition, HPTS has a dual-excitation and a single emission, the ratiometric method can be applied for quantitative determination of pH change. Our pH imaging on leaf epidermis revealed cell-specific pH differences that originated at stomatal complexes, similar to that observed for anthocyanin and peroxidase distribution. We discuss these results in the context of anthocyanin and pH patterns within leaf epidermal cells.

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Although dynamics of molecules or organelles in the local region of a cell can be detectable by a total reflection immunofluorescent microscopy or by a video microscopy, it is not easy to detect these local molecular/particle behaviors and the global cellular events simultaneously. In order to detect local cytoplasmic behaviors and global cellular events in parallel, we have developed a new microscope system. As our system has two optical paths, one equipped with EMCCD camera for high resolution imaging of local cytoplasmic/molecular dynamics, and another equipped with a confocal laser scanning microscope system (C1) for detecting global cellular events in the same cell. Using this system, we could record particle behaviors on the cortical division site and global cell events in parallel during the progression of the cell division of a Tradescantia stamen hair cell without exchanging objectives. As our system controls the positioning of Z-axis of a stage, we could record two different focus areas in parallel. We also show some results using other materials, and tentative problems and the potential application will be discussed. This work was supported by JST SENTAN.

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D-Mannose/L-Galactose pathway for ascorbate (AsA) biosynthesis has been established recently, while the mechanism of AsA metabolism regulation is still unknown. In this work AsA deficient Arabidopsis mutant vtc2-1 was incubated with AsA precursor L-galactone-1,4-lactone (L-GalL) and developed higher AsA content especially under light. Using whole genome DNA microarray, transcriptome abundance of incubated vtc2-1 was analyzed for screening AsA responsible genes. Several genes were sensitive to L-GalL incubation and were up-regulated significantly. After analyzing the genes expression level by real-time PCR, an aspartyl protease (ASP) and a ring zinc finger protein performed synchronous expression responsive to L-GalL supplementation and AsA pool size change, but not to the addition of D-glucose. Transgenic Arabidopsis plants containing the promoter reporter system were developed and verified that the expression of ASP gene accords with real-time PCR results. These identifications suggest novel clues for understanding the regulation mechanism of AsA pool size regulation in higher plants.

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During the maturation of Arabidopsis seeds, a large portion of sugar carbons imported from the source are converted into oil for storage. An AP2-type transcription factor ASML1/WRI1 directly activates genes for fatty acid synthesis in the plastid, while expression of genes for the synthesis of triacylglycerol (TAG) in the endoplasmic reticulum requires other factor(s). To search for factors involved in the regulation of TAG synthesis, we introduced a LUC reporter gene with the promoter of a gene for diacylglycerol acyltransferase (DGAT1). The DGAT1p::LUC transgenic plants showed strong LUC expression in fruits and weaker expression in young leaves, which is similar to the expression pattern of DGAT1. We carried out enhancer activation-tag screening for mutants with enhanced expression of LUC in seedlings. Among lines which showed enhanced LUC expression in seedlings of the second generation, #14-3 also showed enhanced level of DGAT1 mRNA. One of the genes near the enhancer T-DNA in the genome of #14-3 showed expression during seed maturation similar to DGAT1 and overexpression of this gene in the DGAT1p::LUC plant caused enhancement of LUC activity and the level of DGAT1 mRNA.

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In developing Arabidopsis seeds, assembly of triacylglycerols (TAGs) from fatty acids synthesized in chloroplasts takes place in the endoplasmic reticulumn, and expression of genes involved in TAG assembly such as DGAT1 coding for diacylglycerol acyltransferase follows the expression of genes involved in fatty acid synthesis under the control of WRI1 during seed maturation. Among genes coding for transcription factors that show expression patterns similar to that of DGAT1 in the public database, we searched for transcription factors that could trans-activate transient expression of DGAT1p::LUC reporter gene in Arabidopsis protoplasts. A2 in the DREB subfamily of AP2/ERF family proteins consistently trans-activated DGAT1p::LUC, and the DGAT1 promoter contained a potential DREB-binding site. Although developing fruits of a T-DNA insertion line of A2 contained reduced level of DGAT1 mRNA compared to the wild type, seed oil content was not significantly affected. We are analyzing roles of close homologs of A2 in the TAG assembly during seed maturation and expression of genes involved in TAG assembly other than DGAT1 in the mutants of A2 and related genes.

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The 14-3-3 proteins are in the spotlight recently for their roles mediating protein-protein interaction and their extensive range of target proteins. In this study, we focused on one of thirteen 14-3-3 protein homologues, GF14λ, which showed the clearest interaction with Arabidopsis AREB/ABF transcription factors (AREBs) in yeast two-hybrid systems. To analyze dimerization and liganding activities of GF14λ, we employed bimolecular florescence complementation (BiFC) system with Arabidopsis mesophyll cells. GF14λ showed clear interacting signals with GF14λ indicating homo-dimer formation in the whole cells, but not with GF14κ, which is most similar to GF14λ. The GF14λ-AREB interaction was also analyzed by using BiFC, and consequently, AREB1/ABF2, AREB2/ABF4, and ABF3 showed clear dimerization signals in the nuclei of the cells. Furthermore we introduced amino acid substitution at the external ligand region of GF14λ, resulting no interaction signal. Our data indicate that (1) the GF14λ-AREB interaction has highly restricted specificity and (2) the BiFC system is effective to analyze 14-3-3 protein interaction.

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Most effort in expression vector development has been devoted to the identification and characterization of highly expressed and/or regulated promoter. Another important parameter of gene expression is 3'-processing and polyadenylation of pre-mRNA. Most eukaryotic mRNAs are cleaved post-transcriptionally at a specific sites downstream of polyadenylation signal in the 3'-untranslated region. Functional polyadenylation signals are required for efficient transcription termination. Furthermore, the strength of the poly(A) signal correlates with termination efficiency. Previous studies showed that terminators strongly affect the level of gene expression. We isolate the efficient transcription terminator contribute to express a foreign gene in plants more effectively. The terminator of the heat shock protein gene increases the mRNA level approximately 2-fold more than the nopaline synthase (NOS) terminator. When combined with the HSP terminator, a translational enhancer increased gene expression levels approximately 60- to 100-fold in transgenic plants.

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In the protein-coding genes of plants, core promoter elements such as TATA-box and initiator (Inr) are thought to be the cis-determinants of transcription start sites (TSS). However, high-density TSS analysis of Arabidopsis revealed that many protein-coding genes have neither TATA-box nor Inr, but have multiple TSS distributed over tens of nucleotides. Therefore it is likely that some alternative to the classical core promoter elements should determine the TSS of these genes. In this study, we examined how protein-coding genes recruit TSS close to their 5' termini, with the aid of transgenic plants carrying various chimeric constructs and of chromatin immunoprecipitation analysis with antibodies against the components of PIC (transcriptional preinitiation complex) and nucleosomes. The obtained results indicate that the insertion of the protein-coding region in the plant genome could cause the chromatin remodeling around their 5' termini, recruiting the PIC and causes TSS. Analysis of the underlying mechanism is in progress.

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DNA methylation at cytosine residues is an essential event required for the normal development of multicellular eukaryotes. In plant, a role for DNA methylation in pathogen resistance and genome integrity is well established, but the function of almost all DNA methylation in plant genomes remains unclear. Thus, identification of the degree and genome-wide distribution of DNA methylation would provide important clues to epigenetic machineries associated with development in plants and other organisms.
Recently, comprehensive maps of DNA methylation of the genome of Arabidopsis thaliana have been reported using whole-genome tiling arrays. Here we have generated DNA methylation profile (methylome) as well as sense transcription profiles (transcriptome), at 35-base pair resolution, of the genome derived from two accessions of A. thaliana. Some examples of genes which show different expression patterns with different DNA methylation between the two accessions will be shown.

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Gene silencing technology such as RNA interference (RNAi) is commonly used for the reduction of gene expression in plant cells. Exogenous double-stranded RNA (dsRNA) can induce gene silencing in higher plants. Previously we showed that delivery of dsDNA fragments such as PCR products with an endogenous gene sequence into fern Adiantum capillus-veneris gametophytic cells can induce sequence-specific gene silencing named DNAi (Kawai-Toyooka et al. 2004). In this study, we used a NEO1/PHY3 gene that mediates red light-induced chloroplast movement as a model of DNAi. According to microscopic observation and RT-PCR analysis, PHY3 gene expression and phy3 function was suppressed in DNAi-induced Adiantum gametophytes. Interestingly, this suppression effect still remained in the next generation. Hence, we tried to identify how PHY3 suppression happened during the gene silencing process by DNAi over generation. PCR analyses using methylated cytosine-specific nuclease and cytosine methylation-sensitive restriction enzyme showed that the DNAi gene silencing was mediated by DNA methylation. Methylation profiles in PHY3 gene by bisulfite sequencing will be presented.

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We have been investigating molecular mechanisms of organogenesis in vitro with temperature-sensitive mutants of Arabidopsis thaliana. Of these mutants, srd2 and rid1 were characterized by the temperature sensitivity of hypocotyl dedifferentiation and meristem neo-formation. Studies with the srd2 mutant at the molecular level have revealed that SRD2 regulates snRNA level by activating snRNA transcription and that this is a vital part of hypocotyl dedifferentiation.
The RID1 gene was identified as encoding a putative DEAH-box RNA helicase. Sequence analysis showed a high similarity of RID1 to budding yeast Prp22, which is known to participate in pre-mRNA splicing. This implies that the major role of SRD2-mediated activation of snRNA transcription in organogenesis is the increase of pre-mRNA splicing capacity. In favor of this hypothesis, by RT-PCR analysis, we found that the srd2 mutation affects splicing patterns of several genes during hypocotyl dedifferentiation. More comprehensive analysis of splicing patterns in srd2 and rid1 is in progress with the whole genome tiling array.

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We demonstrated that expression of an Arabidopsis splicing factor, atSR30, is transiently induced by high-light stress, suggesting an involvement of atSR30 in regulation of alternative splicing mechanism in response to the stress. To clarify the role of atSR30 as a splicing regulator, we isolated other SR proteins interacted with atSR30 by a yeast two-hybrid assay, suggesting that the atSR30 and these SR proteins function coordinately in the assembling of spliceosomal components. Next, we identified genes, of which alternative splicing efficiencies are regulated by atSR30, using the Arabidopsis thaliana genome tilling array. Changes in the expression levels of 65 genome regions in the knockout atSR30 plants (KO-ar30) compared with those in the wild-type plants were observed. By a semi-quantitative RT-PCR analysis, we verified changes in the alternative splicing efficiencies and/or in the transcriptional levels of genes in the vicinity of the genome regions. These results suggest that atSR30 acts as a regulator of the expression of various genes through control of alternative splicing efficiency of transcriptional regulators.

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Tobacco chloroplast genome contains about 80 protein genes that are expressed by chloroplast gene expression system. Many chloroplast mRNAs lack SD-like sequences, which are found in the 5'-untranslated region of most of bacterial mRNAs. A common or specific trans-acting factor is thought to be involved in translational regulation of such chloroplast mRNAs but translational regulation of chloroplast mRNAs is still unclear. In the present study, in vitro translation analysis of mutagenized psaA and psbD mRNAs, which carry an SD-like sequence at bacterial consensus position, revealed that translation initiation of these mRNAs is highly dependent on their SD-like sequences and that relatively short region of the 5'-UTR was sufficient for efficient translation initiation. In the case of mRNAs such as ycf4 and psbA that lack SD-like sequences, relatively long region of the 5'-UTR was required for efficient translation initiation of these mRNAs. Mechanism of translation initiation in chloroplasts will be discussed.

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The anthocyanins found in the cyanic ray-florets of chrysanthemum (Chrysanthemum morifolium) flower are only cyanidin-based anthocyanins. Because chrysanthemums do not accumulate delphinidin-based anthocyanins, they have no violet- or blue-flowered cultivars. This is due to the deficiency of flavonoid 3',5'-hydroxylase (F3'5'H), the key enzyme for delphinidin biosynthesis. In order to modify flower color toward bluer color by genetic engineering, we tried to express heterologous F3'5'H and make chrysanthemums to accumulate delphinidin-based anthocyanins. Through evaluation of ability to synthesize delphinidin of binary vectors contained pansy F3'5'H driven by various promoters, we found that combination of petal-specific flavanone 3-hydroxylase gene (CmF3H) promoter from chrysanthemum and translational enhancer NtADH-5'UTR was appropriate to delphinidin synthesis. We then tried to introduce various F3'5'H genes controlled by CmF3H promoter combined with NtADH-5'UTR. Dramatic increase of delphinidin-based anthocyanins (up to 75%) was achieved by bellflower (Campanula medium) F3'5'H expression. The flowers of some transgenic plants changed from red to purple/violet hue.

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