Kazuhiro Ohtsu

Iowa State University, Ames, Iowa, United States

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Publications (17)70.69 Total impact

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    ABSTRACT: This protocol is a modified version of DNA isolation using cetyltrimethylammonium bromide (CTAB) and 96-well plates. It is high-throughput, which facilitates the analysis of large mapping populations. The DNA yield is adequate for at least 100-500 polymerase chain reaction (PCR) procedures.
    Cold Spring Harbor Protocols 11/2010; 2010(11):pdb.prot5516. · 4.63 Impact Factor
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    ABSTRACT: Transposable elements (TEs) comprise a substantial portion of many eukaryotic genomes and are typically transcriptionally silenced. RNA-dependent RNA polymerase 2 (RDR2) is a component of the RNA-directed DNA methylation (RdDM) silencing pathway. In maize, loss of mediator of paramutation1 (mop1) encoded RDR2 function results in reactivation of transcriptionally silenced Mu transposons and a substantial reduction in the accumulation of 24 nt short-interfering RNAs (siRNAs) that recruit RNA silencing components. An RNA-seq experiment conducted on shoot apical meristems (SAMs) revealed that, as expected based on a model in which RDR2 generates 24 nt siRNAs that suppress expression, most differentially expressed DNA TEs (78%) were up-regulated in the mop1 mutant. In contrast, most differentially expressed retrotransposons (68%) were down-regulated. This striking difference suggests that distinct silencing mechanisms are applied to different silencing templates. In addition, >6,000 genes (24% of analyzed genes), including nearly 80% (286/361) of genes in chromatin modification pathways, were differentially expressed. Overall, two-thirds of differentially regulated genes were down-regulated in the mop1 mutant. This finding suggests that RDR2 plays a significant role in regulating the expression of not only transposons, but also of genes. A re-analysis of existing small RNA data identified both RDR2-sensitive and RDR2-resistant species of 24 nt siRNAs that we hypothesize may at least partially explain the complex changes in the expression of genes and transposons observed in the mop1 mutant.
    PLoS Genetics 11/2009; 5(11):e1000737. · 8.52 Impact Factor
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    ABSTRACT: Protocols for laser microdissection and linear amplification of RNA from fixed, sectioned plant tissues are described. When combined with quantitative RT-PCR, microarray analysis, or RNA-sequencing, these procedures enable quantitative analyses of transcript accumulation from microscopic quantities of specific plant organs, tissues, or single cells.
    Current protocols in molecular biology / edited by Frederick M. Ausubel ... [et al.] 08/2009; Chapter 25:Unit 25A.3.
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    ABSTRACT: The shoot apical meristem (SAM) maintains a pool of indeterminate cells within the SAM proper, while lateral organs are initiated from the SAM periphery. Laser microdissection-microarray technology was used to compare transcriptional profiles within these SAM domains to identify novel maize genes that function during leaf development. Nine hundred and sixty-two differentially expressed maize genes were detected; control genes known to be upregulated in the initiating leaf (P0/P1) or in the SAM proper verified the precision of the microdissections. Genes involved in cell division/growth, cell wall biosynthesis, chromatin remodeling, RNA binding, and translation are especially upregulated in initiating leaves, whereas genes functioning during protein fate and DNA repair are more abundant in the SAM proper. In situ hybridization analyses confirmed the expression patterns of six previously uncharacterized maize genes upregulated in the P0/P1. P0/P1-upregulated genes that were also shown to be downregulated in leaf-arrested shoots treated with an auxin transport inhibitor are especially implicated to function during early events in maize leaf initiation. Reverse genetic analyses of asceapen1 (asc1), a maize D4-cyclin gene upregulated in the P0/P1, revealed novel leaf phenotypes, less genetic redundancy, and expanded D4-CYCLIN function during maize shoot development as compared to Arabidopsis. These analyses generated a unique SAM domain-specific database that provides new insight into SAM function and a useful platform for reverse genetic analyses of shoot development in maize.
    PLoS Genetics 06/2009; 5(5):e1000476. · 8.52 Impact Factor
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    ABSTRACT: MicroRNAs (miRNAs) and trans-acting siRNAs (ta-siRNAs) are essential to the establishment of adaxial-abaxial (dorsoventral) leaf polarity. Tas3-derived ta-siRNAs define the adaxial side of the leaf by restricting the expression domain of miRNA miR166, which in turn demarcates the abaxial side of leaves by restricting the expression of adaxial determinants. To investigate the regulatory mechanisms that allow for the precise spatiotemporal accumulation of these polarizing small RNAs, we used laser-microdissection coupled to RT-PCR to determine the expression profiles of their precursor transcripts within the maize shoot apex. Our data reveal that the pattern of mature miR166 accumulation results, in part, from intricate transcriptional regulation of its precursor loci and that only a subset of mir166 family members contribute to the establishment of leaf polarity. We show that miR390, an upstream determinant in leaf polarity whose activity triggers tas3 ta-siRNA biogenesis, accumulates adaxially in leaves. The polar expression of miR390 is established and maintained independent of the ta-siRNA pathway. The comparison of small RNA localization data with the expression profiles of precursor transcripts suggests that miR166 and miR390 accumulation is also regulated at the level of biogenesis and/or stability. Furthermore, mir390 precursors accumulate exclusively within the epidermal layer of the incipient leaf, whereas mature miR390 accumulates in sub-epidermal layers as well. Regulation of miR390 biogenesis, stability, or even discrete trafficking of miR390 from the epidermis to underlying cell layers provide possible mechanisms that define the extent of miR390 accumulation within the incipient leaf, which patterns this small field of cells into adaxial and abaxial domains via the production of tas3-derived ta-siRNAs.
    PLoS Genetics 02/2009; 5(1):e1000320. · 8.52 Impact Factor
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    ABSTRACT: The WUSCHEL-related homeobox (WOX) gene PRESSED FLOWER1 (PRS1) performs a conserved function during lateral organ development in Arabidopsis (Arabidopsis thaliana). Expressed in the periphery of the shoot meristem, PRS1 recruits founder cells that form lateral domains of vegetative and floral organs. Null mutations in PRS1 cause the deletion of lateral stipules from leaves and of lateral sepals and stamens from flowers. Although PRS1 expression is described in the L1 layer, PRS1 recruits founder cells from all meristem layers. The mechanism of non-cell autonomous PRS1 function and the evolution of disparate WOX gene functions are investigated herein. Meristem layer-specific promoters reveal that both L1 and L1-L2 expression of PRS1 fail to fully rescue PRS1 function, and PRS1 protein does not traffic laterally or transversely between shoot meristem layers. PRS1 protein accumulates within all meristematic cell layers (L1-L2-L3) when expressed from the native promoter, presumably due to low-level transcription in the L2 and L3 layers. When driven from the PRS1 promoter, full rescue of vegetative and floral prs1 mutant phenotypes is provided by WUSCHEL1 (WUS1), which is normally expressed in the stem cell organizing center of shoot meristems. The data reveal that WUS1 and PRS1 can engage in equivalent protein-protein interactions and direct transcription of conserved target genes, suggesting that their subfunctionalization has evolved primarily via diverse promoter specificity. Unexpectedly, these results also suggest that meristematic stem cells and lateral organ founder cells are intrinsically similar and formed via equivalent processes such that their ultimate fate is dependent upon stage-specific and domain-specific positional signaling.
    Plant physiology 01/2009; 149(2):841-50. · 6.56 Impact Factor
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    ABSTRACT: All above-ground plant organs are derived from shoot apical meristems (SAMs). Global analyses of gene expression were conducted on maize (Zea mays L.) SAMs to identify genes preferentially expressed in the SAM. The SAMs were collected from 14-day-old B73 seedlings via laser capture microdissection (LCM). The RNA samples extracted from LCM-collected SAMs and from seedlings were hybridized to microarrays spotted with 37 660 maize cDNAs. Approximately 30% (10 816) of these cDNAs were prepared as part of this study from manually dissected B73 maize apices. Over 5000 expressed sequence tags (ESTs) (about 13% of the total) were differentially expressed (P < 0.0001) between SAMs and seedlings. Of these, 2783 and 2248 ESTs were up- and down-regulated in the SAM, respectively. The expression in the SAM of several of the differentially expressed ESTs was validated via quantitative RT-PCR and/or in situ hybridization. The up-regulated ESTs included many regulatory genes including transcription factors, chromatin remodeling factors and components of the gene-silencing machinery, as well as about 900 genes with unknown functions. Surprisingly, transcripts that hybridized to 62 retrotransposon-related cDNAs were also substantially up-regulated in the SAM. Complementary DNAs derived from the LCM-collected SAMs were sequenced to identify additional genes that are expressed in the SAM. This generated around 550 000 ESTs (454-SAM ESTs) from two genotypes. Consistent with the microarray results, approximately 14% of the 454-SAM ESTs from B73 were retrotransposon-related. Possible roles of genes that are preferentially expressed in the SAM are discussed.
    The Plant Journal 12/2007; 52(3):391-404. · 6.58 Impact Factor
  • Kazuhiro Ohtsu, Patrick S Schnable
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    ABSTRACT: INTRODUCTIONThe use of RNA for genotyping analysis can be advantageous because transcriptomes are significantly smaller than genomes and typically contain far fewer repetitive sequences. Laser capture microdissection (LCM) has been used successfully to isolate sequences (especially rare transcripts) that accumulate in specific tissues. Where the quantity of isolated material is limiting, amplification can be used to increase the amount of product. Upon conversion to cDNA, the product serves as template for 454 sequencing to produce expressed sequence tags for subsequent SNP analysis and detection.This protocol describes the preparation of acetone-fixed and paraffin-embedded maize seedling tissue sections. Once the sections are prepared, the PALM MicroBeam System, with its Laser Microdissection and Pressure Catapulting (LMPC) technology, is used to cut out cells of interest and "catapult" isolated tissues into collection caps. This tissue can then be used for RNA extraction.
    Cold Spring Harbor Protocols 07/2007; 2007:pdb.prot4784. · 4.63 Impact Factor
  • Kazuhiro Ohtsu, Patrick S Schnable
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    ABSTRACT: INTRODUCTIONThe use of RNA for genotyping analysis can be advantageous because transcriptomes are significantly smaller than genomes and typically contain far fewer repetitive sequences. Laser capture microdissection (LCM) has been used successfully to isolate sequences (especially rare transcripts) that accumulate in specific tissues. The amount of RNA collected in a standard microdissection is often insufficient for global gene expression analysis but can be increased via linear amplification. Upon conversion to cDNA, the product serves as template for 454 sequencing to produce expressed sequence tags for subsequent SNP analysis and detection. This protocol describes how to amplify RNA extracted from laser-dissected and captured tissues and cells.
    Cold Spring Harbor Protocols 07/2007; 2007:pdb.prot4785. · 4.63 Impact Factor
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    ABSTRACT: Laser microdissection (LM) allows for the isolation of specific cells of interest from heterogeneous tissues under direct microscopic visualization with the assistance of a laser beam. By permitting global analyses of gene expression and metabolites in the selected cells, it is a powerful tool for understanding the biological processes in individual cell types during development or in response to various stimuli. Recently, LM technology has been successfully applied to the separation of individual plant cell types. Here, we provide an overview of applications of LM combined with high-throughput technologies including transcript analyses [microarrays, serial analysis of gene expression (SAGE) and 454-sequencing], proteomic analyses and metabolomic profiling, for cell type-specific gene expression analyses in plants.
    Plant and Cell Physiology 02/2007; 48(1):3-7. · 4.98 Impact Factor
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    ABSTRACT: A new type of transposon, named Anaconda (Anac) has been found in rice (Oryza sativa). In this paper, we demonstrate that Anaconda elements have diversified by acquisition of host cellular genes, amplification of the elements, and substitution and deletion of short segments. We identified four Anaconda elements in studies of rice alternative oxidase (AOX) genes, and subsequently isolated an additional 23 elements based on the identity of their terminal inverted repeats (TIRs). The Anaconda elements have long TIRs (114-458 bp). They also have direct repeats of 9 or 10 bp in their flanking regions that are thought to have been generated upon transposition. These structural features reveal that the Anaconda elements belong to the Mu superfamily. The most prominent feature of the Anaconda elements is the high frequency with which they have acquired host cellular genes. Of the 27 elements found here, 19 appear to have sequences presumably derived from rice genes, for example, the genes for AOX1c (four elements), cytochrome P450 (five elements), L: -asparaginase (five elements), and PCF8 (two elements). Four elements, AnacA1-A4, have both the AOX1c and P450 genes. One element, AnacB14, involves a gene similar to mudrA of maize MuDR. Database analyses revealed that the loci of 26 of the 27 Anaconda elements in the subspecies japonica are the same as those in the subspecies indica. This suggests that these elements were incorporated before the divergence of these two subspecies.
    Molecular Genetics and Genomics 01/2006; 274(6):606-15. · 2.88 Impact Factor
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    ABSTRACT: In higher eudicot, the floral organs consist of four different whorls, containing sepals, petals, stamens and carpels. In contrast, the perianths of many monocotyledonous plants, like Liliaceae plants, have almost identical petaloid organs called tepals. The modified ABC model explains this flower morphology; class B genes are expressed in whorl 1 as well as whorls 2 and 3, resulting that the two outer whorls are almost identical petaloid organs. Expression pattern of class B genes in tulip supports this modified ABC model, however, the expression of asparagus class B genes were restricted in the inner tepals and stamens. To investigate whether the class B gene expression pattern of asparagus is shared by related species, the class B gene MaDEF was isolated from Muscari armeniacum, belonging to the order Asparagales. RT-PCR showed that MaDEF is expressed in outer tepals as well as in inner tepals and stamens. This expression pattern is consistent with that of tulip, supporting the modified ABC model in M. armeniacum. Since M. armeniacum flower buds are too small for accurate separation into individual floral organs, the laser microdissection (LMD) system was used to isolate floral organ-specific RNA. We also discuss the suitability of the LMD technique for plant research.
    Plant Science. 01/2006; 170(1):143-150.
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    ABSTRACT: The rice pyruvate decarboxylase 3 gene (PDC3), which has no introns, was previously postulated to be a pseudogene because no PDC3 mRNA had been detected, even under anaerobic conditions. However, in this study, it was found that rice PDC3 transcripts accumulated in panicles after heading. Within anthers obtained from the panicles, PDC3 was shown to be transcribed in mature pollen by in situ hybridization. These results suggest that the rice PDC3 is a functional gene. Its product may play a role in aerobic alcoholic fermentation in mature pollen.
    Journal of Experimental Botany 02/2004; 55(394):145-6. · 5.79 Impact Factor
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    ABSTRACT: A novel gene for alternative oxidase (AOX) was isolated from rice (Oryza sativa L.) and characterized. The deduced amino acid sequence of the novel AOX gene contains features that are conserved among other AOXs. This AOX gene was designated AOX1c based on a phylogenetic analysis of the AOX genes. Northern hybridization analyses revealed that AOX1c and AOX1a/AOX1b transcripts accumulated differently in various rice organs and rice seedlings under low temperature conditions. AOX1c mRNA was mainly present in young leaves under constant light, mature leaves and panicles after heading, but it was not detected in young etiolated leaves and young roots of seedlings or young panicles. On the other hand, the mRNAs of the rice AOX1a and AOX1b genes were mainly present in young roots and mature leaves. Under low temperature conditions, the steady-state mRNA levels of the rice AOX1a and AOX1b genes clearly increased with time but the rice AOX1c gene was apparently not responsive to low temperature. The rice AOX gene family and differences in their regulation are discussed.
    Genes & Genetic Systems 03/2002; 77(1):31-8. · 1.13 Impact Factor
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    ABSTRACT: Many of the subunits of cytochrome c oxidase (COX) in the mitochondria of higher plants are encoded by nuclear genes. These genes are less characterized compared to mitochondrial-encoded genes. We previously isolated a cDNA encoding COX6b (designated OsCOX6b1 in this study) from the rice nuclear genome and analyzed its expression. The deduced protein had an extended N-terminus compared with human and yeast COX6b proteins. In this study, we identified another COX6b gene (OsCOX6b2) in rice and revealed that it was actually expressed. The deduced protein of this gene did not have an extended N-terminus and had about the same size as the human and yeast proteins. Genomic Southern hybridization analysis revealed that there was at least one OsCOX6b-homologus sequences in the rice genome other than OsCOX6b1 and OsCOX6b2. Furthermore, we identified three COX6b genes in a dicotyledonous plant, Arabidopsis thaliana. One of these genes (AtCOX6b1) was relatively long, with a length similar to that of OsCOX6b1, and the other two (AtCOX6b2 and AtCOX6b3) were shorter, with lengths similar to the length of OsCOX6b2. Genomic Southern hybridization analysis indicated there were no additional COX6b genes in the Arabidopsis genome. The coding regions of OsCOX6b1 and AtCOX6b1 were separated by four introns and those of OsCOX6b2, AtCOX6b2 and AtCOX6b3 were separated by three introns. A Northern hybridization analysis showed that OsCOX6b1, AtCOX6b1 and AtCOX6b3 were expressed in all organs examined, although with some differences in the amount of expression among the organs. OsCOX6b2 and AtCOX6b2 were strongly expressed in roots but most of the transcripts of AtCOX6b2 were degraded. The evolution of COX6b genes from rice and Arabidopsis is discussed.
    Gene 03/2001; 264(2):233-9. · 2.20 Impact Factor
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    ABSTRACT: Little is presently known about the nuclear-encoded genes for cytochrome c oxidase (COX) in higher plants. In rice, only the nuclear-encoded COX5b gene has been reported. To understand the relationship between the expression of nuclear-encoded and mitochondrial-encoded COX genes in rice, we first characterized a cDNA encoding one of the other nuclear COX genes, COX5c, which encodes 63 amino acids. The deduced amino acid sequence of COX5c from rice was highly homologous to that from sweet potato. Genomic Southern hybridization indicated that the rice COX5c subunit is encoded by a single copy of the COX5c gene. Furthermore, we compared the expression patterns of the nuclear-encoded COX5c and COX5b genes with the expression pattern of the mitochondrial-encoded COX1 gene among several organs by Northern blot analysis. The results suggested that regulatory systems of expression between the nuclear-encoded and the mitochondrial-encoded COX genes are different among different organs in rice.
    Genes & Genetic Systems 07/1999; 74(3):71-5. · 1.13 Impact Factor
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    ABSTRACT: Laser microdissection (LM) is a new method that makes it possible to obtain large homogeneous populations of cells from tissue sections in one step. LM in combination with microarray analyses can monitor changes in transcript levels in specific cell types, in which morphological or physiological changes are observed. In this study, we used LM and a microarray to monitor genes expressed during aerenchyma formation in maize (Zea mays) roots. In maize, hypoxia stimulates ethylene biosynthesis, which induces cell death in the root cortex, thereby forming aerenchyma in the roots. Roots of 3-d-old maize (inbred line B73) seedlings were waterlogged for 6 h and then were fixed and embedded in paraffin. We isolated root cortex cells from the paraffin-embedded sections using LM, extracted RNA and carried out a maize cDNA microarray. Finally, we identified several genes that were expressed specifically in the root cortex during aerenchyma formation. Possible roles of these genes in aerenchyma formation are discussed.

Publication Stats

333 Citations
70.69 Total Impact Points

Institutions

  • 2007–2009
    • Iowa State University
      • Department of Agronomy
      Ames, Iowa, United States
  • 2001–2006
    • The University of Tokyo
      • Faculty and Graduate School of Agriculture and Life Sceince
      Tokyo, Tokyo-to, Japan