Masaru Ohme-Takagi

Saitama University, Saitama, Saitama, Japan

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Publications (104)528.54 Total impact

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    ABSTRACT: Gene expression profiles in response to inoculation with Magnaporthe oryzae at infected and adjacent cells obtained by microarray coupled with laser microdissection (LMD) were compared with results of microarray expression profiling using RNAs from pathogen infected whole leaves (WL). Genes whose expression was up-regulated following inoculation with the fungus were classified into two categories: group A indicates those whose increased expression was detected both in LMD- and WL-microarrays, while group B indicates genes of which the expression was detected at significantly higher levels in WL-microarray than LMD-microarray. Interestingly, genes encoding enzymes for the biosynthesis of diterpenoid phytoalexins were exclusively found in group A, while pathogenesis-related (PR) genes were found in both groups A and B. Next, the induction of gene expression in the healthy tissue distant from the infected site was analyzed by quantitative reverse-transcription coupled with PCR. Some of PR and WRKY genes of groups A and B were expressed in the healthy tissue. Such distant up-regulation observed for a set of genes was drastically reduced in nahG-rice, whereas that for others was not. A group A gene, OsWRKY19, was demonstrated to be distantly regulated and work as a positive regulator of defense via activation of transcription. These results implied that the defense responses in rice are systemic in some part, which require unknown signal molecules in addition to salicylic acid.
    Journal of Plant Physiology and Pathology. 10/2014; 2(4).
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    ABSTRACT: Plants respond to pathogen attack by transcriptionally regulating defense-related genes via various types of transcription factors. We identified a transcription factor in rice, OsNAC111, belonging to the TERN subgroup of the NAC family that was transcriptionally upregulated after rice blast fungus (Magnaporthe oryzae) inoculation. OsNAC111 was localized in the nucleus of rice cells and had transcriptional activation activity in yeast and rice cells. Transgenic rice plants overexpressing OsNAC111 showed increased resistance to the rice blast fungus. In OsNAC111-overexpressing plants, the expression of several defense-related genes including pathogenesis-related (PR) genes was constitutively high compared with the control. These genes all showed blast disease-responsive expression in leaves. Among them, two chitinase genes and one beta-1,3-glucanase gene showed reduced expression in transgenic rice plants in which OsNAC111 function was suppressed by a chimeric repressor (OsNAC111-SRDX). OsNAC111 activated transcription from the promoters of the chitinase and beta-1,3-glucanase genes in rice cells. In addition, brown pigmentation at the infection sites, a defense response of rice cells to the blast fungus, was lowered in OsNAC111-SRDX plants at the early infection stage. These results indicate that OsNAC111 positively regulates the expression of a specific set of PR genes in the disease response and contributes to disease resistance.
    Molecular Plant-Microbe Interactions 10/2014; 27(10):1027-1034. · 4.46 Impact Factor
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    Miho Ikeda, Masaru Ohme-Takagi
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    ABSTRACT: In contrast to somatic mammalian cells, which cannot alter their fate, plant cells can dedifferentiate to form totipotent callus cells and regenerate a whole plant, following treatment with specific phytohormones. However, the regulatory mechanisms and key factors that control differentiation-dedifferentiation and cell totipotency have not been completely clarified in plants. Recently, several plant transcription factors that regulate meristem formation and dedifferentiation have been identified and include members of the TEOSINTE BRANCHED1/CYCLOIDEA/PROLIFERATING CELL FACTOR (TCP), WUSCHEL (WUS), and WOUND INDUCED DEDIFFERENTIATION (WIND1) families. WUS and WIND positively control plant cell totipotency, while TCP negatively controls it. Interestingly, TCP is a transcriptional activator that acts as a negative regulator of shoot meristem formation, and WUS is a transcriptional repressor that positively maintains totipotency of the stem cells of the shoot meristem. We describe here the functions of TCP, WUS, and WIND transcription factors in the regulation of differentiation-dedifferentiation by positive and negative transcriptional regulators.
    Frontiers in Plant Science 09/2014; 5:427. · 3.64 Impact Factor
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    ABSTRACT: Biological functions of only some plant transcriptional repressors are known owing to the lack of knockout lines or unclear phenotypes because of redundancy. Here we show that strong viral activation domain VP16 fusion to the transcriptional repressor FLOWERING LOCUS C reversed its function and caused a stronger phenotype than that of the multiple-knockout line of redundant genes, suggesting the potential of this technique to identify transcription factor function that cannot be detected in a single-knockout line. Loss-of-function of transcriptional coactivator Mediator25 did not affect VP16 activity despite their in vivo interaction, suggesting the existence of other key mechanism(s) in plants.
    FEBS Letters 08/2014; · 3.34 Impact Factor
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    ABSTRACT: DELLA protein is a key negative regulator of gibberellin (GA) signaling. Although how DELLA regulates downstream gene expression remains unclear, DELLA has been proposed to function as a transcriptional activator. However, because DELLA lacks a DNA-binding domain, intermediate protein(s) mediating the DELLA/DNA interaction are believed to be necessary for activating DELLA target genes. Here, using yeast hybrid screenings, we identified five members of INDETERMINATE DOMAIN (IDD) protein family which bind physically to both DELLA and the promoter sequence of the GA-positive regulator SCARECROW-LIKE 3 (SCL3), which previously was characterized as a DELLA direct target gene. Transient assays using Arabidopsis protoplasts demonstrated that a luciferase reporter controlled by the SCL3 promoter was additively transactivated by REPRESSOR of ga1-3 (RGA) and IDDs. Phenotypic analysis of transgenic plants expressing AtIDD3 (one of the 16 IDDs in the Arabidopsis genome) fused with the plant-specific repression domain (SRDX) supported the possibility that AtIDD3 is positively involved in GA signaling. In addition, we found that SCL3 protein also interacts with IDDs, resulting in the suppression of its target gene expression. In this context, DELLA and SCL3 interact competitively with IDD proteins to regulate downstream gene expression. These results suggest that the coregulators DELLA and SCL3, using IDDs as transcriptional scaffolds for DNA binding, antagonistically regulate the expression of their downstream targets to control the GA signaling pathway.
    Proceedings of the National Academy of Sciences 05/2014; · 9.81 Impact Factor
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    ABSTRACT: Arabidopsis plants transformed with a chimeric repressor for 6 transcription factors (TFs), including ADA2b, Msantd, DDF1, DREB26, AtGeBP, and ATHB23, that were converted by Chimeric REpressor gene Silencing Technology (CRES-T), show elevated salt and osmotic stress tolerance compared with wild type (WT) plants. However, the roles of TFs in salt and osmotic signaling remain largely unknown. Their hyper-osmotic stress tolerance was evaluated using 3 criteria: germination rate, root length, and rate of seedlings with visible cotyledons at the germination stage. All CRES-T lines tested exhibited better performance than WT, at least for one criterion under stress conditions. Under 600 mM mannitol stress, 3-week-old CRES-T lines accumulated proline, which is a major compatible solute involved in osmoregulation, at higher levels than WT. Expression levels of the delta 1-pyrroline-5-carboxylate synthase gene in CRES-T lines were similar to or lower than those in WT. In contrast, expression of the proline dehydrogenase (PHD) gene in DREB26-SRDX was significantly downregulated and that in ADA2b-SRDX and AtGeBP-SRDX was also rather downregulated compared with that in WT. Although plants at different stages were used for stress tolerance test and proline measurement in this study, we previously reported that 4 out of the 6 CRES-T lines showed better growth than WT after 4 weeks of incubation under 400 mM mannitol. These results suggest that proline accumulation caused by PHD gene suppression may be involved in enhanced osmotic stress tolerance in the CRES-T lines, and that these TFs may be involved in regulating proline metabolism in Arabidopsis.
    Plant signaling & behavior 03/2014; 9(2).
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    ABSTRACT: Plant growth and development require proper cell wall organization but little is known about the transcription factors responsible for the regulation of gene expression involved in cell wall organization. Here we show, using Arabidopsis thaliana, that constitutive expression of the chimeric repressor for the MYB87 transcription factor causes suppression of longitudinal elongation, aberrant radial growth, and radially expanded or swollen cells in multiple organs. Microarray analysis revealed that plants expressing the chimeric repressor have altered expression of various cell wall related genes. MYB87 may therefore function as a regulator of genes affecting cell wall organization and remodeling. These findings improve our understanding of cell wall regulation and its roles in plant growth and development and also contribute information that may allow engineering of plant growth and architecture.
    Biotechnology Letters 02/2014; · 1.74 Impact Factor
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    ABSTRACT: In natural habitats, especially in arid areas, plants are often simultaneously exposed to multiple abiotic stresses, such as salt, osmotic and heat stresses. However, most analyses of gene expression in stress responses examine individual stresses. In this report, we compare gene expression in individual and combined stresses. We show that combined stress treatments with salt, mannitol and heat induce a unique pattern of gene expression that is not a simple merge of the individual stress responses. Under multiple stress condition, expression of most heat and salt stress-responsive genes increased to levels similar to or higher than those measured in single stress conditions, but osmotic stress-responsive genes increased to lower levels. Genes upregulated to higher levels under the multiple stress condition than the single stress conditions include genes for heat shock proteins, heat shock regulators and Late Embryogenesis Abundant proteins (LEAs), which protect other proteins from damage caused by stresses, suggesting their importance in multiple stress condition. Based on this analysis, we identify candidate genes for engineering crop plants tolerant to multiple stresses.
    Plant Cell and Environment 01/2014; · 5.91 Impact Factor
  • Masaru Nakata, Masaru Ohme-Takagi
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    ABSTRACT: The jasmonate (JA) plant hormones regulate responses to biotic and abiotic stress and aspects of plant development, including male fertility in Arabidopsis thaliana. The bHLH-type transcription factor JA-ASSOCIATED MYC2-LIKE1 (JAM1) negatively regulates JA signaling and gain-of-function JAM1 transgenic plants have impaired JA-mediated male fertility. Here we report that JAM2 and JAM3, 2 bHLHs closely related to JAM1, also act as transcriptional repressors. Moreover, overexpression of JAM2 and JAM3 also results in reduced male fertility. These results suggest that JAM1, JAM2, and JAM3 act redundantly as negative regulators of JA-mediated male fertility.
    Plant signaling & behavior 09/2013; 8(12).
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    ABSTRACT: Cyclamen persicum (cyclamen) is a commercially valuable, winter-blooming perennial plant. We cloned two cyclamen orthologues of AGAMOUS (AG), CpAG1 and CpAG2, which are mainly expressed in the stamen and carpel, respectively. Cyclamen flowers have 5 petals, but expression of a chimeric repressor of CpAG1 (CpAG1-SRDX) caused stamens to convert into petals, resulting in a flower with 10 petals. By contrast, CpAG2-SRDX only caused incomplete formation of stamens and carpels. Expression in Arabidopsis thaliana showed similar effects on flower organ specification. Simultaneous expression of CpAG1-SRDX and CpAG2-SRDX in cyclamen induced rose-like, multi-petal flowers, a potentially valuable trait in commercial ornamental varieties. Expression of CpAG2-SRDX in a cyclamen mutant lacking expression of CpAG1 more effectively produced multi-petal flowers. Here, we controlled the number of petals in cyclamen by simple genetic engineering with a chimeric repressor. This strategy may be applicable useful for other ornamental plants with two distinct AG orthologues.
    Scientific Reports 09/2013; 3:2641. · 5.08 Impact Factor
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    Francesco Licausi, Masaru Ohme-Takagi, Pierdomenico Perata
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    ABSTRACT: Transcription factors belonging to the APETALA2/Ethylene Responsive Factor (AP2/ERF) family are conservatively widespread in the plant kingdom. These regulatory proteins are involved in the control of primary and secondary metabolism, growth and developmental programs, as well as responses to environmental stimuli. Due to their plasticity and to the specificity of individual members of this family, AP2/ERF transcription factors represent valuable targets for genetic engineering and breeding of crops. In this review, we integrate the evidence collected from functional and structural studies to describe their different mechanisms of action and the regulatory pathways that affect their activity.
    New Phytologist 08/2013; 199(3):639-49. · 6.55 Impact Factor
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    ABSTRACT: The waxy plant cuticle protects cells from dehydration, repels pathogen attack, and prevents organ fusion during development. The transcription factor WAX INDUCER1/SHINE1 (WIN1/SHN1) regulates the biosynthesis of waxy substances in Arabidopsis thaliana. Here, we show that the MIXTA-like MYB transcription factors MYB106 and MYB16, which regulate epidermal cell morphology, also regulate cuticle development coordinately with WIN1/SHN1 in Arabidopsis and Torenia fournieri. Expression of a MYB106 chimeric repressor fusion (35S:MYB106-SRDX) and knockout/down of MYB106 and MYB16 induced cuticle deficiencies characterized by organ adhesion and reduction of epicuticular wax crystals and cutin nanoridges. A similar organ fusion phenotype was produced by expression of a WIN1/SHN1 chimeric repressor. Conversely, the dominant active form of MYB106 (35S:MYB106-VP16) induced ectopic production of cutin nanoridges and increased expression of WIN1/SHN1 and wax biosynthetic genes. Microarray experiments revealed that MYB106 and WIN1/SHN1 regulate similar sets of genes, predominantly those involved in wax and cutin biosynthesis. Furthermore, WIN1/SHN1 expression was induced by MYB106-VP16 and repressed by MYB106-SRDX. These results indicate that the regulatory cascade of MIXTA-like proteins and WIN1/SHN1 coordinately regulate cutin biosynthesis and wax accumulation. This study reveals an additional key aspect of MIXTA-like protein function and suggests a unique relationship between cuticle development and epidermal cell differentiation.
    The Plant Cell 05/2013; · 9.58 Impact Factor
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    ABSTRACT: Jasmonates (JAs) are plant hormones that regulate the balance between plant growth and responses to biotic and abiotic stresses. Although recent studies have uncovered the mechanisms for JA-induced responses in Arabidopsis thaliana, the mechanisms by which plants attenuate the JA-induced responses remain elusive. Here, we report that a basic helix-loop-helix-type transcription factor, ABA-INDUCIBLE BHLH-TYPE TRANSCRIPTION FACTOR/JA-ASSOCIATED MYC2-LIKE1 (JAM1), acts as a transcriptional repressor and negatively regulates JA signaling. Gain-of-function transgenic plants expressing the chimeric repressor for JAM1 exhibited substantial reduction of JA responses, including JA-induced inhibition of root growth, accumulation of anthocyanin, and male fertility. These plants were also compromised in resistance to attack by the insect herbivore Spodoptera exigua. Conversely, jam1 loss-of-function mutants showed enhanced JA responsiveness, including increased resistance to insect attack. JAM1 and MYC2 competitively bind to the target sequence of MYC2, which likely provides the mechanism for negative regulation of JA signaling and suppression of MYC2 functions by JAM1. These results indicate that JAM1 negatively regulates JA signaling, thereby playing a pivotal role in fine-tuning of JA-mediated stress responses and plant growth.
    The Plant Cell 05/2013; · 9.58 Impact Factor
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    ABSTRACT: Leaf senescence is the final process of leaf development that involves the mobilization of nutrients from old leaves to newly growing tissues. Despite the identification of several transcription factors involved in the regulation of this process, the mechanisms underlying the progression of leaf senescence are largely unknown. Herein, we describe the proteasome-mediated regulation of class II ETHYLENE RESPONSE FACTOR (ERF) transcriptional repressors and involvement of these factors in the progression of leaf senescence in Arabidopsis thaliana (Arabidopsis). Based on previous results showing that the Nicotiana tabacum ERF3 (NtERF3) specifically interacts with a ubiquitin-conjugating enzyme, we examined the stability of NtERF3 in vitro and confirmed its rapid degradation by plant protein extracts. Furthermore, NtERF3 accumulated in plants treated with a proteasome inhibitor. The Arabidopsis class II ERFs AtERF4 and AtERF8 were also regulated by the proteasome and increased with plant aging. Transgenic Arabidopsis plants with enhanced expression of NtERF3, AtERF4, or AtERF8 showed precocious leaf senescence. Our gene expression and chromatin immunoprecipitation analyses suggest that AtERF4 and AtERF8 targeted EPITHIOSPECIFIER PROTEIN/EPITHIOSPECIFYING SENESCENCE REGULATOR gene and regulated the expression of many genes involved in the progression of leaf senescence. By contrast, an aterf4 aterf8 double mutant exhibited delayed leaf senescence. Our results provide insight into the important role of class II ERFs in the progression of leaf senescence.
    Plant physiology 04/2013; · 7.39 Impact Factor
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    ABSTRACT: Plant architecture shows a large degree of developmental plasticity. Some of the key determinants are the timing of the floral transition induced by a systemic flowering signal (florigen) and the branching pattern regulated by key factors such as BRANCHED1 (BRC1). Here, we report that BRC1 interacts with the florigen proteins FLOWERING LOCUS T (FT) and TWIN SISTER OF FT (TSF) but not with TERMINAL FLOWER1, a floral repressor. FT protein induced in leaves moves into the subtended bud, suggesting that FT protein also plays a role in promotion of the floral transition in the axillary meristem (AM). The brc1-2 mutant shows an earlier floral transition in the axillary shoots compared with the wild type, suggesting that BRC1 plays a role in delaying the floral transition of the AMs. Genetic and gene expression analyses suggest that BRC1 interferes with florigen (FT and TSF) function in the AMs. Consistent with this, BRC1 ectopically expressed in the shoot apical meristem delays the floral transition in the main shoot. These results taken together suggest that BRC1 protein interacts with FT and TSF proteins and modulates florigen activity in the axillary buds to prevent premature floral transition of the AMs.
    The Plant Cell 04/2013; · 9.58 Impact Factor
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    ABSTRACT: Chimeric REpressor gene-Silencing Technology (CRES-T) is a reverse genetic method that converts transcriptional factors (TFs) to chimeric repressors by fusion with an ethylene-responsive element-binding factor (ERF)-associated amphiphilic repression domain. The plant expressing chimeric repressor is expected to show loss-of-function phenotype of the original TF even in the presence of other functionally redundant TFs. We used the CRES-T system for modification of flower shape in transgenic Ipomoea nil (formally Pharbitis nil). I. nil is emerging as a model plant for ornamental flowers because it has produced a wide variety of historical mutants, and it has been chosen to be a part of the National BioResource Project in Japan. We used cDNAs of TFs of Arabidopsis thaliana with the CRES-T system because A. thaliana TFs are well characterized compared with I. nil TFs. For this study, we selected two TCP (TEOSINTE BRANCHED1, CYCLOIDEA, and PCF) TFs, TCP3 and TCP5, because overexpression of these chimeric repressors TCP3SRDX and TCP5SRDX causes severe morphological alterations in A. thaliana. We found that these chimeric repressors cause morphological alterations, an undeveloped corolla and wavy petals in I. nil. In addition, the sympetalous corolla was easily disrupted to form choripetalous corolla. Although several TCP3SRDX transgenic ornamental flowers were reported, typical sympetalous flowers like I. nil have not yet been presented.
    Plant Biotechnology 02/2013; 29(5):457-463. · 0.94 Impact Factor
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    Miho Ikeda, Nobutaka Mitsuda, Masaru Ohme-Takagi
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    ABSTRACT: We recently demonstrated that cell elongation in plants is regulated by a triantagonistic bHLH system, in which three bHLH proteins, Activator of Cell Elongation 1 (ACE1), Arabidopsis ILI1 binding BHLH 1 (AtIBH1) and Paclobutrazol Resistance 1 (PRE1), competitively regulate the expression of genes for cell elongation. Here we show that ATBS1 Interacting Factor 2 (AIF2), AIF3 and AIF4 interact with PRE1 and ACE1, similar to AtIBH1, and also negatively regulate cell elongation in the triantagonistic bHLH system. The expression of each AIF is constitutive or induced by light, but AtIBH1 expression is dependent on BR signaling and developmental phase. These results indicate that AIFs and AtIBH1 may play different roles in cell elongation in different signaling pathways.
    Plant signaling & behavior 01/2013; 8(3).
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    ABSTRACT: Plant tissues that require structural rigidity synthesize a thick, strong secondary cell wall of lignin, cellulose and hemicelluloses in a complicated bridged structure. Master regulators of secondary wall synthesis were identified in dicots, and orthologs of these regulators have been identified in monocots, but regulation of secondary cell wall formation in monocots has not been extensively studied. Here we demonstrate that the rice transcription factors SECONDARY WALL NAC DOMAIN PROTEINs (SWNs) can regulate secondary wall formation in rice (Oryza sativa) and are potentially useful for engineering the monocot cell wall. The OsSWN1 promoter is highly active in sclerenchymatous cells of the leaf blade and less active in xylem cells. By contrast, the OsSWN2 promoter is highly active in xylem cells and less active in sclerenchymatous cells. OsSWN2 splicing variants encode two proteins; the shorter protein (OsSWN2S) has very low transcriptional activation ability, but the longer protein (OsSWN2L) and OsSWN1 have strong transcriptional activation ability. In rice, expression of an OsSWN2S chimeric repressor, driven by the OsSWN2 promoter, resulted in stunted growth and para-wilting (leaf rolling and browning under normal water conditions) due to impaired vascular vessels. The same OsSWN2S chimeric repressor, driven by the OsSWN1 promoter, caused a reduction of cell wall thickening in sclerenchymatous cells, a drooping leaf phenotype, reduced lignin and xylose contents and increased digestibility as forage. These data suggest that OsSWNs regulate secondary wall formation in rice and manipulation of OsSWNs may enable improvements in monocotyledonous crops for forage or biofuel applications.
    Frontiers in Plant Science 01/2013; 4:383. · 3.64 Impact Factor
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    ABSTRACT: The B3 DNA binding domain is a plant-specific domain, found throughout the plant kingdom from the alga Chlamydomonas to grasses and flowering plants. Over 100 B3 domain-containing proteins are found in the model plant Arabidopsis thaliana and one of these is critical for accelerating flowering in response to prolonged cold treatment; an epigenetic process called vernalization. Despite the specific phenotype of genetic vrn1 mutants, the VERNALIZATION1 (VRN1) protein localizes throughout the nucleus and in vitro shows sequence non-specific binding. In this work we used a dominant repressor tag that overcomes genetic redundancy to show that VRN1 is involved in processes beyond vernalization that are essential for Arabidopsis development. To understand its sequence non-specific binding we crystallized VRN1208-341 and solved its crystal structure to 1.6 Å resolution using Se-SAD methods. The crystallized construct comprises the second VRN1 B3 domain and a preceding region conserved among VRN1 orthologs, but absent in other B3 domains. We established the DNA binding face using NMR then mutated positively charged residues on this surface with a series of 16 Ala and Glu substitutions ensuring the protein fold was not disturbed using HSQC NMR spectra. A triple mutant R249E R289E R296E was almost completely incapable of DNA binding in vitro. Thus, we have revealed that although VRN1 is sequence non-specific in DNA binding, it has a defined DNA binding surface.
    Journal of Biological Chemistry 12/2012; · 4.60 Impact Factor
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    ABSTRACT: The WUSCHEL related homeobox (WOX) genes play key roles in stem cell maintenance, embryonic patterning, and lateral organ development. WOX genes have been categorized into three clades-ancient, intermediate, and modern/WUS-based on phylogenetic analysis, but a functional basis for this classification has not been established. Using the classical bladeless lam1 mutant of Nicotiana sylvestris as a genetic tool, we examined the function of the Medicago truncatula WOX gene, STENOFOLIA (STF), in controlling leaf blade outgrowth. STF and LAM1 are functional orthologs. We found that the introduction of mutations into the WUS-box of STF (STFm1) reduces its ability to complement the lam1 mutant. Fusion of an exogenous repressor domain to STFm1 restores complementation, whereas fusion of an exogenous activator domain to STFm1 enhances the narrow leaf phenotype. These results indicate that transcriptional repressor activity mediated by the WUS-box of STF acts to promote blade outgrowth. With the exception of WOX7, the WUS-box is conserved in the modern clade WOX genes, but is not found in members of the intermediate or ancient clades. Consistent with this, all members of the modern clade except WOX7 can complement the lam1 mutant when expressed using the STF promoter, but members of the intermediate and ancient clades cannot. Furthermore, we found that fusion of either the WUS-box or an exogenous repressor domain to WOX7 or to members of intermediate and ancient WOX clades results in a gain-of-function ability to complement lam1 blade outgrowth. These results suggest that modern clade WOX genes have evolved for repressor activity through acquisition of the WUS-box.
    Proceedings of the National Academy of Sciences 12/2012; · 9.81 Impact Factor

Publication Stats

5k Citations
528.54 Total Impact Points

Institutions

  • 2014
    • Saitama University
      Saitama, Saitama, Japan
  • 2001–2014
    • National Institute of Advanced Industrial Science and Technology
      Tsukuba, Ibaraki, Japan
  • 2006–2012
    • Japan International Research Center for Agricultural Sciences
      Tsukuba, Ibaraki, Japan
  • 2003–2011
    • Kyoto University
      • • Graduate School of Biostudies
      • • Division of Applied Life Sciences
      Kyoto, Kyoto-fu, Japan