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ABSTRACT: Screening of rice full-length cDNA overexpressing (FOX) lines allowed the identification of a TIFY gene, TIFY11b, as a growth-promoting gene whose overexpression increased plant height and seed size. The grains of TIFY11b-overexpressing plants exceeded those of non-transformants in length, width and thickness, resulting in 9-21% increases in grain weight. The increase was achieved by overexpressing the gene in the whole plant body, but not by seed-restricted expression, indicating that seed enlargement is attributable to overexpression in vegetative organs such as the leaf. The whole-body overexpressing plants developed longer leaves along with higher levels of starch and sucrose in the leaf sheath and culm at the heading stage than the non-transformants. Although overexpression of TIFY11b did not alter the photosynthetic rate per leaf area before and after heading, it caused an accumulation of higher levels of the carbohydrate assimilate, probably due to increased photosynthesis per plant, suggesting that the increase in grain size and weight is attained by enhanced accumulation and translocation of the carbohydrate in the culms and leaf sheaths of the transgenic plants. Thus, TIFY11b is a novel grain-size increasing gene.
Bioscience Biotechnology and Biochemistry 11/2012; · 1.28 Impact Factor
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ABSTRACT: The meristem initiates lateral organs in a regular manner, and proper communication between the meristem and the lateral organs ensures the normal development of plants. Here, we show that mutation of the rice (Oryza sativa) gene TONGARI-BOUSHI1 (TOB1) results in pleiotropic phenotypes in spikelets, such as the formation of a cone-shaped organ instead of the lemma or palea, the development of two florets in a spikelet, or premature termination of the floret meristem, in addition to reduced growth of the lemma or palea and elongation of the awn. These phenotypes seem to result from not only failure in growth of the lateral organs, but also defects in maintenance and organization of the meristem. For example, the cone-shaped organ develops as a ring-like primordium from an initial stage, suggesting that regulation of organ initiation in the meristem may be compromised. TOB1 encodes a YABBY protein, which is closely related to FILAMENTOUS FLOWER in Arabidopsis thaliana, and is expressed in the lateral organ primordia without any patterns of polarization. No TOB1 expression is detected in the meristem, so TOB1 may act non-cell autonomously to maintain proper meristem organization and is therefore likely to play an important role in rice spikelet development.
The Plant Cell 01/2012; 24(1):80-95. · 8.99 Impact Factor
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ABSTRACT: We identified a short-grain mutant (Short grain1 (Sg1) Dominant) via phenotypic screening of 13,000 rice (Oryza sativa) activation-tagged lines. The causative gene, SG1, encodes a protein with unknown function that is preferentially expressed in roots and developing panicles. Overexpression of SG1 in rice produced a phenotype with short grains and dwarfing reminiscent of brassinosteroid (BR)-deficient mutants, with wide, dark-green, and erect leaves. However, the endogenous BR level in the SG1 overexpressor (SG1:OX) plants was comparable to the wild type. SG1:OX plants were insensitive to brassinolide in the lamina inclination assay. Therefore, SG1 appears to decrease responses to BRs. Despite shorter organs in the SG1:OX plants, their cell size was not decreased in the SG1:OX plants. Therefore, SG1 decreases organ elongation by decreasing cell proliferation. In contrast to the SG1:OX plants, RNA interference knockdown plants that down-regulated SG1 and a related gene, SG1-LIKE PROTEIN1, had longer grains and internodes in rachis branches than in the wild type. Taken together, these results suggest that SG1 decreases responses to BRs and elongation of organs such as seeds and the internodes of rachis branches through decreased cellular proliferation.
Plant physiology 12/2011; 158(3):1208-19. · 6.53 Impact Factor
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Takayuki Asano,
Nagao Hayashi,
Michie Kobayashi,
Naohiro Aoki,
Akio Miyao,
Ichiro Mitsuhara, Hiroaki Ichikawa,
Setsuko Komatsu,
Hirohiko Hirochika,
Shoshi Kikuchi,
Ryu Ohsugi
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ABSTRACT: Calcium-dependent protein kinases (CDPKs) regulate the downstream components in calcium signaling pathways. We investigated the effects of overexpression and disruption of an Oryza sativa (rice) CDPK (OsCPK12) on the plant's response to abiotic and biotic stresses. OsCPK12-overexpressing (OsCPK12-OX) plants exhibited increased tolerance to salt stress. The accumulation of hydrogen peroxide (H(2) O(2) ) in the leaves was less in OsCPK12-OX plants than in wild-type (WT) plants. Genes encoding reactive oxygen species (ROS) scavenging enzymes (OsAPx2 and OsAPx8) were more highly expressed in OsCPK12-OX plants than in WT plants, whereas the expression of the NADPH oxidase gene, OsrbohI, was decreased in OsCPK12-OX plants compared with WT plants. Conversely, a retrotransposon (Tos17) insertion mutant, oscpk12, and plants transformed with an OsCPK12 RNA interference (RNAi) construct were more sensitive to high salinity than were WT plants. The level of H(2) O(2) accumulation was greater in oscpk12 and OsCPK12 RNAi plants than in the WT. These results suggest that OsCPK12 promotes tolerance to salt stress by reducing the accumulation of ROS. We also observed that OsCPK12-OX seedlings had increased sensitivity to abscisic acid (ABA) and increased susceptibility to blast fungus, probably resulting from the repression of ROS production and/or the involvement of OsCPK12 in the ABA signaling pathway. Collectively, our results suggest that OsCPK12 functions in multiple signaling pathways, positively regulating salt tolerance and negatively modulating blast resistance.
The Plant Journal 08/2011; 69(1):26-36. · 6.16 Impact Factor
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ABSTRACT: Calcium acts as a messenger in various signal transduction pathways in plants. Calcium-dependent protein kinases (CDPKs) play important roles in regulating downstream components in calcium signaling pathways. In rice, the CDPKs constitute a large multigene family consisting of 29 genes, but the biological functions and functional divergence or redundancy of most of these genes remain unclear. Using a mini-scale full-length cDNA overexpressor (FOX) gene hunting system, we generated 250 independent transgenic rice plants overexpressing individual rice CDPKs (CDPK FOX-rice lines). These CDPK FOX-rice lines were screened for salt stress tolerance. The survival rate of the OsCPK21-FOX plants was higher than that of wild-type (WT) plants grown under high salinity conditions. The inhibition of seedling growth by abscisic acid (ABA) treatment was greater in the OsCPK21-FOX plants than in WT plants. Several ABA- and high salinity-inducible genes were more highly expressed in the OsCPK21-FOX plants than in WT plants. These results suggest that OsCPK21 is involved in the positive regulation of the signaling pathways that are involved in the response to ABA and salt stress.
Plant Molecular Biology 01/2011; 75(1-2):179-91. · 4.15 Impact Factor
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ABSTRACT: Genes involved in the differentiation and development of tissues and organs are temporally and spatially regulated in plant development. The DROOPING LEAF (DL) gene, a member of the YABBY gene family, promotes midrib formation in the leaf and carpel specification in the flower. Consistent with these functions, DL is initially expressed in the central region of the leaf primordia (presumptive midrib) and in the presumptive carpel primordia in the meristem. To understand the regulatory mechanism underlying DL expression, we tried to identify cis-regulatory regions required for temporal and spatial expression of this gene. We found that the cis region responsible for the presumptive midrib-specific expression in the leaf primordia is located in intron 2. Next, we confined the region to a sequence of about 200bp, which corresponds to a conserved non-coding sequence (CNS) identified by phylogenetic footprinting. In addition, a sequence termed DG1, incorporating a 5' upstream region of about 7.4kb, and introns 1 and 2, was shown to be sufficient to induce DL in the presumptive midrib, and to suppress it in other regions in the leaf primordia. By contrast, the regulatory region required for carpel-specific expression was not included in the DG1 sequence. We modified Oryza sativa (rice) plant architecture by expressing an activated version of DL (DL-VP16) in a precise manner using the DG1 sequence: the resulting transgenic plant produced a midrib in the distal region of the leaf blade, where there is no midrib in wild type, and formed more upright leaves compared with the wild type.
The Plant Journal 01/2011; 65(1):77-86. · 6.16 Impact Factor
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ABSTRACT: The excessive amounts of nitrogen applied in current farming systems can cause environmental problems. There is therefore a need to improve the ability of crop plants to utilize nitrogenous fertilizers. We screened for nitrogen deficiency-tolerant lines among transgenic rice plants that overexpressed full-length cDNAs (FL-cDNAs) corresponding to low-nitrogen response genes, genes related to nitrogen metabolism, and genes related to carbon metabolism. We found that overexpression of OsCPK12 FL-cDNA, encoding a calcium-dependent protein kinase (CDPK), conferred tolerance to low-nitrogen stress in rice. After two weeks of low-nitrogen treatment, dry weights of shoots from OsCPK12-overexpressing plants were greater than those from control plants. Furthermore, total nitrogen contents of OsCPK12-overexpressing plants were higher than those of the control plants. Our findings suggest that OsCPK12 is involved in the signal transduction pathway(s) in the low-nitrogen stress response and may be useful in engineering crop plants with improved tolerance to low nitrogen levels.
Plant Biotechnology. 01/2010; 27:369-373.
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ABSTRACT: For systematic and genome-wide analyses of rice gene functions, we took advantage of the full-length cDNA overexpresser (FOX) gene-hunting system and generated >12 000 independent FOX-rice lines from >25 000 rice calli treated with the rice-FOX Agrobacterium library. We found two FOX-rice lines generating green calli on a callus-inducing medium containing 2,4-D, on which wild-type rice calli became ivory yellow. In both lines, OsGLK1 cDNA encoding a GARP transcription factor was ectopically overexpressed. Using rice expression-microarray and northern blot analyses, we found that a large number of nucleus-encoded genes involved in chloroplast functions were highly expressed and transcripts of plastid-encoded genes, psaA, psbA and rbcL, increased in the OsGLK1-FOX calli. Transmission electron microscopy showed the existence of differentiated chloroplasts with grana stacks in OsGLK1-FOX calli cells. However, in darkness, OsGLK1-FOX calli did not show a green color or develop grana stacks. Furthermore, we found developed chloroplasts in vascular bundle and bundle sheath cells of coleoptiles and leaves from OsGLK1-FOX seedlings. The OsGLK1-FOX calli exhibited high photosynthetic activity and were able to grow on sucrose-depleted media, indicating that developed chloroplasts in OsGLK1-FOX rice calli are functional and active. We also observed that the endogenous OsGLK1 mRNA level increased synchronously with the greening of wild-type calli after transfer to plantlet regeneration medium. These results strongly suggest that OsGLK1 regulates chloroplast development under the control of light and phytohormones, and that it is a key regulator of chloroplast development.
Plant and Cell Physiology 10/2009; 50(11):1933-49. · 4.70 Impact Factor
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Kiyomi Abe,
Keishi Osakabe,
Yuichi Ishikawa,
Akemi Tagiri,
Hiroaki Yamanouchi,
Toshio Takyuu,
Terutaka Yoshioka,
Takuya Ito,
Masatomo Kobayashi,
Kazuo Shinozaki, Hiroaki Ichikawa,
Seiichi Toki
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ABSTRACT: BRCA2 is a breast tumour susceptibility factor with functions in maintaining genome stability through ensuring efficient double-strand DNA break (DSB) repair via homologous recombination. Although best known in vertebrates, fungi, and higher plants also possess BRCA2-like genes. To investigate the role of Arabidopsis BRCA2 genes in DNA repair in somatic cells, transposon insertion mutants of the AtBRCA2a and AtBRCA2b genes were identified and characterized. atbrca2a-1 and atbrca2b-1 mutant plants showed hypersensitivity to genotoxic stresses compared to wild-type plants. An atbrca2a-1/atbrca2b-1 double mutant showed an additive increase in sensitivity to genotoxic stresses compared to each single mutant. In addition, it was found that atbrca2 mutant plants displayed fasciation and abnormal phyllotaxy phenotypes with low incidence, and that the ratio of plants exhibiting these phenotypes is increased by gamma-irradiation. Interestingly, these phenotypes were also induced by gamma-irradiation in wild-type plants. Moreover, it was found that shoot apical meristems of the atbrca2a-1/atbrca2b-1 double mutant show altered cell cycle progression. These data suggest that inefficient DSB repair in the atbrca2a-1/atbrca2b-1 mutant leads to disorganization of the programmed cell cycle of apical meristems.
Journal of Experimental Botany 06/2009; 60(9):2751-61. · 5.36 Impact Factor
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ABSTRACT: The DMC1 protein, a meiosis-specific DNA recombinase, catalyzes strand exchange between homologous chromosomes. In rice, two Dmc1 genes, Dmc1A and Dmc1B, have been reported. Although the Oryza sativa DMC1A protein has been partially characterized, however the biochemical properties of the DMC1B protein have not been defined. In the present study, we expressed the Oryza sativa DMC1A and DMC1B proteins in bacteria and purified them. The purified DMC1A and DMC1B proteins formed helical filaments along single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA), and promoted robust strand exchange between ssDNA and dsDNA over five thousand base pairs in the presence of RPA, as a co-factor. The DMC1A and DMC1B proteins also promoted strand exchange in the absence of RPA with long DNA substrates containing several thousand base pairs. In contrast, the human DMC1 protein strictly required RPA to promote strand exchange with these long DNA substrates. The strand-exchange activity of the Oryza sativa DMC1A protein was much higher than that of the DMC1B protein. Consistently, the DNA-binding activity of the DMC1A protein was higher than that of the DMC1B protein. These biochemical differences between the DMC1A and DMC1B proteins may provide important insight into their functional differences during meiosis in rice.
Nucleic Acids Research 07/2008; 36(13):4266-76. · 8.03 Impact Factor
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Hidemitsu Nakamura,
Makoto Hakata,
Kou Amano,
Akio Miyao,
Naoko Toki,
Mariko Kajikawa,
Jinhuan Pang,
Naokuni Higashi,
Shigeko Ando,
Seiichi Toki,
Miki Fujita,
Akiko Enju,
Motoaki Seki,
Miki Nakazawa,
Takanari Ichikawa,
Kazuo Shinozaki,
Minami Matsui,
Yoshiaki Nagamura,
Hirohiko Hirochika, Hiroaki Ichikawa
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ABSTRACT: The latest report has estimated the number of rice genes to be approximately 32,000. To elucidate the functions of a large population of rice genes and to search efficiently for agriculturally useful genes, we have been taking advantage of the Full-length cDNA Over-eXpresser (FOX) gene-hunting system. This system is very useful for analyzing various gain-of-function phenotypes from large populations of transgenic plants overexpressing cDNAs of interest and others with unknown or important functions. We collected the plasmid DNAs of 13,980 independent full-length cDNA (FL-cDNA) clones to produce a FOX library by placing individual cDNAs under the control of the maize Ubiquitin-1 promoter. The FOX library was transformed into rice by Agrobacterium-mediated high-speed transformation. So far, we have generated approximately 12,000 FOX-rice lines. Genomic PCR analysis indicated that the average number of FL-cDNAs introduced into individual lines was 1.04. Sequencing analysis of the PCR fragments carrying FL-cDNAs from 8615 FOX-rice lines identified FL-cDNAs in 8225 lines, and a database search classified the cDNAs into 5462 independent ones. Approximately 16.6% of FOX-rice lines examined showed altered growth or morphological characteristics. Three super-dwarf mutants overexpressed a novel gibberellin 2-oxidase gene,confirming the importance of this system. We also show here the other morphological alterations caused by individual FL-cDNA expression. These dominant phenotypes should be valuable indicators for gene discovery and functional analysis.
Plant Molecular Biology 12/2007; 65(4):357-71. · 4.15 Impact Factor
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ABSTRACT: Rice EL5 is an ATL family gene characterized by a transmembrane domain at the N-terminal and a RING-H2 finger domain (RFD), which exhibits ubiquitin ligase (E3) activity. To elucidate the physiological roles of EL5, we analyzed transgenic rice plants overexpressing mutant EL5 proteins that are impaired in E3 activity to various degrees. Plants expressing EL5C153A and EL5W165A, which encode an inactive E3, showed a rootless phenotype accompanied by cell death in root primordia, and those expressing EL5V162A, with moderately impaired E3 activity, formed short crown roots with necrotic lateral roots. The dominant-negative phenotype was specifically observed in root meristems where EL5 is expressed, and not recovered by exogenous auxin. When wild-type EL5 was transcriptionally overexpressed, the EL5 protein was barely detected by Western blotting. Neither treatment with a proteasome inhibitor nor mutation of the sole lysine residue, a potential target of ubiquitination, resulted in increased EL5 accumulation, whereas mutations in the RFD led to increased EL5 accumulation. The stabilized EL5 without the RFD was localized in the plasma membrane. Deletion of the transmembrane domain prevented the EL5 from localizing in the membrane and from exerting an inhibitory effect on root formation. Deletion of the C-terminal region also neutralized the negative effect. We concluded that EL5 plays a major role as a membrane-anchored E3 for the maintenance of cell viability after the initiation of root primordial formation. In addition, we propose that EL5 is an unstable protein, of which degradation is regulated by the RFD in a proteasome-independent manner.
The Plant Journal 08/2007; 51(1):92-104. · 6.16 Impact Factor
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ABSTRACT: Members of the YABBY gene family have a general role that promotes abaxial cell fate in a model eudicot, Arabidopsis thaliana. To understand the function of YABBY genes in monocots, we have isolated all YABBY genes in Oryza sativa (rice), and revealed the spatial and temporal expression pattern of one of these genes, OsYABBY1. In rice, eight YABBY genes constitute a small gene family and are classified into four groups according to sequence similarity, exon-intron structure, and organ-specific expression patterns. OsYABBY1 shows unique spatial expression patterns that have not previously been reported for other YABBY genes, so far. OsYABBY1 is expressed in putative precursor cells of both the mestome sheath in the large vascular bundle and the abaxial sclerenchyma in the leaves. In the flower, OsYABBY1 is specifically expressed in the palea and lemma from their inception, and is confined to several cell layers of these organs in the later developmental stages. The OsYABBY1-expressing domains are closely associated with cells that subsequently differentiate into sclerenchymatous cells. These findings suggest that the function of OsYABBY1 is involved in regulating the differentiation of a few specific cell types and is unrelated to polar regulation of lateral organ development.
Molecular and General Genetics 06/2007; 277(5):457-68. · 2.63 Impact Factor
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ABSTRACT: OsMUS81, a rice homolog of the yeast MUS81 endonuclease gene, produced two alternative transcripts, OsMUS81alpha and OsMUS81beta. OsMus81alpha contained a Helix-hairpin-Helix (HhH) motif at the N- and C-termini, and a conserved XPF-like motif in the center, while the OsMus81beta isoform lacked the second HhH motif by alternative splicing of a cryptic intron generating a truncated protein. The two transcripts were induced after DNA-damaging treatments such as high intensity light, UV-C and gamma-radiation. The yeast two-hybrid assay detected a strong interaction between OsMus81 and OsRad54 recombinational repair proteins. These findings suggest that OsMus81 functions in maintaining genome integrity through homologous recombination.
Plant and Cell Physiology 05/2007; 48(4):648-54. · 4.70 Impact Factor
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ABSTRACT: Homologous recombination (HR) is an essential process in maintaining genome integrity and variability. In eukaryotes, the Rad52 epistasis group proteins are involved in meiotic recombination and/or HR repair. One member of this group, Rad54, belongs to the SWI2/SNF2 family of DNA-stimulated ATPases. Recent studies indicate that Rad54 has important functions in HR, both as a chromatin remodelling factor and as a mediator of the Rad51 nucleoprotein filament. Despite the importance of Rad54 in HR, no study of Rad54 from plants has yet been performed. Here, we cloned the full-length AtRAD54 cDNA sequence; an open reading frame of 910 amino acids encodes a protein with a predicted molecular mass of 101.9 kDa. Western blotting analysis showed that the AtRad54 protein was indeed expressed as a protein of approximately 110 kDa in Arabidopsis. The predicted protein sequence of AtRAD54 contains seven helicase domains, which are conserved in all other Rad54s. Yeast two-hybrid analysis revealed an interaction between Arabidopsis Rad51 and Rad54. AtRAD54 transcripts were found in all tissues examined, with the highest levels of expression in flower buds. Expression of AtRAD54 was induced by gamma-irradiation. A T-DNA insertion mutant of AtRAD54 devoid of full-length AtRAD54 expression was viable and fertile; however, it showed increased sensitivity to gamma-irradiation and the cross-linking reagent cisplatin. In addition, the efficiency of somatic HR in the mutant plants was reduced relative to that in wild-type plants. Our findings point to an important role for Rad54 in HR repair in higher plants.
The Plant Journal 01/2007; 48(6):827-42. · 6.16 Impact Factor
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Masaki Endo,
Yuichi Ishikawa,
Keishi Osakabe,
Shigeki Nakayama,
Hidetaka Kaya,
Takashi Araki,
Kei-ichi Shibahara,
Kiyomi Abe, Hiroaki Ichikawa,
Lisa Valentine,
Barbara Hohn,
Seiichi Toki
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ABSTRACT: Chromatin assembly factor 1 (CAF-1) is involved in nucleo some assembly following DNA replication and nucleotide excision repair. In Arabidopsis thaliana, the three CAF-1 subunits are encoded by FAS1, FAS2 and, most likely, MSI1, respectively. In this study, we asked whether genomic stability is altered in fas1 and fas2 mutants that are lacking CAF-1 activity. Depletion of either subunit increased the frequency of somatic homologous recombination (HR) in planta approximately 40-fold. The frequency of transferred DNA (T-DNA) integration was also elevated. A delay in loading histones onto newly replicated or repaired DNA might make these DNA stretches more accessible, both to repair enzymes and to foreign DNA. Furthermore, fas mutants exhibited increased levels of DNA double-strand breaks, a G2-phase retardation that accelerates endoreduplication, and elevated levels of mRNAs coding for proteins involved in HR-all factors that could also contribute to upregulation of HR frequency in fas mutants.
The EMBO Journal 12/2006; 25(23):5579-90. · 9.20 Impact Factor
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Yukihiro Kitanaga,
Cui Jian,
Morifumi Hasegawa,
Junshi Yazaki,
Naoki Kishimoto,
Shoshi Kikuchi,
Hidemitsu Nakamura, Hiroaki Ichikawa,
Tadao Asami,
Shigeo Yoshida,
Isomaro Yamaguchi,
Yoshihito Suzuki
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ABSTRACT: Transcripts of thionin genes encoding antimicrobial peptides were present at a high level in rice coleoptiles just after germination, and decreased to an undetectable level after about 3 d, but this decline was suppressed by co-treatment with gibberellic acid (GA(3)) and brassinolide (BL). The temporal expression patterns of key enzyme genes for the biosyntheses of gibberellins (GAs) and brassinosteroids (BRs) were correlated with the fluctuation of thionin mRNAs. Jasmonic acid (JA) replaced the effect of GA3 and BL, and its change in endogenous level was parallel to that of the thionin genes. These results strongly suggest that thionin gene expression was positively regulated by JA, whose endogenous level was synergistically regulated by GAs and BRs. In contrast, thionin gene expression in etiolated seedlings remained high while the endogenous level of JA was low, suggesting the presence of another signaling pathway in the dark to maintain the thionin level.
Bioscience Biotechnology and Biochemistry 11/2006; 70(10):2410-9. · 1.28 Impact Factor
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ABSTRACT: Brassinosteroids (BRs) are a group of plant hormones involved in a wide range of plant growth and developmental processes. To investigate the mechanism of BR action in monocots, a brassinolide (BL) upregulated gene designated OsBLE3 was identified, cloned and characterized in rice. It was mainly expressed in roots and leaf sheaths with levels of expression directly dependent on the dose of BL. In situ hybridization detected OsBLE3 mRNA in the shoot apical meristem, organ primordia and vascular tissue. Furthermore, its expression was enhanced by co-treatment with BL and low concentrations of IAA. These results, and the existence of auxin response elements in the 5'-flanking region of the OsBLE3 gene, indicate that OsBLE3 expression is under control of both BR and auxin. The GUS reporter gene driven by a 2277 bp OsBLE3 putative promoter was mainly expressed in vascular tissues, branch root primordia and was responsive to exogenous BL treatment. OsBLE3 transcript levels were greatly reduced in brd1 plants, a BL deficient mutant, compared to the wild type control. In OsBRI1 antisense transgenic rice and OsBLE3, the BR-insensitive mutant expression of OsBLE3 in response to exogenous BL treatment was significantly lower compared to that in control plants transformed with a vacant vector. Reduced OsBLE3 expression and growth retardation was also observed in OsBLE3 antisense transgenic rice plants. Internode cell length of the OsBLE3 antisense transgenic lines was about 70% of that in the vacant vector transformed control lines. These results suggest that OsBLE3 is involved in cell elongation in rice through dual regulation by BL and IAA.
Phytochemistry 08/2006; 67(14):1442-54. · 3.35 Impact Factor
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ABSTRACT: Precise modification of plant genomes via gene targeting (GT) is important for the study of gene function in vivo. A reliable GT system using the protoporphyrinogen oxidase (PPO) gene in Arabidopsis was reported 4 years ago; however, there are no subsequent successful reports of GT in Arabidopsis. A previous study showed ectopic gene targeting (EGT) of the endogenous gene in two-thirds of GT plants, which was an obstacle to efficient true gene targeting (TGT). The endogenous acetolactate synthase (ALS) gene is involved in the biosynthesis of branched chain amino acids in plants and is the site of action of several herbicides. To confirm the generality of the GT system in Arabidopsis, and to characterize the EGT event in plants in detail, we converted ALS from a herbicide (imazapyr)-susceptible to a -resistant form by GT. We obtained two imazapyr-resistant plants following GT. One of the targeting events was TGT while the other was EGT. After detailed Southern blotting, PCR and nucleotide sequence analysis of the EGT plant, we determined the genomic position and structure of the ectopically targeted site. Based on our findings, we discuss the possible mechanisms of EGT in plants.
Plant and Cell Physiology 04/2006; 47(3):372-9. · 4.70 Impact Factor
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ABSTRACT: Pyruvate dehydrogenase kinase (PDK) is a negative regulator of the mitochondrial pyruvate dehydrogenase complex (mtPDC) that plays a key role in intermediary metabolism. OsPDK1 was identified as a gibberellin-up-regulated gene using a cDNA microarray. The full-length cDNA for OsPDK1 was 1498 bp and encoded a predicted polypeptide of 363 amino acids. Genomic DNA analysis showed the presence of another isoform of PDK, OsPDK2, in rice. Reverse transcriptase-PCR analysis revealed differential expression of the two isoforms. OsPDK1 was expressed in leaf blade and leaf sheath but not in callus and root, while OsPDK2 was expressed constitutively in all tissues examined. Maximum expression of OsPDK1 in leaf sheath was detected by Northern blot analysis when seedlings were treated with 5 microM GA3 for 24 h. OsPDK1 expression was up-regulated by GA3, and there was little effect of other plant hormones. Mitochondrial pyruvate dehydrogenase (PDH) activity was reduced compared with control plants in 2-week-old seedlings treated with GA3. The beta-glucuronidase (GUS) reporter gene, driven by a 2,067 bp OsPDK1 promoter region fragment, was mainly expressed in the aleurone layer of germinating seed and leaf sheath. Transgenic rice expressing PDK1 RNAi had altered vegetative growth with reduced accumulation of vegetative tissues. These results suggest that gibberellin modulates the activity of mtPDC by regulating OsPDK1 expression and subsequently controlling plant growth and development.
Plant and Cell Physiology 03/2006; 47(2):244-53. · 4.70 Impact Factor
