Codon usage in nuclear genes of four monocot and three dicot species was analyzed to find general patterns in codon choice of plant species. Codon bias was correlated with GC content at the third codon position. GC contents were higher in monocot species than in dicot species at all codon positions. The high GC contents of monocot species might be the result of relatively strong mutational bias that occurred in the lineage of the Poaceae species. In both dicot and monocot species, the effective number of codons (ENCs) for most genes was similar to that for the expected ENCs based on the GC content at the third codon positions. G and C ending codons were detected as the "preferred" codons in monocot species, as in Drosophila. Also, many "preferred" codons are the same in dicot species. Pyrimidine (C and T) is used more frequently than purine (G and A) in four-fold degenerate codon groups.

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Introns have an enhancing effect on gene expression known as intron-mediated enhancement in various organisms, including plants. Although the mechanism of the enhancement is largely unknown, most enhancing introns are first introns. In this study, we examined whether the first intron of rice superoxide dismutase sodCc2 gene has an enhancing effect in rice and other plant species. A transient expression assay revealed that the sodCc2 intron elevated reporter gene expression in rice, wheat, and maize, but not in Arabidopsis and tobacco, indicating that the sodCc2 intron has an enhancing effect in monocot but not in dicot plants. To find the putative signal sequences responsible for the enhancement, we carried out an in silico search and found two motifs conserved among the sodCc2 intron and 11 enhancing introns previously known in rice. The motifs contain a consensus sequence, GATCTG, which also exists in the conserved motif found in Arabidopsis enhancing introns. Our results suggest that common motifs are conserved between rice and Arabidopsis enhancing introns.

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アブチロンモザイクウイルス(AbMV),タバコ巻葉ウイルス(TbLCV),サツマイモ葉巻ウイルス(SPLCV)の感染植物から全核酸を抽出し,Rojas et al.の5組のプライマーおよびBriddon & Markhamが報告した1組のプライマー(BMプライマー)を用いてPCR増幅した。AbMV感染葉からは6組すべてのプライマーにより特異的断片が増幅され,TbLCVおよびSPLCV感染葉からはBMプライマーでのみ特異的断片が得られた。BMプライマーによるPCR産物をクローニングし,遺伝子間領域(IR)の一部塩基配列を決定した。3種ウイルスのIR中にはジェミニウイルスに特微的なTAATATTACの9塩基の配列が認められた。また,IR中の反復配列(iteron)の配置からAbMVはサブグループIII,西半球型,TbLCVおよびSPLCVはサブグループIII,東半球型のジェミニウイルスであることが判明した。

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NADP-ME型C₄単子葉植物であるトウモロコシと, 同型のC₄双子葉植物であるマツバボタンについて, グラナの重なり (スタッキング) に注目して葉緑体の発達過程を観察した. 両植物種において, 維管束鞘葉緑体では1グラナを形成するチラコイド数の平均値は, 葉緑体発達過程を通して3末満に抑えられていたのに対し, 葉肉葉緑体では発達にともない徐々に増大した. したがって, 系統発生的に異なる2種類のNADP-ME型C₄植物において, 維管束鞘緑体ではグラナの発達は共に葉緑体発達初期から抑えられていると考えられた.

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Ornithogalum mosaic virus (OrMV) occurred in Akita Prefecture. An OrMV isolate obtained from a diseased O. thyrsoides could infect not only O. thyrsoides and O. dubium but also five dicot plants (Nicotiana clevelandii, Gomphrena globosa, Chenopodium amaranticolor, C. quinoa and Tetragonia tetragonoides). Another isolate from a diseased O. dubium, by contrast, was transmissible via sap inoculation to the two Ornithogalum plants but not to any dicot plants tested. The two isolates caused mosaic symptoms on the two Ornithogalum plants. This is the first report on the occurrence of OrMV in Japan.

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Recently, the complete chloroplast genome sequences of many important crop plants were determined, and this can be considered a major step forward toward exploiting the usefulness of chloroplast genetic engineering technology. Economically, cotton is one of the most important crop plants for many countries. To further our understanding of this important crop, we determined the complete nucleotide sequence of the chloroplast genome from cotton (Gossypium barbadense L.). The chloroplast genome of cotton is 160,317 base pairs (bp) in length, and is composed of a large single copy (LSC) of 88,841 bp, a small single copy (SSC) of 20,294 bp, and two identical inverted repeat (IR) regions of 25,591 bp each. The genome contains 114 unique genes, of which 17 genes are duplicated in the IRs. In addition, many open reading frames (ORFs) and hypothetical chloroplast reading frames (ycfs) with unknown functions were deduced. Compared to the chloroplast genomes from 8 other dicot plants, the cotton chloroplast genome showed a high degree of similarity of the overall structure, gene organization, and gene content. Furthermore, the sequences of the genes showed high degrees of identity at the DNA and amino acid levels. The cotton chloroplast genome was somewhat longer than the chloroplast genomes of most of the other dicot plants compared here. However, this elongation of the cotton chloroplast genome was found to be due mainly to expansions of the intergenic regions and introns (non-coding DNA). Moreover, these expansions occurred predominantly in the LSC and SSC regions.

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A ferredoxin-nitrite reductase (EC 1.7.7.1) cDNA was isolated and sequenced from a λgt 11 cDNA library constructed from nitrate-induced greening shoots of rice (Oryza sativa L.) seedlings. The nucleotide sequence of the cDNA clone contains an open reading frame of 1788 nucleotides. There exists a strong bias for the third codon usage of G/C (95.5%) as in the case of the maize enzyme. The deduced amino acid sequence shows an overall homology to the maize (81%) and the dicot enzymes (70-74%), suggesting that the primary structure of ferredoxin-nitrite reductase is highly conserved in higher plants.

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We have cloned a full length cDNA for the small subunit of ribulose-1, 5-bisphosphate carboxylase from C₄ monocot maize, determined the complete nucleotide sequence of this cDNA and deduced its amino acid sequence. The cDNA insert included 513 by of the coding region, and 65 and 252 nucleotides of the 5' and 3' untranslated regions, respectively. The transit and mature peptides have, respectively, 47 and 123 amino acids. Comparison with the small subunit genes from other plants revealed that the maize small subunit is similar to the wheat one, there being 73 % homology between the transit peptides and 64 % between the mature proteins. This indicates that there is no noteworthy difference between the C₃ and C₄ small subunit structures. Extreme codon bias was observed for this gene, and similar codon preferences are observed for other proteins highly expressed in maize leaf, light harvesting chlorophyll binding protein and phosphoenolpyruvate carboxylase. The results indicate that preferential codon usage for highly expressed genes occurs in maize leaf

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植物のstarch synthaseは，デンプン合成においてアミロースならびにアミロペクチンのグルカン鎖伸長を司る酵素であり，その特性はデンプンの微細構造に影響する．本研究では，インゲンマメ(Phaseolus vulgaris L.)種子におけるstarch synthasesの詳細な特性を明らかにするため，starch synthase II(PvSSII-2)をコードするcDNAクローン(pvss22)をreverse transcriptase-mediated PCR(RT-PCR)，5´-rapid amplification of cDNA end(RACE)および3´-RACE法により単離した(Fig. 1)．pvss22 cDNAは，2486 bpよりなり，738アミノ酸残基のオープンリーディングフレームを含んでいた．推定アミノ酸配列は，他の双子葉由来starch synthase IIと高い同一性(58%以上)を示した(Fig. 2)．pvss22転写産物は登熟中期から後期の種子に顕著に蓄積したが，登熟初期および完熟種子や葉では非常に低いレベルで存在した(Fig. 3)．大腸菌で発現させた組換えPvSSII-2タンパク質は，封入体の主要ポリペプチドとして生産された(Fig. 4A)．封入体から抽出したポリペプチドを抗原として，抗体を調製した．この抗体は，登熟および完熟種子のデンプン粒結合画分の少なくとも7種のポリペプチドと反応した(Fig. 4B)．このうち3種のN末端配列を解析したところ，いずれの配列もPvSSII-2の一次構造中に見出された(Fig. 1)．これらのことから，インゲンマメ登熟種子デンプン粒中には，pvss22遺伝子にコードされるN末端領域の異なる複数のisoformsが存在することが明らかとなった．

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To obtain cDNAs encoding oxidosqualene cyclase (OSC), we cloned two cDNAs, KcCAS and RsCAS, from roots of Kandelia candel (L.) Druce and leaves of Rhizophora stylosa Griff. by homology based PCR method respectively. The deduced amino acid sequences of both OSCs showed 82% homology to cycloartenol synthases from Lotus japonicus (OSC5) and Ricinus cummunis (RcCAS), suggesting that these are cycloartenol synthases of K. candel and R. stylosa. The genes obtained were expressed in a lanosterol synthase deficient Saccharomyces cerevisiae (ERG7) strain, GIL77. GC–MS analysis identified the accumulated reaction product in the yeast transformant to be cycloartenol, indicating that both KcCAS and RsCAS encode cycloartenol synthase.

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To confirm the involvement of galactose oxidase/kelch repeat-containing protein (Glx) in response to iron (Fe) deficiency in Hyoscyamus albus, we cloned a putative full-length HaGlx cDNA, which contained an open reading frame (975 bp, 324 amino acids). HaGlx was confirmed by homology searches, molecular phylogeny analysis, and domain search. HaGlx was expressed in the roots but not in the leaves, and the expression significantly increased under Fe deficiency. Sequencing of ∼1.9 kb of the 5'-upstream region of the HaGlx gene, followed by the analysis of promoter elements, resulted in the identification of multiple root-specific elements together with stress-induced elements, including the Fe deficiency-induced element (IDE1) core motif. This suggests that HaGlx plays a key role in stress responses induced under Fe deficiency in the roots. To our knowledge, this is the first report confirming, in a plant other than Arabidopsis thaliana, that Glx is involved in the stress response to Fe deficiency.

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Ferredoxin-nitrite reductase [EC 1.7.7.1] has been purified to apparent homogeneity from rice (Oryza sativa cv. Kinmaze) leaves by a procedure used for the spinach enzyme [S. Ida and B. Mikami, Biochitn. Biophys. Acta, 681, 167 (1986)]. The rice enzyme consists of a single polypeptide of molecular weight of 60, 000 with 536 amino acid residues. The enzyme showed nearly identical absorption, circular dichroism, and magnetic circular dichroism spectra to those of the spinach enzyme, indicating the presence of the same prosthetic groups and protein conformation in both enzymes. The apparent Km values for nitrite and methyl viologen were 360μM and 63μM, respectively. The pH optimum was 7.6. These kinetic parameters are indistinguishable from those reported for spinach nitrite reductase. Monospecific antiserum against purified rice enzyme cross-reacted with nitrite reductases from a variety of higher plants and some phylogenetically divergent plants. Immunological comparisons indicated the rice enzyme is much more closely related to the other monocot enzymes in antigenic structure than to the dicot enzyme proteins. The results lend further support to our previous study [S. Ida, Plant Sci., 49, 111 (1987)] that spinach ferredoxin-nitrite reductase is serologically more related to the dicot enzymes than to the monocot nitrite reductases. Conspicuous differences between the rice and spinach enzymes were found in their molecular sizes and antigenicity. Relatedness of amino acid compositions of the enzyme proteins is discussed in relation to antigenic properties of ferredoxin-nitrite reductase.

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　我々は、植物遺伝子組換え技術を応用したファイトレメディエーションに関する研究を行っている。これまでの研究では、土壌中の環境ホルモンを分解する目的で、リグニン分解酵素の一種であるラッカーゼを植物で発現させることを試みた。その際、ラッカーゼ遺伝子をCaMV35Sプロモーターの下流に連結させてイネに導入して発現させたところ、顕著な生育傷害がみられた。そこで本研究では、植物遺伝子組換え技術を応用した土壌中の有害物質分解を合目的的に効率よく行うため、根で特異的に発現するプロモーターの開発を行った。
　開発には、シロイヌナズナ由来リン酸トランスポーター遺伝子（PHT1）のプロモーターを用いた。PHT1プロモーターの下流にレポーター遺伝子GUSを連結したPHT1::GUS融合遺伝子をシロイヌナズナとイネに導入し、PHT1プロモーターの発現を調べた。その結果、シロイヌナズナ、イネともに根で強いGUS活性が検出され、リン酸濃度に対する負の応答を示した。根以外の器官でのGUS活性は低かった。
　双子葉植物由来のPHT1プロモーターが単子葉植物であるイネにおいても機能することから、同プロモーターは植物が共通に持っている発現機構によって制御されていることが示唆された。このことから、現在は実用植物であるユーカリとポプラでPHT1プロモーターが機能するかを解析している。

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The differences in the rates of ethylene production and growth between the calluses derived from the seeds of rice (16 cultivars of Oryza sativa L., Japonica and Indica types) and soybean (10 cultivars of Glycine max (L.) Merr.) were investigated. On the medium containing 2, 4-dichlorophenoxyacetic acid (2, 4-D) and benzylaminopurine (BAP), soybean calluses produced a larger amount of ethylene (208-1541 nLg^>-1< FW 24 h^>-1<) than rice calluses (8-37 nLg^>-1< FW 24 h^>-1<), and the rate of ethylene production from the calluses of both species greatly varied with the cultivar. The combined application of 2, 4-D and BAP greatly increased the ethylene production rate and the growth of calluses in soybean, but not in rice. Moreover, the addition of BAP to the medium strikingly promoted ethylene production in soybean calluses cultured on the medium containing 2, 4-D, but not in those cultured on the medium containing indolebutyric acid (IBA). The rate of ethylene production in both soybean and rice negatively correlated with the growth of calluses. The ethylene production and growth of calluses cultured under various conditions greatly differed between soybean and rice, and also varied with the cultivar of each species. The different aspects of the growth and ethylene production between soybean and rice calluses may represent the difference between dicots and monocots.

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The effect of NaCl stress on the structure of leaf chloroplasts was investigated in several NAD-Malic enzyme (NAD-ME) and phosphoenolpyruvate carboxykinase (PCK) type C₄ plant species. Seedlings of the monocot species, except Zoysia japonica, grown in 300 mL pots were subjected to salt stress by adding 50 mL of 3% NaCl solution per day to the soil for 5 d after the fourth leaf blades were fully developed. Z. japonica and the dicot species, Amaranthus tricolor, were also treated with 3% NaCl in a similar manner from 5 wk after germination. Salt stress negatively affected the growth, chlorophyll content and chloroplast structure in all the species. At the ultrastructure level, swelling of thylakoids and disruption of envelopes were more or less observed in mesophyll cell (MC) chloroplasts after salt treatment. The structure of bundle sheath cell (BSC) chloroplasts, on the other hand, was hardly damaged under salt condition although stromal and starch areas were considerably decreased. Furthermore, salinity induced granal development in BSC chloroplasts in most species; the number of thylakoids per granum, granal indices and appressed thylakoid density in salt-treated plants were generally higher than those in control. Since the similar responses have also been reported in all NADP-ME type C₄ species investigated in our previous study, the high sensitivity to salt stress in MC chloroplasts and the granal development in BSC chloroplasts by salinity were considered to be common phenomena in all three C₄ subtypes.

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In plants, the NADP malic enzymes (NADP-MEs) are encoded by small gene families. These NADP-ME gene families are relatively well described in C4 plants but not well studied in C3 plants. In this study, we investigated the NADP-ME gene family in a model C3 monocot plant (rice, Oryza sativa) based on its recently released genomic DNA sequence. We found that the rice NADP-ME family is composed of four members, one plastidic NADP-ME and three cytosolic versions. Although the rice NADP-ME genes identified share a high degree of similarity with one another, one cytosolic NADP-ME (OscytME3) contains several unique amino acid substitutions within highly conserved amino acid regions. Phylogenetic analysis showed that OscytME3 might be derived from a different evolutionary branch than the other three rice genes. Expression analysis of the four rice NADP-ME genes indicated that each had a different tissue-specific and developmental profile, although all four responded to stress stimuli.

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Tabacco leaf curl geminivirus (TLCV)のAC1遺伝子とAV1遺伝子の間の領域(1R)を含む1.1kbpの塩基配列をもとに構造解析と分子系統解析を行った。IR中にはTLCVに特徴的な反復配列ATTGGTACCAが存在し,その配置は他の旧世界型ジェミニウイルスと似ていた。TLCVのIRの塩基配列を,双子葉植物に感染しかつタバココナジラミによって伝搬される22種類のジェミニウイルスのIRと比較したところ,旧世界型ジェミニウイルスに分類され,相同性の高かったtomato leaf curl virusのオーストラリア分離株およびイスラエル株でもそれぞれ57.1%および56.5%であった。以上の結果から,TLCVは,他のジェミニウイルスの系統でなく,独立した種であると考えられる。

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Many eukaryotic mRNAs contain one or more upstream open reading frames (uORFs) in their 5′ untranslated regions (5′-UTRs). Some uORFs encode regulatory peptides that repress translation of the main ORF. To comprehensively identify uORFs encoding regulatory peptides, genome-wide searches for uORFs with evolutionarily conserved amino acid sequences, referred to as conserved peptide uORFs (CPuORFs), have been conducted using bioinfomatic approaches. To date, more than 40 homology groups of CPuORFs have been identified in dicotyledonous plants. The Arabidopsis thaliana ANAC096 gene is one of the CPuORF-containing genes; however, the ANAC096 CPuORF exerts only little peptide sequence-dependent effect on expression of the main ORF. Here, we investigated the effect of the CPuORF sequence of a tomato ANAC096 homologue on expression of the main ORF, because it has a more highly conserved amino acid sequence than the ANAC096 CPuORF. Mutational analyses revealed that the CPuORF of the tomato ANAC096 homologue represses main ORF expression in a peptide sequence-dependent manner, and determined the critical amino acid residues of the CPuORF peptide responsible for the repression. This study identified a novel peptide sequence-dependent regulatory uORF and demonstrated that the level of uORF peptide-mediated repression can differ among closely related homologues.

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