A two-point mutated gene of acetolactate synthase (ALS) was isolated from rice cells, which was cultured together with an ALS-inhibiting herbicide, bispyribac-sodium (BS). The mutations involved residues of tryptophan at position 548 to leucine (W548L) and serine at position 627 to isoleucine (S627I). The ALS expressed in Escherichia coli from this gene showed resistance to multiple herbicides including pyrimidinylcarboxylate (PC), sulfonylurea and imidazolinone herbicides, and showed stronger resistance to PC herbicides than to other herbicides. BS, a PC herbicide, had almost no effect on the enzyme even at 100 mM, which is an approximately 10,000-fold higher concentration than the concentration required for 50% inhibition of the wild-type. The resistance level of W548L/S627I mutating ALS to BS was stronger than the additive effect predicted from the degree of resistance of each single amino acid mutated ALS. Transformed rice cells carrying this gene and a regenerated rice plant expressed resistance to BS, suggesting that this gene is useful as a selectable marker for introducing foreign traits into rice when used with PC herbicides.

Mutated acetolactate synthase (ALS) genes of rice and Arabidopsis, which confer resistance to ALS-inhibiting herbicides, were generated using PCR and overlap extension. Recombinant ALSs from these mutated genes were prepared as glutathione S-transferase-fused proteins, and sensitivities of the proteins to ALS-inhibiting herbicides were examined. Nine kinds of rice ALSs that have mutations at the P171 position (each amino acid is represented by one letter) showed high resistance to a herbicide, bensulfuron-methyl, but the resistance level to a herbicide, bispyribac-sodium (BS), changed among ALSs. S627I ALS of rice expressed high resistance to herbicides: pyriminobac and pyrithiobac-sodium. P171H/R172S ALS of rice showed greater resistance to a herbicide, chlorsulfuron than the additive effect predicted from the resistance of each single mutated ALS. P171H/W548L ALS and P171H/S627I ALS of rice showed similar synergistic resistance to BS. On the other hand, P197S, W574L, S653I, P197H/R198S and W574L/S653I-mutated ALSs of Arabidopsis expressed similar sensitivities to herbicides as those of rice ALSs with the corresponding mutations. These results proposed that P171-mutated ALSs of rice can be used as model enzymes for resistant weed management to ALS-inhibiting herbicides, and rice-mutated ALS genes encoding mutated ALSs such as S627I, P171H/R172S, P171H/W548L, P171H/S627I, and Arabidopsis-mutated ALS genes encoding mutated ALSs, such as S653I, P197H/R198S and W574L/S653I, are useful as new selectable marker genes for genetic transformation of plants when used together with ALS-inhibiting herbicides to which mutated ALSs express high resistance.

Strategies employed for the production of genetically modified (GM) crops are premised on (1) the avoidance of gene transfer in the field; (2) the use of genes derived from edible organisms such as plants; (3) preventing the appearance of herbicideresistant weeds; and (4) maintaining transgenes without obstructing plant cell propagation. To this end, we developed a novel vector system for chloroplast transformation with acetolactate synthase (ALS). ALS catalyzes the first step in the biosynthesis of the branched amino acids, and its enzymatic activity is inhibited by certain classes of herbicides. We generated a series of Arabidopsis (Arabidopsis thaliana) mutated ALS (mALS) genes and introduced constructs with mALS and the aminoglycoside 3#-adenyltransferase gene (aadA) into the tobacco (Nicotiana tabacum) chloroplast genome by particle bombardment. Transplastomic plants were selected using their resistance to spectinomycin. The effects of herbicides on transplastomic mALS activity were examined by a colorimetric assay using the leaves of transplastomic plants. We found that transplastomic G121A, A122V, and P197S plants were specifically tolerant to pyrimidinylcarboxylate, imidazolinon, and sulfonylurea/pyrimidinylcarboxylate herbicides, respectively. Transplastomic plants possessing mALSs were able to grow in the presence of various herbicides, thus affirming the relationship between mALSs and the associated resistance to herbicides. Our results show that mALS genes integrated into the chloroplast genome are useful sustainable markers that function to exclude plants other than those that are GM while maintaining transplastomic crops. This investigation suggests that the resistance management of weeds in the field amid growing GM crops is possible using (1) a series of mALSs that confer specific resistance to herbicides and (2) a strategy that employs herbicide rotation.

Trichoderma asperellum SKT-1 and Gibberella fujikuroi, known as causal agents of "Bakanae" disease, were both transformed with genes encoding green fluorescent protein (GFP) and hygromycin B (hygB) by restriction enzyme-mediated integration (REMI). Rice seeds inoculated with GFP-tagged G. fujikuroi showed "Bakanae" symptoms. GFP-tagged SKT-1 maintained biocontrol activity against the pathogen by soaking seeds in SKT-1 spore suspension. Then, we monitored in situ interactions between SKT-1 and G. fujikuroi on rice seeds using GFP-tagged transformations under confocal scanning laser stereomicroscopy. G. fujikuroi disappeared from the embryo of rice seeds after treatment with SKT-1, whereas SKT-1 was observed on the embryo 24 hr after initiation of germination. In addition, the hyphae of G. fujikuroi were penetrated by the hyphae of SKT-1, and degradation of the cell walls of G. fujikuroi was observed under SEM in co-culture. The cell wall of G. fujikuroi on the embryo of rice seeds was lysed, suggesting that mycoparasitism is the mode of action of T. asperellum SKT-1.

To develop a screening system for plant activators, which are novel substances that protect plants by enhancing their inherent disease-resistance mechanisms, we utilized a GUS reporter gene system using promoters of the defenserelated genes, PR-1 and PR-4. To validate the strategy, we performed subsequent analysis using an Arabidopsis microarray consisting of 1200 full-length cDNA clones representing putative defense-related and regulatory genes. Benzo(1,2,3)thiadiazole-7-carbothioic acid S-methyl ester and 2,6-dichloroisonicotinic acid activated plant defense responses via the salicylic acid (SA)-dependent signaling pathway, and b-aminobutyric acid triggered a primed state in the plant that enables more efficient activation of the SA-, jasmonic acid-and ethylene-signaling pathway. These results suggest that this novel system can be used to screen for candidate plant activators.

SigA-binding protein (SIB A) is a nuclearencoded chloroplast-targeted protein that interacts with the plastid-encoded plastid RNA polymerase r-factor SigA (Sig1). In this study, the SIB A gene responded rapidly to salicylic acid (SA) treatment, but responded slowly to ethylene (ET) and jasmonic acid (JA) treatments as determined with microarray and quantitative real-time RT-PCR analyses. Expression of the SIB A gene increased rapidly, with a peak at 2 h after SA treatment and then decreased gradually. In contrast, expression of the PR-1 gene, a marker gene on the SA defense signaling pathway, increased gradually between 0 and 24 h after SA treatment. In addition, transcription levels of SIB A and PR-1 decreased between 0 and 24 h in the benzothiadiazole (BTH)-treated npr1-1 plants. We suggest that the SIB A gene is downstream of NPR1 on the SA signaling pathway. From the results of our analysis of the 1.5-kbp promoter region of the SIB A gene in Arabidopsis, the isolated promoter region of the SIB A gene seems able to drive the transcription of the GUS gene to elevated levels more rapidly in transgenic Arabidopsis plants undergoing SA treatment compared with levels of PR-1. To analyze biological function of SIB A and investigate SIB A-regulated genes, we generated transgenic Arabidopsis plants in which SIB A was overexpressed. As a result, ROS-related genes, such as glutathione transferase, were up-regulated in plants overexpressing SIB A. However, SIB A-overexpressing plants were not resistant to Pseudomonas syringae pv. tomato DC3000 strain. The SIB A gene has promise as a new tool to analyze the SA-signaling pathway and the role of the chloroplast in plant defense responses.

Previously, we showed that four mutated acetolactate synthase (ALS) genes derived from rice and Arabidopsis (W548L/S627IOsALS, S627IOsALS, W574L/S653IAtALS and S653IAtALS) confer high levels of resistance to pyrimidinylcarboxylate type ALS inhibitors (Kawai et al. 2008). Mutated ALS genes of rice were obtained from rice cells cultured in the presence of an ALS-inhibitor. The mutated ALS genes of Arabidopsis, which have the same amino acid substitutions as those of rice, have been generated artificially. Here, we demonstrate that these mutated genes function as effective selectable markers for transformation of Arabidopsis. Specifically, we studied expression of the mutated ALSs in Arabidopsis and their effect on the sensitivity of transgenic Arabidopsis plants to the ALS inhibitors. Our results show that the degree of resistance to the ALS inhibitors of transformants expressing Arabidopsis mutated ALSs was greater than those of transformants expressing rice mutated ALSs. The amino acid sequences of ALSs derived from monocotyledonous plants and those derived from dicotyledonous plants were clearly divided into two clusters in a phylogenetic tree. Based on these results, it would be preferable to use rice and Arabidopsis mutated ALS genes for generating monocotyledonous and dicotyledonous transgenic plants, respectively. Moreover, our findings are particularly useful when generating transgenic plants with a known ALS nucleotide sequence. In such cases, their own ALS gene carrying these mutations could be used as a selectable marker because amino acid residues at the point of mutation are conserved among plant species.Key words: Acetolactate synthase, pyrimidinylcarboxylate, selectable marker gene.Plant Biotechnology 27, 75-84 (2010) Original PaperAbbreviations: ALS, acetolactate synthase; BS, bispyribac-sodium; IM, imidazolinone; PC, pyrimidinylsalicylate; PS, pyrithiobac-sodium; SU, sulfonylurea This article can be found at http://www.jspcmb.jp/ inhibitor, bispyribac-sodium (BS), and demonstrated that it could be used as a selectable marker for generating transgenic rice plants (Kawai et al. 2007a;Kawai et al. 2007b). We also found that a single amino acid substitution (S627I) in the ALS gene (S627IOsALS) displays high levels of resistance to other PC-type ALS inhibitors; pyrithiobac-sodium (PS) and pyriminobac (PM). In addition, artificially generated W574L/S653I and S653I Arabidopsis ALS genes (W574L/S653IAtALS and S653IAtALS) corresponding to the rice mutated ALS genes showed similar sensitivity to PC-type ALS inhibitors (Kawai et al. 2008). We postulated that these mutated ALS genes coupled with the PC-type ALS inhibitors might be promising selectable markers for various plant species. Indeed, previous reports have shown that W548L/S627IOsALS works as an effective selectable marker gene for transformation of wheat (Ogawa et al. 2008) and soybean (Tougou M et al. 2009).In this paper, we generated transgenic Arabidopsis via agrobacterium-mediated transformation with rice mutated ALS genes (W548L/S6...

Fava beans are eaten all over the world and recently, marketing for their sprouts began in Japan. Fava bean sprouts contain more polyphenols and l-3,4-dihydroxyphenylalanine (l-DOPA) than the bean itself. Our antioxidant screening program has shown that fava bean sprouts also possess a higher antioxidant activity than other commercially available sprouts and mature beans. However, the individual constituents of fava bean sprouts are not entirely known. In the present study, we investigated the phenolic compounds of fava bean sprouts and their antioxidant activity. Air-dried fava bean sprouts were treated with 80% methanol and the extract was partitioned in water with chloroform and ethyl acetate. HPLC analysis had shown that the ethyl acetate-soluble parts contained phenolic compounds, separated by preparative HPLC to yield 5 compounds (1-5). Structural analysis using NMR and MS revealed that the compounds isolated were kaempferol glycosides. All isolated compounds had an α-rhamnose at the C-7 position with different sugars attached at the C-3 position. Compounds 1-5 had β-galactose, β-glucose, α-rhamnose, 6-acetyl-β-galactose and 6-acetyl-β-glucose, respectively, at the C-3 position. The amount of l-DOPA in fava bean sprouts was determined by the quantitative (1) H NMR technique. The l-DOPA content was 550.45 mg ± 11.34 /100 g of the raw sprouts. The antioxidant activities of compounds 2-5 and l-DOPA were evaluated using the 2,2-diphenyl-1-picrylhydrazyl scavenging assay. l-DOPA showed high antioxidant activity, but the isolated kaempferol glycosides showed weak activity. Therefore, it can be suggested that l-DOPA contributed to the antioxidant activity of fava bean sprouts.