Takaaki Nishioka

Herbivory-induced volatiles elicit defence genes in lima bean leaves

In response to herbivore damage, several plant species emit volatiles that attract natural predators of the attacking herbivores. Using spider mites (Tetranychus urticae) and predatory mites (Phytoseiulus persimilis), it has been shown that not only the attacked plant but also neighbouring plants are affected, becoming more attractive to predatory mites and less susceptible to spider mites. The mechanism involved in such interactions, however, remains elusive. Here we show that uninfested lima bean leaves activate five separate defence genes when exposed to volatiles from conspecific leaves infested with T. urticae, but not when exposed to volatiles from artificially wounded leaves. The expression pattern of these genes is similar to that produced by exposure to jasmonic acid. At least three terpenoids in the volatiles are responsible for this gene activation; they are released in response to herbivory but not artificial wounding. Expression of these genes requires calcium influx and protein phosphorylation/dephosphorylation.

LIGAND: database of chemical compounds and reactions in biological pathways

LIGAND is a composite database comprising three sections: COMPOUND for the information about metabolites and other chemical compounds, REACTION for the collection of substrate-product relations representing metabolic and other reactions, and ENZYME for the information about enzyme molecules. The current release (as of September 7, 2001) includes 7298 compounds, 5166 reactions and 3829 enzymes. In addition to the keyword search provided by the DBGET/LinkDB system, a substructure search to the COMPOUND and REACTION sections is now available through the World Wide Web (http://www.genome.ad.jp/ligand/). LIGAND may be also downloaded by anonymous FTP (ftp://ftp.genome.ad.jp/pub/kegg/ligand/).

Changing green leaf volatile biosynthesis in plants: An approach for improving plant resistance against both herbivores and pathogens

Green leaf volatiles (GLVs) are commonly emitted by green plants, and their production is drastically enhanced when they are under biotic stress. To clarify the ecological function of naturally emitted GLVs, we studied the response of Arabidopsis, whose GLV biosynthesis had been modified, when subjected to herbivory or a pathogenic infection. There was a significant increase in GLV production after herbivory by cabbage white butterfly larvae and pathogen (gray mold) infection in hydroperoxide lyase (HPL) sense Arabidopsis compared with WT controls. The HPL sense modification resulted in the plant being more attractive to the parasitic wasp Cotesia glomerata, leading to higher mortality of the herbivores. The HPL sense modification also resulted in greater inhibition of growth of the fungus. By contrast, HPL antisense Arabidopsis produced fewer GLVs, attracted fewer parasitoids, and was more susceptible to the pathogens than the WT control. These data show that (i) one of the ecological functions of GLV biosynthesis related to resistance against both herbivores and pathogens, and (ii) the genetic modification of GLV biosynthesis could be a unique approach for improving plant resistance against such biotic stresses.

Plant–plant interactions mediated by volatiles emitted from plants infested by spider mites

In an earlier study, we demonstrated plant–plant interactions mediated by volatiles released from lima bean leaves infested by spider mites (Tetranychus urticae) (Nature 406 (2000a) 512, Biochem. Biophys. Res. Commun. 277 (2000b) 305). In the present study, we further show that, under laboratory conditions, volatiles emitted from T. urticae-infested lima bean plants activate transcription of genes encoding pathogenesis-related proteins and phenylalanine ammonia-lyase in leaves of intact neighboring plants. This finding indicates that intact lima bean plants may be responsive to volatile signals. Further, as green leaf volatiles (GLVs) are released from green plants in response to mechanical damage caused by herbivores, we studied possible involvement of GLVs in plant–plant interaction. We found that (Z)-3-hexenol, (E)-2-hexenal, and (Z)-3-hexenyl acetate, induced the expression of defense genes in uninfested leaves. This finding suggests that GLVs may act as signal compounds in plant–plant interactions.

LIGAND: chemical database for enzyme reactions.

MOTIVATION The existing molecular biology databases focus on the sequence and structural aspects of biological macromolecules, i.e. DNAs, RNAs and proteins. However, in order to understand the functional aspects, it is essential to computerize the interaction of these molecules. Furthermore, living cells contain additional molecules, such as metabolic compounds and metal ions, that may also be considered as parts of the basic building blocks of life, but are not well organized in public databases. LIGAND chemical database is our attempt to solve these problems, at least for enzymatic reactions. RESULTS LIGAND consists of two sections: ENZYME and COMPOUND. The ENZYME section is an extension of previous studies (Suyama et al. , Comput. Applic. Biosci., 9, 9-15, 1993), and it is a flat-file representation of 3303 enzymes and 2976 enzymatic reactions in the chemical equation format that can be parsed by machine. The COMPOUND section has been newly constructed for information on the nomenclature and chemical structures of compounds. It contains 5383 chemical compounds. Both ENZYME and COMPOUND entries contain rich cross-reference information, most of which is automatically generated by the DBGET/LinkDB system, thus providing the linkage between chemical and biological databases. LIGAND is updated daily, tightly coupled with the KEGG metabolic pathway database, and forms the basis for reconstruction and computation of pathways. AVAILABILITY LIGAND can be accessed through the DBGET/LinkDB and KEGG systems in the Japanese GenomeNet database service via http://www.genome.ad.jp/. The flat-file format of the LIGAND database can be downloaded by anonymous FTP via ftp://kegg. genome.adjp/molecules/ligand/. CONTACT goto@kuicr.kyoto-u.ac.jp; nishioka@scl.kyoto-u.ac.jp; kanehisa@kuicr.kyoto-u.ac.jp

The tryptophan pathway is involved in the defense responses of rice against pathogenic infection via serotonin production

The upregulation of the tryptophan (Trp) pathway in rice leaves infected by Bipolaris oryzae was indicated by: (i) enhanced enzyme activity of anthranilate synthase (AS), which regulates metabolic flux in the Trp pathway; (ii) elevated levels of the AS (OASA2, OASB1, and OASB2) transcripts; and (iii) increases in the contents of anthranilate, indole, and Trp. The measurement of the contents of Trp-derived metabolites by high-performance liquid chromatography coupled with tandem mass spectrometry revealed that serotonin and its hydroxycinnamic acid amides were accumulated in infected leaves. Serotonin accumulation was preceded by a transient increase in the tryptamine content and by marked activation of Trp decarboxylase, indicating that enhanced Trp production is linked to the formation of serotonin from Trp via tryptamine. Feeding of radiolabeled serotonin to inoculated leaves demonstrated that serotonin is incorporated into the cell walls of lesion tissue. The leaves of a propagating-type lesion mimic mutant (sl, Sekiguchi lesion) lacked both serotonin production and deposition of unextractable brown material at the infection sites, and showed increased susceptibility to B. oryzae infection. Treating the mutant with serotonin restored deposition of brown material at the lesion site. In addition, the serotonin treatment suppressed the growth of fungal hyphae in the leaf tissues of the sl mutant. These findings indicated that the activation of the Trp pathway is involved in the establishment of effective physical defenses by producing serotonin in rice leaves.

Identification of receptors of main sex-pheromone components of three Lepidopteran species

Male moths discriminate conspecific female‐emitted sex pheromones. Although the chemical components of sex pheromones have been identified in more than 500 moth species, only three components in Bombyx mori and Heliothis virescens have had their receptors identified. Here we report the identification of receptors for the main sex‐pheromone components in three moth species, Plutella xylostella, Mythimna separata and Diaphania indica. We cloned putative sex‐pheromone receptor genes PxOR1, MsOR1 and DiOR1 from P. xylostella, M. separata and D. indica, respectively. Each of the three genes was exclusively expressed with an Or83b orthologous gene in male olfactory receptor neurons (ORNs) that are surrounded by supporting cells expressing pheromone‐binding‐protein (PBP) genes. By two‐electrode voltage‐clamp recording, we tested the ligand specificity of Xenopus oocytes co‐expressing PxOR1, MsOR1 or DiOR1 with an OR83b family protein. Among the seven sex‐pheromone components of the three moth species, the oocytes dose‐dependently responded only to the main sex‐pheromone component of the corresponding moth species. In our study, PBPs were not essential for ligand specificity of the receptors. On the phylogenetic tree of insect olfactory receptors, the six sex‐pheromone receptors identified in the present and previous studies are grouped in the same subfamily but have no relation with the taxonomy of moths. It is most likely that sex‐pheromone receptors have randomly evolved from ancestral sex‐pheromone receptors before the speciation of moths and that their ligand specificity was modified by mutations of local amino acid sequences after speciation.

Hemocyte components in crustaceans convert hemocyanin into a phenoloxidase-like enzyme

The functional conversion of hemocyanin (Hc), an oxygen transporter, into an enzyme was investigated in crustaceans. Hc is converted into a phenoloxidase-like enzyme by hemocyte components, which is triggered by beta-1,3-glucan. This activation is severely hampered with leupeptin and E-64 treatment, indicating that the serine/cysteine proteases in the hemocytes are involved in the activation. In a SDS-PAGE-analysis, no change was observed between normal and activated Hc under reduced conditions. However, under non-reduced condition of normal Hc, several minor bands were observed at oligomeric position of Hc subunit, which disappeared upon activation. These results indicate that a split of the reductive bond, such as the disulfide bond between subunits, is essential for Hc activation. This is the first report to show the enzymatic conversion of Hc and the presence of the covalent bond in the Hc subunit of crustaceans.

Quantitative structure-reactivity analysis of the inclusion mechanism by cyclodextrins

The applications of quantitative structure-reactivity analysis to cyclodextrin complexation and cyclodextrin catalysis, mostly from our laboratories, as well as the experimental and theoretical backgrounds of these approaches, are reviewed. These approaches enable us to separate several intermolecular interactions, acting simultaneously, from one another in terms of physicochemical parameters, to evaluate the extent to which each interaction contributes, and to predict thermodynamic stabilities and/or kinetic rate constants experimentally undetermined. Conclusions obtained are mostly consistent with those deduced from experimental measurements.

Exogenous ACC enhances volatiles production mediated by jasmonic acid in lima bean leaves

We report the synergistic effects of exogenous 1‐aminocyclopropane‐1‐carboxylic acid (ACC) and jasmonic acid (JA) on production of induced volatiles by excised lima bean leaves. Application of ACC alone to leaves induced trace amounts of volatiles. ACC positively affected three JA‐induced volatiles, (E)‐ and (Z)‐β‐ocimene, and (Z)‐3‐hexenyl acetate. The ethylene inhibitor, silver thiosulfate, inhibited the production of these compounds. The results suggest synergistic effects of JA and ACC on inducible volatile production by lima bean leaves. Furthermore, lima bean leaves treated with JA plus ACC became more attractive to predatory mites, Phytoseiulus persimilis, than those treated with JA alone.