Goro Taguchi

RNAi Suppression of Arogenate Dehydratase1 Reveals That Phenylalanine Is Synthesized Predominantly via the Arogenate Pathway in Petunia Petals

This study analyzed the l-Phe biosynthetic pathways in Petunia hybrida flowers, which emit high levels of Phe-derived volatiles. RNA interference suppression of petunia arogenate dehydratase1 provides in planta evidence that l-Phe is synthesized via arogenate and uncovers a novel posttranscriptional regulation of the shikimate pathway. l-Phe, a protein building block and precursor of numerous phenolic compounds, is synthesized from prephenate via an arogenate and/or phenylpyruvate route in which arogenate dehydratase (ADT) or prephenate dehydratase, respectively, plays a key role. Here, we used Petunia hybrida flowers, which are rich in Phe-derived volatiles, to determine the biosynthetic routes involved in Phe formation in planta. Of the three identified petunia ADTs, expression of ADT1 was the highest in petunia petals and positively correlated with endogenous Phe levels throughout flower development. ADT1 showed strict substrate specificity toward arogenate, although with the lowest catalytic efficiency among the three ADTs. ADT1 suppression via RNA interference in petunia petals significantly reduced ADT activity, levels of Phe, and downstream phenylpropanoid/benzenoid volatiles. Unexpectedly, arogenate levels were unaltered, while shikimate and Trp levels were decreased in transgenic petals. Stable isotope labeling experiments showed that ADT1 suppression led to downregulation of carbon flux toward shikimic acid. However, an exogenous supply of shikimate bypassed this negative regulation and resulted in elevated arogenate accumulation. Feeding with shikimate also led to prephenate and phenylpyruvate accumulation and a partial recovery of the reduced Phe level in transgenic petals, suggesting that the phenylpyruvate route can also operate in planta. These results provide genetic evidence that Phe is synthesized predominantly via arogenate in petunia petals and uncover a novel posttranscriptional regulation of the shikimate pathway.

Contribution of CoA Ligases to Benzenoid Biosynthesis in Petunia Flowers

Biochemical and genetic characterization of two petunia CoA ligases reveals that subcellular compartmentalization of enzymes determines their involvement in the benzenoid metabolic network. Additional evidence shows that formation of cinnamoyl-CoA in peroxisomes is the committed step in the synthesis of benzoyl-CoA via the β-oxidative pathway. Biosynthesis of benzoic acid from Phe requires shortening of the side chain by two carbons, which can occur via the β-oxidative or nonoxidative pathways. The first step in the β-oxidative pathway is cinnamoyl-CoA formation, likely catalyzed by a member of the 4-coumarate:CoA ligase (4CL) family that converts a range of trans-cinnamic acid derivatives into the corresponding CoA thioesters. Using a functional genomics approach, we identified two potential CoA-ligases from petunia (Petunia hybrida) petal-specific cDNA libraries. The cognate proteins share only 25% amino acid identity and are highly expressed in petunia corollas. Biochemical characterization of the recombinant proteins revealed that one of these proteins (Ph-4CL1) has broad substrate specificity and represents a bona fide 4CL, whereas the other is a cinnamate:CoA ligase (Ph-CNL). RNA interference suppression of Ph-4CL1 did not affect the petunia benzenoid scent profile, whereas downregulation of Ph-CNL resulted in a decrease in emission of benzylbenzoate, phenylethylbenzoate, and methylbenzoate. Green fluorescent protein localization studies revealed that the Ph-4CL1 protein is localized in the cytosol, whereas Ph-CNL is in peroxisomes. Our results indicate that subcellular compartmentalization of enzymes affects their involvement in the benzenoid network and provide evidence that cinnamoyl-CoA formation by Ph-CNL in the peroxisomes is the committed step in the β-oxidative pathway.

Effects of methyl jasmonate and elicitor on the activation of phenylalanine ammonia-lyase and the accumulation of scopoletin and scopolin in tobacco cell cultures

Abstract The effects of methyl jasmonate (MJ) and elicitor on the phenylpropanoid metabolism in tobacco was studied. It was found that suspended cells of tobacco, responded to MJ and elicitor from the plant pathogenic fungus Fusarium solani with differential formation of phenolic compounds. Both MJ and elicitor induced phenylalanine ammonia-lyase (PAL, EC 4.3.1.5) activity to comparable levels. Northern hybridization showed that the increase in PAL activity was preceded by PAL mRNA accumulation. PAL was induced transcriptionally and enzymatically faster in elicitor-treated cells as compared to MJ-treated cells. HPLC of extracts from tobacco cells and their culture filtrate treated with MJ, showed an induction of the syntheses of coumarin-scopoletin and scopolin. This effect was not seen in elicitor-treated or control cell cultures. Scopolin was mainly found within the cell (99%), whereas scopoletin accumulated in the culture filtrate (90%). We conclude that though MJ mimics stress, indicating its possible role in the plant defense metabolism, responses of tobacco cells to MJ and elicitor are distinct.

Mapping of isolate-specific QTLs for clubroot resistance in Chinese cabbage (Brassica rapa L. ssp. pekinensis)

A number of clubroot resistant (CR) Chinese cabbage cultivars have been developed in Japan using resistant genes from CR European fodder turnips (B. rapa ssp. rapifera). Clubroot resistance in European fodder turnips are known to be controlled by the combined action of several dominant resistance genes. We have developed three Chinese cabbage clubroot-resistant doubled haploid (DH) lines—T136-8, K10, and C9—which express resistance in different manners against two isolates of Plasmodiophora brassicae, M85 and K04. Depending on the isolates, we identified two CR loci, CRk and CRc. CRk was identified by quantitative trait loci (QTL) analysis of an F2 population derived from a cross between K10 and Q5. This locus showed resistance to both isolates and is located close to Crr3 in linkage group R3. The other locus, CRc was identified by QTL analysis of an F2 population derived from a cross between C9 and susceptible DH line, 6R. This locus was mapped to linkage group R2 and is independent from any published CR loci. We developed sequence-tagged site markers linked to this locus.

Scopoletin uptake from culture medium and accumulation in the vacuoles after conversion to scopolin in 2,4-D-treated tobacco cells.

Tobacco (Nicotiana tabacum L. Bright Yellow) T-13 cell line has the ability to produce scopoletin endogenously and release some of it into the culture medium. We investigated the mechanism of scopoletin uptake following treatment of a tobacco culture with 2,4-dichlorophenoxyacetic acid (2,4-D). Addition of [14C]-labeled scopoletin showed that scopoletin was taken up by 2,4-D-treated cells and converted to scopolin, a 7-O-glucoside of scopoletin. This uptake of scopoletin began 6 h after 2,4-D addition to the cells. Experiments using several inhibitors showed that this uptake was energy-dependent. The phenomenon of 2,4-D-stimulated uptake was observed only for 7-hydroxycoumarins, such as scopoletin, umbelliferone and esculetin. To further investigate the site for scopoletin accumulation, we separated the vacuoles from T-13 cells and quantified the coumarin contents in this fraction. Most of the scopoletin in the vacuoles was present as glucoconjugate, scopolin. Moreover, glucosylation activity was absent from isolated vacuoles and, therefore, is likely to be located in the cytosol. Therefore, we can state that 2,4-D treatment of tobacco cells stimulated scopoletin uptake. The scopoletin was converted into scopolin in the cytoplasm, and then transferred into the vacuoles.

Isolation and characterization of a gene coding for chitin deacetylase specifically expressed during fruiting body development in the basidiomycete Flammulina velutipes and its expression in the yeast Pichia pastoris

Fv-pda, a gene coding for chitin deacetylase (CDA), was isolated from the basidiomycete Flammulina velutipes by differential display targeted for genes specifically expressed during fruiting body development. The fv-pda ORF comprises 250 amino acid residues and is interrupted by 10 introns. The fv-pda cDNA was expressed in the yeast Pichia pastoris, and the resulting recombinant FV-PDA was used for enzymatic characterization. The recombinant FV-PDA catalyses deacetylation of N-acetyl-chitooligomers, from dimer to pentamer, glycol chitin and colloidal chitin. The fv-pda was specifically expressed through the entire stage of fruiting body development, and the transcript was abundant in stipes of mature fruiting bodies. These results suggest that CDA plays an important role in the process of fruiting of F. velutipes.

Purification and characterization of UDP-glucose: hydroxycoumarin 7-O-glucosyltransferase, with broad substrate specificity from tobacco cultured cells.

The enzyme UDP-glucose: hydroxycoumarin 7-O-glucosyltransferase (CGTase), which catalyzes the formation of scopolin from scopoletin, was purified approximately 1200-fold from a culture of 2,4-D-treated tobacco cells (Nicotiana tabacum L. cv. Bright Yellow T-13) with a yield of 7%. Purification to apparent homogeneity, as judged by SDS-PAGE, was achieved by sequential anion-exchange chromatography, hydroxyapatite chromatography, gel filtration, a second round of anion-exchange chromatography, and affinity chromatography on UDP-glucuronic acid agarose. The purified enzyme had a pH optimum of 7.5, an isoelectric point (pI) of 5.0, and a molecular mass of 49 kDa. The enzyme did not require metal cofactors for activity. Its activity was inhibited by Zn(2+), Co(2+) and Cu(2+) ions, as well as by SH-blocking reagents. The K(m) values for UDP-glucose, scopoletin and esculetin were 43, 150 and 25 µM, respectively. A study of the initial rate of the reaction suggested that the reaction proceeded via a sequential mechanism. The purified enzyme preferred hydroxycoumarins as substrates but also exhibited significant activity with flavonoids. A database search using the amino terminus amino acid sequence of CGTase revealed strong homology to the amino acid sequences of other glucosyltransferases in plants.

Exogenously added naphthols induce three glucosyltransferases, and are accumulated as glucosides in tobacco cells

Abstract Plants detoxify and accumulate several compounds as glucosides. In this work, detoxification of the exogenously added harmful compound naphthol in tobacco cells (Nicotiana tabacum L. Bright Yellow) was studied. When 250 μM of 1-naphthol or 2-naphthol was added to the tobacco cells, most of the naphthol was accumulated in the cell as glucosides and in further modified forms. The glucosylation activities against naphthols were increased in proportion to the concentration of naphthols in the culture medium. Addition of 1 mM naphthols caused cell death. Three glucosyltransferase genes, namely NtGT1a, NtGT1b and NtGT3 were isolated and characterized. The recombinant enzymes encoded by these genes showed glucosylation activity against naphthols and other phenolic compounds. It was also shown that these genes were induced following the addition of naphthols to the tobacco cells. These results suggest that naphthols are metabolized by glucosyltransferases whose production is inducible by naphthol itself.

Plant hormone regulation on scopoletin metabolism from culture medium into tobacco cells.

Tobacco (Nicotiana tabacum L. Bright Yellow) T-13 cell line has an ability for production of scopoletin. In this cell culture, scopoletin is taken up from culture medium and accumulated in vacuoles after conversion to scopolin when cells are treated with 2,4-dichlorophenoxyacetic acid (2,4-D) (Taguchi et al. (2000)). To clarify the effect of 2,4-D on tobacco cells, its interaction with several other plant hormones was investigated. Other auxins also stimulated the uptake in the same manner as 2,4-D did, although higher concentrations were required than that of 2,4-D. When p-chlorophenoxyisobutyric acid (PCIB), an antiauxin, was added to the cell culture before 2,4-D, it inhibited 2,4-D-stimulated scopoletin uptake. This result suggests that the stimulation of scopoletin uptake was one of the auxin effects on tobacco cells. Among other classes of plant hormones that were tested, only salicylic acid stimulated the uptake. When these hormones were added to the cell cultures before 2,4-D, methyl jasmonate and kinetin reduced scopoletin uptake. These results suggest that this scopoletin uptake by tobacco cells is regulated by the interaction between different plant hormones.

Analysis of a change in bacterial community in different environments with addition of chitin or chitosan

The temporal changes of a bacterial community in soil with chitin or chitosan added were analyzed by PCR-denaturing gradient gel electrophoresis (DGGE) targeting the 16S rRNA gene using total DNAs prepared from the community. Band patterns of PCR-DGGE confirmed that 31 species become predominant after the addition of chitin or chitosan. The determination of the nucleotide sequences of the bands of the 31 species indicated that 20 species belonged to the division Proteobacteria, and that the genus Cellvibrio was apparently predominant among them (7/20). The 16S rRNA sequences of the 16 deduced species (16/31) showed less than 98% similarities to those of previously identified bacteria, indicating that the species were derived from unidentified bacteria. The total community DNAs extracted from bacterial cells adsorbed on the surface of flakes of chitin and chitosan placed in a river, a moat, or soil were subjected to PCR-DGGE to examine the extent of diversity of chitinolytic bacteria among different environments. The predominant species significantly differed between the chitin and chitosan placed in the river and moat, but not so much between those placed in the soil. The large difference between the diversities of the three bacterial communities indicated that a wide variety of bacteria including unidentified ones are involved in the degradation of chitin and chitosan in the above-mentioned natural environments.