Showing papers by "Shin-ichi Ayabe published in 2005"

Molecular and biochemical characterization of 2-hydroxyisoflavanone dehydratase. Involvement of carboxylesterase-like proteins in leguminous isoflavone biosynthesis.

Abstract: Isoflavonoids are ecophysiologically active secondary metabolites of the Leguminosae and known for health-promoting phytoestrogenic functions. Isoflavones are synthesized by 1,2-elimination of water from 2-hydroxyisoflavanones, the first intermediate with the isoflavonoid skeleton, but details of this dehydration have been unclear. We screened the extracts of repeatedly fractionated Escherichia coli expressing a Glycyrrhiza echinata cDNA library for the activity to convert a radiolabeled precursor into formononetin (7-hydroxy-4′-methoxyisoflavone), and a clone of 2-hydroxyisoflavanone dehydratase (HID) was isolated. Another HID cDNA was cloned from soybean (Glycine max), based on the sequence information in its expressed sequence tag library. Kinetic studies revealed that G. echinata HID is specific to 2,7-dihydroxy-4′-methoxyisoflavanone, while soybean HID has broader specificity to both 4′-hydroxylated and 4′-methoxylated 2-hydroxyisoflavanones, reflecting the structures of isoflavones contained in each plant species. Strikingly, HID proteins were members of a large carboxylesterase family, of which plant proteins form a monophyletic group and some are assigned defensive functions with no intrinsic catalytic activities identified. Site-directed mutagenesis with soybean HID protein suggested that the characteristic oxyanion hole and catalytic triad are essential for the dehydratase as well as the faint esterase activities. The findings, to our knowledge, represent a new example of recruitment of enzymes of primary metabolism during the molecular evolution of plant secondary metabolism.

A comprehensive analysis of six dihydroflavonol 4-reductases encoded by a gene cluster of the Lotus japonicus genome

Abstract: Dihydroflavonol 4-reductase (DFR) is the first committed enzyme of the anthocyanin and condensed tannin pathways. Several DFR cDNAs have been cloned, and different specificities of DFR isozymes in the substrate hydroxylation patterns have been reported, but only fragmentary knowledge of DFR gene organization is available. Reported here is a comprehensive analysis of DFRs of a model legume, Lotus japonicus. A total of five DFR genes were found to form a cluster within a 38 kb region in the L. japonicus genome, whereas six cDNAs, including two splicing variants resulting from a transversion at a splicing acceptor site, were cloned. All the genes were expressed, with different organ specificities, in the mature plant. Three of the DFR proteins heterologously expressed in Escherichia coli showed catalytic activity, and their substrate preferences agreed with the variation of a specific active site residue (Asp or Asn) reported to control the specificity. The hydroxylation patterns of anthocyanidins and condensed tannin units in the stems did not reflect the substrate specificity of the expressed isozymes, implying complex regulation mechanisms in the biosynthesis. The two splicing variants and one DFR with Ser at the specificity-controlling position failed to show the activity, but a revertant protein replacing the unusual splicing restored the activity. The phylogenetic tree, constructed with known DFR sequences, showed evolutionary divergence of some of the DFR genes prior to the plant speciation. This work affords the basis for genetic and biochemical studies on the diversity of DFR and the flavonoid products.

Suppression of root nodule formation by artificial expression of the TrEnodDR1 (coat protein of White clover cryptic virus 1) gene in Lotus japonicus.

Abstract: TrEnodDR1 (Trifolium repens early nodulin downregulation 1) encodes a coat protein of White clover cryptic virus 1. Its expression in white clover was down-regulated at the time when root nodules formed. We surmised that its artificial expression would interfere with root nodulation. Therefore, we investigated the effects of its artificial expression on the growth and root nodulation of Lotus japonicus (a model legume). Transformants were prepared by Agrobacterium spp.-mediated transformation. The growth of transformants was reduced and the number of root nodules per unit root length was greatly decreased relative to control. The concentration of endogenous abscisic acid (ABA), which controls nodulation, increased in plants containing TrEnodDR1. These phenotypes clearly were canceled by treatment with abamine, a specific inhibitor of ABA biosynthesis. The increase in endogenous ABA concentration explained the reduced stomatal aperture and the deformation of root hairs in response to inoculation of transgenic L. japonicus with Mesorhizobium loti. Transcriptome comparison between TrEnodDR1 transformants and control plants showed clearly enhanced expression levels of various defense response genes in transformants. These findings suggest that TrEnodDR1 suppresses nodulation by increasing the endogenous ABA concentration, perhaps by activating the plant's innate immune response. This is the first report of the suppression of nodulation by the artificial expression of a virus coat protein gene.

Activation tagging approach in a model legume, Lotus japonicus.

Abstract: We constructed T-DNA insertional lines of a model legume, Lotus japonicus, using a multifunctional vector for gene and exon activation tagging. The vector had the CaMV 35S promoter together with two additional enhancer elements, the start codon, and splice donor and acceptor sites facing the left border, in anticipation of the activation of T-DNA flanking genes and forced expression of flanking exons. The improved transformation technique yielded more than 3,500 lines, including 45 dominant mutant candidates with abnormal phenotypes with respect to aerial parts, nodules, and roots. Among the 44 selected lines, one copy of T-DNA was inserted into the genome of 37 lines (84%). The T-DNA flanking regions of seven lines were isolated by thermal asymmetric interlaced (TAIL)-PCR or reverse transcription (RT)-PCR, and the corresponding genomic clones were analyzed. The transcripts of four genes adjacent to T-DNA out of 11 genes tested were increased in the T(1) generation, demonstrating that gene and exon activation effects by the newly developed tagging vector are heritable. The T-DNA insertional population of L. japonicus will provide legume-specific dominant mutants.

Isoflavonoid production by adventitious-root cultures of Iris germanica (Iridaceae)

Abstract: We established the adventitious-root cultures of Iris germanica, a monocotyledonous plant known for isoflavonoid production. Irigenin (5,7,3′-trihydroxy-6,4′,5′-trimethoxyisoflavone) and iristectorigenin A (5,7,3′-trihydroxy-6,4′-dimethoxyisoflavone) along with their 7-O-β-D-glucosides, iridin and iristectorin A, respectively, were found as the major components in adventitious roots in the liquid medium, and the total isoflavone content was about 3.6 µmol per g fresh weight in 3-week-old cultures, which was much higher than the 0.7 µmol per g dry weight in the rhizome previously reported (Ali et al. 1983 Phytochemistry 22: 2061). Abiotic stress was applied by addition of 3 mM cupric chloride (CuCl2) to the liquid medium. The isoflavone glucoside content was increased during the initial 6 h of CuCl2 treatment, which was followed by a decrease; the aglycone level continued to increase throughout the 48 h of treatment. The decrease in glucoside content was negatively correlated with the increase in aglycone content between 6 and 48 h. The total isoflavone content (glucosides+aglycones) at 6 h after the start of CuCl2 treatment was 1.4-fold the initial value, and nearly the same content was maintained for 48 h. Thus, the main effect of CuCl2 treatment appeared to be the induction of hydrolysis of isoflavone glucosides. A cDNA of chalcone synthase was cloned, and the mRNA was expressed in the culture producing isoflavones.