Norimoto Shimada

A Cluster of Genes Encodes the Two Types of Chalcone Isomerase Involved in the Biosynthesis of General Flavonoids and Legume-Specific 5-Deoxy(iso)flavonoids in Lotus japonicus

Leguminous plants produce 5-deoxyflavonoids and 5-deoxyisoflavonoids that play essential roles in legume-microbe interactions. Together with chalcone polyketide reductase and cytochrome P450 2-hydroxyisoflavanone synthase, the chalcone isomerase (CHI) of leguminous plants is fundamental in the construction of these ecophysiologically active flavonoids. Although CHIs of nonleguminous plants isomerize only 6′-hydroxychalcone to 5-hydroxyflavanone (CHIs with this function are referred to as type I), leguminous CHIs convert both 6′-deoxychalcone and 6′-hydroxychalcone to 5-deoxyflavanone and 5-hydroxyflavanone, respectively (referred to as type II). In this study, we isolated multiple CHI cDNAs (cCHI1–cCHI3) from a model legume,Lotus japonicus. In contrast to previous observations, the amino acid sequence of CHI2 was highly homologous to nonleguminous CHIs, whereas CHI1 and CHI3 were the conventional leguminous type. Furthermore, genome sequence analysis revealed that fourCHI genes (CHI1–3 and a putative gene, CHI4) form a tandem cluster within 15 kb. Biochemical analysis with recombinant CHIs expressed inEscherichia coli confirmed that CHI1 and CHI3 are type II CHIs and that CHI2 is a type I CHI. The occurrence of both types of CHIs is probably common in leguminous plants, and it was suggested that type II CHIs evolved from an ancestral CHI by gene duplication and began to produce 5-deoxy(iso)flavonoids along with the establishment of the Fabaceae.

A single-base deletion in soybean flavonol synthase gene is associated with magenta flower color

The Wm locus of soybean [Glycine max (L.) Merr.] controls flower color. Dominant Wm and recessive wm allele of the locus produce purple and magenta flower, respectively. A putative full-length cDNA of flavonol synthase (FLS), gmfls1 was isolated by 5′ RACE and end-to-end PCR from a cultivar Harosoy with purple flower (WmWm). Sequence analysis revealed that gmfls1 consisted of 1,208 nucleotides encoding 334 amino acids. It had 59–72% homology with FLS proteins of other plant species. Conserved dioxygenase domains A and B were found in the deduced polypeptide. Sequence comparison between Harosoy and Harosoy-wm (magenta flower mutant of Harosoy; wmwm) revealed that they differed by a single G deletion in the coding region of Harosoy-wm. The deletion changed the subsequent reading frame resulting in a truncated polypeptide consisting of 37 amino acids that lacked the dioxygenase domains A and B. Extracts of E. coli cells expressing gmfls1 of Harosoy catalyzed the formation of quercetin from dihydroquercetin, whereas cell extracts expressing gmfls1 of Harosoy-wm had no FLS activity. Genomic Southern analysis suggested the existence of three to four copies of the FLS gene in the soybean genome. CAPS analysis was performed to detect the single-base deletion. Harosoy and Clark (WmWm) exhibited longer fragments, while Harosoy-wm had shorter fragments due to the single-base deletion. The CAPS marker co-segregated with genotypes at Wm locus in a F2 population segregating for the locus. Linkage mapping using SSR markers revealed that the Wm and gmfls1 were mapped at similar position in the molecular linkage group F. The above results strongly suggest that gmfls1 represents the Wm gene and that the single-base deletion may be responsible for magenta flower color.

Differential gene expression profiles of red and green forms of Perilla frutescens leading to comprehensive identification of anthocyanin biosynthetic genes

Differential screening by PCR‐select subtraction was carried out for cDNAs from leaves of red and green perilla, two chemovarietal forms of Perilla frutescens regarding anthocyanin accumulation. One hundred and twenty cDNA fragments were selected as the clones preferentially expressed in anthocyanin‐accumulating red perilla over the nonaccumulating green perilla. About half of them were the cDNAs encoding the proteins related presumably to phenylpropanoid‐derived metabolism. The cDNAs encoding glutathione S‐transferase (GST), PfGST1, and chalcone isomerase (CHI), PfCHI1, were further characterized. The expression of PfGST1 in an Arabidopsis thaliana tt19 mutant lacking the GST‐like gene involved in vacuole transport of anthocyanin rescued the lesion of anthocyanin accumulation in tt19, indicating a function of PfGST1 in vacuole sequestration of anthocyanin in perilla. The recombinant PfCHI1 could stereospecifically convert naringenin chalcone to (2S)‐naringenin. PfGST1 and PfCHI1 were preferentially expressed in the leaves of red perilla, agreeing with the accumulation of anthocyanin and expression of other previously identified genes for anthocyanin biosynthesis. These results suggest that the genes of the whole anthocyanin biosynthetic pathway are regulated in a coordinated manner in perilla.

Genome-wide Analyses of the Structural Gene Families Involved in the Legume-specific 5-Deoxyisoflavonoid Biosynthesis of Lotus japonicus

Abstract A model legume Lotus japonicus (Regel) K. Larsen is one of the subjects of genome sequencing and functional genomics programs. In the course of targeted approaches to the legume genomics, we analyzed the genes encoding enzymes involved in the biosynthesis of the legume-specific 5-deoxyisoflavonoid of L. japonicus, which produces isoflavan phytoalexins on elicitor treatment. The paralogous biosynthetic genes were assigned as comprehensively as possible by biochemical experiments, similarity searches, comparison of the gene structures, and phylogenetic analyses. Among the 10 biosynthetic genes investigated, six comprise multigene families, and in many cases they form gene clusters in the chromosomes. Semi-quantitative reverse transcriptase–PCR analyses showed coordinate up-regulation of most of the genes during phytoalexin induction and complex accumulation patterns of the transcripts in different organs. Some paralogous genes exhibited similar expression specificities, suggesting their genetic redundancy. The molecular evolution of the biosynthetic genes is discussed. The results presented here provide reliable annotations of the genes and genetic markers for comparative and functional genomics of leguminous plants.

Transcriptional control of the dihydroflavonol 4-reductase multigene family in Lotus japonicus

In the genome of the model legume Lotus japonicus, dihydroflavonol 4-reductase (DFR), which is the first committed enzyme of the anthocyanin and proanthocyanidin (PA) pathways, is encoded as a tandemly arrayed five-gene family. Expression analysis revealed that both organ specificity and stress responsiveness differ among the DFRs. To elucidate the regulatory mechanisms underlying the expression of DFRs, we investigated the transcriptional control of each member of the DFR multigene family. Ectopic expression of a combination of the transcription factors MYB, bHLH, and WDR showed that only the DFR2 promoter was activated, indicating that each member of the DFR gene family is regulated independently.

The soybean F3′H protein is localized to the tonoplast in the seed coat hilum

We previously isolated a soybean (Glycine max (L.) Merr.) flavonoid 3′-hydroxylase (F3′H) gene (sf3′h1) corresponding to the T locus, which controls pubescence and seed coat color, from two near-isogenic lines (NILs), To7B (TT) and To7G (tt). The T allele is also associated with chilling tolerance. Here, Western-blot analysis shows that the sf3′h1 protein was predominantly detected in the hilum and funiculus of the immature seed coat in To7B, whereas sf3′h1 was not detected in To7G. A truncated sf3′h1 protein isolated from To7G was detected only upon enrichment by immunoprecipitation. An analysis using diphenylboric acid 2-aminoethyl ester (DBPA) staining revealed that flavonoids accumulated in the hilum and the funiculus in both To7B and To7G. Further, the scavenging activity of the 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical in methanol extracts from the funiculus and hilum of To7B was higher than that of To7G. Moreover, the enzymatic activity of F3′H was detected using microsomal fractions from yeast transformed with sf3′h1 from To7B, but not from To7G. These results indicate that sf3′h1 is involved in flavonoid biosynthesis in the seed coat and affects the antioxidant properties of those tissues. As shown by immunofluorescence microscopy, the sf3′h1 protein was detected primarily around the vacuole in the parenchymatic cells of the hilum in To7B. Further immunoelectron microscopy detected sf3′h1 protein on the membranous structure of the vacuole. Based on these observations, we conclude that F3′H, which is a cytochrome P450 monooxygenase and has been found to be localized to the ER in other plant systems, is localized in the tonoplast in soybean.

Pathway-specific metabolome analysis with 18O2-labeled Medicago truncatula via a mass spectrometry-based approach

IntroductionOxygen from carbon dioxide, water or molecular oxygen, depending on the responsible enzyme, can lead to a large variety of metabolites through chemical modification.ObjectivesPathway-specific labeling using isotopic molecular oxygen (18O2) makes it possible to determine the origin of oxygen atoms in metabolites and the presence of biosynthetic enzymes (e.g., oxygenases). In this study, we established the basis of 18O2-metabolome analysis.Methods18O2 labeled whole Medicago truncatula seedlings were prepared using 18O2-air and an economical sealed-glass bottle system. Metabolites were analyzed using high-accuracy and high-resolution mass spectrometry. Identification of the metabolite was confirmed by NMR following UHPLC–solid-phase extraction (SPE).ResultsA total of 511 peaks labeled by 18O2 from shoot and 343 peaks from root were annotated by untargeted metabolome analysis. Additionally, we identified a new flavonoid, apigenin 4′-O-[2′-O-coumaroyl-glucuronopyranosyl-(1–2)-O-glucuronopyranoside], that was labeled by 18O2. To the best of our knowledge, this is the first report of apigenin 4′-glucuronide in M. truncatula. Using MSn analysis, we estimated that 18O atoms were specifically incorporated in apigenin, the coumaroyl group, and glucuronic acid. For apigenin, an 18O atom was incorporated in the 4′-hydroxy group. Thus, non-specific incorporation of an 18O atom by recycling during one month of labeling is unlikely compared with the more specific oxygenase-catalyzing reaction.ConclusionOur finding indicated that 18O2 labeling was effective not only for the mining of unknown metabolites which were biosynthesized by oxygenase-related pathway but also for the identification of metabolites whose oxygen atoms were derived from oxygenase activity.