Phenolic Acids Induce Nod Factor Production in Lotus japonicus-Mesorhizobium Symbiosis.

In legume-rhizobia symbiosis, partner recognition and the initiation of symbiosis processes require the mutual exchange of chemical signals. Chemicals, generally (iso)flavonoids, in the root exudates of the host plant induce the expression of nod genes in rhizobia, and, thus, are called nod gene inducers. The expression of nod genes leads to the production of lipochitooligosaccharides (LCOs) called Nod factors. Natural nod gene inducer(s) in Lotus japonicus-Mesorhizobium symbiosis remain unknown. Therefore, we developed an LCO detection method based on ultra-high-performance liquid chromatography-tandem-quadrupole mass spectrometry (UPLC-TQMS) to identify these inducers and used it herein to screen 40 phenolic compounds and aldonic acids for their ability to induce LCOs in Mesorhizobium japonicum MAFF303099. We identified five phenolic acids with LCO-inducing activities, including p-coumaric, caffeic, and ferulic acids. The induced LCOs caused root hair deformation, and nodule numbers in L. japonicus inoculated with M. japonicum were increased by these phenolic acids. The three phenolic acids listed above induced the expression of the nodA, nodB, and ttsI genes in a strain harboring a multicopy plasmid encoding NodD1, but not that encoding NodD2. The presence of p-coumaric and ferulic acids in the root exudates of L. japonicus was confirmed by UPLC-TQMS, and the induction of ttsI::lacZ in the strain harboring the nodD1 plasmid was detected in the rhizosphere of L. japonicus. Based on these results, we propose that phenolic acids are a novel type of nod gene inducer in L. japonicus-Mesorhizobium symbiosis.

Mutants of Lotus japonicus deficient in flavonoid biosynthesis.

Spatiotemporal features of anthocyanin accumulation in a model legume Lotus japonicus (Regel) K.Larsen were elucidated to develop criteria for the genetic analysis of flavonoid biosynthesis. Artificial mutants and wild accessions, with lower anthocyanin accumulation in the stem than the standard wild type (B-129 'Gifu'), were obtained by ethyl methanesulfonate (EMS) mutagenesis and from a collection of wild-grown variants, respectively. The loci responsible for the green stem of the mutants were named as VIRIDICAULIS (VIC). Genetic and chemical analysis identified two loci, namely, VIC1 and VIC2, required for the production of both anthocyanins and proanthocyanidins (condensed tannins), and two loci, namely, VIC3 and VIC4, required for the steps specific to anthocyanin biosynthesis. A mutation in VIC5 significantly reduced the anthocyanin accumulation. These mutants will serve as a useful system for examining the effects of anthocyanins and proanthocyanidins on the interactions with herbivorous pests, pathogenic microorganisms and nitrogen-fixing symbiotic bacteria, Mesorhizobium loti.

Identification of protein-protein interactions of isoflavonoid biosynthetic enzymes with 2-hydroxyisoflavanone synthase in soybean (Glycine max (L.) Merr.).

Metabolic enzymes, including those involved in flavonoid biosynthesis, are proposed to form weakly bound, ordered protein complexes, called "metabolons". Some hypothetical models of flavonoid biosynthetic metabolons have been proposed, in which metabolic enzymes are believed to anchor to the cytoplasmic surface of the endoplasmic reticulum (ER) via ER-bound cytochrome P450 isozymes (P450s). However, no convincing evidence for the interaction of flavonoid biosynthetic enzymes with P450s has been reported previously. Here, we analyzed binary protein-protein interactions of 2-hydroxyisoflavanone synthase 1 (GmIFS1), a P450 (CYP93C), with cytoplasmic enzymes involved in isoflavone biosynthesis in soybean. We identified binary interactions between GmIFS1 and chalcone synthase 1 (GmCHS1) and between GmIFS1 and chalcone isomerases (GmCHIs) by using a split-ubiquitin membrane yeast two-hybrid system. These binary interactions were confirmed in planta by means of bimolecular fluorescence complementation (BiFC) using tobacco leaf cells. In these BiFC analyses, fluorescence signals that arose from the interaction of these cytoplasmic enzymes with GmIFS1 generated sharp, network-like intracellular patterns, which was very similar to the ER-localized fluorescence patterns of GmIFS1 labeled with a fluorescent protein. These observations provide strong evidence that, in planta, interaction of GmCHS1 and GmCHIs with GmIFS1 takes place on ER on which GmIFS1 is located, and also provide important clues to understand how enzymes and proteins form metabolons to establish efficient metabolic flux of (iso)flavonoid biosynthesis.

Discriminative phytoalexin accumulation in Lotus japonicus against symbiotic and non-symbiotic microorganisms and related chemical signals.

The phytoalexin response of Lotus japonicus seedlings to selected microbes and chemical signals was analyzed. The symbiotic rhizobium induced vestitol production weakly, while non-symbiotic rhizobia and potential pathogens led to increases in its accumulation. Whereas chitin-related molecules were ineffective, a flagellin-derived peptide not of symbiont origin induced phytoalexin production, indicating discriminative antibiotic production by the plant host.

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.

Molecular characterization of an oxidosqualene cyclase that yields shionone, a unique tetracyclic triterpene ketone of Aster tataricus.

Shionone is the major triterpenoid component of Aster tataricus possessing a unique all six-membered tetracyclic skeleton and 3-oxo-4-monomethyl structure. To clarify its biosynthetic process, an oxidosqualene cyclase cDNA was isolated from A. tataricus, and the function of the enzyme was determined in lanosterol synthase-deficient yeast. The cyclase yielded ca. 90% shionone and small amounts of ß-amyrin, friedelin, dammara-20,24-dienol, and 4-epishionone and was designated as a shionone synthase (SHS). Transcripts of SHS were detected in A. tataricus organs, confirming its involvement in shionone biosynthesis. SHS was shown to have evolved in the Asteraceae from ß-amyrin synthase lineages and acquired characteristic species- and product-specificities.

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.

Molecular cloning and characterization of a cDNA for pterocarpan 4-dimethylallyltransferase catalyzing the key prenylation step in the biosynthesis of glyceollin, a soybean phytoalexin.

Glyceollins are soybean (Glycine max) phytoalexins possessing pterocarpanoid skeletons with cyclic ether decoration originating from a C5 prenyl moiety. Enzymes involved in glyceollin biosynthesis have been thoroughly characterized during the early era of modern plant biochemistry, and many genes encoding enzymes of isoflavonoid biosynthesis have been cloned, but some genes for later biosynthetic steps are still unidentified. In particular, the prenyltransferase responsible for the addition of the dimethylallyl chain to pterocarpan has drawn a large amount of attention from many researchers due to the crucial coupling process of the polyphenol core and isoprenoid moiety. This study narrowed down the candidate genes to three soybean expressed sequence tag sequences homologous to genes encoding homogentisate phytyltransferase of the tocopherol biosynthetic pathway and identified among them a cDNA encoding dimethylallyl diphosphate: (6aS, 11aS)-3,9,6a-trihydroxypterocarpan [(-)-glycinol] 4-dimethylallyltransferase (G4DT) yielding the direct precursor of glyceollin I. The full-length cDNA encoding a protein led by a plastid targeting signal sequence was isolated from young soybean seedlings, and the catalytic function of the gene product was verified using recombinant yeast microsomes. Expression of the G4DT gene was strongly up-regulated in 5 to 24 h after elicitation of phytoalexin biosynthesis in cultured soybean cells similarly to genes associated with isoflavonoid pathway. The prenyl part of glyceollin I was demonstrated to originate from the methylerythritol pathway by a tracer experiment using [1-(13)C]Glc and nuclear magnetic resonance measurement, which coincided with the presumed plastid localization of G4DT. The first identification of a pterocarpan-specific prenyltransferase provides new insights into plant secondary metabolism and in particular those reactions involved in the disease resistance mechanism of soybean as the penultimate gene of glyceollin biosynthesis.

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.

2-Hydroxyisoflavanone dehydratase is a critical determinant of isoflavone productivity in hairy root cultures of Lotus japonicus.

Hairy root cultures of a model legume, Lotus japonicus, were established to characterize two heterologous cDNAs encoding enzymes involved in isoflavone biosynthesis, i.e. licorice 2-hydroxyisoflavanone synthase (IFS) and soybean 2-hydroxyisoflavanone dehydratase (HID) catalyzing sequential reactions to yield isoflavones. While the control and the IFS overexpressor did not accumulate detectable isoflavones, the HID overexpressors did accumulate daidzein and genistein, showing that HID is a critical determinant of isoflavone productivity. Production of coumestrol in all the genotypes and isoliquiritigenin/liquiritigenin in IFS + HID-overexpressing lines was also noted. These results provide insight into the regulatory mechanism that controls isoflavonoid biosynthesis.

Genome-wide analyses of the structural gene families involved in the legume-specific 5-deoxyisoflavonoid biosynthesis of Lotus japonicus.

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.

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 F(2) 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.

Catalytic specificity of pea O-methyltransferases suggests gene duplication for (+)-pisatin biosynthesis.

S-adenosyl-l-methionine: 2-hydroxyisoflavanone 4'-O-methyltransferase (HI4'OMT) methylates 2,7, 4'-trihydroxyisoflavanone to produce formononetin, an essential intermediate in the synthesis of isoflavonoids with methoxy or methylenedioxy groups at carbon 4' (isoflavone numbering). HI4'OMT is highly similar (83% amino acid identity) to (+)-6a-hydroxymaackiain 3-O-methyltransferase (HMM), which catalyzes the last step of (+)-pisatin biosynthesis in pea. Pea contains two linked copies of HMM with 96% amino acid identity. In this report, the catalytic activities of the licorice HI4'OMT protein and of extracts of Escherichia coli containing the pea HMM1 or HMM2 protein are compared on 2,7,4'-trihydroxyisoflavanone and enantiomers of 6a-hydroxymaackiain. All these enzymes produced radiolabelled 2,7-dihydroxy-4'-methoxyisoflavanone or (+)-pisatin from 2,7,4'-trihydroxyisoflavanone or (+)-6a-hydroxymaakiain when incubated with [methyl-(14)C]-S-adenosyl-l-methionine. No product was detected when (-)-6a-hydroxymaackiain was used as the substrate. HI4'OMT and HMM1 showed efficiencies (relative V(max)/K(m)) for the methylation of 2,7,4'-trihydroxyisoflavanone 20 and 4 times higher than for the methylation of (+)-6a-hydroxymaackiain, respectively. In contrast, HMM2 had a higher V(max) and lower K(m) on (+)-6a-hydroxymaackiain, and had a 67-fold higher efficiency for the methylation of (+)-6a-hydroxymaackiain than that for 2,7,4'-trihydroxyisoflavanone. Among the 15 sites at which HMM1 and HMM2 have different amino acid residues, 11 of the residues in HMM1 are the same as found in HI4'OMTs from three plant species. Modeling of the HMM proteins identified three or four putative active site residues responsible for their different substrate preferences. It is proposed that HMM1 is the pea HI4'OMT and that HMM2 evolved by the duplication of a gene encoding a general biosynthetic enzyme (HI4'OMT).

Identification of cDNAs encoding pterocarpan reductase involved in isoflavan phytoalexin biosynthesis in Lotus japonicus by EST mining.

Isoflavans and pterocarpans are the major biosynthetically connected phytoalexins in legumes. A search of the expressed sequence tag library of a model legume Lotus japonicus, which produces an (-)-isoflavan, for homologs of phenylcoumaran benzylic ether reductase catalyzing the reductive cleavage of dihydrofurans, yielded seven full-length cDNAs, and the encoded proteins were analyzed in vitro. Four of them cleaved the dihydrofuran of a pterocarpan medicarpin to yield an isoflavan (-)-vestitol and were designated pterocarpan reductase (PTR). Two PTRs displayed enantiospecificity to (-)-medicarpin, representing genuine L. japonicus PTRs, while the other two lacked enantiospecificity and were presumed to be evolutionarily primitive types.

Expression of the 1-aminocyclopropane-1-carboxylic acid deaminase gene requires symbiotic nitrogen-fixing regulator gene nifA2 in Mesorhizobium loti MAFF303099.

Many soil bacteria contain 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase, which degrades ACC, a precursor of the phytohormone ethylene. In order to examine the regulation of the acdS gene encoding ACC deaminase in Mesorhizobium loti MAFF303099 during symbiosis with the host legume Lotus japonicus, we introduced the beta-glucuronidase (GUS) gene into acdS so that GUS was expressed under control of the acdS promoter, and we also generated disruption mutants with mutations in a nitrogen fixation regulator gene, nifA. The histochemical GUS assay showed that there was exclusive expression of acdS in mature root nodules. Two homologous nifA genes, mll5857 and mll5837, were found in the symbiosis island of M. loti and were designated nifA1 and nifA2, respectively. Quantitative reverse transcription-PCR demonstrated that nifA2 disruption resulted in considerably diminished expression of acdS, nifH, and nifA1 in bacteroid cells. In contrast, nifA1 disruption slightly enhanced expression of the acdS transcripts and suppressed nifH to some extent. These results indicate that the acdS gene and other symbiotic genes are positively regulated by the NifA2 protein, but not by the NifA1 protein, in M. loti. The mode of gene expression suggests that M. loti acdS participates in the establishment and/or maintenance of mature nodules by interfering with the production of ethylene, which induces negative regulation of nodulation.

Plant lanosterol synthase: divergence of the sterol and triterpene biosynthetic pathways in eukaryotes.

Sterols, essential eukaryotic constituents, are biosynthesized through either cyclic triterpenes, lanosterol (fungi and animals) or cycloartenol (plants). The cDNA for OSC7 of Lotus japonicus was shown to encode lanosterol synthase (LAS) by the complementation of a LAS-deficient mutant yeast and structural identification of the accumulated lanosterol. A double site-directed mutant of OSC7, in which amino acid residues crucial for the reaction specificity were changed to the cycloartenol synthase (CAS) type, produced parkeol and cycloartenol. The multiple amino acid sequence alignment of a conserved region suggests that the LAS of different eukaryotic lineages emerged from the ancestral CAS by convergent evolution.

Activation tagging approach in a model legume, Lotus japonicus.

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.

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

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.

Morphometric evaluation of posterior funiculus nerve fibers in relation to aging.

We morphologically evaluated the size of axons in the posterior funiculus in different age groups and examined the changes due to aging. In the past, such studies have been conducted at the cervical spinal cord (C6) level, and a decrease in the size and number of axons due to aging has been noted. The current study was conducted at the lower lumbar spinal cord (L2) level.