Summary Several recent studies describe the influence of the gut microbiota on host brain and behavior. However, the mechanisms responsible for microbiota-nervous system interactions are unknown. Using a combination of genetics, biochemistry, and crystallography, we identify and characterize two phylogenetically distinct enzymes found in the human microbiome that decarboxylate tryptophan to form the β-arylamine neurotransmitter tryptamine. Although this enzymatic activity is exceedingly rare among bacteria more broadly, analysis of the Human Microbiome Project data demonstrates that at least 10% of the human population harbors at least one bacterium encoding a tryptophan decarboxylase in their gut community. Our results uncover a previously unrecognized enzymatic activity that can give rise to host-modulatory compounds and suggests a potential direct mechanism by which gut microbiota can influence host physiology, including behavior.

SummaryThe 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 highperformance 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.

Hexaploid wheat (Triticum aestivum) accumulates benzoxazinones (Bxs) as defensive compounds. Previously, we found that five Bx biosynthetic genes, TaBx1-TaBx5, are located on each of the three genomes (A, B, and D) of hexaploid wheat. In this study, we isolated three homoeologous cDNAs of each TaBx gene to estimate the contribution of individual homoeologous TaBx genes to the biosynthesis of Bxs in hexaploid wheat. We analyzed their transcript levels by homoeolog-or genome-specific quantitative RT-PCR and the catalytic properties of their translation products by kinetic analyses using recombinant TaBX enzymes. The three homoeologs were transcribed differentially, and the ratio of the individual homoeologous transcripts to total homoeologous transcripts also varied with the tissue, i.e., shoots or roots, as well as with the developmental stage. Moreover, the translation products of the three homoeologs had different catalytic properties. Some TaBx homoeologs were efficiently transcribed, but the translation products showed only weak enzymatic activities, which inferred their weak contribution to Bx biosynthesis. Considering the transcript levels and the catalytic properties collectively, we concluded that the homoeologs on the B genome generally contributed the most to the Bx biosynthesis in hexaploid wheat, especially in shoots. In tetraploid wheat and the three diploid progenitors of hexaploid wheat, the respective transcript levels of the TaBx homoeologs were similar in ratio to those observed in hexaploid wheat. This result indicates that the genomic bias in the transcription of the TaBx genes in hexaploid wheat originated in the diploid progenitors and has been retained through the polyploidization.biosynthetic genes ͉ homoeolog ͉ polyploidization

Hydroxycinnamic acid amides (HCAAs) are secondary metabolites involved in the defense of plants against pathogens. Here, we report the first identification of HCAAs, p-coumaroylagmatine, feruloylagmatine, p-coumaroylputrescine and feruloylputrescine, in Arabidopsis thaliana rosette leaves infected with Alternaria brassicicola and the assignment of At5g61160 as the agmatine coumaroyltransferase (AtACT) that catalyzes the last reaction in the biosynthesis of the HCAAs. Feeding experiments with putative labeled precursors revealed that the four HCAAs were synthesized from hydroxycinnamic acids and agmatine or putrescine. AtACT gene function was identified from an analysis of a mutant that did not accumulate HCAAs. In wild-type Arabidopsis, AtACT transcripts markedly increased in response to A. brassicicola infection. Enzymatic activity that catalyzes the synthesis of the HCAAs was confirmed in vitro by using a recombinant AtACT expressed in Escherichia coli. The Atact mutant was susceptible to infection by A. brassicicola, indicating that HCAAs are responsible for defense against pathogens in A. thaliana.

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Gramineous plants, including the major agricultural crops wheat (Triticum aestivum L.), rye (Secale cereale L.) and maize (Zea mays L.), accumulate benzoxazinones (Bxs) as defensive compounds. Previously, we isolated cDNAs of the Bx biosynthetic genes in wheat, TaBx2- TaBx5, that encode the enzymes catalyzing the sequential hydroxylation of indole to Bxs. In this study we isolated a cDNA of TaBx1, which encodes the first enzyme of the Bx pathway of wheat. The level of identity (80%) in deduced amino-acid sequence between TaBx1 and the corresponding maize gene Bx1 was as high as those shown between TaBx2- TaBx5 and the corresponding maize genes Bx2- Bx5, respectively. Southern blot analysis using the TaBx1- TaBx5 cDNAs as probes was conducted with aneuploid lines of hexaploid wheat in order to determine sub-chromosomal locations of the five Bx biosynthetic genes in Triticeae species. In wheat, TaBx1 and TaBx2 co-existed in specific regions of chromosomes 4A, 4B and 4D, and TaBx3- TaBx5 were localized together in the distal regions of the short arms of chromosomes 5A, 5B and 5D. TaBx3 and TaBx5 were found to have duplicated loci in the long arm and the short arm of chromosome 5B, respectively. In rye, homoeoloci of TaBx1 and TaBx2 were located on chromosome 7R and those for TaBx3- TaBx5 were located on chromosome 5R. In barley, no Southern blot band was detected with any of the probes under the highly stringent hybridization conditions, suggesting that the non-Bx phenotype of barley is attributable to the loss of Bx biosynthetic genes.

The inhibitor binding domain in bovine complex I is believed to be constructed by multisubunits, but it remains to be learned how the binding positions of chemically diverse inhibitors relate to each other. To get insight into the inhibitor binding domain in complex I, we synthesized a photoreactive acetogenin [[125I](trifluoromethyl)phenyldiazirinylacetogenin, [125I]TDA], in which an aryldiazirine group serves as both a photoreactive group and a substitute for the gamma-lactone ring that is a common toxophore of numerous natural acetogenins, and carried out photoaffinity labeling to identify the labeled subunit using bovine heart submitochondrial particles (SMP). When SMP were UV-irradiated in the presence of [125I]TDA, radioactivity was predominantly incorporated into an approximately 30 kDa band on a SDS gel. Blue native gel electrophoresis of the [125I]TDA-labeled SMP revealed that the majority of radioactivity was observed in complex I. Analysis of complex I on a SDS gel showed a predominant peak of radioactivity at approximately 30 kDa. Immnoprecipitation of the [125I]TDA-labeled complex I with anti-bovine ND1 antibody indicated that the labeled protein is the ND1 subunit. A variety of complex I inhibitors such as piericidin A and rotenone efficiently suppressed the specific binding of [125I]TDA to ND1, indicating that they share a common binding domain. However, the suppression efficiency of Deltalac-acetogenin, a new type of complex I inhibitor synthesized in our laboratory, was much lower than that of the traditional inhibitors. Our results unequivocally reveal that the ND1 subunit constructs the inhibitor binding domain, though the contribution of this subunit has been challenged. Further, the present study corroborates our previous proposition that the inhibition site of Deltalac-acetogenins differs from that of traditional inhibitors.

Two oat genes encoding hydroxycinnamoyl-CoA:hydroxyanthranilate N-hydroxycinnamoyltransferase (HHT) and S-adenosyl-L-methionine:trans-caffeoyl-CoA 3-O-methyltransferase (CCoAOMT), both of which are possibly involved in the biosynthesis of oat avenanthramide phytoalexins, were cloned and their expression profiles in response to biological stress were studied. Four distinct cDNAs of oat HHT (AsHHT1-4) were isolated with the degenerative polymerase chain reaction method. The enzymatic activity of AsHHT1 expressed in E. coli was found using hydroxyanthranilate and hydroxycinnamoyl-CoAs as cosubstrates. Cloned oat CCoAOMT (AsCCoAOMT) encoded a polypeptide of 130 amino acid residues with 77.7 to 80.8% identities to the CCoAOMT sequences from other plant species. The accumulation of AsHHT1 and AsCCoAOMT transcripts increased concomitantly with phytoalexin accumulation by the treatment of victorin, a specific elicitor in oat lines carrying the Pc-2/Vb gene. Pharmacological approaches indicated the involvement of Ca2+, NO, and protein kinases in the signaling pathways of AsHHT1 and AsCCoAOMT mRNA induction. When oat leaves were inoculated with Puccinia coronata, the mRNA expression of AsHHT1 and AsCCOAOMT increased in both incompatible and compatible interactions but more rapidly in incompatible interaction. Interestingly, however, significant phytoalexin accumulation was observed only in incompatible interaction during the experimental period, suggesting that phytoalexin accumulation may be inhibited in one or more posttranscriptional processes in the compatible interaction.