Inventory of ATP-binding cassette proteins in Lithospermum erythrorhizon as a model plant producing divergent secondary metabolites.

ATP-binding cassette (ABC) proteins are the largest membrane transporter family in plants. In addition to transporting organic substances, these proteins function as ion channels and molecular switches. The development of multiple genes encoding ABC proteins has been associated with their various biological roles. Plants utilize many secondary metabolites to adapt to environmental stresses and to communicate with other organisms, with many ABC proteins thought to be involved in metabolite transport. Lithospermum erythrorhizon is regarded as a model plant for studying secondary metabolism, as cells in culture yielded high concentrations of meroterpenes and phenylpropanoids. Analysis of the genome and transcriptomes of L. erythrorhizon showed expression of genes encoding 118 ABC proteins, similar to other plant species. The number of expressed proteins in the half-size ABCA and full-size ABCB subfamilies was ca. 50% lower in L. erythrorhizon than in Arabidopsis, whereas there was no significant difference in the numbers of other expressed ABC proteins. Because many ABCG proteins are involved in the export of organic substances, members of this subfamily may play important roles in the transport of secondary metabolites that are secreted into apoplasts.

Production of raspberry ketone by redirecting the metabolic flux to the phenylpropanoid pathway in tobacco plants.

Raspberry ketone is one of the characteristic flavors of raspberry fruits, and it is an important and expensive ingredient in the flavor and fragrance industries. It is present at low levels in plant tissues, and its occurrence is limited to a few taxa. In this context, the stable production of nature-identical raspberry ketone using heterologous synthesis in plants hosts has recently garnered the attention of plant biochemists. In this study, we demonstrate the rational switching of the metabolic flow from anthocyanin pigments to volatile phenylbutanoid production via the phenylpropanoid pathway. This shift led to the efficient and stable production of raspberry ketone and its glycosides via heterologous expression of the biosynthetic enzymes benzalacetone synthase (BAS) and raspberry ketone/zingerone synthase 1 (RZS1) in the transgenic tobacco (Nicotiana tabacum 'Petit Havana SR-1'). Additionally, we achieved improved product titers by activating the phenylpropanoid pathway with the transcriptional factor, production of anthocyanin pigment 1 (PAP1), from Arabidopsis thaliana. We further demonstrated another metabolic-flow switching by RNA interference (RNAi)-mediated silencing of chalcone synthase (CHS) to increase pathway-intermediate p-coumaroyl-CoA in transgenic tobacco for raspberry-ketone production. The redirection of metabolic flux resulted in transgenic lines producing 0.45 µg/g of raspberry ketone and 4.5 µg/g, on the fresh weight basis, of its glycosides in the flowers. These results suggest that the intracellular enforcement of endogenous substrate supply is an important factor while engineering the phenylpropanoid pathway. This strategy might be useful for the production of other phenylpropanoids/polyketides that are produced via the pathway-intermediate p-coumaroyl-CoA, in tobacco plants.

Tobacco Root Endophytic Arthrobacter Harbors Genomic Features Enabling the Catabolism of Host-Specific Plant Specialized Metabolites.

Plant roots constitute the primary interface between plants and soilborne microorganisms and harbor microbial communities called the root microbiota. Recent studies have demonstrated a significant contribution of plant specialized metabolites (PSMs) to the assembly of root microbiota. However, the mechanistic and evolutionary details underlying the PSM-mediated microbiota assembly and its contribution to host specificity remain elusive. Here, we show that the bacterial genus Arthrobacter is predominant specifically in the tobacco endosphere and that its enrichment in the tobacco endosphere is partially mediated by a combination of two unrelated classes of tobacco-specific PSMs, santhopine and nicotine. We isolated and sequenced Arthrobacter strains from tobacco roots as well as soils treated with these PSMs and identified genomic features, including but not limited to genes for santhopine and nicotine catabolism, that are associated with the ability to colonize tobacco roots. Phylogenomic and comparative analyses suggest that these genes were gained in multiple independent acquisition events, each of which was possibly triggered by adaptation to particular soil environments. Taken together, our findings illustrate a cooperative role of a combination of PSMs in mediating plant species-specific root bacterial microbiota assembly and suggest that the observed interaction between tobacco and Arthrobacter may be a consequence of an ecological fitting process. IMPORTANCE Host secondary metabolites have a crucial effect on the taxonomic composition of its associated microbiota. It is estimated that a single plant species produces hundreds of secondary metabolites; however, whether different classes of metabolites have distinctive or common roles in the microbiota assembly remains unclear. Here, we show that two unrelated classes of secondary metabolites in tobacco play a cooperative role in the formation of tobacco-specific compositions of the root bacterial microbiota, which has been established as a consequence of independent evolutionary events in plants and bacteria triggered by different ecological effects. Our findings illustrate mechanistic and evolutionary aspects of the microbiota assembly that are mediated by an arsenal of plant secondary metabolites.

Comparative Proteomic Analysis of Lithospermum erythrorhizon Reveals Regulation of a Variety of Metabolic Enzymes Leading to Comprehensive Understanding of the Shikonin Biosynthetic Pathway.

Plants produce a large variety of specialized (secondary) metabolites having a wide range of hydrophobicity. Shikonin, a red naphthoquinone pigment, is a highly hydrophobic metabolite produced in the roots of Lithospermum erythrorhizon, a medicinal plant in the family Boraginaceae. The shikonin molecule is formed by the coupling of p-hydroxybenzoic acid and geranyl diphosphate, catalyzed by a membrane-bound geranyltransferase LePGT at the endoplasmic reticulum, followed by cyclization of the geranyl chain and oxidations; the latter half of this biosynthetic pathway, however, has not yet been clarified. To shed light on these steps, a proteome analysis was conducted. Shikonin production in vitro was specifically regulated by illumination and by the difference in media used to culture cells and hairy roots. In intact plants, however, shikonin is produced exclusively in the root bark of L. erythrorhizon. These features were utilized for comparative transcriptome and proteome analyses. As the genome sequence is not known for this medicinal plant, sequences from de novo RNA-seq data with 95,861 contigs were used as reference for proteome analysis. Because shikonin biosynthesis requires copper ions and is sensitive to blue light, this methodology identified strong candidates for enzymes involved in shikonin biosynthesis, such as polyphenol oxidase, cannabidiolic acid synthase-like and neomenthol dehydrogenase-like proteins. Because acetylshikonin is the main end product of shikonin derivatives, an O-acetyltransferase was also identified. This enzyme may be responsible for end product formation in these plant species. Taken together, these findings suggest a putative pathway for shikonin biosynthesis.

Molecular Characterization of LjSWEET3, a Sugar Transporter in Nodules of Lotus japonicus.

Symbiotic nitrogen fixation in legumes contributes greatly to the global nitrogen cycle on the earth. In nodules, resident rhizobia supply nitrogen nutrient fixed from atmospheric N2 to the host plant; in turn, the plant provides photosynthetic metabolites to bacteroids as a carbon source. In this process, various transporters are involved at different membrane systems; however, little is known at the molecular level about the flow of carbon from the host cells to the symbiotic bacteria. We have been studying transporters functioning in nodules of Lotus japonicus, and found that out of 13 SWEET genes in the L. japonicus genome LjSWEET3, a member of the SWEET transporter family, is highly expressed in nodules. The SWEET family was first identified in Arabidopsis, where members of the family are involved in phloem loading, nectar secretion, pollen nutrition and seed filling. The expression of LjSWEET3 strongly increased during nodule development and reached the highest level in mature nodules. Histochemical analysis using L. japonicus plants transformed with LjSWEET3 promoter:GUS (ß-glucuronidase) showed strong expression in the vascular systems of nodules. Analysis of an LjSWEET3-green fluorescent protein (GFP) fusion expressed in Nicotiana banthamiana and Coptis japonica indicates that LjSWEET3 localizes to the plasma membrane. Together these data are consistent with a role for LjSWEET3 in sugar translocation towards nodules and also suggest the possible existence of multiple routes of carbon supply into nodules.

The Full-Size ABCG Transporters Nb-ABCG1 and Nb-ABCG2 Function in Pre- and Postinvasion Defense against Phytophthora infestans in Nicotiana benthamiana.

The sesquiterpenoid capsidiol is the major phytoalexin produced by Nicotiana and Capsicum species. Capsidiol is produced in plant tissues attacked by pathogens and plays a major role in postinvasion defense by inhibiting pathogen growth. Using virus-induced gene silencing-based screening, we identified two Nicotiana benthamiana (wild tobacco) genes encoding functionally redundant full-size ABCG (PDR-type) transporters, Nb-ABCG1/PDR1 and Nb-ABCG2/PDR2, which are essential for resistance to the potato late blight pathogen Phytophthora infestans Silencing of Nb-ABCG1/2 compromised secretion of capsidiol, revealing Nb-ABCG1/2 as probable exporters of capsidiol. Accumulation of plasma membrane-localized Nb-ABCG1 and Nb-ABCG2 was observed at the site of pathogen penetration. Silencing of EAS (encoding 5-epi-aristolochene synthase), a gene for capsidiol biosynthesis, reduced resistance to P. infestans, but penetration by P. infestans was not affected. By contrast, Nb-ABCG1/2-silenced plants showed reduced penetration defense, indicating that Nb-ABCG1/2 are involved in preinvasion defense against P. infestans Plastidic GGPPS1 (geranylgeranyl diphosphate synthase) was also found to be required for preinvasion defense, thereby suggesting that plastid-produced diterpene(s) are the antimicrobial compounds active in preinvasion defense. These findings suggest that N. benthamiana ABCG1/2 are involved in the export of both antimicrobial diterpene(s) for preinvasion defense and capsidiol for postinvasion defense against P. infestans.

Molecular evolution of parsnip (Pastinaca sativa) membrane-bound prenyltransferases for linear and/or angular furanocoumarin biosynthesis.

In Apiaceae, furanocoumarins (FCs) are plant defence compounds that are present as linear or angular isomers. Angular isomers appeared during plant evolution as a protective response to herbivores that are resistant to linear molecules. Isomeric biosynthesis occurs through prenylation at the C6 or C8 position of umbelliferone. Here, we report cloning and functional characterization of two different prenyltransferases, Pastinaca sativa prenyltransferase 1 and 2 (PsPT1 and PsPT2), that are involved in these crucial reactions. Both enzymes are targeted to plastids and synthesize osthenol and demethylsuberosin (DMS) using exclusively umbelliferone and dimethylallylpyrophosphate (DMAPP) as substrates. Enzymatic characterization using heterologously expressed proteins demonstrated that PsPT1 is specialized for the synthesis of the linear form, demethylsuberosin, whereas PsPT2 more efficiently catalyses the synthesis of its angular counterpart, osthenol. These results are the first example of a complementary prenyltransferase pair from a single plant species that is involved in synthesizing defensive compounds. This study also provides a better understanding of the molecular mechanisms governing the angular FC biosynthetic pathway in apiaceous plants, which involves two paralogous enzymes that share the same phylogenetic origin.

Involvement of the leaf-specific multidrug and toxic compound extrusion (MATE) transporter Nt-JAT2 in vacuolar sequestration of nicotine in Nicotiana tabacum.

Alkaloids play a key role in higher plant defense against pathogens and herbivores. Following its biosynthesis in root tissues, nicotine, the major alkaloid of Nicotiana species, is translocated via xylem transport toward the accumulation sites, leaf vacuoles. Our transcriptome analysis of methyl jasmonate-treated tobacco BY-2 cells identified several multidrug and toxic compound extrusion (MATE) transporter genes. In this study, we characterized a MATE gene, Nicotiana tabacum jasmonate-inducible alkaloid transporter 2 (Nt-JAT2), which encodes a protein that has 32% amino acid identity with Nt-JAT1. Nt-JAT2 mRNA is expressed at a very low steady state level in whole plants, but is rapidly upregulated by methyl jasmonate treatment in a leaf-specific manner. To characterize the function of Nt-JAT2, yeast cells were used as the host organism in a cellular transport assay. Nt-JAT2 was localized at the plasma membrane in yeast cells. When incubated in nicotine-containing medium, the nicotine content in Nt-JAT2-expressing cells was significantly lower than in control yeast. Nt-JAT2-expressing cells also showed lower content of other alkaloids like anabasine and anatabine, but not of flavonoids, suggesting that Nt-JAT2 transports various alkaloids including nicotine. Fluorescence assays in BY-2 cells showed that Nt-JAT2-GFP was localized to the tonoplast. These findings indicate that Nt-JAT2 is involved in nicotine sequestration in leaf vacuoles following the translocation of nicotine from root tissues.

Molecular cloning and characterization of a geranyl diphosphate-specific aromatic prenyltransferase from lemon.

Prenyl residues confer divergent biological activities such as antipathogenic and antiherbivorous activities on phenolic compounds, including flavonoids, coumarins, and xanthones. To date, about 1,000 prenylated phenolics have been isolated, with these compounds containing various prenyl residues. However, all currently described plant prenyltransferases (PTs) have been shown specific for dimethylallyl diphosphate as the prenyl donor, while most of the complementary DNAs encoding these genes have been isolated from the Leguminosae. In this study, we describe the identification of a novel PT gene from lemon (Citrus limon), ClPT1, belonging to the homogentisate PT family. This gene encodes a PT that differs from other known PTs, including flavonoid-specific PTs, in polypeptide sequence. This membrane-bound enzyme was specific for geranyl diphosphate as the prenyl donor and coumarin as the prenyl acceptor. Moreover, the gene product was targeted to plastid in plant cells. To our knowledge, this is the novel aromatic PT specific to geranyl diphosphate from citrus species.

A coumarin-specific prenyltransferase catalyzes the crucial biosynthetic reaction for furanocoumarin formation in parsley.

Furanocoumarins constitute a sub-family of coumarin compounds with important defense properties against pathogens and insects, as well as allelopathic functions in plants. Furanocoumarins are divided into two sub-groups according to the alignment of the furan ring with the lactone structure: linear psoralen and angular angelicin derivatives. Determination of furanocoumarin type is based on the prenylation position of the common precursor of all furanocoumarins, umbelliferone, at C6 or C8, which gives rise to the psoralen or angelicin derivatives, respectively. Here, we identified a membrane-bound prenyltransferase PcPT from parsley (Petroselinum crispum), and characterized the properties of the gene product. PcPT expression in various parsley tissues is increased by UV irradiation, with a concomitant increase in furanocoumarin production. This enzyme has strict substrate specificity towards umbelliferone and dimethylallyl diphosphate, and a strong preference for the C6 position of the prenylated product (demethylsuberosin), leading to linear furanocoumarins. The C8-prenylated derivative (osthenol) is also formed, but to a much lesser extent. The PcPT protein is targeted to the plastids in planta. Introduction of this PcPT into the coumarin-producing plant Ruta graveolens showed increased consumption of endogenous umbelliferone. Expression of PcPT and a 4-coumaroyl CoA 2'-hydroxylase gene in Nicotiana benthamiana, which does not produce furanocoumarins, resulted in formation of demethylsuberosin, indicating that furanocoumarin production may be reconstructed by a metabolic engineering approach. The results demonstrate that a single prenyltransferase, such as PcPT, opens the pathway to linear furanocoumarins in parsley, but may also catalyze the synthesis of osthenol, the first intermediate committed to the angular furanocoumarin pathway, in other plants.

LjMATE1: a citrate transporter responsible for iron supply to the nodule infection zone of Lotus japonicus.

Symbiotic nitrogen fixation by intracellular rhizobia within legume root nodules requires the exchange of nutrients between host plant cells and their resident bacteria. While exchanged molecules imply nitrogen compounds, carbohydrates and also various minerals, knowledge of the molecular basis of plant transporters that mediate those metabolite exchanges is still limited. In this study, we have shown that a multidrug and toxic compound extrusion (MATE) protein, LjMATE1, is specifically induced during nodule formation, which nearly paralleled nodule maturation, in a model legume Lotus japonicus. Reporter gene experiments indicated that the expression of LjMATE1 was restricted to the infection zone of nodules. To characterize the transport function of LjMATE1, we conducted a biochemical analysis using a heterologous expression system, Xenopus oocytes, and found that LjMATE1 is a specific transporter for citrate. The physiological role of LjMATE1 was analyzed after generation of L. japonicus RNA interference (RNAi) lines. One RNAi knock-down line revealed limited growth under nitrogen-deficient conditions with inoculation of rhizobia compared with the controls (the wild type and an RNAi line in which LjMATE1 was not suppressed). It was noteworthy that Fe localization was clearly altered in nodule tissues of the knock-down line. These results strongly suggest that LjMATE1 is a nodule-specific transporter that assists the translocation of Fe from the root to nodules by providing citrate.

Characterization of coumarin-specific prenyltransferase activities in Citrus limon peel.

Coumarins, a large group of polyphenols, play important roles in the defense mechanisms of plants, and they also exhibit various biological activities beneficial to human health, often enhanced by prenylation. Despite the high abundance of prenylated coumarins in citrus fruits, there has been no report on coumarin-specific prenyltransferase activity in citrus. In this study, we detected both O- and C-prenyltransferase activities of coumarin substrates in a microsome fraction prepared from lemon (Citrus limon) peel, where large amounts of prenylated coumarins accumulate. Bergaptol was the most preferred substrate out of various coumarin derivatives tested, and geranyl diphosphate (GPP) was accepted exclusively as prenyl donor substrate. Further enzymatic characterization of bergaptol 5-O-geranyltransferase activity revealed its unique properties: apparent K(m) values for GPP (9 µM) and bergaptol (140 µM) and a broad divalent cation requirement. These findings provide information towards the discovery of a yet unidentified coumarin-specific prenyltransferase gene.

Metabolic engineering for the production of prenylated polyphenols in transgenic legume plants using bacterial and plant prenyltransferases.

Prenylated polyphenols are secondary metabolites beneficial for human health because of their various biological activities. Metabolic engineering was performed using Streptomyces and Sophora flavescens prenyltransferase genes to produce prenylated polyphenols in transgenic legume plants. Three Streptomyces genes, NphB, SCO7190, and NovQ, whose gene products have broad substrate specificity, were overexpressed in a model legume, Lotus japonicus, in the cytosol, plastids or mitochondria with modification to induce the protein localization. Two plant genes, N8DT and G6DT, from Sophora flavescens whose gene products show narrow substrate specificity were also overexpressed in Lotus japonicus. Prenylated polyphenols were undetectable in these plants; however, supplementation of a flavonoid substrate resulted in the production of prenylated polyphenols such as 7-O-geranylgenistein, 6-dimethylallylnaringenin, 6-dimethylallylgenistein, 8-dimethylallynaringenin, and 6-dimethylallylgenistein in transgenic plants. Although transformants with the native NovQ did not produce prenylated polyphenols, modification of its codon usage led to the production of 6-dimethylallylnaringenin and 6-dimethylallylgenistein in transformants following naringenin supplementation. Prenylated polyphenols were not produced in mitochondrial-targeted transformants even under substrate feeding. SCO7190 was also expressed in soybean, and dimethylallylapigenin and dimethylallyldaidzein were produced by supplementing naringenin. This study demonstrated the potential for the production of novel prenylated polyphenols in transgenic plants. In particular, the enzymatic properties of prenyltransferases seemed to be altered in transgenic plants in a host species-dependent manner.

Homoglutathione confers tolerance to acifluorfen in transgenic tobacco plants expressing soybean homoglutathione synthetase.

Homoglutathione (hGSH), which is present in some leguminous plants, is preferred over GSH in in vitro conjugation of acifluorfen and fomesafen by glutathione S-transferase. To investigate the function of hGSH in in vivo detoxification of xenobiotics, we evaluated herbicide tolerance of transgenic tobacco plants expressing soybean homoglutathione synthetase in the cytosol or chloroplasts. Transgenic plants synthesizing hGSH in the cytosol were more tolerant to acifluorfen than wild-type plants, whereas enhanced tolerance to fomesafen was not observed. Transgenic plants synthesizing hGSH in the chloroplasts showed no enhanced tolerance to acifluorfen or fomesafen.