RESUMEN
4-Coumaroyl-CoA ligase (4CL) is a key enzyme in the phenylpropanoid pathway, which is involved in the biosynthesis of various specialized metabolites such as flavonoids, coumarins, lignans, and lignin. Plants have several 4CLs showing divergence in sequence: Class I 4CLs involved in lignin metabolism, Class II 4CLs associated with flavonoid metabolism, and atypical 4CLs and 4CL-like proteins of unknown function. Shikonin, a Boraginaceae-specific specialized metabolite in red gromwell (Lithospermum erythrorhizon), is biosynthesized from p-hydroxybenzoic acid, and the involvement of 4CL in its biosynthesis has long been debated. In this study, we demonstrated the requirement of 4CL for shikonin biosynthesis using a 4CL-specific inhibitor. In silico analysis of the L. erythrorhizon genome revealed the presence of at least 8 4CL genes, among which the expression of 3 (Le4CL3, Le4CL4, and Le4CL5) showed a positive association with shikonin production. Phylogenetic analysis indicated that Le4CL5 belongs to Class I 4CLs, while Le4CL3 and Le4CL4 belong to clades that are distant from Class I and Class II. Interestingly, both Le4CL3 and Le4CL4 have peroxisome targeting signal 1 in their C-terminal region, and subcellular localization analysis revealed that both localize to the peroxisome. We targeted each of the 3 Le4CL genes by CRISPR/Cas9-mediated mutagenesis and observed remarkably lower shikonin production in Le4CL3-ge and Le4CL4-ge genome-edited lines compared with the vector control. We, therefore, conclude that peroxisomal Le4CL3 and Le4CL4 are responsible for shikonin production and propose a model for metabolite-specific 4CL distribution in L. erythrorhizon.
Asunto(s)
Coenzima A Ligasas , Lithospermum , Naftoquinonas , Peroxisomas , Naftoquinonas/metabolismo , Lithospermum/genética , Lithospermum/metabolismo , Peroxisomas/metabolismo , Coenzima A Ligasas/genética , Coenzima A Ligasas/metabolismo , Proteínas de Plantas/metabolismo , Proteínas de Plantas/genética , Regulación de la Expresión Génica de las Plantas , FilogeniaRESUMEN
Shikonin and its enantiomer, alkannin, are bioactive naphthoquinones produced in several plants of the family Boraginaceae. The structures of these acylated derivatives, which have various short-chain acyl moieties, differ among plant species. The acylation of shikonin and alkannin in Lithospermum erythrorhizon was previously reported to be catalyzed by two enantioselective BAHD acyltransferases, shikonin O-acyltransferase (LeSAT1) and alkannin O-acyltransferase (LeAAT1). However, the mechanisms by which various shikonin and alkannin derivatives are produced in Boraginaceae plants remain to be determined. In the present study, evaluation of six Boraginaceae plants identified 23 homologs of LeSAT1 and LeAAT1, with 15 of these enzymes found to catalyze the acylation of shikonin or alkannin, utilizing acetyl-CoA, isobutyryl-CoA or isovaleryl-CoA as an acyl donor. Analyses of substrate specificities of these enzymes for both acyl donors and acyl acceptors and determination of their subcellular localization using Nicotiana benthamiana revealed a distinct functional differentiation of BAHD acyltransferases in Boraginaceae plants. Gene expression of these acyltransferases correlated with the enantiomeric ratio of produced shikonin/alkannin derivatives in L. erythrorhizon and Echium plantagineum. These enzymes showed conserved substrate specificities for acyl donors among plant species, indicating that the diversity in acyl moieties of shikonin/alkannin derivatives involved factors other than the differentiation of acyltransferases. These findings provide insight into the chemical diversification and evolutionary processes of shikonin/alkannin derivatives.
Asunto(s)
Boraginaceae , Naftoquinonas , Boraginaceae/genética , Boraginaceae/química , Boraginaceae/metabolismo , Aciltransferasas/genética , Naftoquinonas/metabolismoRESUMEN
Plants produce â¼300 aromatic compounds enzymatically linked to prenyl side chains via C-O bonds. These O-prenylated aromatic compounds have been found in taxonomically distant plant taxa, with some of them being beneficial or detrimental to human health. Although their O-prenyl moieties often play crucial roles in the biological activities of these compounds, no plant gene encoding an aromatic O-prenyltransferase (O-PT) has been isolated to date. This study describes the isolation of an aromatic O-PT gene, CpPT1, belonging to the UbiA superfamily, from grapefruit (Citrus × paradisi, Rutaceae). This gene was shown responsible for the biosynthesis of O-prenylated coumarin derivatives that alter drug pharmacokinetics in the human body. Another coumarin O-PT gene encoding a protein of the same family was identified in Angelica keiskei, an apiaceous medicinal plant containing pharmaceutically active O-prenylated coumarins. Phylogenetic analysis of these O-PTs suggested that aromatic O-prenylation activity evolved independently from the same ancestral gene in these distant plant taxa. These findings shed light on understanding the evolution of plant secondary (specialized) metabolites via the UbiA superfamily.
Asunto(s)
Angelica/genética , Citrus paradisi/genética , Evolución Molecular , Furocumarinas/biosíntesis , Proteínas de Plantas/genética , Prenilación , Angelica/metabolismo , Citrus paradisi/metabolismo , Filogenia , Proteínas de Plantas/metabolismoRESUMEN
Plant specialized metabolites (PSMs) are often stored as glycosides within cells and released from the roots with some chemical modifications. While isoflavones are known to function as symbiotic signals with rhizobia and to modulate the soybean rhizosphere microbiome, the underlying mechanisms of root-to-soil delivery are poorly understood. In addition to transporter-mediated secretion, the hydrolysis of isoflavone glycosides in the apoplast by an isoflavone conjugate-hydrolyzing ß-glucosidase (ICHG) has been proposed but not yet verified. To clarify the role of ICHG in isoflavone supply to the rhizosphere, we have isolated two independent mutants defective in ICHG activity from a soybean high-density mutant library. In the root apoplastic fraction of ichg mutants, the isoflavone glycoside contents were significantly increased, while isoflavone aglycone contents were decreased, indicating that ICHG hydrolyzes isoflavone glycosides into aglycones in the root apoplast. When grown in a field, the lack of ICHG activity considerably reduced isoflavone aglycone contents in roots and the rhizosphere soil, although the transcriptomes showed no distinct differences between the ichg mutants and wild-types (WTs). Despite the change in isoflavone contents and composition of the root and rhizosphere of the mutants, root and rhizosphere bacterial communities were not distinctive from those of the WTs. Root bacterial communities and nodulation capacities of the ichg mutants did not differ from the WTs under nitrogen-deficient conditions either. Taken together, these results indicate that ICHG elevates the accumulation of isoflavones in the soybean rhizosphere but is not essential for isoflavone-mediated plant-microbe interactions.
Asunto(s)
Isoflavonas , Isoflavonas/química , Glycine max/genética , Glycine max/metabolismo , beta-Glucosidasa/genética , beta-Glucosidasa/química , Rizosfera , Glicósidos/metabolismo , Bacterias/metabolismo , SueloRESUMEN
Plants produce a large variety of lipophilic metabolites, many of which are secreted by cells and accumulated in apoplasts. These compounds often play a role to protect plants from environmental stresses. However, little is known about how these lipophilic compounds are secreted into apoplastic spaces. In this study, we used shikonin-producing cultured cells of Lithospermum erythrorhizon as an experimental model system to analyze the secretion of lipophilic metabolites, taking advantage of its high production rate and the clear inducibility in culture. Shikonin derivatives are lipophilic red naphthoquinone compounds that accumulate exclusively in apoplastic spaces of these cells and also in the root epidermis of intact plants. Microscopic analysis showed that shikonin is accumulated in the form of numerous particles on the cell wall. Lipidomic analysis showed that L. erythrorhizon cultured cells secrete an appreciable portion of triacylglycerol (24-38% of total triacylglycerol), composed predominantly of saturated fatty acids. Moreover, in vitro reconstitution assay showed that triacylglycerol encapsulates shikonin derivatives with phospholipids to form lipid droplet-like structures. These findings suggest a novel role for triacylglycerol as a matrix lipid, a molecular component involved in the secretion of specialized lipophilic metabolites.
Asunto(s)
Naftoquinonas , Proteínas de Plantas , Proteínas de Plantas/metabolismo , Regulación de la Expresión Génica de las Plantas , Naftoquinonas/metabolismo , LípidosRESUMEN
Saponins are the group of plant specialized metabolites which are widely distributed in angiosperm plants and have various biological activities. The present study focused on α-tomatine, a major saponin present in tissues of tomato (Solanum lycopersicum) plants. α-Tomatine is responsible for defense against plant pathogens and herbivores, but its biological function in the rhizosphere remains unknown. Secretion of tomatine was higher at the early growth than the green-fruit stage in hydroponically grown plants, and the concentration of tomatine in the rhizosphere of field-grown plants was higher than that of the bulk soil at all growth stages. The effects of tomatine and its aglycone tomatidine on the bacterial communities in the soil were evaluated in vitro, revealing that both compounds influenced the microbiome in a concentration-dependent manner. Numerous bacterial families were influenced in tomatine/tomatidine-treated soil as well as in the tomato rhizosphere. Sphingomonadaceae species, which are commonly observed and enriched in tomato rhizospheres in the fields, were also enriched in tomatine- and tomatidine-treated soils. Moreover, a jasmonate-responsive ETHYLENE RESPONSE FACTOR 4 mutant associated with low tomatine production caused the root-associated bacterial communities to change with a reduced abundance of Sphingomonadaceae. Taken together, our results highlight the role of tomatine in shaping the bacterial communities of the rhizosphere and suggest additional functions of tomatine in belowground biological communication.
Asunto(s)
Microbiota/fisiología , Raíces de Plantas/metabolismo , Rizosfera , Solanum lycopersicum/metabolismo , Tomatina/metabolismo , Bacterias/metabolismo , Fenómenos Fisiológicos Bacterianos , Solanum lycopersicum/microbiología , Raíces de Plantas/microbiologíaRESUMEN
Plants interact with microorganisms in the phyllosphere and rhizosphere. Here the roots exude plant specialized metabolites (PSMs) that have diverse biological and ecological functions. Recent reports have shown that these PSMs influence the rhizosphere microbiome, which is essential for the plant's growth and health. This review summarizes several specialized metabolites secreted into the rhizosphere of the tomato plant (Solanum lycopersicum), which is an important model species for plant research and a commercial crop. In this review, we focused on the effects of such plant metabolites on plant-microbe interactions. We also reviewed recent studies on improving the growth of tomatoes by analyzing and reconstructing the rhizosphere microbiome and discussed the challenges to be addressed in establishing sustainable agriculture.
Asunto(s)
Solanum lycopersicum , Rizosfera , Microbiología del Suelo , Plantas , Agricultura , Raíces de PlantasRESUMEN
MAIN CONCLUSION: Genes of the PLAT protein family, including PLAT and ATS3 subfamilies of higher plants and homologs of liverwort, are involved in plant defense against insects. Laticifer cells in plants contain large amounts of anti-microbe or anti-insect proteins and are involved in plant defense against biotic stresses. We previously found that PLAT proteins accumulate in laticifers of fig tree (Ficus carica) at comparable levels to those of chitinases, and the transcript level of ATS3, another PLAT domain-containing protein, is highest in the transcriptome of laticifers of Euphorbia tirucalli. In this study, we investigated whether the PLAT domain-containing proteins are involved in defense against insects. Larvae of the lepidopteran Spodoptera litura showed retarded growth when fed with Nicotiana benthamiana leaves expressing F. carica PLAT or E. tirucalli ATS3 genes, introduced by agroinfiltration using expression vector pBYR2HS. Transcriptome analysis of these leaves indicated that ethylene and jasmonate signaling were activated, leading to increased expression of genes for PR-1, ß-1,3-glucanase, PR5 and trypsin inhibitors, suggesting an indirect mechanism of PLAT- and ATS3-induced resistance in the host plant. Direct cytotoxicity of PLAT and ATS3 to insects was also possible because heterologous expression of the corresponding genes in Drosophila melanogaster caused apoptosis-mediated cell death in this insect. Larval growth retardation of S. litura occurred when they were fed radish sprouts, a good host for agroinfiltration, expressing any of nine homologous genes of dicotyledon Arabidopsis thaliana, monocotyledon Brachypodium distachyon, conifer Picea sitchensis and liverwort Marchantia polymorpha. Of these nine genes, the heterologous expression of A. thaliana AT5G62200 and AT5G62210 caused significant increases in larval death. These results indicated that the PLAT protein family has largely conserved anti-insect activity in the plant kingdom (249 words).
Asunto(s)
Arabidopsis , Regulación de la Expresión Génica de las Plantas , Insectos , Proteínas de Plantas , Plantas , Animales , Arabidopsis/metabolismo , Quitinasas/metabolismo , Drosophila melanogaster/efectos de los fármacos , Ficus/genética , Ficus/parasitología , Insectos/efectos de los fármacos , Larva/efectos de los fármacos , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Proteínas de Plantas/farmacología , Plantas/genética , Plantas/parasitología , Spodoptera/efectos de los fármacos , TranscriptomaRESUMEN
Several Boraginaceae plants produce biologically active red naphthoquinone pigments, derivatives of the enantiomers shikonin and alkannin, which vary in acyl groups on their side chains. Compositions of shikonin/alkannin derivatives vary in plant species, but the mechanisms generating the diversity of shikonin/alkannin derivatives are largely unknown. This study describes the identification and characterization of two BAHD acyltransferases, shikonin O-acyltransferase (LeSAT1) and alkannin O-acyltransferase (LeAAT1), from Lithospermum erythrorhizon, a medicinal plant in the family Boraginaceae that primarily produces the shikonin/alkannin derivatives acetylshikonin and ß-hydroxyisovalerylshikonin. Enzyme assays using Escherichia coli showed that the acylation activity of LeSAT1 was specific to shikonin, whereas the acylation activity of LeAAT1 was specific to alkannin. Both enzymes recognized acetyl-CoA, isobutyryl-CoA, and isovaleryl-CoA as acyl donors to produce their corresponding shikonin/alkannin derivatives, with both enzymes showing the highest activity for acetyl-CoA. These findings were consistent with the composition of shikonin/alkannin derivatives in intact L erythrorhizon plants and cell cultures. Genes encoding both enzymes were preferentially expressed in the roots and cell cultures in the dark in pigment production medium M9, conditions associated with shikonin/alkannin production. These results indicated that LeSAT1 and LeAAT1 are enantiomer-specific acyltransferases that generate various shikonin/alkannin derivatives.
Asunto(s)
Aciltransferasas/metabolismo , Lithospermum/enzimología , Naftoquinonas/metabolismo , Aciltransferasas/genética , Escherichia coli , Lithospermum/genética , Especificidad por SustratoRESUMEN
Geranyl diphosphate (GPP) is the direct precursor of all monoterpenoids and is the prenyl source of many meroterpenoids, such as geranylated coumarins. GPP synthase (GPPS) localized in plastids is responsible for providing the substrate for monoterpene synthases and prenyltransferases for synthesis of aromatic substances that are also present in plastids, but GPPS activity in Lithospermum erythrorhizon localizes to the cytosol, in which GPP is utilized for the biosynthesis of naphthoquinone pigments, which are shikonin derivatives. This study describes the identification of the cytosol-localized GPPS gene, LeGPPS, through EST- and homology-based approaches followed by functional analyses. The deduced amino acid sequence of the unique LeGPPS showed greater similarity to that of farnesyl diphosphate synthase (FPPS), which generally localizes to the cytosol, than to plastid-localized conventional GPPS. Biochemical characterization revealed that recombinant LeGPPS predominantly produces GPP along with a trace amount of FPP. LeGPPS expression was mainly detected in root bark, in which shikonin derivatives are produced, and in shikonin-producing cultured cells. The GFP fusion protein in onion (Allium cepa) cells localized to the cytosol. Site-directed mutagenesis of LeGPPS and another FPPS homolog identified in this study, LeFPPS1, showed that the His residue at position 100 of LeGPPS, adjacent to the first Asp-rich motif, contributes to substrate preference and product specificity, leading to GPP formation. These results suggest that LeGPPS, which is involved in shikonin biosynthesis, is recruited from cytosolic FPPS and that point mutation(s) result in the acquisition of GPPS activity.
Asunto(s)
Citosol/metabolismo , Geraniltranstransferasa/metabolismo , Lithospermum/metabolismo , Cumarinas/metabolismo , Geraniltranstransferasa/genética , Monoterpenos/metabolismo , Mutagénesis Sitio-Dirigida , Naftoquinonas/metabolismo , Plastidios/genética , Plastidios/metabolismoRESUMEN
Analyses of metabolite secretions by field-grown plants remain scarce. We analyzed daidzein secretion by field-grown soybean. Daidzein secretion was higher during early vegetative stages than reproductive stages, a trend that was also seen for hydroponically grown soybean. Daidzein secretion was up to 10 000-fold higher under field conditions than hydroponic conditions, leading to a more accurate simulation of rhizosphere daidzein content.
Asunto(s)
Glycine max/metabolismo , Isoflavonas/biosíntesis , Hojas de la Planta/metabolismo , Raíces de Plantas/metabolismo , Genisteína/aislamiento & purificación , Genisteína/metabolismo , Glucósidos/biosíntesis , Glucósidos/aislamiento & purificación , Hidroponía/métodos , Isoflavonas/aislamiento & purificación , Especificidad de Órganos , Hojas de la Planta/crecimiento & desarrollo , Raíces de Plantas/crecimiento & desarrollo , Rizosfera , Glycine max/crecimiento & desarrolloRESUMEN
Shikonin derivatives are red naphthoquinone pigments produced by several boraginaceous plants, such as Lithospermum erythrorhizon. These compounds are biosynthesized from p-hydroxybenzoic acid and geranyl diphosphate. The coupling reaction that yields m-geranyl-p-hydroxybenzoic acid has been actively characterized, but little is known about later biosynthetic reactions. Although 3â³-hydroxy-geranylhydroquinone produced from geranylhydroquinone by CYP76B74 has been regarded as an intermediate of shikonin derivatives, the next intermediate has not yet been identified. This study describes a novel alcohol dehydrogenase activity in L. erythrorhizon cell cultures. This enzyme was shown to oxidize the 3â³-alcoholic group of (Z)-3â³-hydroxy-geranylhydroquinone to an aldehyde moiety concomitant with the isomerization at the C2'-C3' double bond from the Z-form to the E-form. An enzyme oxidizing this substrate was not detected in other plant cell cultures, suggesting that this enzyme is specific to L. erythrorhizon. The reaction product, (E)-3â³-oxo-geranylhydroquinone, was further converted to deoxyshikonofuran, another meroterpenoid metabolite produced in L. erythrorhizon cells. Although nonenzymatic cyclization occurred slowly, it was more efficient in the presence of crude enzymes of L. erythrorhizon cells. This activity was detected in both shikonin-producing and nonproducing cells, suggesting that the aldehyde intermediate at the biosynthetic branch point between naphthalene and benzo/hydroquinone ring formation likely constitutes a key common intermediate in the synthesis of shikonin and benzoquinone products, respectively.
Asunto(s)
Alcohol Deshidrogenasa/metabolismo , Aldehídos/metabolismo , Benzoquinonas/metabolismo , Lithospermum/enzimología , Naftoquinonas/metabolismo , Terpenos/metabolismo , Lithospermum/metabolismo , Redes y Vías MetabólicasRESUMEN
Furanocoumarins (FCs) are plant-specialized metabolites with potent allelochemical properties. The distribution of FCs is scattered with a chemotaxonomical tendency towards four distant families with highly similar FC pathways. The mechanism by which this pathway emerged and spread in plants has not been elucidated. Furanocoumarin biosynthesis was investigated in Ficus carica (fig, Moraceae), focusing on the first committed reaction catalysed by an umbelliferone dimethylallyltransferase (UDT). Comparative RNA-seq analysis among latexes of different fig organs led to the identification of a UDT. The phylogenetic relationship of this UDT to previously reported Apiaceae UDTs was evaluated. The expression pattern of F. carica prenyltransferase 1 (FcPT1) was related to the FC contents in different latexes. Enzymatic characterization demonstrated that one of the main functions of FcPT1 is UDT activity. Phylogenetic analysis suggested that FcPT1 and Apiaceae UDTs are derived from distinct ancestors, although they both belong to the UbiA superfamily. These findings are supported by significant differences in the related gene structures. This report describes the identification of FcPT1 involved in FC biosynthesis in fig and provides new insights into multiple origins of the FC pathway and, more broadly, into the adaptation of plants to their environments.
Asunto(s)
Dimetilaliltranstransferasa , Ficus , Furocumarinas , Dimetilaliltranstransferasa/genética , Ficus/genética , Látex , FilogeniaRESUMEN
Plant roots nurture a wide variety of microbes via exudation of metabolites, shaping the rhizosphere's microbial community. Despite the importance of plant specialized metabolites in the assemblage and function of microbial communities in the rhizosphere, little is known of how far the effects of these metabolites extend through the soil. We employed a fluid model to simulate the spatiotemporal distribution of daidzein, an isoflavone secreted from soybean roots, and validated using soybeans grown in a rhizobox. We then analysed how daidzein affects bacterial communities using soils artificially treated with daidzein. Simulation of daidzein distribution showed that it was only present within a few millimetres of root surfaces. After 14 days in a rhizobox, daidzein was only present within 2 mm of root surfaces. Soils with different concentrations of daidzein showed different community composition, with reduced α-diversity in daidzein-treated soils. Bacterial communities of daidzein-treated soils were closer to those of the soybean rhizosphere than those of bulk soils. This study highlighted the limited distribution of daidzein within a few millimetres of root surfaces and demonstrated a novel role of daidzein in assembling bacterial communities in the rhizosphere by acting as more of a repellant than an attractant.
Asunto(s)
Glycine max/microbiología , Isoflavonas/metabolismo , Rizosfera , Microbiología del Suelo , Modelos Biológicos , Raíces de Plantas/metabolismo , Raíces de Plantas/microbiología , Glycine max/metabolismoRESUMEN
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.
Asunto(s)
Vías Biosintéticas , Lithospermum/enzimología , Lithospermum/metabolismo , Naftoquinonas/metabolismo , Proteómica , Análisis por Conglomerados , Regulación de la Expresión Génica de las Plantas , Lithospermum/genética , Naftoquinonas/química , Proteínas de Plantas/metabolismo , Proteoma/metabolismo , Reproducibilidad de los Resultados , Análisis de Secuencia de ARNRESUMEN
Rhododendron dauricum produces daurichromenic acid, an anti-HIV meroterpenoid, via oxidative cyclization of the farnesyl group of grifolic acid. The prenyltransferase (PT) that synthesizes grifolic acid is a farnesyltransferase in plant specialized metabolism. In this study, we demonstrated that the isoprenoid moiety of grifolic acid is derived from the 2-C-methyl-d-erythritol-4-phosphate pathway that takes place in plastids. We explored candidate sequences of plastid-localized PT homologs and identified a cDNA for this PT, RdPT1, which shares moderate sequence similarity with known aromatic PTs. RdPT1 is expressed exclusively in the glandular scales, where daurichromenic acid accumulates. In addition, the gene product was targeted to plastids in plant cells. The recombinant RdPT1 regiospecifically synthesized grifolic acid from orsellinic acid and farnesyl diphosphate, demonstrating that RdPT1 is the farnesyltransferase involved in daurichromenic acid biosynthesis. This enzyme strictly preferred orsellinic acid as a prenyl acceptor, whereas it had a relaxed specificity for prenyl donor structures, also accepting geranyl and geranylgeranyl diphosphates with modest efficiency to synthesize prenyl chain analogs of grifolic acid. Such a broad specificity is a unique catalytic feature of RdPT1 that is not shared among secondary metabolic aromatic PTs in plants. We discuss the unusual substrate preference of RdPT1 using a molecular modeling approach. The biochemical properties as well as the localization of RdPT1 suggest that this enzyme produces meroterpenoids in glandular scales cooperatively with previously identified daurichromenic acid synthase, probably for chemical defense on the surface of R. dauricum plants.
Asunto(s)
Fármacos Anti-VIH/metabolismo , Cromanos/metabolismo , Dimetilaliltranstransferasa/metabolismo , Farnesiltransferasa/metabolismo , VIH/efectos de los fármacos , Rhododendron/enzimología , Fármacos Anti-VIH/química , Cromanos/química , Clonación Molecular , Ciclización , Dimetilaliltranstransferasa/genética , Farnesiltransferasa/genética , Modelos Moleculares , Oxidación-Reducción , Filogenia , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plastidios/enzimología , Rhododendron/genética , Sesterterpenos/química , Sesterterpenos/metabolismoRESUMEN
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.
Asunto(s)
Transportador de Casetes de Unión a ATP, Subfamilia G/metabolismo , Nicotiana/metabolismo , Nicotiana/microbiología , Phytophthora infestans/patogenicidad , Proteínas de Plantas/metabolismo , Transportador de Casetes de Unión a ATP, Subfamilia G/genética , Regulación de la Expresión Génica de las Plantas/genética , Enfermedades de las Plantas/genética , Enfermedades de las Plantas/microbiología , Proteínas de Plantas/genética , Nicotiana/genéticaRESUMEN
Purine permeases (PUPs) mediate the proton-coupled uptake of nucleotide bases and their derivatives into cytosol. PUPs facilitate uptake of adenine, cytokinins and nicotine. Caffeine, a purine alkaloid derived from xanthosine, occurs in only a few eudicot species, including coffee, cacao, and tea. Although caffeine is not an endogenous metabolite in Arabidopsis and rice, AtPUP1 and OsPUP7 were suggested to transport caffeine. In this study, we identified 15 PUPs in the genome of Coffea canephora. Direct uptake measurements in yeast demonstrated that CcPUP1 and CcPUP5 facilitate adenine - but not caffeine - transport. Adenine uptake was pH-dependent, with increased activity at pH 3 and 4, and inhibited by nigericin, a potassium-proton ionophore, suggesting that CcPUP1 and CcPUP5 function as proton-symporters. Furthermore, adenine uptake was not competitively inhibited by an excess amount of caffeine, which implies that PUPs of C. canephora have evolved to become caffeine-insensitive to promote efficient uptake of adenine into cytosol.
Asunto(s)
Adenina/metabolismo , Coffea/metabolismo , Proteínas de Transporte de Nucleobases/metabolismo , Arabidopsis/metabolismo , Cafeína/metabolismo , Coffea/efectos de los fármacos , Concentración de Iones de Hidrógeno , Nigericina/farmacología , Oryza/metabolismoRESUMEN
Root exudates are plant metabolites secreted from the roots into the soil. These exudates are involved in many important biological processes, including acquisition of nutrients, defense and signaling to rhizosphere bacteria, such as isoflavones of soybean crucial for the symbiosis with rhizobium. Less is known, however, about other types of root exudates. This study shows that soybean roots secrete large amounts of soyasaponins (triterpenoid glycosides) as root exudates. The soyasaponins are classified into four groups, with group A being the most secreted of these compounds, whereas DDMP (2,3-dihydro-2,5-dihydroxy-6-methyl-4H-pyran-4-one) soyasaponins is the group showing greatest accumulation in root tissues, suggesting a selection system for secreted compounds. Time-course experiments showed that the soyasaponin secretion peaked during early vegetative stages. In particular, soyasaponin Ah was the major compound secreted by soybean roots, whereas the deacetylated derivative Af was the major compound secreted specifically during the VE stage. The secretion of soyasaponins containing glycosyl moieties is an apparent loss of photosynthates. This phenomenon has been also observed in other legume species, although the composition of secreted soyasaponins is plant species dependent. The identification of triterpenoid saponins as major metabolites in legume root exudates will provide novel insights into chemical signaling in the rhizosphere between plants and other organisms.