ABSTRACT
KEY MESSAGE: LeBAHD56 is preferentially expressed in tissues where shikonin and its derivatives are biosynthesized, and it confers shikonin acylation in vivo. Two WRKY transcriptional factors might regulate LeBAHD56's expression. Shikonin and its derivatives, found in the roots of Lithospermum erythrorhizon, have extensive application in the field of medicine, cosmetics, and other industries. Prior research has demonstrated that LeBAHD1(LeSAT1) is responsible for the biochemical process of shikonin acylation both in vitro and in vivo. However, with the exception of its documented in vitro biochemical function, there is no in vivo genetic evidence supporting the acylation function of the highly homologous gene of LeSAT1, LeBAHD56(LeSAT2), apart from its reported role. Here, we validated the critical acylation function of LeBAHD56 for shikonin using overexpression (OE) and CRISPR/Cas9-based knockout (KO) strategies. The results showed that the OE lines had a significantly higher ratio of acetylshikonin, isobutyrylshikonin or isovalerylshikonin to shikonin than the control. In contrast, the KO lines had a significantly lower ratio of acetylshikonin, isobutyrylshikonin or isovalerylshikonin to shikonin than controls. As for its detailed expression patterns, we found that LeBAHD56 is preferentially expressed in roots and callus cells, which are the biosynthesis sites for shikonin and its derivatives. In addition, we anticipated that a wide range of putative transcription factors might control its transcription and verified the direct binding of two crucial WRKY members to the LeBAHD56 promoter's W-box. Our results not only confirmed the in vivo function of LeBAHD56 in shikonin acylation, but also shed light on its transcriptional regulation.
Subject(s)
Gene Expression Regulation, Plant , Lithospermum , Naphthoquinones , Plant Proteins , Plants, Genetically Modified , Naphthoquinones/metabolism , Lithospermum/genetics , Lithospermum/metabolism , Acylation , Plant Proteins/genetics , Plant Proteins/metabolism , Plant Roots/genetics , Plant Roots/metabolism , Transcription Factors/metabolism , Transcription Factors/genetics , CRISPR-Cas Systems , AnthraquinonesABSTRACT
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.
Subject(s)
Coenzyme A Ligases , Lithospermum , Naphthoquinones , Peroxisomes , Naphthoquinones/metabolism , Lithospermum/genetics , Lithospermum/metabolism , Peroxisomes/metabolism , Coenzyme A Ligases/genetics , Coenzyme A Ligases/metabolism , Plant Proteins/metabolism , Plant Proteins/genetics , Gene Expression Regulation, Plant , PhylogenyABSTRACT
The unambiguous identification of plant material is a prerequisite of rational phytotherapy. Misidentification can even cause serious health problems, as in the case of the Chinese medicinal herb Zicao. Commercial material labelled "Zicao" may be derived from the roots of Arnebia euchroma (ruan zicao), Lithospermum erythrorhizon (ying zicao), or Onosma paniculata (dian zicao). All of these roots contain shikonin derivatives as main bioactive constituents, but ying zicao and dian zicao contain also hepatotoxic pyrrolizidine alkaloids in high amounts. Therefore, the use of A. euchroma with a very low pyrrolizidine alkaloid content is desirable. Confusions of the species occur quite often, indicating an urgent need for an unambiguous identification method. Discrimination of 23 zicao samples has been achieved by analyses of the nuclear internal transcribed spacer ITS2 and trnL-F intergenic spacer of the chloroplast DNA. Data were analyzed using Bioedit, ClustalX, Mega 11 and BLAST. Results indicate that ITS2 barcoding can accurately distinguish Arnebia euchroma from their adulterants. Subsequently, an HPTLC method has been developed allowing a chemical discrimination of the most widely used species. (22E)-Ergosta-4,6,8(14),22-tetraen-3-one has been identified as characteristic marker compound, allowing an unambiguous discrimination of A. euchroma and L. erythrorhizon.
Subject(s)
DNA Barcoding, Taxonomic , Lithospermum , DNA Barcoding, Taxonomic/methods , DNA, Chloroplast , Lithospermum/genetics , DNA, Plant/geneticsABSTRACT
Alkannin/shikonin and their derivatives are specialised metabolites of high pharmaceutical and ecological importance exclusively produced in the periderm of members of the plant family Boraginaceae. Previous studies have shown that their biosynthesis is induced in response to methyl jasmonate but not salicylic acid, two phytohormones that play important roles in plant defence. However, mechanistic understanding of induction and non-induction remains largely unknown. In the present study, we generated the first comprehensive transcriptomic dataset and metabolite profiles of Lithospermum officinale plants treated with methyl jasmonate and salicylic acid to shed light on the underlying mechanisms. Our results highlight the diverse biological processes activated by both phytohormones and reveal the important regulatory role of the mevalonate pathway in alkannin/shikonin biosynthesis in L. officinale. Furthermore, by modelling a coexpression network, we uncovered structural and novel regulatory candidate genes connected to alkannin/shikonin biosynthesis. Besides providing new mechanistic insights into alkannin/shikonin biosynthesis, the generated methyl jasmonate and salicylic acid elicited expression profiles together with the coexpression networks serve as important functional genomic resources for the scientific community aiming at deepening the understanding of alkannin/shikonin biosynthesis.
Subject(s)
Lithospermum , Naphthoquinones , Acetates , Cyclopentanes , Lithospermum/genetics , Mevalonic Acid/metabolism , Naphthoquinones/metabolism , Oxylipins , Pharmaceutical Preparations/metabolism , Plant Growth Regulators/metabolism , Plant Growth Regulators/pharmacology , Salicylic Acid/metabolism , Salicylic Acid/pharmacologyABSTRACT
Increasing genome data are coming out. Genome size estimation plays an essential role in guiding genome assembly. Several months ago, other researchers were the first to publish a draft genome of the red gromwell (i.e. Lithospermum erythrorhizon). However, we considered that the genome size they estimated and assembled was incorrect. This study meticulously estimated the L. erythrorhizon genome size to should be â¼708.74 Mb and further provided a reliable genome version (size ≈ 693.34 Mb; contigN50 length ≈ 238.08 Kb) to support our objection. Furthermore, according to our genome, we identified a gene family of the alkannin/shikonin O-acyltransferases (i.e. AAT/SAT) that catalysed enantiomer-specific acylations in the alkannin/shikonin biosynthesis (a characteristic metabolic pathway in L. erythrorhizon's roots) and further explored its evolutionary process. The results indicated that the existing AAT/SAT were not generated from only one round of gene duplication but three rounds; after different rounds of gene duplication, the existing AAT/SAT and their recent ancestors were under positive selection at different amino acid sites. These suggested that a combined power from gene duplication plus positive selection plausibly propelled AAT/SAT's functional differentiation in evolution.
Subject(s)
Lithospermum , Naphthoquinones , Acyltransferases , Lithospermum/geneticsABSTRACT
Lithospermum erythrorhizon is a medicinal plant that produces shikonin, a red lipophilic naphthoquinone derivative that accumulates exclusively in roots. The biosynthetic steps required to complete the naphthalene ring of shikonin and its mechanism of secretion remain unclear. Multiple omics studies identified several candidate genes involved in shikonin production. The functions of these genes can be evaluated using virus-induced gene silencing (VIGS) systems, which have been shown advantageous in introducing iRNA genes into non-model plants. This study describes the development of a VIGS system using an apple latent spherical virus (ALSV) vector and a target gene, phytoene desaturase (LePDS1). Virus particles packaged in Nicotiana benthamiana were inoculated into L. erythrorhizon seedlings, yielding new leaves with albino phenotype but without disease symptoms. The levels of LePDS1 mRNAs were significantly lower in the albino plants than in mock control or escape plants. Virus-derived mRNA was detected in infected plants but not in escape and mock plants. Quantitative PCR and deep sequencing analysis indicated that transcription of another hypothetical PDS gene (LePDS2) also decreased in the defective leaves. Virus infection, however, had no effect on shikonin production. These results suggest that virus-based genetic transformation and the VIGS system silence target genes in soil-grown L. erythrorhizon.
Subject(s)
Gene Expression Regulation, Plant , Gene Silencing , Lithospermum/genetics , Plant Diseases/genetics , Plant Leaves/genetics , Plant Proteins/antagonists & inhibitors , Plants, Medicinal/genetics , Secoviridae/genetics , Lithospermum/virology , Plant Diseases/virology , Plant Leaves/virology , Plant Proteins/genetics , Plants, Medicinal/virology , Secoviridae/pathogenicityABSTRACT
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.
Subject(s)
Acyltransferases/metabolism , Lithospermum/enzymology , Naphthoquinones/metabolism , Acyltransferases/genetics , Escherichia coli , Lithospermum/genetics , Substrate SpecificityABSTRACT
Lithospermum arvense is a troublesome dicotyledonous winter annual weed of wheat in China. A L. arvense population (HN01) suspected of being resistant to acetolactate synthase (ALS) inhibitors was found in Henan Province, China. This study aimed to testify the sensitivity of this HN01 population to eight herbicides from 3 different modes of action, and to explore the potential target-site-resistance mechanism to tribenuron-methyl. The whole-plant bioassays indicated that the population was highly resistant to tribenuron-methyl (SU, 350-fold), pyrithiobac sodium (PTB, 151-fold), pyroxsulam (TP, 62.7-fold), florasulam (TP, 80.6-fold), and imazethapyr (IMI, 136-fold), but was sensitive to carfentrazone-ethyl and fluroxypyr-meptyl. ALS gene sequencing revealed that the Trp (TGG) was substituted by Leu (TTG) at codon 574 in resistant plants. In in vitro ALS assays, the concentration of tribenuron-methyl required to inhibit 50% ALS activity (I50) for HN01 was 117-fold greater than that required to inhibit a susceptible population (HN05), indicating that resistance was due to reduced sensitivity of the ALS enzyme to tribenuron-methyl. To the best of our knowledge, this is the first report of ALS gene Trp-574-Leu amino acid mutation confer resistance to tribenuron-methyl in L. arvense.
Subject(s)
Acetolactate Synthase/genetics , Lithospermum/drug effects , Lithospermum/enzymology , Mutation/genetics , Arylsulfonates/toxicity , Benzoates/toxicity , Herbicide Resistance/genetics , Herbicides/toxicity , Lithospermum/genetics , Nicotinic Acids/toxicity , Plant Proteins/genetics , Pyrimidines/toxicity , Sulfonamides/toxicityABSTRACT
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.
Subject(s)
Biosynthetic Pathways , Lithospermum/enzymology , Lithospermum/metabolism , Naphthoquinones/metabolism , Proteomics , Cluster Analysis , Gene Expression Regulation, Plant , Lithospermum/genetics , Naphthoquinones/chemistry , Plant Proteins/metabolism , Proteome/metabolism , Reproducibility of Results , Sequence Analysis, RNAABSTRACT
Lithospermum officinale is a valuable source of bioactive metabolites with medicinal and industrial values. However, little is known about genes involved in the biosynthesis of these metabolites, primarily due to the lack of genome or transcriptome resources. This study presents the first effort to establish and characterize de novo transcriptome assembly resource for L. officinale and expression analysis for three of its tissues, namely leaf, stem, and root. Using over 4Gbps of RNA-sequencing datasets, we obtained de novo transcriptome assembly of L. officinale, consisting of 77,047 unigenes with assembly N50 value as 1524 bps. Based on transcriptome annotation and functional classification, 52,766 unigenes were assigned with putative genes functions, gene ontology terms, and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. KEGG pathway and gene ontology enrichment analysis using highly expressed unigenes across three tissues and targeted metabolome analysis showed active secondary metabolic processes enriched specifically in the root of L. officinale. Using co-expression analysis, we also identified 20 and 48 unigenes representing different enzymes of lithospermic/chlorogenic acid and shikonin biosynthesis pathways, respectively. We further identified 15 candidate unigenes annotated as cytochrome P450 with the highest expression in the root of L. officinale as novel genes with a role in key biochemical reactions toward shikonin biosynthesis. Thus, through this study, we not only generated a high-quality genomic resource for L. officinale but also propose candidate genes to be involved in shikonin biosynthesis pathways for further functional characterization.
Subject(s)
Benzofurans/metabolism , Chlorogenic Acid/metabolism , Depsides/metabolism , Lithospermum/genetics , Metabolome , Naphthoquinones/metabolism , Transcriptome , Biosynthetic Pathways , Gene Ontology , Lithospermum/chemistry , Lithospermum/metabolism , Plant Leaves/chemistry , Plant Leaves/genetics , Plant Leaves/metabolism , Plant Roots/chemistry , Plant Roots/genetics , Plant Roots/metabolism , Plant Stems/chemistry , Plant Stems/genetics , Plant Stems/metabolismABSTRACT
Shikonin and its derivatives are important medicinal secondary metabolites accumulating in roots of Lithospermum erythrorhizon. Although some membrane proteins have been identified as transporters of secondary metabolites, the mechanisms underlying shikonin transport and accumulation in L. erythrorhizon cells still remain largely unknown. In this study, we isolated a cDNA encoding LeMRP, an ATP-binding cassette transporter from L. erythrorhizon, and further investigated its functions in the transport and biosynthesis of shikonin using the yeast transformation and transgenic hairy root methods, respectively. Real-time PCR was applied for expression analyses of LeMRP and shikonin biosynthetic enzyme genes. Functional analysis of LeMRP using the heterologous yeast cell expression system showed that LeMRP could be involved in shikonin transport. Transgenic hairy roots of L. erythrorhizon demonstrated that LeMRP overexpressing hairy roots produced more shikonin than the empty vector (EV) control. Real-time PCR results revealed that the enhanced shikonin biosynthesis in the overexpression lines was mainly caused by highly up-regulated expression of genes coding key enzymes (LePAL, HMGR, Le4CL and LePGT) involved in shikonin biosynthesis. Conversely, LeMRP RNAi decreased the accumulation of shikonin and effectively down-regulated expression level of the above genes. Typical inhibitors of ABC proteins, such as azide and buthionine sulphoximine, dramatically inhibited accumulation of shikonin in hairy roots. Our findings provide evidence for the important direct or indirect role of LeMRP in transmembrane transport and biosynthesis of shikonin.
Subject(s)
ATP-Binding Cassette Transporters/metabolism , Lithospermum/metabolism , Naphthoquinones/metabolism , Plant Proteins/metabolism , ATP-Binding Cassette Transporters/genetics , Cloning, Molecular , Gene Expression Regulation, Plant , Lithospermum/genetics , Membrane Transport Proteins/metabolism , Phylogeny , Plant Proteins/genetics , Plants, Genetically Modified , Real-Time Polymerase Chain Reaction , Sequence Analysis, DNAABSTRACT
Shikonin and its derivatives extracted from Lithospermeae plants' red roots have current applications in food and pharmaceutical industries. Previous studies have cloned some genes related to shikonin biosynthesis. However, most genes related to shikonin biosynthesis remain unclear, because the lack of the genome/transcriptome of the Lithospermeae plants. Therefore, in order to provide a new understanding of shikonin biosynthesis, we obtained transcriptome data and unigenes expression profiles in three shikonin-producing Lithospermeae plants, i.e., Lithospermum erythrorhizon, Arnebia euchroma and Echium plantagineum. As a result, two unigenes (i.e., G10H and 12OPR) that are involved in "shikonin downstream biosynthesis" and "methyl jasmonate biosynthesis" were deemed to relate to shikonin biosynthesis in this study. Furthermore, we conducted a Lamiids phylogenetic model and identified orthologous unigenes under positive selection in above three Lithospermeae plants. The results indicated Boraginales was more relative to Solanales/Gentianales than to Lamiales.
Subject(s)
Biological Evolution , Biosynthetic Pathways/genetics , Gene Expression Regulation, Plant , Lithospermum/genetics , Lithospermum/metabolism , Naphthoquinones/metabolism , Transcriptome , Boraginaceae/genetics , Boraginaceae/metabolism , Chromatography, High Pressure Liquid , Computational Biology/methods , Gene Expression Profiling , High-Throughput Nucleotide Sequencing , Lithospermum/classification , Molecular Sequence Annotation , Naphthoquinones/analysis , Phylogeny , Selection, GeneticABSTRACT
BACKGROUND: The phytohormone ethylene (ET) is a key signaling molecule for inducing the biosynthesis of shikonin and its derivatives, which are secondary metabolites in Lithospermum erythrorhizon. Although ETHYLENE INSENSITIVE3 (EIN3)/EIN3-like proteins (EILs) are crucial transcription factors in ET signal transduction pathway, the possible function of EIN3/EIL1 in shikonin biosynthesis remains unknown. In this study, by targeting LeEIL-1 (L. erythrorhizon EIN3-like protein gene 1) at the expression level, we revealed the positive regulatory effect of LeEIL-1 on shikonin formation. RESULTS: The mRNA level of LeEIL-1 was significantly up-regulated and down-regulated in the LeEIL-1-overexpressing hairy root lines and LeEIL-1-RNAi hairy root lines, respectively. Specifically, LeEIL-1 overexpression resulted in increased transcript levels of the downstream gene of ET signal transduction pathway (LeERF-1) and a subset of genes for shikonin formation, excretion and/or transportation (LePAL, LeC4H-2, Le4CL-1, HMGR, LePGT-1, LeDI-2, and LePS-2), which was consistent with the enhanced shikonin contents in the LeEIL-1-overexpressing hairy root lines. Conversely, LeEIL-1-RNAi dramatically repressed the expression of the above genes and significantly reduced shikonin production. CONCLUSIONS: The results revealed that LeEIL-1 is a positive regulator of the biosynthesis of shikonin and its derivatives in L. erythrorhizon hairy roots. Our findings gave new insights into the molecular regulatory mechanism of ET in shikonin biosynthesis. LeEIL-1 could be a crucial target gene for the genetic engineering of shikonin biosynthesis.
Subject(s)
Gene Expression Regulation, Plant , Lithospermum/genetics , Naphthoquinones/metabolism , Plant Proteins/genetics , Transcription Factors/genetics , Lithospermum/metabolism , Plant Proteins/metabolism , Plant Roots/metabolism , Plants, Genetically Modified/genetics , Plants, Genetically Modified/metabolism , RNA Interference , Transcription Factors/metabolismABSTRACT
A novel strategy to finely control the electron transfer chain (ETC) activity of Escherichia coli was established. In this study, the fine-tuning of the ubiquinone biosynthesis pathway was applied to further controlling ETC function in coenzyme Q8 biosynthesis-deficient E. coli strains, HW108 and HW109, which contain mutations in ubiE and ubiG, respectively. A competing pathway on the intermediate substrates of the Q8 synthesis pathway, catalyzed by diphosphate:4-hydroxybenzoate geranyltransferase (PGT-1) of Lithospermum erythrorhizon, was introduced into these mutant strains. A nearly theoretical yield of lactate production can be achieved under fully aerobic conditions via an in vivo, genetically fine-tunable means to further control the activity of the ETC of the Q8 biosynthesis-deficient E. coli strains.
Subject(s)
Escherichia coli/metabolism , Lactic Acid/metabolism , Metabolic Engineering/methods , Ubiquinone/biosynthesis , Ubiquinone/deficiency , Aerobiosis , Alkyl and Aryl Transferases/genetics , Alkyl and Aryl Transferases/metabolism , Electron Transport , Escherichia coli/genetics , Fermentation , Lithospermum/enzymology , Lithospermum/genetics , Recombinant Proteins/genetics , Recombinant Proteins/metabolismABSTRACT
Membrane-bound type prenyltransferases for aromatic substrates play crucial roles in the biosynthesis of various natural compounds. Lithospermum erythrorhizon p-hydroxybenzoate: geranyltransferase (LePGT1), which contains multiple transmembrane α-helices, is involved in the biosynthesis of a red naphthoquinone pigment, shikonin. Taking LePGT1 as a model membrane-bound aromatic substrate prenyltransferase, we utilized a baculovirus-Sf9 expression system to generate a high yield LePGT1 polypeptide, reaching ~ 1000-fold higher expression level compared with a yeast expression system. Efficient solubilization procedures and biochemical purification methods were developed to extract LePGT1 from the membrane fraction of Sf9 cells. As a result, 80 µg of LePGT1 was purified from 150 mL culture to almost homogeneity as judged by SDS/PAGE. Using purified LePGT1, enzymatic characterization, e.g. substrate specificity, divalent cation requirement and kinetic analysis, was done. In addition, inhibition experiments revealed that aromatic compounds having two phenolic hydroxyl groups effectively inhibited LePGT1 enzyme activity, suggesting a novel recognition mechanism for aromatic substrates. As the first example of solubilization and purification of this membrane-bound protein family, the methods established in this study will provide valuable information for the precise biochemical characterization of aromatic prenyltransferases as well as for crystallographic analysis of this novel enzyme family.
Subject(s)
Alkyl and Aryl Transferases/isolation & purification , Alkyl and Aryl Transferases/metabolism , Dimethylallyltranstransferase/isolation & purification , Dimethylallyltranstransferase/metabolism , Lithospermum/enzymology , Animals , Dimethylallyltranstransferase/genetics , Kinetics , Lithospermum/genetics , Plant Proteins/genetics , Plant Proteins/isolation & purification , Plant Proteins/metabolism , Point Mutation , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/isolation & purification , Recombinant Fusion Proteins/metabolism , Sf9 Cells , Spodoptera , Substrate SpecificityABSTRACT
We previously showed that ethylene might be involved in the process of shikonin biosynthesis regulated by light signals. Here, we cloned a full-length cDNA of LeERF-1, a putative ethylene response factor gene, from Lithospermum erythrorhizon using the RACE (rapid amplification of cDNA ends) method. Phylogenetic analysis revealed that LeERF-1 was classified in the B3 subfamily, together with ERF1 and ORA59 of Arabidopsis. Heterologous expression of LeERF-1 in Arabidopsis showed that LeERF-1:eGFP fusion protein was precisely localised to the nucleus, implying that it might function as a transcription factor. Detailed expression analysis with real-time PCR showed that LeERF-1 was significantly down-regulated by white, blue and red light, although the inhibitory effect of red light was relatively weak compared to other light conditions. Tissue-specific expression analysis also indicated that LeERF-1 was dominantly expressed in the roots, which grow in soil in darkness. These patterns are all consistent with the effects of different light signals on regulating formation of shikonin and its derivatives, indicating that LeERF-1 might be a crucial positive regulator, like other B3 subfamily proteins (such as ORCA3 and ORA59), in regulating biosynthesis of secondary metabolites.
Subject(s)
Gene Expression Regulation, Plant/radiation effects , Light , Lithospermum/genetics , Plant Proteins/metabolism , Transcription Factors/metabolism , Arabidopsis/genetics , Arabidopsis/metabolism , Base Sequence , Cloning, Molecular , DNA, Complementary/genetics , DNA, Plant/genetics , Down-Regulation , Genes, Regulator , Lithospermum/metabolism , Lithospermum/radiation effects , Molecular Sequence Data , Multigene Family , Naphthoquinones/metabolism , Plant Proteins/genetics , Plants, Genetically Modified/genetics , Plants, Genetically Modified/metabolism , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Sequence Alignment , Transcription Factors/geneticsABSTRACT
Phylogenetic relationships are complex within the Lithospermeae, a large subgroup of the Boraginaceae s.str. The relationships of New World Lasiarrhenum, Macromeria, Nomosa, Onosmodium, Perittostoma, and Psilolaemus to subcosmopolitan and much larger Lithospermum have not been critically investigated in the recent past. No molecular data on the phylogeny of these genera and Lithospermum have so far been published. We investigated the relationships within Lithospermeae using three loci (nuclear ITS plus 5.8S rRNA, chloroplast trnL-F-spacer, and trnS-G-spacer) and micromorphological character traits (pollen, nutlets). Lithospermums.l. constitutes the sistergroup of Asian Ulugbekia and is monophyletic only when its American segregates "Macromeria", monotypic Nomosa, and Onosmodium are included. Both the African and the South American species groups of Lithospermum are monophyletic, but North American representatives are not resolved in a single clade. Morphological characters that have been considered as important for generic delimitation in the past (such as large, yellow corollas without faucal scales, particular pollen types, coarsely veined leaves, shrubby habit) have evolved in at least two only distantly related lineages within Lithospermums.l. The reduction of American "Macromeria", Nomosa, and Onosmodium as well as Asian Ulugbekia under Lithospermum is proposed to render the latter monophyletic. This redefined Lithospermum s.l. appears to have undergone a type of recent "island radiation" in the Americas, reflected in a morphological diversity far exceeding that found in the Old World.
Subject(s)
Evolution, Molecular , Lithospermum/genetics , Phylogeny , DNA, Chloroplast/genetics , DNA, Plant/genetics , DNA, Ribosomal Spacer/genetics , Fruit/ultrastructure , Lithospermum/classification , Lithospermum/ultrastructure , Microscopy, Electron, Scanning , Pollen/ultrastructure , RNA, Ribosomal, 5.8S/genetics , Sequence Alignment , Sequence Analysis, DNAABSTRACT
A cDNA (LEPS-2) encoding a novel cell wall protein was cloned from shikonin-producing callus tissues of Lithospermum erythrorhizon by differential display between a shikonin-producing culture strain and a non-producing strain. The LEPS-2 cDNA encoded a polypeptide of 184 amino acids. The deduced amino acid sequence exhibited no significant homology with known proteins. Expression of LEPS-2 gene as well as accumulation of LEPS-2 protein was highly correlated with shikonin production in L. erythrorhizon cells in culture. In the intact plant, expression of LEPS-2 was detected only in the roots where shikonin pigments accumulated. Cell fractionation experiments and immunocytochemical analysis showed that the protein was localized in the apoplast fraction of the cell walls. The shikonin pigments were also stored on the cell walls as oil droplets. These results indicate that expression of the LEPS-2 is closely linked with shikonin biosynthesis and the LEPS-2 protein may be involved in the intra-cell wall trapping of shikonin pigments.
Subject(s)
Lithospermum/genetics , Naphthoquinones/metabolism , Plant Proteins/genetics , Amino Acid Sequence , Base Sequence , Cloning, Molecular , Culture Techniques , DNA, Complementary/chemistry , DNA, Complementary/genetics , Enzymes/genetics , Enzymes/metabolism , Gene Expression Regulation, Enzymologic , Gene Expression Regulation, Plant , Lithospermum/metabolism , Molecular Sequence Data , Plant Proteins/metabolism , Propanols/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , Sequence Analysis, DNA , Substrate SpecificityABSTRACT
Shikonin, a red naphthoquinone pigment, is produced by cell cultures of Lithospermum erythrorhizon (Boraginaceae). It is biosynthetically derived from two key precursors, 4-hydroxybenzoate (4HB) and geranyldiphosphate (GPP). The bacterial ubiC gene, encoding chorismate pyruvate-lyase (CPL) which converts chorismate to 4-hydroxybenzoate, was expressed in L. erythrorhizon under the control of the strong (ocs)(3)mas-promoter. This introduced an efficient biosynthetic pathway to 4HB, i.e. a one-step reaction from chorismate, in addition to the endogeneous multi-step phenylpropanoid pathway. Feeding experiments with [1,7-(13)C(2)]shikimic acid showed that in the most active transgenic line, 73% of 4HB was synthesized via the genetically introduced pathway. However, there was no correlation between CPL activity and 4HB glucoside or shikonin accumulation in the transgenic lines. HMG-CoA reductase (HMGR) is involved in the biosynthesis of GPP in L. erythrorhizon. Two forms of HMGR1 of Arabidopsis thaliana were expressed in Lithospermum under control of the (ocs)(3)mas promoter. Only moderate increases in enzyme activity were obtained with the complete enzyme, but high activity was achieved using the soluble cytosolic domain of HMGR1. Shikonin accumulation remained unchanged even upon high expression of soluble HMGR.
Subject(s)
Hydroxymethylglutaryl CoA Reductases/genetics , Lithospermum/enzymology , Oxo-Acid-Lyases/genetics , Plant Roots/enzymology , Arabidopsis/enzymology , Blotting, Northern , Blotting, Southern , Carbon Isotopes , Cell Surface Extensions/enzymology , Cloning, Molecular , Culture Techniques , Gene Expression Regulation, Enzymologic , Gene Expression Regulation, Plant , Genetic Vectors/genetics , Glucosides/biosynthesis , Glucosides/chemistry , Hydroxymethylglutaryl CoA Reductases/metabolism , Lithospermum/genetics , Magnetic Resonance Spectroscopy , Naphthoquinones/chemistry , Naphthoquinones/metabolism , Oxo-Acid-Lyases/metabolism , Parabens/chemistry , Parabens/metabolism , Plant Roots/genetics , Plants, Genetically Modified , Polyisoprenyl Phosphates/chemistry , Polyisoprenyl Phosphates/metabolism , Shikimic Acid/metabolismABSTRACT
Two cDNAs encoding geranyl diphosphate:4-hy- droxybenzoate 3-geranyltransferase were isolated from Lithospermum erythrorhizon by nested PCR using the conserved amino acid sequences among polyprenyl- transferases for ubiquinone biosynthesis. They were functionally expressed in yeast COQ2 disruptant and showed a strict substrate specificity for geranyl diphosphate as the prenyl donor, in contrast to ubiquinone biosynthetic enzymes, suggesting that they are involved in the biosynthesis of shikonin, a naphthoquinone secondary metabolite. Regulation of their expression by various culture conditions coincided with that of geranyltransferase activity and the secondary metabolites biosynthesized via this enzyme. This is the first established plant prenyltransferase that transfers the prenyl chain to an aromatic substrate.