Identification of novel flavin-dependent monooxygenase from Strobilanthes Cusia reveals molecular basis of indoles' biosynthetic logic.

BACKGROUND: Strobilanthes cusia (Nees) Kuntze is a traditional medical plant distributed widely in south China. The indole compounds that originated from the plant are responsible for its pharmacological activities. However, the reason why indole ingredients are accumulated in this herb and how it is biosynthesized has remained largely unknown. RESULTS: In this study, metabolic and transcriptional profiling measurement experiments of different S. cusia organs were carried out to understand the underlying molecular basis of indoles' biosynthetic logic. A metabolic investigation demonstrated that the indoles are primarily accumulated mainly in aerial parts, particularly in leaves. RNA-seq was employed to reveal the organ specific accumulation of indoles in different S. cusia organs. Meanwhile, a flavin-dependent monooxygenase gene (ScFMO1) was found in S. cusia, and it has capacity to produce indoxyl from indole by the fermentation assay. Finally, we assessed the outcomes of transient expression experiment in tobacco and confirmed that ScFMO1 localizes in cytoplasm. CONCLUSIONS: Our results suggest that ScFMO1 plays a key role in biosynthesis of indoles (Indigo, indirubin, indican, etc.), it will be useful for illuminating the molecular basis of the medicinal indoles' biosynthesis and developing strategies for improving their yields.

Effects of exogenous indole-3-acetic acid on the density of trichomes, expression of artemisinin biosynthetic genes, and artemisinin biosynthesis in Artemisia annua.

Artemisinin is the most practical medication for the treatment of malaria, but is only very minimally synthesized in Artemisia annua, significantly less than the market needs. In this study, indole-3-acetic acid (IAA) was used to investigate its effects on trichomes, artemisinin accumulation, and biosynthetic gene expression in A. anuua. The results showed that exogenous IAA could contribute to the growth and development of A. annua and increase the density of trichomes. Analysis using liquid chromatography-tandem mass spectrometry (LC-MS/MS) indicated that artemisinin and dihydroartemisinic acid (DHAA) contents were increased by 1.9-fold (1.1 mg/g) and 2.1-fold (0.51 mg/g) after IAA treatment in comparison with control lines (CK), respectively. Furthermore, quantitative real-time PCR results showed that AaADS, AaCYP71AV1, AaALDH1, and AaDBR2, four critical enzyme genes for the biosynthesis of artemisinin, had relatively high transcription levels in leaves of A. annua treated with IAA. In summary, this study indicated that exogenous IAA treatment was a feasible strategy to enhance artemisinin production, which paves the way for further metabolic engineering of artemisinin biosynthesis.

Molecular cloning and functional characterization of BcTSA in the biosynthesis of indole alkaloids in Baphicacanthus cusia.

Baphicacanthus cusia (Nees) Bremek (B. cusia) is an essential traditional Chinese herb that is commonly used to treat colds, fever, and influenza. Indole alkaloids, such as indigo and indirubin, are the primary active constituents of B. cusia. The indole-producing reaction is crucial for regulating the flow of indole alkaloids metabolites along the pathways and coordinating primary and secondary product biosynthesis in plants. The tryptophan synthase alpha-subunit (TSA) can catalyse a process that produces indole, which is free to enter secondary metabolite pathways; however, the underlying potential mechanism of regulating indigo alkaloids synthesis remains unknown. Here, a BcTSA was cloned from the transcriptome of B. cusia. The BcTSA has a significant degree of similarity with other plant TSAs according to bioinformatics and phylogenetic analyses. Quantitative real-time PCR (RT-qPCR) research showed that BcTSA was dramatically enhanced in response to treatment with methyl jasmonate (MeJA), salicylic acid (SA), and abscisic acid (ABA), and was predominantly expressed in the stems as opposed to the leaves and rhizomes. Subcellular localization revealed that BcTSA is localized in chloroplasts, which is compatible with the fact that the conversion of indole-3-glycerol phosphate (IGP) to indole occurs in chloroplasts. The complementation assay results showed that BcTSA was functional, demonstrating that it was capable of catalyzing the conversion of IGP to indole. BcTSA was shown to stimulate the manufacture of indigo alkaloids including isatin, indigo, and indirubin when the gene was overexpressed in the hairy roots of Isatis indigotica. In conclusion, our research provides novel perspectives that might be applied to manipulating the indole alkaloid composition of B. cusia.

Genome-wide identification and characterisation of bHLH transcription factors in Artemisia annua.

BACKGROUND: A. annua (also named Artemisia annua, sweet wormwood) is the main source of the anti-malarial drug artemisinin, which is synthesised and stored in its trichomes. Members of the basic Helix-Loop-Helix (bHLH) family of transcription factors (TFs) have been implicated in artemisinin biosynthesis in A. annua and in trichome development in other plant species. RESULTS: Here, we have systematically identified and characterised 226 putative bHLH TFs in A. annua. All of the proteins contain a HLH domain, 213 of which also contain the basic motif that mediates DNA binding of HLH dimers. Of these, 22 also contained a Myc domain that permits dimerisation with other families of TFs; only two proteins lacking the basic motif contained a Myc domain. Highly conserved GO annotations reflected the transcriptional regulatory role of the identified TFs, and suggested conserved roles in biological processes such as iron homeostasis, and guard cell and endosperm development. Expression analysis revealed that three genes (AabHLH80, AabHLH96, and AaMyc-bHLH3) exhibited spatiotemporal expression patterns similar to genes encoding key enzymes in artemisinin synthesis. CONCLUSIONS: This comprehensive analysis of bHLH TFs provides a new resource to direct further analysis into key molecular mechanisms underlying and regulating artemisinin biosynthesis and trichome development, as well as other biological processes, in the key medicinal plant A. annua.

IiWRKY34 positively regulates yield, lignan biosynthesis and stress tolerance in Isatis indigotica.

Yield potential, pharmaceutical compounds production and stress tolerance capacity are 3 classes of traits that determine the quality of medicinal plants. The autotetraploid Isatis indigotica has greater yield, higher bioactive lignan accumulation and enhanced stress tolerance compared with its diploid progenitor. Here we show that the transcription factor IiWRKY34, with higher expression levels in tetraploid than in diploid I. indigotica, has large pleiotropic effects on an array of traits, including biomass growth rates, lignan biosynthesis, as well as salt and drought stress tolerance. Integrated analysis of transcriptome and metabolome profiling demonstrated that IiWRKY34 expression had far-reaching consequences on both primary and secondary metabolism, reprograming carbon flux towards phenylpropanoids, such as lignans and flavonoids. Transcript-metabolite correlation analysis was applied to construct the regulatory network of IiWRKY34 for lignan biosynthesis. One candidate target Ii4CL3, a key rate-limiting enzyme of lignan biosynthesis as indicated in our previous study, has been demonstrated to indeed be activated by IiWRKY34. Collectively, the results indicate that the differentially expressed IiWRKY34 has contributed significantly to the polyploidy vigor of I. indigotica, and manipulation of this gene will facilitate comprehensive improvements of I. indigotica herb.

TRICHOME AND ARTEMISININ REGULATOR 2 positively regulates trichome development and artemisinin biosynthesis in Artemisia annua.

Glandular secretory trichomes (GSTs) are regarded as biofactories for synthesizing, storing, and secreting artemisinin. It is necessary to figure out the initiation and development regulatory mechanism of GSTs to cultivate high-yielding Artemisia annua. Here, we identified an MYB transcription factor, AaTAR2, from bioinformatics analysis of the A. annua genome database and Arabidopsis trichome development-related genes. AaTAR2 is mainly expressed in young leaves and located in the nucleus. Repression and overexpression of AaTAR2 resulted in a decrease and increase, respectively, in the GSTs numbers, leaf biomass, and the artemisinin content in transgenic plants. Furthermore, the morphological characteristics changed obviously in trichomes, suggesting AaTAR2 plays a key role in trichome formation. In addition, the expression of flavonoid biosynthesis genes and total flavonoid content increased dramatically in AaTAR2-overexpressing transgenic plants. Owing to flavonoids possibly counteracting emerging resistance to artemisinin in Plasmodium species, AaTAR2 is a potential target to improve the effect of artemisinin in clinical therapy. Taken together, AaTAR2 positively regulates trichome development and artemisinin and flavonoid biosynthesis. A better understanding of this 'multiple functions' transcription factor may enable enhanced artemisinin and flavonoids yield. AaTAR2 is a potential breeding target for cultivating high-quality A. annua.

Author Correction: SmMYC2a and SmMYC2b played similar but irreplaceable roles in regulating the biosynthesis of tanshinones and phenolic acids in Salvia miltiorrhiza.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Molecular cloning and metabolomic characterization of the 5-enolpyruvylshikimate-3-phosphate synthase gene from Baphicacanthus cusia.

BACKGROUND: Indigo alkaloids, such as indigo, indirubin and its derivatives, have been identified as effective antiviral compounds in Baphicacanthus cusia. Evidence suggests that the biosynthesis of indigo alkaloids in plants occurs via the shikimate pathway. The enzyme 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) is involved in plant metabolism; however, its underlying putative mechanism of regulating the production of indigo alkaloids is currently unknown. RESULTS: One gene encoding EPSPS was isolated from B. cusia. Quantitative real-time PCR analysis revealed that BcEPSPS was expressed at the highest level in the stem and upregulated by methyl jasmonate (MeJA), salicylic acid (SA) and abscisic acid (ABA) treatment. The results of subcellular localization indicated that BcEPSPS is mainly expressed in both the plastids and cytosol, which has not been previously reported. An enzyme assay revealed that the heterogeneously expressed BcEPSPS protein catalysed the generation of 5-enolpyruvyl shikimate-3-phosphate. The overexpression of BcEPSPS in Isatis indigotica hairy roots resulted in the high accumulation of indigo alkaloids, such as indigo, secologanin, indole and isorhamnetin. CONCLUSIONS: The function of BcEPSPS in catalysing the production of EPSP and regulating indigo alkaloid biosynthesis was revealed, which provided a distinct view of plant metabolic engineering. Our findings have practical implications for understanding the effect of BcEPSPS on active compound biosynthesis in B. cusia.

Transcriptome analysis reveals novel enzymes for apo-carotenoid biosynthesis in saffron and allows construction of a pathway for crocetin synthesis in yeast.

Crocus sativus is generally considered the source of saffron spice which is rich in apo-carotenoid compounds such as crocins, crocetin, picrocrocin, and safranal, which possess effective pharmacological activities. However, little is known about the exact genes involved in apo-carotenoid biosynthesis in saffron and the potential mechanism of specific accumulation in the stigma. In this study, we integrated stigmas at different developmental stages to perform in-depth transcriptome and dynamic metabolomic analyses to discover the potential key catalytic steps involved in apo-carotenoid biosynthesis in saffron. A total of 61 202 unigenes were obtained, and 28 regulators and 32 putative carotenogenic genes were captured after the co-expression network analysis. Moreover, 15 candidate genes were predicted to be closely related to safranal and crocin production, in which one aldehyde dehydrogenase (CsALDH3) was validated to oxidize crocetin dialdehyde into crocetin and a crocetin-producing yeast strain was created. In addition, a new branch pathway that catalyses the conversion of geranyl-geranyl pyrophosphate to copalol and ent-kaurene by the class II diterpene synthase CsCPS1 and three class I diterpene synthases CsEKL1/2/3 were investigated for the first time. Such gene to apo-carotenoid landscapes illuminate the synthetic charactersistics and regulators of apo-carotenoid biosynthesis, laying the foundation for a deep understanding of the biosynthesis mechanism and metabolic engineering of apo-carotenoids in plants or microbes.

Genome-Wide Identification and Characterization of Salvia miltiorrhiza Laccases Reveal Potential Targets for Salvianolic Acid B Biosynthesis.

Laccases are widely distributed in plant kingdom catalyzing the polymerization of lignin monolignols. Rosmarinic acid (RA) has a lignin monolignol-like structure and is converted into salvianolic acid B (SAB), which is a representatively effective hydrophilic compound of a well-known medicinal plant Salvia miltiorrhiza and also the final compound of phenolic acids metabolism pathway in the plant. But the roles of laccases in the biosynthesis of SAB are poorly understood. This work systematically characterizes S. miltiorrhiza laccase (SmLAC) gene family and identifies the SAB-specific candidates. Totally, 29 laccase candidates (SmLAC1-SmLAC29) are found to contain three signature Cu-oxidase domains. They present relatively low sequence identity and diverse intron-exon patterns. The phylogenetic clustering of laccases from S. miltiorrhiza and other ten plants indicates that the 29 SmLACs can be divided into seven groups, revealing potential distinct functions. Existence of diverse cis regulatory elements in the SmLACs promoters suggests putative interactions with transcription factors. Seven SmLACs are found to be potential targets of miR397. Putative glycosylation sites and phosphorylation sites are identified in SmLAC amino acid sequences. Moreover, the expression profile of SmLACs in different organs and tissues deciphers that 5 SmLACs (SmLAC7/8/20/27/28) are expressed preferentially in roots, adding the evidence that they may be involved in the phenylpropanoid metabolic pathway. Besides, silencing of SmLAC7, SmLAC20 and SmLAC28, and overexpression of SmLAC7 and SmLAC20 in the hairy roots of S. miltiorrhiza result in diversification of SAB, signifying that SmLAC7 and SmLAC20 take roles in SAB biosynthesis. The results of this study lay a foundation for further elucidation of laccase functions in S. miltiorrhiza, and add to the knowledge for SAB biosynthesis in S. miltiorrhiza.

Targeted expression of Vitreoscilla hemoglobin improves the production of tropane alkaloids in Hyoscyamus niger hairy roots.

Under hypoxic conditions, the expression of Vitreoscilla hemoglobin (VHb) in plants is proposed to increase the productivity of certain oxygen-requiring metabolic pathways by promoting the delivery of oxygen. Tropane alkaloids (TAs) are a class of important plant secondary metabolites with significant medicinal value; the final step in their biosynthesis requires oxygen. Whether heterologous expression of VHb, especially in different subcellular compartments, can accelerate the accumulation of TAs is not known. Herein, the effect of heterologous expression of VHb in different subcellular locations on the TA profile of H. niger hairy roots was investigated. The targeted expression of VHb in the plastids (using pVHb-RecA construct), led to the accumulation of 197.68 µg/g hyoscyamine in the transgenic H. niger hairy roots, which was 1.25-fold of the content present in the lines in which VHb expression was not targeted, and 3.66-fold of that present in the wild type (WT) lines. The content of scopolamine was increased by 2.20- and 4.70-fold in the pVHb-RecA transgenic lines compared to that in the VHb transgenic and WT lines. Our results demonstrate that VHb could stimulate the accumulation of TAs in the transgenic H. niger hairy roots. Quantitative RT-PCR analysis revealed that the expression of key genes involved in TA biosynthesis increased significantly in the VHb transgenic lines. We present the first description of a highly efficient strategy to increase TA content in H. niger. Moreover, our results also shed light on how the production of desired metabolites can be efficiently enhanced by using more accurate and appropriate genetic engineering strategies.

Integrated Transcript and Metabolite Profiles Reveal That EbCHI Plays an Important Role in Scutellarin Accumulation in Erigeron breviscapus Hairy Roots.

Scutellarin, a flavonoid 7-O-glucuronide, is an essential bioactive compound of Erigeron breviscapus (Vaniot) Hand.-Mazz. used for the treatment of cerebrovascular diseases. However, due to overexploitation and overuse, E. breviscapus is facing the problems of extinction and habitat degradation. In this study, a correlation analysis between the transcript and metabolite profiles of methyl jasmonate (MeJA)-treated E. breviscapus at different time points indicated that chalcone isomerase (EbCHI) was the primary contributor to scutellarin accumulation during flavonoid biosynthesis. EbCHI was then further characterized as a chalcone isomerase that efficiently converted chalcone to naringenin in vitro. Optimal parameters derived by comparing different culture conditions were successfully used to establish hairy root cultures of E. breviscapus with a maximum transformation rate of 60% in B5 medium. Furthermore, overexpression of EbCHI significantly enhanced scutellarin accumulation in E. breviscapus hairy roots with a maximum content of 2.21 mg g-1 (dw), 10-fold higher than that of natural roots (0.21 mg g-1 dw). This study sheds new light on a method of effective gene-based metabolic engineering by accurate and appropriate strategies and provides a protocol for hairy root cultures that accumulate high levels of scutellarin, providing a promising prospect for relieving the overexploitation and unavailability of E. breviscapus in the future.

Rice actin binding protein RMD controls crown root angle in response to external phosphate.

Root angle has a major impact on acquisition of nutrients like phosphate that accumulate in topsoil and in many species; low phosphate induces shallower root growth as an adaptive response. Identifying genes and mechanisms controlling root angle is therefore of paramount importance to plant breeding. Here we show that the actin-binding protein Rice Morphology Determinant (RMD) controls root growth angle by linking actin filaments and gravity-sensing organelles termed statoliths. RMD is upregulated in response to low external phosphate and mutants lacking of RMD have steeper crown root growth angles that are unresponsive to phosphate levels. RMD protein localizes to the surface of statoliths, and rmd mutants exhibit faster gravitropic response owing to more rapid statoliths movement. We conclude that adaptive changes to root angle in response to external phosphate availability are RMD dependent, providing a potential target for breeders.

[Bioinformation analysis of chorismate synthase in Baphicacantus cusia and other 78 species of plants].

Chorismate synthase(CS, EC:4.2.3.5) catalyses 5-enolpyruvy-shikimate-3-phosphate to form chorismate, which is the essential enzyme for chorismate biosynthesis in organisms. The amino acid sequences of CS from 79 species of higher plants were reported in GenBank at present. 125 amino acid sequences of CS from Baphicacanthus cusia and other 78 species of plants were predicted and analyzed by using various bioinformatics software, including the composition of amino acid sequences, signal peptide, leader peptide, hydrophobic/hydrophilic, transmembrane structure, coiled-coil domain, protein secondary structure, tertiary structure and functional domains. The phylogenetic tree of CS protein family was constructed and divided into eight groups by phylogenetic analysis. The homology comparison indicated that B. cusia shared a high homology with several plants such as Sesamum indicum, Nicotiana tabacum, Solanum tuberosum and so on. The open reading frame(ORF) of all samples is about 1 300 bp, the molecular weight is about 50 kDa, the isoelectric point(pI) is 5.0-8.0 which illustrated that CS protein is slightly basic. The ORF of CS we cloned in B. cusia is 1 326 bp, the amino acid residues are 442, the molecular weight is 47 kDa and pI is 8.11. The CS in B.cusia showed obvious hydrophobicity area and hydrophilicity area, no signal peptide, and may exists transmembrane structure areas. The main secondary structures of CS protein are random coil and Alpha helix, also contain three main structural domains which are an active structural domain, a PLN02754 conserved domain and a FMN binding site. The acquired information in this study would provide certain scientific basis for further study on structure-activity relationship and structure modification of CS in plants in the future.

CRISPR/Cas9-mediated efficient targeted mutagenesis of RAS in Salvia miltiorrhiza.

The CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas9 (CRISPR-associated) system is a powerful genome editing tool that has been used in many species. In this study, we focused on the phenolic acid metabolic pathway in the traditional Chinese medicinal herb Salvia miltiorrhiza, using the CRISPR/Cas9 system to edit the rosmarinic acid synthase gene (SmRAS) in the water-soluble phenolic acid biosynthetic pathway. The single guide RNA (sgRNA) was designed to precisely edit the most important SmRAS gene, which was selected from 11 family members through a bioinformatics analysis. The sequencing results showed that the genomes of 50% of the transgenic regenerated hairy roots had been successfully edited. Five biallelic mutants, two heterozygous mutants and one homozygous mutant were obtained from 16 independent transgenic hairy root lines when the sgRNA was driven by the Arabidopsis U6 promoter, while no mutants were obtained from 13 independent transgenic hairy root lines when the sgRNA was driven by the rice U3 promoter. Subsequently, expression and metabolomics analysis showed that the contents of phenolic acids, including rosmarinic acid (RA) and lithospermic acid B, and the RAS expression level were decreased in the successfully edited hairy root lines, particularly in the homozygous mutants. In addition, the level of the RA precursor 3,4-dihydroxyphenyllactic acid clearly increased. These results indicated that the CRISPR/Cas9 system can be utilized to identify important genes in a gene family with the assistance of bioinformatics analysis and that this new technology is an efficient and specific tool for genome editing in S. miltiorrhiza. This new system presents a promising potential method to regulate plant metabolic networks and improve the quality of traditional Chinese medicinal herbs.

[Molecular mechanism of artemisinin biosynthesis and regulation in Artemisia annua].

Artemisinin-based combination therapy (ACT) is the best available treatment, particularly for Plasmodium falciparum malaria. Artemisinin, whose main source is Artemisia annua, has large demand and shortsupply every year.Artemisininis synthesized,stored, and secreted by the glandular secretory trichomes of A. annua(AaGSTs).In general, the population and morphology of AaGSTs are often positively correlated with artemisinin content.This review article introduces the molecular mechanism of biosynthesis and regulation of artemisininin A. annua. Furthermore, this article will refresh the classification of trichomes in A. annua and provide anoverview of the recent achievements regarding AaGSTs and artemisinin.These will shed light on exploring the method for increasing plant-derived artemisinin.

AP2/ERF Transcription Factor, Ii049, Positively Regulates Lignan Biosynthesis in Isatis indigotica through Activating Salicylic Acid Signaling and Lignan/Lignin Pathway Genes.

Lignans, such as lariciresinol and its derivatives, have been identified as effective antiviral ingredients in Isatis indigotica. Evidence suggests that the APETALA2/ethylene response factor (AP2/ERF) family might be related to the biosynthesis of lignans in I. indigotica. However, the special role played by the AP2/ERF family in the metabolism and its underlying putative mechanism still need to be elucidated. One novel AP2/ERF gene, named Ii049, was isolated and characterized from I. indigotica in this study. The quantitative real-time PCR analysis revealed that Ii049 was expressed highest in the root and responded to methyl jasmonate, salicylic acid (SA) and abscisic acid treatments to various degrees. Subcellular localization analysis indicated that Ii049 protein was localized in the nucleus. Knocking-down the expression of Ii049 caused a remarkable reduction of lignan/lignin contents and transcript levels of genes involved in the lignan/lignin biosynthetic pathway. Ii049 bound to the coupled element 1, RAV1AAT and CRTAREHVCBF2 motifs of genes IiPAL and IiCCR, the key structural genes in the lignan/lignin pathway. Furthermore, Ii049 was also essential for SA biosynthesis, and SA induced lignan accumulation in I. indigotica. Notably, the transgenic I. indigotica hairy roots overexpressing Ii049 showed high expression levels of lignan/lignin biosynthetic genes and SA content, resulting in significant accumulation of lignan/lignin. The best-engineered line (OVX049-10) produced 425.60 µg·g-1 lariciresinol, an 8.3-fold increase compared with the wild type production. This study revealed the function of Ii049 in regulating lignan/lignin biosynthesis, which had the potential to increase the content of valuable lignan/lignin in economically significant medicinal plants.

Stable Internal Reference Genes for Normalizing Real-Time Quantitative PCR in Baphicacanthus cusia under Hormonal Stimuli and UV Irradiation, and in Different Plant Organs.

Baphicacanthus cusia (Nees) Bremek, the plant source for many kinds of drugs in traditional Chinese medicine, is widely distributed in South China, especially in Fujian. Recent studies about B. cusia mainly focus on its chemical composition and pharmacological effects, but further analysis of the plant's gene functions and expression is required to better understand the synthesis of its effective compounds. Real-time quantitative polymerase chain reaction (RT-qPCR) is a powerful method for gene expression analysis. It is necessary to select a suitable reference gene for expression normalization to ensure the accuracy of RT-qPCR results. Ten candidate reference genes were selected from the transcriptome datasets of B. cusia in this study, and the expression stability was assessed across 60 samples representing different tissues and organs under various conditions, including ultraviolet (UV) irradiation, hormonal stimuli (jasmonic acid methyl ester and abscisic acid), and in different plant organs. By employing different algorithms, such as geNorm, NormFinder, and BestKeeper, which are complementary approaches based on different statistical procedures, 18S rRNA was found to be the most stable gene under UV irradiation and hormonal stimuli, whereas ubiquitin-conjugating enzyme E2 was the best suitable gene for different plant organs. This novel study aimed to screen for suitable reference genes and corresponding primer pairs specifically designed for gene expression studies in B. cusia, in particular for RT-qPCR analyses.

HPPR encodes the hydroxyphenylpyruvate reductase required for the biosynthesis of hydrophilic phenolic acids in Salvia miltiorrhiza.

Salvia miltiorrhiza is a medicinal plant widely used in the treatment of cardiovascular and cerebrovascular diseases. Hydrophilic phenolic acids, including rosmarinic acid (RA) and lithospermic acid B (LAB), are its primary medicinal ingredients. However, the biosynthetic pathway of RA and LAB in S. miltiorrhiza is still poorly understood. In the present study, we accomplished the isolation and characterization of a novel S. miltiorrhiza Hydroxyphenylpyruvate reductase (HPPR) gene, SmHPPR, which plays an important role in the biosynthesis of RA. SmHPPR contained a putative catalytic domain and a NAD(P)H-binding motif. The recombinant SmHPPR enzyme exhibited high HPPR activity, converting 4-hydroxyphenylpyruvic acid (pHPP) to 4-hydroxyphenyllactic acid (pHPL), and exhibited the highest affinity for substrate 4-hydroxyphenylpyruvate. SmHPPR expression could be induced by various treatments, including SA, GA3, MeJA and Ag+, and the changes in SmHPPR activity were correlated well with hydrophilic phenolic acid accumulation. SmHPPR was localized in cytoplasm, most likely close to the cytosolic NADPH-dependent hydroxypyruvate reductase active in photorespiration. In addition, the transgenic S. miltiorrhiza hairy roots overexpressing SmHPPR exhibited up to 10-fold increases in the products of hydrophilic phenolic acid pathway. In conclusion, our findings provide a new insight into the synthesis of active pharmaceutical compounds at molecular level.