Elsevier

Biochemical Engineering Journal

Volume 74, 15 May 2013, Pages 115-120
Biochemical Engineering Journal

Metabolic pools of phenolic acids in Salvia miltiorrhiza are enhanced by co-expression of Antirrhinum majus Delila and Rosea1 transcription factors

https://doi.org/10.1016/j.bej.2013.02.014Get rights and content

Abstract

Phenolic acids are universally distributed in plant species, where they participate in numerous bioactivities. However, their low abundance in Salvia miltiorrhiza is a perplexing problem. We examined the heterologous expression of two transcription factors, Delila (DEL) and Rosea1 (ROS1), from Antirrhinum majus in genetically modified S. miltiorrhiza. Productions of rosmarinic acid (RA) and salvianolic acid B (Sal B) were significantly elevated during normal growth stages. Contents of other phenylpropanoid metabolites were enhanced and antioxidant activity was markedly stronger. Both SmCHS and SmRAS were stimulated by co-expression of DEL and ROS1. Because levels of phenylpropanoid metabolites were increased while both were co-expressed in S. miltiorrhiza, we believe that this will be a useful strategy for improving the contents of phenolic acids (especially RA and Sal B) in this species.

Highlights

► The increased phenylpropane metabolites resulted in the enhancements of antioxidant activities. ► Rosmarinic acid and salvianolic acid B were significantly accumulated during different growth stages. ► Transgenic lines with the similar transcription profiles of AnRosea1 and AnDelila had higher contents of metabolites.

Introduction

The root of Salvia miltiorrhiza Bunge (Dansen) is a well-known traditional herbal medicine in China, where it is used mainly for treating cardiovascular and cerebrovascular diseases [1], [2]. Dansen contains two major types of biologically active compounds, lipid-soluble tanshinones and water-soluble phenolic acids. Salvianolic acid B, an important phenolic ingredient, has a wide spectrum of bioactivities, including powerful antioxidation, prevention of brain injury [3], [4], promotion of angiogenesis and reduction in myocardial ischemia [2], [5], antiatherosclerosis [6] and inhibition of platelet aggregation [7]. As one of the most effective secondary metabolites in this species, Sal B is a dimer of RA that is thought to be derived from both the phenylpropanoid and tyrosine pathways (Fig. 1) [8]. Since RA and Sal B are at low levels in this species under natural environment, the genetically engineering strategy of over-expression transcription factors (TFs) increases their production as desirable pharmaceuticals in some medicinal plants during their metabolic biosynthetic pathways [9]. The discovery of TFs that can improve the expression or activity of some genes involved in a desired pathway had opened new avenues toward controlling secondary metabolism [9]. Thus, over-expression of select TFs that stimulated the expression of the key enzyme genes involved in entire metabolic pathways was a feasible tool for engineering high levels of desirable metabolites. For example, Zhang et al. [10] had shown that a MYB-type TF, AtPAP1 (Production of Anthocyanin Pigmentation 1), was a positive transcriptional activator of phenolic acid biosynthesis. Furthermore, the contents of total phenolics, total flavonoids, anthocyanin, and lignin were significantly enhanced in S. miltiorrhiza when all were biosynthesized via core phenylpropanoid metabolism that was shared upstream [8], [11].

Because these TFs are positive regulators of the phenylpropanoid metabolic pathway and other related pathways, they are likely to modulate secondary metabolism and cause Sal B to accumulate in S. miltiorrhiza. To date, many MYB TFs had been shown to participate in the regulation of phenylpropanoid metabolism. These included ZmC1/ZmP1 (COLORED ALEURONE1/PURPLE PLANT1) in Zea mays (maize) [12], [13], PyAN2 (ANTHOCYANIN2 in Petunia hybrida) [14], AtPAP1/AtPAP2 in Arabidopsis thaliana [15], and AmRosea1/AmRosea2 in Antirrhinum majus [16]. All of them induced the accumulation of phenylpropanoid metabolites, e.g., hydroxycinnamic acids, anthocyanins, proanthocyanins, isoflavones, flavonols, lignins, and some other species-specific metabolites. Other types of TFs, such as bHLH (basic helix-loop-helix), regulated the key structural genes of secondary metabolism and positively triggered the accumulation of secondary metabolic products. Likewise, ZmLc controlled anthocyanin synthesis in Medicago sp. (alfalfa) [17], while transient expression of AtGL3 (a bHLH-type TF, Glabra3) induced the accumulation of anthocyanin in a white-flowered Matthiola incana mutant [18]. Two bHLH TFs – OsRc (Rc, brown pericarp and seed coat), and OsRd (Rd, red pericarp and seed coat) – were positive regulators of proanthocyanidin involved in developing the red pericarp of rice [19], [20]. Other bHLH TFs participated in the regulation of secondary metabolism, e.g., AmDelila in A. majus [21], PfMYC-RP in Perilla frutescens [22], and PhAN1 and PhJAF13 in Petunia hybrida [23], [24].

Although it had been considered that only MYB or bHLH could regulate phenylpropanoid metabolism and enhance the production of secondary metabolites, many studies had confirmed that they can play more prominent roles when in the form of a complex (e.g., MBW, MYB/bHLH/WD40 protein complex) [25], [26], [27]. Whereas the WD40 protein did not directly participate in specific recognition of the target gene promoter, it might stabilize expression of the complex and enhance the transcription of target gene expression [28]. The MYB/bHLH complex may significantly elevate the production of secondary metabolites. In tomato, flavonol contents were increased 60 times by the heterologous co-expression of the maize transcription factor genes LC (Leaf color) and C1 compared with the wild type (WT), while those levels were not significantly improved by separate transformation with C1 or LC [29]. Heterologous expression had been examined in two genes from A. majus: DEL (Delila, a bHLH TF) and ROS1 (Rosea1, a MYB TF). The DEL/ROS1 protein complex activated expression of the key genes for enzymes in the phenylpropanoid pathway (especially phenylalanine ammonia lyase, PAL). Anthocyanins were accumulated at high levels in the fruit of transgenic “purple tomato”, which had strong antioxidant activity and can significantly extend the life expectancy of Trp53−/− knockout mice. Although anthocyanin contents were also risen when plants were transformed by DEL or ROS1 separately, those levels were not as high as what was combination used [30].

In this study, AmDEL and AmROS1 were constitutively co-expressed under the control of two separate strong constitutive cauliflower mosaic virus (CaMV) 35S promoters in S. miltiorrhiza. The contents of different secondary products from phenylpropanoid metabolism were evaluated in seedling and mature transgenic plants. We also evaluated the antioxidant capacity of extracts and monitored several key enzyme genes. Our objective was to determine whether these TFs can be used to improve the potential production of RA, Sal B, and other phenolic acids in this species.

Section snippets

Plant material and culture conditions

Mature seeds of S. miltiorrhiza were surface-sterilized with 0.1% mercuric chloride (HgCl2) and germinated on an MS basal medium. Cultures were maintained at 25 ± 2 °C and 60% relative humidity with 16/8 h light-dark photoperiod provided by cool white fluorescent lamps (25 μmol m−2 s−1). One-month-old seedlings were used for Agrobacterium-mediated transformation.

Vector construction and plant transformation

Both the full-length cDNA coding sequence for AmDEL (NCBI reference sequence: M84913) and AmROS1 (NCBI reference sequence: DQ275529) were

Generation of transgenic plants

Explant was transformed by Agrobacterium mediation with either control construct pBC or co-expression construct pDR (Fig. 2A and B). After selective culturing on a hygromycin medium, PCR-amplification was conducted with genomic DNA as templates. Approximately 30 independent pDR-transgenic lines (DR) were obtained, all transgenic plants were further evaluated via RT-PCR and RT-Q-PCR (Fig. 2C and D). DEL and ROS1 were proved to be stably expressed, with each line having a different transcription

Conclusion

This study confirmed that the contents of both RA and Sal B in S. miltiorrhiza were enriched by the co-expression of DEL and ROS1 and contents of each phenolic varied with growth stage. Even though RA is extensively distributed in many members of Lamiaceae, such metabolite is not normally found at high levels in these commonly culinary herbs. Therefore, our goal here was to demonstrate that large quantities of RA can be produced by propagating transgenic plants of S. miltiorrhiza via tissue

Acknowledgments

This work was supported by the Fundamental Research Funds for the Central Universities (Program No. 2010ZYGX028), the National Natural Science Foundation of China (Grant No. 31270338), and the 10–11th “Five-year-technique-project” by the Ministry of Technique and Science (2006BAI06A12-04), PR China. We thank all of our colleagues in the laboratory for constructive discussions and technical support.

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