Metabolic Engineering of the Phenylpropanoid Pathway Enhances the Antioxidant Capacity of Saussurea involucrata

et al. (2013) Metabolic Engineering of the Phenylpropanoid Pathway Enhances the Antioxidant Capacity of Saussurea involucrata. PLoS ONE 8(8): e70665. doi:10.1371/journal.pone.0070665 Metabolic Engineering of the Phenylpropanoid Pathway Enhances the Antioxidant Capacity of Saussurea involucrata Jian Qiu 0 Fenghua Gao 0 Guoan Shen 0 Chonghui Li 0 Xiaoyan Han 0 Qiao Zhao 0 Dexiu Zhao 0 Xuejun Hua 0 Yongzhen Pang 0 Turgay Unver, Cankiri Karatekin University, Turkey 0 1 The Key Laboratory of Plant Resources/Beijing Botanical Garden, Institute of Botany, the Chinese Academy of Sciences , Beijing , China , 2 The Key Laboratory of Biology and Genetic Resources of Rubber Tree, Rubber Research Institute, the Chinese Academy of Tropical Agricultural Sciences , Danzhou, Hainan , China , 3 Plant Biology Division, the Samuel Roberts Noble Foundation , Ardmore, Oklahoma , United States of America The rare wild species of snow lotus Saussurea involucrata is a commonly used medicinal herb with great pharmacological value for human health, resulting from its uniquely high level of phenylpropanoid compound production. To gain information on the phenylpropanid biosynthetic pathway genes in this critically important medicinal plant, global transcriptome sequencing was performed. It revealed that the phenylpropanoid pathway genes were well represented in S. involucrata. In addition, we introduced two key phenylpropanoid pathway inducing transcription factors (PAP1 and Lc) into this medicinal plant. Transgenic S. involucrata co-expressing PAP1 and Lc exhibited purple pigments due to a massive accumulation of anthocyanins. The over-expression of PAP1 and Lc largely activated most of the phenylpropanoid pathway genes, and increased accumulation of several phenylpropanoid compounds significantly, including chlorogenic acid, syringin, cyanrine and rutin. Both ABTS (2,29-azinobis-3-ethylbenzotiazo-line-6-sulfonic acid) and FRAP (ferric reducing antioxidant power) assays revealed that the antioxidant capacity of transgenic S. involucrata lines was greatly enhanced over controls. In addition to providing a deeper understanding of the molecular basis of phenylpropanoid metabolism, our results potentially enable an alternation of bioactive compound production in S. involucrata through metabolic engineering. - Funding: This work was supported by the National Science Foundation of China (Grant No. 30873452 and 31200228). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. . These authors contributed equally to this work. Snow lotus is a well-known and rare traditional Chinese medicinal herb, the aerial part of which has been used commonly to treat a wide spectrum of clinical diseases, including arthritis, stomachache, and gynecological diseases [1,2]. The main bioactive compounds found in snow lotus are polyphenols, including phenolic acids, flavonoids and lignans. The potent antioxidant effects of these compounds are responsible for anti-inflammatory, antimutagenic and anti-tumor activities [13]. Our previous studies have shown that total polyphenol/flavonoid content correlates with assayed antioxidant activity in a variety of wild snow lotus species [4]. Among the few available endangered and rare wild snow lotus species in China, S. involucrata is of particularly high quality for medicinal purposes due to its relatively higher total polyphenol/flavonoid content and antioxidant activity [4]. Polyphenol/flavonoid compounds are important plant secondary metabolites for both plant defense and human health. They are derived from the general phenylpropanoid biosynthetic pathway, which is comprised of several biosynthetic branches, including lignin, stilbenes, flavonoids, and anthocyanins [5]. The biosynthesis of polyphenol/flavonoid has been extensively studied in the model plants Arabidopsis, petunia and maize [6,7]. A ubiquitous regulation complex comprising MYB, bHLH and WD40, is structurally and functionally conserved among species for flavonoid biosynthesis [8,9]. The PAP1 (Production of Anthocyanin Pigment 1) gene encoding a MYB transcription factor from Arabidopsis is a key player involved in the anthocyanin biosynthetic pathway [10]. Over-expression of PAP1 can effectively induce anthocyanin production in tobacco, hops, rose, Salvia miltiorrhiza and canola by regulating the related pathway genes, chalcone synthase (CHS), anthocyanidin synthase (ANS), flavonol synthase (FLS) etc [1116]. The Lc (Leaf color) gene encodes a bHLH-type transcription factor identified from maize, which plays an important role in regulating anthocyanin production [17]. The ectopic expression of Lc can enhance anthocyanin and other flavonoid accumulation in Lycopersicon esculentum [18], petunia [19], Medicago sativa [20], Caladium bicolor [21] and Malus domestica [22]. In addition to expression studies of the individual genes, PAP1 and Lc were also expressed simultaneously in an effort to increase anthocyandin production in Arabidopsis [23]. Given that PAP1 and Lc have been shown to successfully induce phenylpropanoid biosynthesis, are of two distinct gene families, and are derived from different species, these two transcription factors were here selected as high potential candidates for use as metabolic engineering tools in S. involucrata, which has not been modified genetically to generate whole transgenic plant previously. In the present study, two key transcription factors (PAP1 and Lc) were selected to enhance phenylpropanoid production in S. involucrata. Expression of these transcription factors in transgenic S. involucrata greatly increased the levels of individual phenylpropanoids (including phenolic acids, lignins, flavonoids and anthocyanins), total phenolics, total flavonoids and antioxidant capacity. The transcriptome data of S. involucrata obtained in this study can push forward the efforts for the metabolic engineering of medicinal plants in terms of high-value products. Furthermore, our study demonstrates that S. involucrata would be an ideal system for studying natural product biosynthesis in medicinal plants. Transcriptome sequencing of wild S. involucrata In order to obtain global transcriptome information for further investigation of the phenylpropanoid pathway in S. involucrata, Illumina sequencing was performed with pooled cDNA samples from leaf, stem and inflorescence organs (the medicinal parts) of wild S. involucrata. From 1.33 Gb of primary sequencing data, 56,151 unigenes with more than 200 bp in length (covering 28.3 Mb) were obtained after sequence cleaning and assembling with the SOAP de novo program (http://soap.genomics.org.cn). The raw transcriptome sequence information from the present study was deposited at the Sequence Read Archive (SRA) with accession No. SRA094934. Assembled unigenes were deposited at the NCBI Transcri (...truncated)