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

Abstract

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 (2,2′-azinobis-3-ethylbenzotiazo-line-6-sulfonic acid) and FRAP (ferric reducing anti-oxidant 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.

MYB Transcription Factors as Regulators of Phenylpropanoid Metabolism in Plants

“…When constitutively expressed in maize, ZmMYB-IF35 promotes the accumulation of ferulic and chlorogenic acids (Dias and Grotewold, 2003). Meanwhile, constitutive expression in tobacco, potato, tomato, and snow lotus (Saussurea involucrata) of the potato genes StAN1, StMTF1, and PAP1, as well as that of AtMYB12, increases chlorogenic acid content (Luo et al, 2008;Rommens et al, 2008;Payyavula et al, 2013;Qiu et al, 2013). Both A. thaliana AtMYB4 and S. miltiorrhiza SmMYB39 belong to R2R3-MYB subgroup 4 ( Figure 3).…”

“…Some R2R3-MYB transcription factors regulate the synthesis of more than one class of phenylpropanoid-derived compounds. Examples include MdMYB3 (anthocyanin and flavonol), AtMYB4 (monolignol and phenolic acid), AtMYB12 (flavonol and phenolic acid), PAP1 (monolignol, anthocyanin, proanthocyanidin, flavonols, and phenolic acid), FaMYB1 (anthocyanin, proanthocyanidins, and flavonol), VvMYB5a (anthocyanin, proanthocyanidin, flavonol), VvMYB5b (anthocyanin and proanthocyanidin), StAN1 (anthocyanin, flavonols, and phenolic acid), StMTF1 (anthocyanin, flavonols, and phenolic acid), and ROSEA1 (anthocyanin and phenolic acid) (Aharoni et al, 2001;Deluc et al, 2006Deluc et al, , 2008Stracke et al, 2007;Luo et al, 2008;Rommens et al, 2008;Zuluaga et al, 2008;Li et al, 2010;Zhang et al, 2010;Paolocci et al, 2011;Payyavula et al, 2013;Qiu et al, 2013;Vimolmangkang et al, 2013;Wang et al, 2013). MdMYB3, AtMYB12, FaMYB1, and VvMYB5b regulate the expression of different phenylpropanoid-derived compound structural genes such as CHS, CHI, F3H, FLS, F3 0 5 0 H (encoding flavonoid 3 0 5 0hydroxylase), DFR, ANS, ANR, LAR, GT (encoding glycosyl transferase), HCT (encoding hydroxycinnamoyl CoA shikimate/ quinate transferase), C3H (encoding p-coumaroyl ester 3-hydroxylase), and HQT (encoding hydroxycinnamoyl CoA quinate transferase) (Aharoni et al, 2001;Stracke et al, 2007;Deluc et al, 2008;Luo et al, 2008;Paolocci et al, 2011;Vimolmangkang et al, 2013).…”

“…MdMYB3, AtMYB12, FaMYB1, and VvMYB5b regulate the expression of different phenylpropanoid-derived compound structural genes such as CHS, CHI, F3H, FLS, F3 0 5 0 H (encoding flavonoid 3 0 5 0hydroxylase), DFR, ANS, ANR, LAR, GT (encoding glycosyl transferase), HCT (encoding hydroxycinnamoyl CoA shikimate/ quinate transferase), C3H (encoding p-coumaroyl ester 3-hydroxylase), and HQT (encoding hydroxycinnamoyl CoA quinate transferase) (Aharoni et al, 2001;Stracke et al, 2007;Deluc et al, 2008;Luo et al, 2008;Paolocci et al, 2011;Vimolmangkang et al, 2013). Overexpression of PAP1, ROSEA1, VvMYB5a, or StAN1 results in elevated expression of the genes encoding enzymes of the GPP such as PAL, C4H, 4CL, and the genes encoding enzymes committed to different phenylpropanoid-derived compounds such as CHS, CHI, F3H, FLS, F3 0 H (encoding flavonoid 3 0 -hydroxylase), F3 0 5 0 H, DFR, ANS, LAR, GT, HCT, C3H, RAS (encoding rosmarinic acid synthase), CCR (encoding cinnamoyl-CoA reductase), COMT (encoding caffeic acid O-methyltransferase), and CAD (encoding cinnamyl alcohol dehydrogenase) (Deluc et al, 2006;Zuluaga et al, 2008;Li et al, 2010;Zhang et al, 2010;Payyavula et al, 2013;Qiu et al, 2013;Wang et al, 2013). StMTF1 increases expression of both the chlorogenic acid biosynthetic HQT gene and the upstream chorismate mutase and prephenate dehydratase genes involving in the formation of phenylalanine, the important precursor for phenylpropanoidderived compounds (Rommens et al, 2008).…”

Optimization of Ultrasonic Flavonoid Extraction from Saussurea involucrate, and the Ability of Flavonoids to Block Melanin Deposition in Human Melanocytes

“…belongs to the Asteraceae family of the genus Saussurea, which is used to treat rheumatoid arthritis and regulate the menstrual cycle [1]. Pharmacologic studies show that many medicinal values of S. involucrate are derived from its flavonoids, and these include anti-neoplastic [2], anti-arthritic [3], anti-oxidative, anti-aging [4,5], and anti-fatigue [6] properties. Flavonoids also play an important role in blocking melanin deposition and skin whitening products.…”

Comparative temporal metabolomics studies to investigate interspecies variation in three Ocimum species

“…Considering the diverse functions of these TFs it becomes extremely difficult to give explanations to the intricate role of bHLH, MYB, MYC and TTG1 TFs. PAP1 (production of anthocyanin pigment1) being well described for its involvement in the anthocyanin biosynthetic pathway 82 but Sekhon et al 83 and Pourtau et al 84 have also reported PAP1 and PAP2 to be involved in senescence induced by pollination prevention in maize and sugar application in Arabidopsis, respectively. Hence, the expression pattern of PAP1 in O. sanctum may be due to some stress induced at the onset of winter season in November and December as Ocimum is a plant of tropics.…”