CN: 32-1845/R
ISSN: 2095-6975
Cite this paper:
MO Guo-Yan, HUANG Fang, FANG Yin, HAN Lin-Tao, Kayla K. Pennerman, BU Li-Jing, DU Xiao-Wei, Joan W. Bennett, YIN Guo-Hua. Transcriptomic analysis in Anemone flaccida rhizomes reveals ancillary pathway for triterpene saponins biosynthesis and differential responsiveness to phytohormones[J]. Chinese Journal of Natural Medicines, 2019, 17(2): 131-144

Transcriptomic analysis in Anemone flaccida rhizomes reveals ancillary pathway for triterpene saponins biosynthesis and differential responsiveness to phytohormones

MO Guo-Yan1, HUANG Fang1, FANG Yin1, HAN Lin-Tao1, Kayla K. Pennerman2, BU Li-Jing3, DU Xiao-Wei4, Joan W. Bennett2, YIN Guo-Hua2
1 China Key Laboratory of TCM Resource and Prescription, Ministry of Education, Hubei University of Chinese Medicine, Wuhan 430065, China;
2 Department of Plant Biology and Pathology, Rutgers, The State University of New Jersey, New Jersey 08901, USA;
3 Department of Biology, University of New Mexico, Albuquerque, New Mexico 87131, USA;
4 Department of Pharmacy, Heilongjiang University of Chinese Medicine, Harbin 150040, China
Anemone flaccida Fr. Schmidt is a perennial medicinal herb that contains pentacyclic triterpenoid saponins as the major bioactive constituents. In China, the rhizomes are used as treatments for a variety of ailments including arthritis. However, yields of the saponins are low, and little is known about the plant’s genetic background or phytohormonal responsiveness. Using one-quarter of the 454 pyrosequencing information from the Roche GS FLX Titanium platform, we performed a transcriptomic analysis to identify 157 genes putatively encoding 26 enzymes involved in the synthesis of the bioactive compounds. It was revealed that there are two biosynthetic pathways of triterpene saponins in A. flaccida. One pathway depends on β-amyrin synthase and is similar to that found in other plants. The second, subsidiary (“backburner”) pathway is catalyzed by camelliol C synthase and yields β-amyrin as minor byproduct. Both pathways used cytochrome P450-dependent monooxygenases (CYPs) and family 1 uridine diphosphate glycosyl­transferases (UGTs) to modify the triterpenoid backbone. The expression of CYPs and UGTs were quite different in roots treated with the phytohormones methyl jasmonate, salicylic acid and indole-3-acetic acid. This study provides the first large-scale transcriptional dataset for the biosynthetic pathways of triterpene saponins and their phytohormonal responsiveness in the genus Anemone.
Key words:    Anemone flaccida Fr. Schmidt    Triterpenoid saponins    Biosynthetic pathways    Transcriptomic analysis    Phytohormonal responsiveness   
Received: 2018-09-29   Revised:
PDF (1673 KB) Free
Print this page
Email this article to others
Articles by MO Guo-Yan
Articles by HUANG Fang
Articles by FANG Yin
Articles by HAN Lin-Tao
Articles by Kayla K. Pennerman
Articles by BU Li-Jing
Articles by DU Xiao-Wei
Articles by Joan W. Bennett
Articles by YIN Guo-Hua
[1] Hao DC, Gu XJ, Xiao PG. Anemone medicinal plants:ethnopharmacology, phytochemistry and biology[J]. Acta Pharm Sin B, 2017, 7(2):146-158.
[2] Editorial Committee of Flora of China. Flora of China[M]. Beijing:Science Press, 1980, 28:16-18.
[3] Liu Q, Zhu XZ, Feng RB, et al. Crude triterpenoid saponins from Anemone flaccida (Di Wu) exert anti-arthritic effects on type Ⅱ collagen-induced arthritis in rats[J]. Chin Med, 2015, 10:20.
[4] Cao P, Wu FE, Ding LS. Advances in the studies on the chemical constituents and biologic activities for Anemone species[J]. Nat Prod Res Dev, 2004, 16:581-584.
[5] Sun YX, Liu JC, Liu DY. Phytochemicals and bioactivities of Anemone raddeana Regel:a review[J]. Pharmazie, 2011, 66(11):813-821.
[6] Zou ZJ, LH, Yang JS. Phytochemicals components and pharmacological activities of the genus Anemone[J]. Chin Pharm J, 2004, 39:493-495.
[7] Hao DC, Xiao PG, Ma HY, et al. Mining chemodiversity from biodiversity:pharmacophylogeny of medicinal plants of Ranunculaceae[J]. Chin J Nat Med, 2015, 13(7):507-520.
[8] Hao DC, Gu XJ, Xiao PG. Medicinal plants:chemistry, biology and omics[M]. Oxford,Elsevier-Woodhead, 2015.
[9] Hao DC, Gu XJ, Xiao PG, et al. Chemical and biological research of Clematis medicinal resources[J]. Chin Sci Bull, 2013, 58(10):1120-1129.
[10] Hao DC, Gu XJ, Xiao PG, et al. Recent advance in chemical and biological studies on Cimicifugeae pharmaceutical resources[J]. Chin Herb Med, 2013, 5(2):81-95.
[11] Han LT, Li J, Huang F, et al. Triterpenoid saponins from Anemone flaccida induce apoptosis activity in HeLa cells[J]. J Asian Nat Prod Res, 2009, 11(2):122-127.
[12] Liu J. Pharmacology of oleanolic acid and ursolic acid[J]. J Ethnopharmacol, 1995, 49(2):57-68.
[13] Han LT, Fang Y, Li MM, et al. The antitumor effects of triterpenoid saponins from the Anemone flaccida and the underlying mechanism[J]. Evid Based Complement Alternat Med, 2013, 2013:517931.
[14] Han LT, Fang Y, Cao Y, et al. Triterpenoid saponin flaccidoside Ⅱ from Anemone flaccida triggers apoptosis of NF1-associated malignant peripheral nerve sheath tumors via the MAPK-HO-1 pathway[J]. Onco Targets Ther, 2016, 9:1969-1979.
[15] Huang XJ, Tang JQ, Li MM, et al. Triterpenoid saponins from the rhizomes of Anemone flaccida and their inhibitory activities on LPS-induced NO production in macrophage RAW264.7 cells[J]. J Asian Nat Prod Res, 2014,16(9):910-921.
[16] Kong X, Wu W, Yang Y, et al. Total saponin from Anemone flaccida Fr. Schmidt abrogates osteoclast differentiation and bone resorption via the inhibition of RANKL-induced NF-kappaB, JNK and p38 MAPKs activation[J]. J Transl Med, 2015, 13:91.
[17] Liu C, Yang Y, Sun D, et al. Total saponin from Anemone flaccida Fr. schmidt prevents bone destruction in experimental rheumatoid arthritis via inhibiting osteoclastogenesis[J]. Rejuvenation Res, 2015, 18(6):528-542.
[18] Kong X, Yang Y, Wu W, et al. Triterpenoid saponin W3 from Anemone flaccida suppresses osteoclast differentiation through inhibiting activation of MAPKs and NF-kappaB pathways[J]. Int J Biol Sci, 2015, 11(10):1204-1214.
[19] Liu Q, Xiao XH, Hu LB, et al. Anhuienoside C ameliorates collagen-induced arthritis through inhibition of MAPK and NF-kappaB signaling pathways[J]. Front Pharmacol, 2017, 8:299.
[20] Pollier J, Goossens A. Oleanolic acid[J]. Phytochemistry, 2012, 77:10-15.
[21] Altschul SF, Gish W, Miller W, et al. Basic local alignment search tool[J]. J Mol Biol, 1990, 215:403-410.
[22] Kanehisa M, Araki M, Goto S, et al. KEGG for linking genomes to life and the environment[J]. Nucleic Acids Res, 2008, 36:D480-484.
[23] Kanehisa M, Goto S. KEGG:kyoto encyclopedia of genes and genomes[J]. Nucleic Acids Res, 2000, 28(1):27-30.
[24] Yin G, Sun Z, Liu N, et al. Production of double-stranded RNA for interference with TMV infection utilizing a bacterial prokaryotic expression system[J]. Appl Microbiol Biotechnol, 2009, 84(2):323-333.
[25] Huang L, Yan H, Jiang X, et al. Identification of candidate reference genes in perennial ryegrass for quantitative RT-PCR under various abiotic stress conditions[J]. PLos One, 2014, 9(4):e93724.
[26] Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2-△△CT method[J]. Methods, 2001, 25:402-408.
[27] Pop M, Salzberg SL. Bioinformatics challenges of new sequencing technology[J]. Trends Genet, 2008, 24(3):142-149.
[28] Tholl D. Biosynthesis and biological functions of terpenoids in plants[J]. Adv Biochem Eng Biotechnol, 2015, 148:63-106.
[29] Haralampidis K, Trojanowska M, Osbourn AE. Biosynthesis of triterpenoid saponins in plants[J]. Adv Biochem Eng/Biotechnol, 2002, 75:31-49.
[30] Rohdich F, Kis K, Bacher A, et al. The non-mevalonate pathway of isoprenoids:genes, enzymes and intermediates[J]. Curr Opin Chem Biol, 2001, 5(5):535-540.
[31] Zheng X, Xu H, Ma X, et al. Triterpenoid saponin biosynthetic pathway profiling and candidate gene mining of the Ilex asprella root using RNA-Seq[J]. Int J Mol Sci, 2014, 15(4):5970-5987.
[32] Pollier J, Moses T, Gonzalez-Guzman M, et al. The protein quality control system manages plant defence compound synthesis[J]. Nature, 2013, 504(7478):148-152.
[33] Chang WC, Song H, Liu HW, et al. Current development in isoprenoid precursor biosynthesis and regulation[J]. Curr Opin Chem Biol, 2013, 17(4):571-579.
[34] Phillips DR, Rasbery JM, Bartel B, et al. Biosynthetic diversity in plant triterpene cyclization[J]. Curr Opin Plant Biol, 2006, 9(3):305-314.
[35] Kalinowska M, Zimowski J, Pączkowski C, et al. The formation of sugar chains in triterpenoid saponins and glycoalkaloids[J]. Phytochem Rev, 2005, 4:237-257.
[36] Vincken JP, Heng L, de Groot A, et al. Saponins, classification and occurrence in the plant kingdom[J]. Phytochemistry, 2007, 68(3):275-297.
[37] Abe I. Enzymatic synthesis of cyclic triterpenes[J]. Nat Prod Rep, 2007, 24(6):1311-1331.
[38] Xu R, Fazio GC, Matsuda SP. On the origins of triterpenoid skeletal diversity[J]. Phytochemistry, 2004, 65(3):261-291.
[39] Thoma R, Schulz-Gasch T, D'Arcy B, et al. Insight into steroid scaffold formation from the structure of human oxidosqualene cyclase[J]. Nature, 2004, 432:118-122.
[40] Corey EJ, Matsuda SP, Bartel B. Isolation of an Arabidopsis thaliana gene encoding cycloartenol synthase by functional expression in a yeast mutant lacking lanosterol synthase by the use of a chromatographic screen[J]. Proc Natl Acad Sci USA, 1993, 90(24):11628-11632.
[41] Kushiro T, Shibuya M, Ebizuka Y. β-amyrin synthase:cloning of oxidosqualene cyclase that catalyzes the formation of the most popular triterpene among higher plants[J]. Eur J Biochem, 1998, 256(1):238-244.
[42] Kolesnikova M, Wilson W, Lynch D, et al. Arabidopsis camelliol C synthase evolved from enzymes that make pentacycles[J]. Org Lett, 2007, 9(25):5223-5226.
[43] Zhao L, Chen W, Fang Q. Triterpenoid saponins from Anemone flaccida[J]. Planta Med, 1990, 56:92-93.
[44] Huhman DV, Berhow MA, Sumner LW. Quantification of saponins in aerial and subterranean tissues of Medicago truncatula[J]. J Agric Food Chem, 2005, 53(6):1914-1920.
[45] Hayashi H, Sakai T, Fukui H, et al. Formation of soyasaponins in liquorice cell suspension cultures[J]. Phytochemistry, 1990, 29:3.
[46] Croteau R, Kutchan TM, Lewis NG. Biochemistry & Molecular Biology of Plants[M]. American Society of Plant Biologists, 2000:1250-1318.
[47] Ross J, Li Y, Lim EK, et al. Higher plant glycosyltransferases. genome biology 2:reviews[J]. Genome Biol, 2001, 2(2):reviews3004.1
[48] Augustin JM, Kuzina V, Andersen SB, et al. Molecular activities, biosynthesis and evolution of triterpenoid saponins[J]. Phytochemistry, 2011, 72(6):435-457.
[49] Luo M, Brown RL, Chen ZY, et al. Transcriptional profiles uncover Aspergillus flavus-induced resistance in maize kernels[J]. Toxins, 2011, 3(7):766-786.
[50] Campbell JA, Davies GJ, Bulone V, et al. A classification of nucleotide-diphospho-sugar glycosyltranszferases based on amino acid sequence similarities[J]. Biochem J, 1998, 329(Pt 3):719.
[51] Vogt T, Jones P. Glycosyltransferases in plant natural product synthesis:characterization of a supergene family[J]. Trends Plant Sci, 2000, 5(9):380-386.
[52] Bowles D, Lim EK, Poppenberger B, et al. Glycosyltransferases of lipophilic small molecules[J]. Ann Rev Plant Biol, 2006, 57:567-597.
[53] Yonekura-Sakakibara K, Hanada K. An evolutionary view of functional diversity in family 1 glycosyltransferases[J]. Plant J, 2011, 66(1):182-193.
[54] Achnine L, Huhman DV, Farag MA, et al. Genomics-based selection and functional characterization of triterpene glycosyltransferases from the model legume Medicago truncatula[J]. Plant J, 2005, 41(6):875-887.
[55] Meesapyodsuk D, Balsevich J, Reed DW, et al. Saponin biosynthesis in Saponaria vaccaria. cDNAs encoding β-amyrin synthase and a triterpene carboxylic acid glucosyltransferase[J]. Plant Physiol, 2007, 143(2):959-969.
[56] Shibuya M, Nishimura K, Yasuyama N, et al. Identification and characterization of glycosyltransferases involved in the biosynthesis of soyasaponin I in Glycine max[J]. FEBS Lett, 2010, 584(11):2258-2264.
[57] Naoumkina MA, Modolo LV, Huhman DV, et al. Genomic and coexpression analyses predict multiple genes involved in triterpene saponin biosynthesis in Medicago truncatula[J]. The Plant Cell, 2010, 22(3):850-866.
[58] Seo HS, Song JT, Cheong JJ, et al. Jasmonic acid carboxyl methyltransferase:a key enzyme for jasmonate-regulated plant responses[J]. Proc Natl Acad Sci USA, 2001, 98(8):4788-4793.
[59] Wani SH KV, Shriram V, Sah SK. Phytohormones and their metabolic engineering for abiotic stress tolerance in crop plants[J]. Crop J, 2016, 4(3):162-176.
[60] Kazan K. Auxin and the integration of environmental signals into plant root development[J]. Ann Bot, 2013, 112(9):1655-1665.
[61] Duca D, Lorv J, Patten CL, et al. Indole-3-acetic acid in plant-microbe interactions[J]. Antonie Van Leeuw, 2014, 106(1):85-125.
[62] Rivas-San Vicente M, Plasencia J. Salicylic acid beyond defence:its role in plant growth and development[J]. J Exp Bot, 2011, 62(10):3321-3338.
[63] Strickler SR, Bombarely A, Mueller LA. Designing a transcriptome next-generation sequencing project for a nonmodel plant species[J]. Am J Bot, 2012, 99(2):257-266.
[64] Sui C, Zhang J, Wei J, et al. Transcriptome analysis of Bupleurum chinense focusing on genes involved in the biosynthesis of saikosaponins[J]. BMC Genomics, 2011, 12:539.