Cloning and Characterization of Key Synthase FAS Gene Involved in Terpenoids Pathway of Chrysanthemum morifolium

doi:10.16420/j.issn.0513-353x.2020-0232

Acta Horticulturae Sinica ›› 2021, Vol. 48 ›› Issue (2): 313-324.doi: 10.16420/j.issn.0513-353x.2020-0232

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Cloning and Characterization of Key Synthase FAS Gene Involved in Terpenoids Pathway of Chrysanthemum morifolium

HU Hao¹^,², YANG Ting³, GAO Liping³, Maarten A. Jongsma³, WANG Caiyun¹^,²^,^*()

¹Key Laboratory for Biology of Horticultural Plants,Ministry of Education,Huazhong Agricultural University,Wuhan 430070,China
²Key Laboratory of Urban Agriculture in Central China,Ministry of Agriculture and Rural Affairs,P. R. China,Wuhan 430070,China
³Wageningen Plant Research，Wageningen University and Research，P.O. Box 619，6700 AP，Wageningen，The Netherlands

Received:2020-09-22 Revised:2020-12-07 Online:2021-02-25 Published:2021-03-09
Contact: WANG Caiyun E-mail:wangcy@mail.hzau.edu.cn

Abstract

Abstract:

The secondary metabolite composition and relative abundance in leaves and ovaries of the Chrysanthemum morifolium model cultivar‘1581’were analyzed by SPME extraction and GC-MS analysis. The terpenoids represented the dominant group of volatiles. Based on conserved sequences FDS-like genes identified in a transcriptome database derived from chrysanthemum,a farnesol synthase gene（CmFAS）was cloned with an 1 197 bp open reading frame（ORF）,encoding 398 amino acids and putatively located in the plastids. Phylogenetic analysis showed that CmFAS belongs to the FDS gene family in Asteraceae and has higher sequence similarity with the monoterpene CDS genes of pyrethrum （Tanacetum cinerariifolium）and Artemisia. Multiple alignment of FDS proteins of several different plant species demonstrated that CmFAS had the five typical conserved regions of chain elongation prenyltransferases. The gene expression of CmFAS was the highest in young leaves and lower in ovaries and mature leaves. The expression level of CmFAS was lower than that of CmFDS1 and CmFDS2 in chrysanthemum. By enzyme assay,it showed that CmFAS not only catalyzed DMAPP and IPP to farnesol but also converted CPP to chrysanthemol. CmFAS is a bifunctional enzyme generating both monoterpene and sesquiterpene products. CmFAS is the first sesquiterpene synthase located in plastids of chrysanthemum.

Key words: Chrysanthemum, farnesol synthase, terpenoids, gene expression, prokaryotic expression

CLC Number:

S682.1 ⁺1

HU Hao, YANG Ting, GAO Liping, Maarten A. Jongsma, WANG Caiyun. Cloning and Characterization of Key Synthase FAS Gene Involved in Terpenoids Pathway of Chrysanthemum morifolium[J]. Acta Horticulturae Sinica, 2021, 48(2): 313-324.

Figures/Tables 7

Table 1 Primers for cloning,construction of vector and gene expression of CmFAS in Chrysanthemum

引物用途 Primer use	引物名称 Primer name	引物序列（5′-3′） Primer sequence
基因ORF克隆 Cloning the full-length ORF	CmFAS LF	ATGGCATTCTGTAATAGTCTCGTAGG
基因ORF克隆 Cloning the full-length ORF	CmFAS LR	TTACTTATGTCCTTTGTATATCTTTCCG
原核表达载体构建 For prokaryotic expression vector	CmFAS_AscⅠ	TATAGGCGCGCCTGACTACGACAATGAGCAGCGA
原核表达载体构建 For prokaryotic expression vector	CmFAS_NotⅠ	TATAGCGGCCGCTTACTTATGTCCTTTGTATATCTTTCC
基因表达分析 Gene expression	CmFAS F	CCGAGGTTGGTATGACTGCTG
	CmFAS R	TCGTTGAATAGGTCCACTAGATGC
	CmActin F	CCTCTTAATCCTAAGGCTAATCAG
	CmActin R	CCAGGAATCCAGCACAATACC

Fig. 1 The relative abundance of secondary metabolites in different tissues of Chrysanthemum‘1581’

Fig. 2 The phylogenetic analysis of CmFAS and FDS family with other plants The number in the tree represents boots value. The scale bar represents branch length.

Fig. 3 Protein alignment of CmFAS with FDS proteins from other plant species Ⅰ-Ⅴ underline：Five conserved regions in chain elongation prenyltransferases.

Fig. 4 Expression levels of CmFAS and other homologous genes in different tissues and development stages of Chrysanthemum‘1581’ P < 0.05.

Fig. 5 The catalytic function of CmFAS A：GC-MS detection of product of CmFAS protein with different substrates. B：Schematic pathway of synthesis of terpenoids catalyzed by CmFAS.

Fig. 6 The putative schematic of CmFAS involved in MEP pathway

References 34

[1]	Aharoni A, Giri A P, Deuerlein S, Griepink F, de Kogel W J, Verstappen F W, Verhoeven H A, Jongsma M A, Schwab W, Bouwmeester H J. 2003. Terpenoid metabolism in wild-type and transgenic Arabidopsis plants. Plant Cell, 15(12):2866-2884. pmid: 14630967
[2]	Aharoni A, Giri A P, Verstappen F W A, Bertea C M, Sevenier R, Sun Z K, Jongsma M A, Schwab W, Bouwmeester H J. 2004. Gain and loss of fruit flavor compounds produced by wild and cultivated strawberry species. Plant Cell, 16(11):3110-3131. pmid: 15522848
[3]	Barkman T J, Martins T R, Sutton E, Stout J T. 2007. Positive selection for single amino acid change promotes substrate discrimination of a plant volatile producing enzyme. Molecular Biology Evolution, 24(6):1320-1329. doi: 10.1093/molbev/msm053 URL
[4]	Bleeker P M, Mirabella R, Diergaarde P J, Vandoorn A, Tissier A, Kant M R, Prins M, de Vos M, Haring M A, Schuurink R C. 2012. Improved herbivore resistance in cultivated tomato with the sesquiterpene biosynthetic pathway from a wild relative. Proceedings of the National Academy of Sciences of the USA, 109(49):20124-20129.
[5]	Chen F, Tholl D, D'Auria J C, Farooq A, Pichersky E, Gershenzon J. 2003. Biosynthesis and emission of terpenoid volatiles from Arabidopsis flowers. Plant Cell, 15(2):481-494. doi: 10.1105/tpc.007989 URL
[6]	Davidovich-Rikanati R, Lewinsohn E, Bar E, Iijima Y, Pichersky E, Sitrit Y. 2008. Overexpression of the lemon basil alpha-zingiberene synthase gene increases both mono-and sesquiterpene contents in tomato fruit. Plant Journal, 56(2):228-238. doi: 10.1111/j.1365-313X.2008.03599.x URL
[7]	Dudareva N, Pichersky E. 2008. Metabolic engineering of plant volatiles. Current Opinion in Biotechnology, 19(2):181-189. doi: 10.1016/j.copbio.2008.02.011 URL
[8]	Guti E Rrez C, Fereres A, Reina M I A, Cabrera R, Gonz A Lez-Coloma A. 1997. Behavioral and sublethal effects of structurally related lower terpenes on Myzus persicae. Journal of Chemical Ecology, 23(6):1641-1650. doi: 10.1023/B:JOEC.0000006428.00568.c5 URL
[9]	Halbert S E, Corsini D, Wiebe M, Vaughn S F. 2009. Plant-derived compounds and extracts with potential as aphid repellents. Annals of Applied Biology, 154(2):303-307. doi: 10.1111/aab.2009.154.issue-2 URL
[10]	Hemmerlin A, Rivera S B, Erickson H K, Poulter C D. 2003. Enzymes encoded by the farnesyl diphosphate synthase gene family in the big sagebrush Artemisia tridentata ssp. spiciformis. Journal of Biological Chemistry, 278(34):32132-32140. doi: 10.1074/jbc.M213045200 URL
[11]	Holstein S A, Hohl R J. 2003. Monoterpene regulation of Ras and Ras-related protein expression. The Journal of Lipid Research, 44:1209-1215. doi: 10.1194/jlr.M300057-JLR200 URL
[12]	Hosfield D J, Zhang Y M, Dougan D R, Broun A, Tari L W, Swanson R V, Finn J. 2004. Structural basis for bisphosphonate-mediated inhibition of isoprenoid biosynthesis. Journal of Biological Chemistry, 279(10):8526-8529. doi: 10.1074/jbc.C300511200 URL
[13]	Hu H, Li J, Delatte T, Vervoort J, Gao L, Verstappen F, Xiong W, Gan J, Jongsma M, Wang C. 2018. Modification of chrysanthemum odour and taste with chrysanthemol synthase induces strong dual resistance against cotton aphids. Plant Biotechnology Journal, 16(8):1434-1445. doi: 10.1111/pbi.2018.16.issue-8 URL
[14]	Hughes A L, Ota T, Nei M. 1990. Positive Darwinian selection promotes charge profile diversity in the antigen-binding cleft of class-I major-histocompatibility-complex molecules. Molecular Biology and Evolution, 7(6):515-524.
[15]	Li Xiaoying, Wu Junkai, Wang Haijing, Zhang Hongxia, Guo Xuemin. 2019. Analysis of volatile components in whorl tepals of Magnolia denudata‘Feihuang’during its development. Acta Horticulturae Sinica, 46(10):2009-2020. (in chinese)
	李晓颍, 武军凯, 王海静, 张红霞, 郭学民. 2019. ‘飞黄’玉兰花发育期各轮花被片挥发性成分分析. 园艺学报, 46(10):2009-2020.
[16]	Liu P, Wan J, Guo Y, Ge S, Rao G. 2012. Adaptive evolution of the chrysanthemyl diphosphate synthase gene involved in irregular monoterpene metabolism. BMC Evolutionary Biology, 12(1):214. doi: 10.1186/1471-2148-12-214 URL
[17]	Loza-Tavera H. 1999. Monoterpenes in essential oils: biosynthesis and properties. In Advances in Experimental Medicine and Biology, 464:49-62.
[18]	Luo C, Chen D, Cheng X, Liu H, Li Y, Huang C L. 2018. SSR analysis of genetic relationship and classification in chrysanthemum germplasm collection. Horticultural Plant Journal, 4(2):73-82. doi: 10.1016/j.hpj.2018.01.003 URL
[19]	Mcgarvey D J, Croteau R. 1995. Terpenoid metabolism. Plant Cell, 7(7):1015-1026.
[20]	Nagegowda D A, Gutensohn M, Wilkerson C G, Dudareva N. 2008. Two nearly identical terpene synthases catalyze the formation of nerolidol and linalool in snapdragon flowers. Plant Journal, 55(2):224-239. doi: 10.1111/tpj.2008.55.issue-2 URL
[21]	Phillips M A, D'Auria J C, Gershenzon J, Pichersky E. 2008. The Arabidopsis thaliana type 1 isopentenyl diphosphate isomerase are targeted to multiple subcellular compartments and gave overlapping functions in isoprenoid biosynthesis. Plant Cell, 20(3):677-696. doi: 10.1105/tpc.107.053926 pmid: 18319397
[22]	Pickett J A, Allemann R K, Birkett M A. 2013. The semiochemistry of aphids. Natural Product Reports, 30(10):1277-1283. pmid: 24156096
[23]	Pupko T, Sharan R, Hasegawa M, Shamir R, Graur D. 2003. Detecting excess radical replacements in phylogenetic trees. Gene, 319(1):127-135. doi: 10.1016/S0378-1119(03)00802-3 URL
[24]	Rondeau J M, Bitsch F, Bourgier E, Geiser M, Hemmig R, Kroemer M, Lehmann S, Ramage P, Rieffel S, Strauss A, Green J R, Jahnke W. 2006. Structural basis for the exceptional in vivo efficacy of bisphosphonate drugs. ChemMedChem, 1(2):267-273. doi: 10.1002/(ISSN)1860-7187 URL
[25]	Sacchettini J C, Poulter C D. 1997. Biochemistry-Creating isoprenoid diversity. Science, 277(5333):1788-1789. doi: 10.1126/science.277.5333.1788 URL
[26]	Sallaud C, Rontein D, Onillon S, Jabes F, Duffe P, Giacalone C, Thoraval S, Escoffier C, Herbette G, Leonhardt N, Causse M, Tissier A. 2009. A novel pathway for sesquiterpene biosynthesis from Z,Z-farnesyl pyrophosphate in the wild tomato Solanum habrochaites. Plant Cell, 21(1):301-317. doi: 10.1105/tpc.107.057885 URL
[27]	Schilmiller A L, Schauvinhold I, Larson M, Xu R, Charbonneau A L, Schmidt A, Wilkerson C, Last R L, Pichersky E. 2009. Monoterpenes in the glandular trichomes of tomato are synthesized from a neryl diphosphate precursor rather than geranyl diphosphate. Proceedings of the National Academy of Sciences of the USA, 106(26):10865-10870.
[28]	Steele C L, Crock J, Bohlmann J, Croteau R. 1998. Sesquiterpene synthases from grand fir(Abies grandis)-Comparison of constitutive and wound-induced activities,and CDNA isolation,characterization and bacterial expression of delta-selinene synthase and gamma-humulene synthase. Journal of Biological Chemistry, 273(4):2078-2089. doi: 10.1074/jbc.273.4.2078 URL
[29]	Tholl D. 2006. Terpene synthases and the regulation,diversity and biological roles of terpene metabolism. Current Opinion in Plant Biology, 9(3):297-304. doi: 10.1016/j.pbi.2006.03.014 URL
[30]	Wang K, Ohnuma S. 1999. Chain-length determination mechanism of isoprenyl diphosphate synthases and implications for molecula revolution. Trends in Biochemical Sciences, 24(11):445-451. pmid: 10542413
[31]	Yang T, Gao L, Hu H, Stoopen G, Wang C, Jongsma M A. 2014. Chrysanthemyl diphosphate synthase operates in planta as a bifunctional enzyme with chrysanthemol synthase activity. Journal of Biological Chemistry, 289(52):36325-36335. doi: 10.1074/jbc.M114.623348 URL
[32]	Yu X, Jones H D, Ma Y, Wang G, Xu Z, Zhang B, Zhang Y, Ren G, Pickett J A, Xia L. 2012 . ( E)-β-Farnesene synthase genes affect aphid(Myzus persicae)infestation in tobacco(Nicotiana tabacum). Functional & Integrative Genomics, 12(1):207-213.
[33]	Yue Yue-chong, Fan Yan-ping. 2011. The terpene synthases and regulation of terpene metabolism in plants. Acta Horticulturae Sinica, 38(2):379-388. (in Chinese)
	岳跃冲, 范燕萍. 2011. 植物萜类合成酶及其代谢调控的研究进展. 园艺学报, 38(2):379-388.
[34]	Zhang J, Zhang Y, Rosenberg H F. 2002. Adaptive evolution of a duplicated pancreatic ribonuclease gene in a leaf-eating monkey. Nature Genet, 30(4):411-415. doi: 10.1038/ng852 URL

Cloning and Characterization of Key Synthase FAS Gene Involved in Terpenoids Pathway of Chrysanthemum morifolium

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