Genome-wide identification, characterization, expression and enzyme activity analysis of coniferyl alcohol acetyltransferase genes involved in eugenol biosynthesis in Prunus mume

Autoři: Tengxun Zhang aff001;  Tingting Huo aff001;  Anqi Ding aff001;  Ruijie Hao aff001;  Jia Wang aff001;  Tangren Cheng aff001;  Fei Bao aff001;  Qixiang Zhang aff001
Působiště autorů: Beijing Key Laboratory of Ornamental Plants Germplasm Innovation & Molecular Breeding, Beijing Forestry University, Beijing, China aff001;  National Engineering Research Center for Floriculture, Beijing Forestry University, Beijing, China aff002;  Beijing Laboratory of Urban and Rural Ecological Environment, Beijing Forestry University, Beijing, China aff003;  Key Laboratory of Genetics and Breeding in Forest Trees and Ornamental Plants of Ministry of Education, Beijing Forestry University, Beijing, China aff004;  School of Landscape Architecture, Beijing Forestry University, Beijing, China aff005
Vyšlo v časopise: PLoS ONE 14(10)
Kategorie: Research Article
doi: 10.1371/journal.pone.0223974


Prunus mume, a traditional Chinese flower, is the only species of Prunus known to produce a strong floral fragrance, of which eugenol is one of the principal components. To explore the molecular mechanism of eugenol biosynthesis in P. mume, patterns of dynamic, spatial and temporal variation in eugenol were analysed using GC-MS. Coniferyl alcohol acetyltransferase (CFAT), a member of the BAHD acyltransferase family, catalyses the substrate of coniferyl alcohol to coniferyl acetate, which is an important substrate for synthesizing eugenol. In a genome-wide analysis, we found 90 PmBAHD genes that were phylogenetically clustered into five major groups with motif compositions relatively conserved in each cluster. The phylogenetic tree showed that the PmBAHD67-70 proteins were close to the functional CFATs identified in other species, indicating that these four proteins might function as CFATs. In this work, 2 PmCFAT genes, named PmCFAT1 and PmCFAT2, were cloned from P. mume ‘Sanlunyudie’, which has a strong fragrance. Multiple sequences indicated that PmCFAT1 contained two conserved domains, HxxxD and DFGWG, whereas DFGWG in PmCFAT2 was changed to DFGFG. The expression levels of PmCFAT1 and PmCFAT2 were examined in different flower organs and during the flowering stages of P. mume ‘Sanlunyudie’. The results showed that PmCFAT1 was highly expressed in petals and stamens, and this expression increased from the budding stage to the full bloom stage and decreased in the withering stage, consistent with the patterns of eugenol synthesis and emission. However, the peak of gene expression appeared earlier than those of eugenol synthesis and emission. In addition, the expression level of PmCFAT2 was higher in pistils and sepals than in other organs and decreased from the budding stage to the blooming stage and then increased in the withering stage, which was not consistent with eugenol synthesis. Subcellular localization analysis indicated that PmCFAT1 and PmCFAT2 were located in the cytoplasm and nucleus, while enzyme activity assays showed that PmCFAT1 is involved in eugenol biosynthesis in vitro. Overall, the results suggested that PmCFAT1, but not PmCFAT2, contributed to eugenol synthesis in P. mume.

Klíčová slova:

Alcohols – Biosynthesis – Flowers – Leaves – Petals – Sequence alignment – Sequence motif analysis – Stamens


1. Pasay C, Mounsey K, Stevenson G, Davis R, Arlian L, Morgan M, et al. Acaricidal activity of eugenol based compounds against scabies mites. PLoS One. 2010;5(8):e12079. doi: 10.1371/journal.pone.0012079 20711455

2. Atkinson RG. Phenylpropenes: Occurrence, Distribution, and Biosynthesis in Fruit. Journal of Agricultural and Food Chemistry. 2018;66(10):2259–72. doi: 10.1021/acs.jafc.6b04696 28006900

3. Seskar M, Shulaev V, Raskin I. Endogenous Methyl Salicylate in Pathogen-Inoculated Tobacco Plants. Plant Physiology. 1998;116(1):387–92. doi: 10.1104/pp.116.1.387

4. Vainstein A, Lewinsohn E, Pichersky E, Weiss D. Floral fragrance. New inroads into an old commodity. Plant Physiology. 2001;127(4):1383–9. doi: 10.1104/pp.010706 11743078

5. Zhang J, Yang WR, Hao RJ, Zhang QX. Cloning and expression of PmCBF gene from Prunus mume. Acta Agriculturae Boreali-Sinica. 2012;27(3):91–5. doi: 10.3969/j.issn.1000-7091.2012.03.018

6. Hao RJ, Du DL, Wang T, Yang WR, Wang J, Zhang QX. A comparative analysis of characteristic floral scent compounds in Prunus mume and related species. Bioscience, Biotechnology, and Biochemistry. 2014;78(10):1640–7. doi: 10.1080/09168451.2014.936346 25273130

7. Anand A, Jayaramaiah RH, Beedkar SD, Singh PA, Joshi RS, Mulani FA, et al. Comparative functional characterization of eugenol synthase from four different Ocimum species: Implications on eugenol accumulation. Biochimica et Biophysica Acta. 2016;1864(11):1539–47. doi: 10.1016/j.bbapap.2016.08.004 27519164

8. Sanchez-Palomo E, Garcia-Carpintero EG, Alonso-Villegas R, Gonzalez-Vinas MA. Characterization of aroma compounds of Verdejo white wines from the La Mancha region by odour activity values. Flavour and Fragrance Journal. 2010;25(6):456–62. doi: 10.1002/ffj.2005

9. Dexter R, Qualley A, Kish CM, Ma CJ, Koeduka T, Nagegowda DA, et al. Characterization of a petunia acetyltransferase involved in the biosynthesis of the floral volatile isoeugenol. Plant Journal. 2007;49(2):265–75. doi: 10.1111/j.1365-313X.2006.02954.x 17241449

10. Muhlemann JK, Woodworth BD, Morgan JA, Dudareva N. The monolignol pathway contributes to the biosynthesis of volatile phenylpropenes in flowers. New Phytologist. 2014;204(3):661–70. doi: 10.1111/nph.12913 24985707

11. Araguez I, Hoffmann T, Osorio S, Rambla JL, Medina-Escobar N, Granell A, et al. Eugenol 批roduction in achenes and receptacles of strawberry fruits is catalyzed by synthases exhibiting distinct kinetics. Plant Physiology. 2013;163(2):946–58. doi: 10.1104/pp.113.224352 23983228

12. Gupta AK, Schauvinhold I, Pichersky E, Schiestl FP. Eugenol synthase genes in floral scent variation in Gymnadenia species. Functional & Integrative Genomics. 2014;14(4):779–88. doi: 10.1007/s10142-014-0397-9 25239559

13. Kim SJ, Vassao DG, Moinuddin SGA, Bedgar DL, Davin LB, Lewis NG. Allyl/propenyl phenol synthases from the creosote bush and engineering production of specialty/commodity chemicals, eugenol/isoeugenol, in Escherichia coli. Archives of Biochemistry and Biophysics. 2014;541:37–46. doi: 10.1016/ 24189289

14. Hao RJ, Zhang Q, Yang WR, Wang J, Cheng TR, Pan HT, et al. Emitted and endogenous floral scent compounds of Prunus mume and hybrids. Biochemical Systematics and Ecology. 2014;54:23–30. doi: 10.1016/j.bse.2013.12.007

15. Bao F, Ding AQ, Zhang TX, Luo L, Wang J, Cheng TR, et al. Expansion of PmBEAT genes in the Prunus mume genome induces characteristic floral scent production. Horticulture Research-England. 2019;6. doi: 10.1038/s41438-018-0104-4 30729014

16. Larkin MA, Blackshields G, Brown NP, Chenna R, McGettigan PA, McWilliam H, et al. Clustal W and Clustal X version 2.0. Bioinformatics. 2007;23(21):2947–8. doi: 10.1093/bioinformatics/btm404 17846036

17. Kumar S, Stecher G, Tamura K. MEGA7: Molecular Evolutionary Genetics Analysis Version 7.0 for Bigger Datasets. Molecular Biology and Evolution. 2016;33(7):1870–4. doi: 10.1093/molbev/msw054 27004904

18. Deng W, Wang Y, Liu Z, Cheng H, Xue Y. HemI: A Toolkit for Illustrating Heatmaps. PLoS ONE. 2014;9(11):e111988. doi: 10.1371/journal.pone.0111988 25372567

19. Wang T, Lu JX, Xu ZD, Yang WR, Wang J, Cheng TR, et al. Selection of suitable reference genes for miRNA expression normalization by qRT-PCR during flower development and different genotypes of Prunus mume. Scientia Horticulturae-Amsterdam. 2014;169:130–7. doi: 10.1016/j.scienta.2014.02.006

20. Yu XH, Gou JY, Liu CJ. BAHD superfamily of acyl-CoA dependent acyltransferases in Populus and Arabidopsis: bioinformatics and gene expression. Plant Molecular Biology. 2009;70(4):421–42. doi: 10.1007/s11103-009-9482-1 19343509

21. Dudareva N, D’Auria JC, Nam KH, Raguso RA, Pichersky E. Acetyl-CoA:benzylalcohol acetyltransferase—an enzyme involved in floral scent production in Clarkia breweri. Plant Journal. 1998;14(3):297–304. doi: 10.1046/j.1365-313x.1998.00121.x 9628024

22. Aharoni A, Keizer LCP, Bouwmeester HJ, Sun ZK, Alvarezhuerta M, Verhoeven HA, et al. Identification of the SAAT gene involved in strawberry flavor biogenesis by use of DNA microarrays. Plant Cell. 2000;12(5):647–61. doi: 10.1105/tpc.12.5.647 10810141

23. Shalit M, Guterman I, Volpin H, Bar E, Tamari T, Menda N, et al. Volatile ester formation in roses. Identification of an acetyl-coenzyme A. Geraniol/Citronellol acetyltransferase in developing rose petals. Plant Physiology. 2003;131(4):1868–76. doi: 10.1104/pp.102.018572 12692346

24. El-Sharkawy I, Manriquez D, Flores FB, Regad F, Bouzayen M, Latche A, et al. Functional characterization of a melon alcohol acyl-transferase gene family involved in the biosynthesis of ester volatiles. Identification of the crucial role of a threonine residue for enzyme activity. Plant Molecular Biology. 2005;59(2):345–62. doi: 10.1007/s11103-005-8884-y 16247561

25. Feng L, Chen C, Li T, Wang M, Tao J, Zhao D, et al. Flowery odor formation revealed by differential expression of monoterpene biosynthetic genes and monoterpene accumulation in rose (Rosa rugosa Thunb.). Plant Physiology & Biochemistry. 2014;75:80–8. doi: 10.1016/j.plaphy.2013.12.006 24384414

26. Souleyre EJF, Greenwood DR, Friel EN, Karunairetnam S, Newcomb RD. An alcohol acyl transferase from apple (cv. Royal Gala), MpAAT1, produces esters involved in apple fruit flavor. Febs Journal. 2005;272(12):3132–44. doi: 10.1111/j.1742-4658.2005.04732.x 15955071

27. Li D, Xu Y, Xu G, Gu L, Li D, Shu H. Molecular cloning and expression of a gene encoding alcohol acyltransferase (MdAAT2) from apple (cv. Golden Delicious). Phytochemistry. 2006;67(7):658–67. doi: 10.1016/j.phytochem.2006.01.027 16524607

28. Gonzalez-Aguero M, Troncoso S, Gudenschwager O, Campos-Vargas R, Moya-Leon MA, Defilippi BG. Differential expression levels of aroma-related genes during ripening of apricot (Prunus armeniaca L.). Plant physiology and biochemistry. 2009;47(5):435–40. doi: 10.1016/j.plaphy.2009.01.002 19233665

29. Cao Y, Hu SL, Zhang HY, Tang XF, Liu YX. Cloning, sequence analysis and prokaryotic expression of an alcohol acyltransferase (AAT) gene in tomato (Solanum lycopersicum). Bulletin of Botanical Research. 2012;32(6):731–6.

30. Muhlemann JK, Klempien A, Dudareva N. Floral volatiles: from biosynthesis to function. Plant, Cell & Environment. 2014;37(8):1936–49. doi: 10.1111/pce.12314 24588567

31. Koeduka T, Suzuki S, Iijima Y, Ohnishi T, Suzuki H, Watanabe B, et al. Enhancement of production of eugenol and its glycosides in transgenic aspen plants via genetic engineering. Biochemical and Biophysical Research Communications. 2013;436(1):73–8. doi: 10.1016/j.bbrc.2013.05.060 23707945

32. Gao F, Liu B, Li M, Gao X, Fang Q, Liu C, et al. Identification and characterization of terpene synthase genes accounting for volatile terpene emissions in flowers of Freesia x hybrida. Journal of Experimental Botany. 2018;69(18):4249–65. doi: 10.1093/jxb/ery224 29901784

33. Zhang T, Sun M, Guo Y, Shi X, Yang Y, Chen J, et al. Overexpression of LiDXS and LiDXR from Lily (Lilium ‘Siberia’) enhances the terpenoid content in tobacco flowers. Frontiers in Plant Science. 2018;9(909). doi: 10.3389/fpls.2018.00909 30038631

34. Bergougnoux V, Caissard JC, Jullien F, Magnard JL, Scalliet G, Cock JM, et al. Both the adaxial and abaxial epidermal layers of the rose petal emit volatile scent compounds. Planta. 2007;226(4):853–66. doi: 10.1007/s00425-007-0531-1 17520281

35. Fan ZQ, Li JY, Li XL, Yin HF. Composition analysis of floral scent within genus Camellia uncovers substantial interspecific variations. Scientia Horticulturae-Amsterdam. 2019;250:207–13. doi: 10.1016/j.scienta.2019.02.050

36. Zhao K, Yang W, Zhou Y, Zhang J, Li Y, Ahmad S, et al. Comparative transcriptome reveals benzenoid biosynthesis regulation as inducer of floral scent in the woody plant Prunus mume. Frontiers in Plant Science. 2017;8(319). doi: 10.3389/fpls.2017.00319 28344586

37. D’Auria JC. Acyltransferases in plants: a good time to be BAHD. Current Opinion in Plant Biology. 2006;9(3):331–40. doi: 10.1016/j.pbi.2006.03.016 16616872

38. Tuominen LK, Johnson VE, Tsai CJ. Differential phylogenetic expansions in BAHD acyltransferases across five angiosperm taxa and evidence of divergent expression among Populus paralogues. Bmc Genomics. 2011;12(1):236. doi: 10.1186/1471-2164-12-236 21569431

39. Okada T, Hirai MY, Suzuki H, Yamazaki M, Saito K. Molecular characterization of a novel quinolizidine alkaloid O-tigloyltransferase: cDNA cloning, catalytic activity of recombinant protein and expression analysis in Lupinus plants. Plant Cell Physiology. 2005;46(1):233–44. doi: 10.1093/pcp/pci021 15659437

40. Wang J, De Luca V. The biosynthesis and regulation of biosynthesis of Concord grape fruit esters, including ‘oxy’ethylanthranilate. Plant Journal. 2005;44(4):606–19. doi: 10.1111/j.1365-313X.2005.02552.x 16262710

41. Walker K, Croteau R. Taxol biosynthesis: molecular cloning of a benzoyl-CoA:taxane 2alpha-O-benzoyltransferase cDNA from taxus and functional expression in Escherichia coli. Proceedings of the National Academy of Sciences of the United States of America. 2000;97(25):13591–6. doi: 10.1073/pnas.250491997 11095755

42. D’Auria JC, Chen F, Pichersky E. Characterization of an acyltransferase capable of synthesizing benzylbenzoate and other volatile esters in flowers and damaged leaves of Clarkia breweri. Plant Physiology. 2002;130(1):466–76. doi: 10.1104/pp.006460 12226525

43. Boatright J, Negre F, Chen XL, Kish CM, Wood B, Peel G, et al. Understanding in vivo benzenoid metabolism in petunia petal tissue. Plant Physiology. 2004;135(4):1993–2011. doi: 10.1104/pp.104.045468 15286288

44. Zang L, Zheng T, Chu Y, Ding C, Zhang W, Huang Q, et al. Genome-Wide analysis of the fasciclin-Like arabinogalactan protein gene family reveals differential expression patterns, localization, and aalt stress response in Populus. Frontiers in Plant Science. 2015;6:1140. doi: 10.3389/fpls.2015.01140 26779187

45. Fan ZQ, Li JY, Li XL, Wu B, Wang JY, Liu ZC, et al. Genome-wide transcriptome profiling provides insights into floral bud development of summer-flowering Camellia azalea. Scientific Reports-Uk. 2015;5. doi: 10.1038/srep09729 25978548

46. Lavid N, Wang J, Shalit M, Guterman I, Bar E, Beuerle T, et al. O-methyltransferases involved in the biosynthesis of volatile phenolic derivatives in rose petals. Plant Physiology. 2002;129(4):1899–907. doi: 10.1104/pp.005330 12177504

47. Pott MB, Pichersky E, Piechulla B. Evening specific oscillations of scent emission, SAMT enzyme activity, and SAMT mRNA in flowers of Stephanotis floribunda. Journal of Plant Physiology. 2002;159(8):925–34.

Článek vyšel v časopise


2019 Číslo 10