Promoter variations in DBR2-like affect artemisinin production in different chemotypes of Artemisia annua

Abstract

Artemisia annua is the only known plant source of the potent antimalarial artemisinin, which occurs as the low- and high-artemisinin producing (LAP and HAP) chemotypes. Nevertheless, the different mechanisms of artemisinin producing between these two chemotypes were still not fully understood. Here, we performed a comprehensive analysis of genome resequencing, metabolome, and transcriptome data to systematically compare the difference in the LAP chemotype JL and HAP chemotype HAN. Metabolites analysis revealed that 72.18% of sesquiterpenes was highly accumulated in HAN compared to JL. Integrated omics analysis found a DBR2-Like (DBR2L) gene may be involved in artemisinin biosynthesis. DBR2L was highly homologous with DBR2, belonged to ORR3 family, and had the DBR2 activity of catalyzing artemisinic aldehyde to dihydroartemisinic aldehyde. Genome resequencing and promoter cloning revealed that complicated variations existed in DBR2L promoters among different varieties of A. annua and were clustered into three variation types. The promoter activity of diverse variant types showed obvious differences. Furthermore, the core region (−625 to 0) of the DBR2L promoter was identified and candidate transcription factors involved in DBR2L regulation were screened. Thus, the result indicates that DBR2L is another key enzyme involved in artemisinin biosynthesis. The promoter variation in DBR2L affects its expression level, and thereby may result in the different yield of artemisinin in varieties of A. annua. It provides a novel insight into the mechanism of artemisinin-producing difference in LAP and HAP chemotypes of A. annua, and will assist in a high yield of artemisinin in A. annua.

Acknowledgements

This work was supported by the Fundamental Research Funds for the Central Public Welfare Research Institutes (ZZ13-YQ-101, ZZ16-ND-10-09, ZZ16-ND-10-28), the CACMS Innovation Fund (CI2021A04107), the National Natural Science Foundation of China (U1812403-1), the National Key Research and Development Project (2019YFE0108700, China), and the Fundamental Research Funds for the Central Universities (3332022051).

Author contributions

X.W., L.W., and L.X. performed this work. X.W. analysed data and wrote the manuscript. L.W. and M.W. helped to determine artemisinin-related compounds. R.G. and Q.Y. helped to perform enzyme assays. Z.L. and L.L. analysed the genome resequencing data. Y.S., H.W., and T.M. performed part of the experiments. L.X. and S.C. reviewed the manuscript and Y.S. designed the experiment and supervised this work.

Data availability

17 cloned DBR2L promoter sequences have been submitted to NCBI by BankIt tool; the accession numbers are OR268936-OR268940, OR348401-OR348410, OR354319-OR354320. The bioproject number of RNA sequencing data is PRJNA924664.

Conflict of interest statement

The authors declare no conflicts of interest.

References

[1] World Health Organization . World Malaria Report 2022. Geneva, WHO, 2022. https://biznakenya.com/world-malaria-report-2022 (8 December 2022, date last accessed).

[2] White NJ . Assessment of the pharmacodynamic properties of antimalarial drugs in vivo. Antimicrob Agents Chemother. 1997; 41: 1413-22

[3] Ma N, Zhang Z, Liao F et al. The birth of artemisinin. Pharmacol Ther. 2020; 216: 107658

[4] Shi C, Li H, Yang Y et al. Anti-inflammatory and immunoregulatory functions of artemisinin and its derivatives. Mediat Inflamm. 2015; 2015: 435713

[5] Agrawal PK, Agrawal C, Blunden G . Artemisia extracts and artemisinin-based antimalarials for COVID-19 management: could these be effective antivirals for COVID-19 treatment? Molecules. 2022; 27: 3828

[6] Ikram NKBK, Simonsen HT . A review of biotechnological artemisinin production in plants. Front Plant Sci. 2017; 8: 1-10

[7] Paddon CJ, Westfall PJ, Pitera DJ et al. High-level semi-synthetic production of the potent antimalarial artemisinin. Nature. 2013; 496: 528-32

[8] Bouwmeester HJ, Wallaart TE, Janssen MHA et al. Amorpha-4,11-diene synthase catalyses the first probable step in artemisinin biosynthesis. Phytochemistry. 1999; 52: 843-54

[9] Mercke P, Bengtsson M, Bouwmeester HJ et al. Molecular cloning, expression, and characterization of amorpha-4,11-diene synthase, a key enzyme of artemisinin biosynthesis in Artemisia annua L. Arch Biochem Biophys. 2000; 381: 173-80

[10] Teoh KH, Polichuk DR, Reed DW et al. Artemisia annua L. (Asteraceae) trichome-specific cDNAs reveal CYP71AV1, a cytochrome P450 with a key role in the biosynthesis of the antimalarial sesquiterpene lactone artemisinin . FEBS Lett. 2006; 580: 1411-6

[11] Zhang Y, Teoh KH, Reed DW et al. The molecular cloning of artemisinic aldehyde Δ11(13) reductase and its role in glandular trichome-dependent biosynthesis of artemisinin in Artemisia annua. J Biol Chem. 2008; 283: 21501-8

[12] Teoh KH, Polichuk DR, Reed DW et al. Molecular cloning of an aldehyde dehydrogenase implicated in artemisinin biosynthesis in Artemisia annua. Botany. 2009; 87: 635-42

[13] Czechowski T, Larson TR, Catania TM et al. Detailed phytochemical analysis of high- and low artemisinin-producing chemotypes of Artemisia annua. Front Plant Sci. 2018; 9: 1-14

[14] Tang Y, Xiang L, Zhang F et al. Metabolic regulation and engineering of artemisinin biosynthesis in A. annua. Medicinal Plant Biol. 2023; 2: 4

[15] Tan HX, Xiao L, Gao SH et al. Trichome and artemisinin regulator 1 is required for trichome development and artemisinin biosynthesis in Artemisia annua. Mol Plant. 2015; 8: 1396-411

[16] Shen Q, Lu X, Yan T et al. The jasmonate-responsive AaMYC2 transcription factor positively regulates artemisinin biosynthesis in Artemisia annua. New Phytol. 2016; 210: 1269-81

[17] Zhang F, Fu X, Lv Z et al. A basic leucine zipper transcription factor, AabZIP1, connects abscisic acid signaling with artemisinin biosynthesis in Artemisia annua. Mol Plant. 2015; 8: 163-75

[18] Tang Y, Li L, Yan T et al. AaEIN3 mediates the downregulation of artemisinin biosynthesis by ethylene signaling through promoting leaf senescence in Artemisia annua. Front Plant Sci. 2018; 9: 413

[19] Shen Q, Huang H, Xie L et al. Basic helix-loop-helix transcription factors AabHLH2 and AabHLH3 function antagonistically with AaMYC2 and are negative regulators in artemisinin biosynthesis. Front Plant Sci. 2022; 13: 885622

[20] Wallaart TE, Pras N, Beekman AC et al. Seasonal variation of artemisinin and its biosynthetic precursors in plants of Artemisia annua of different geographical origin: proof for the existence of chemotypes . Planta Med. 2000; 66: 57-62

[21] Liao B, Shen X, Xiang L et al. Allele-aware chromosome-level genome assembly of Artemisia annua reveals the correlation between ADS expansion and artemisinin yield . Mol Plant. 2022; 15: 1310-28

[22] Ting HM, Wang B, Rydén AM et al. The metabolite chemotype of Nicotiana benthamiana transiently expressing artemisinin biosynthetic pathway genes is a function of CYP71AV1 type and relative gene dosage . New Phytol. 2013; 199: 352-66

[23] Yang K, Monfared SR, Wang HZ et al. The activity of the artemisinic aldehyde Delta 11(13) reductase promoter is important for artemisinin yield in different chemotypes of Artemisia annua L. Plant Mol Biol. 2015; 88: 325-40

[24] Chen M, Yan T, Ji L et al. Comprehensive map of the Artemisia annua proteome and quantification of differential protein expression in chemotypes producing high versus low content of artemisinin . Proteomics. 2020; 20: e1900310

[25] Liang D, Li W, Yan X et al. Molecular and functional evolution of the spermatophyte sesquiterpene synthases. Int J Mol Sci. 2021; 22: 6348

[26] Gao F, Sun Z, Kong F et al. Artemisinin-derived hybrids and their anticancer activity. Eur J Med Chem. 2020; 188: 112044

[27] Picaud S, Brodelius M, Brodelius PE . Expression, purification and characterization of recombinant (E)- β-farnesene synthase from Artemisia annua. Phytochemistry. 2005; 66: 961-7

[28] Cai Y, Jia JW, Crock J et al. A cDNA clone for β-caryophyllene synthase from Artemisia annua. Phytochemistry. 2002; 61: 523-9

[29] Mercke P, Crock J, Croteau R et al. Cloning, expression, and characterization of epi-cedrol synthase, a sesquiterpene cyclase from Artemisia annua L. Arch Biochem Biophys. 1999; 369: 213-22

[30] Li JX, Fang X, Zhao Q et al. Rational engineering of plasticity residues of sesquiterpene synthases from Artemisia annua: product specificity and catalytic efficiency . Biochem J. 2013; 451: 417-26

[31] Shen Q, Zhang L, Liao Z et al. The genome of Artemisia annua provides insight into the evolution of Asteraceae family and artemisinin biosynthesis . Mol Plant. 2018; 11: 776-88

[32] Breithaupt C, Kurzbauer R, Lilie H et al. Crystal structure of 12-oxophytodienoate reductase 3 from tomato: self-inhibition by dimerization. Proc Natl Acad Sci. 2006; 103: 14337-42

[33] Zheng X, Zhu K, Sun Q et al. Natural variation in CCD4 promoter underpins species-specific evolution of red coloration in citrus peel. Mol Plant. 2019; 12: 1294-307

[34] Liu L, Adrian J, Pankin A et al. Induced and natural variation of promoter length modulates the photoperiodic response of FLOWERING LOCUS T. Nat Commun. 2014; 5: 4558

[35] Adrian J, Farrona S, Reimer JJ et al. Cis-regulatory elements and chromatin state coordinately control temporal and spatial expression of FLOWERING LOCUS T in Arabidopsis. Plant Cell. 2010; 22: 1425-40

[36] Wang J, Hopkins CJ, Hou J et al. Promoter variation and transcript divergence in Brassicaceae lineages of FLOWERING LOCUS T. PLoS One. 2012; 7: e47127

[37] Jiang WM, Lu X, Qiu B et al. Molecular cloning and characterization of a trichome-specific promoter of artemisinic aldehyde delta 11(13) reductase (DBR2) in Artemisia annua. Plant Mol Biol Report. 2014; 32: 82-91

[38] Butelli E, Licciardello C, Zhang Y et al. Retrotransposons control fruit-specific, cold-dependent accumulation of anthocyanins in blood oranges. Plant Cell. 2012; 24: 1242-55

[39] Liu CL, Gao ZY, Shang LG et al. Natural variation in the promoter of OsHMA3 contributes to differential grain cadmium accumulation between Indica and japonica rice . J Integr Plant Biol. 2020; 62: 314-29

[40] Ye J, Li W, Ai G et al. Genome-wide association analysis identifies a natural variation in basic helix-loop-helix transcription factor regulating ascorbate biosynthesis via D-mannose/L-galactose pathway in tomato. PLoS Genet. 2019; 15: e1008149

[41] Ruan B, Shang L, Zhang B et al. Natural variation in the promoter of TGW2 determines grain width and weight in rice . New Phytol. 2020; 227: 629-40

[42] Shi CL, Dong NQ, Guo T et al. A quantitative trait locus GW6 controls rice grain size and yield through the gibberellin pathway . Plant J. 2020; 103: 1174-88

[43] Xu Q, Wu C, Xiao D et al. Ecological function of key volatiles in Vitex negundo infested by Aphis gossypii. Front Plant Sci. 2023; 13: 1-11

[44] Su X, Yang L, Wang D et al. 1K medicinal plant genome database: an integrated database combining genomes and metabolites of medicinal plants. Hortic Res. 2022; 9: 1-3

[45] Yi XZ, Wang XW, Wu L et al. Integrated analysis of basic helix loop helix transcription factor family and targeted terpenoids reveals candidate AarbHLH genes involved in terpenoid biosynthesis in Artemisia argyi. Front Plant Sci. 2022; 12: 1-18

[46] Nie WF, Chen Y, Tao J et al. Identification of the 12-oxophytoeienoic acid reductase (OPR) gene family in pepper (Capsicum annuum L.) and functional characterization of CaOPR6 in pepper fruit development and stress response . Genome. 2022; 65: 537-45

[47] Shen Q, Huang H, Zhao Y et al. The transcription factor AabZIP9 positively regulates the biosynthesis of artemisinin in Artemisia annua. Front Plant Sci. 2019; 10: 1-13