Abstract
Sanguinarine (SAN) and chelerythrine (CHE) have significant anti-inflammatory and growth-promoting activities and have been widely used in medicine and stockbreeding. However, SAN and CHE are present in only small amounts (0.5% capsule dry weight) in the Chinese herbal medicine Macleaya cordata, resulting in a consistently high price of their derivative products. Here, we generated transgenic plants overexpressing the key SAN and CHE biosynthetic pathway gene, protopine-6-hydroxylase (P6H), via genetic transformation. Quantitative detection by ultra-performance liquid chromatography tandem/triple quadrupole mass spectrometry (UPLC-QQQ-MS) showed that the contents of SAN and CHE increased in the overexpressing plants compared with the wild-type plants. Moreover, these plants had a higher relative expression of level of McP6H in the roots, stems and leaves, and the relative expression of the downstream enzyme dihydrobenzophenanthridine oxidase (McDBOX) also increased significantly. These results indicate that overexpression of the P6H gene is a promising approach to improve SAN and CHE production and is significant in the study of the synthesis pathways of benzylisoquinoline alkaloids (BIAs) to obtain germplasm resources with high alkaloid contents.
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Data availability
The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.
References
Basu P, Kumar GS (2016) Sanguinarine and its role in chronic diseases. Adv Exp Med Biol 928:155–172. https://doi.org/10.1007/978-3-319-41334-1_7
Beaudoin GAW, Facchini PJ (2014) Benzylisoquinoline alkaloid biosynthesis in opium poppy. Planta 240:19–32. https://doi.org/10.1007/s00425-014-2056-8
Brazier M, Cole DJ, Edwards R (2002) O-Glucosyltransferase activities toward phenolic natural products and xenobiotics in wheat and herbicide-resistant and herbicide-susceptible black-grass (Alopecurus myosuroides). Phytochemistry 59:149–156. https://doi.org/10.1016/S0031-9422(01)00458-7
Chávez MLD, Rolf M, Gesell A, Kutchan TM (2011) Characterization of two methylenedioxy bridge-forming cytochrome P450-dependent enzymes of alkaloid formation in the Mexican prickly poppy Argemone mexicana. Arch Biochem Biophys 507:186–193. https://doi.org/10.1016/j.abb.2010.11.016
Gesell A, Rolf M, Ziegler J, Díaz Chávez ML, Huang FC, Kutchan TM (2009) CYP719B1 is salutaridine synthase, the C-C phenol-coupling enzyme of morphine biosynthesis in opium poppy. J Biol Chem 284:24432–24442. https://doi.org/10.1074/jbc.M109.033373
Hawkins KM, Smolke CD (2008) Production of benzylisoquinoline alkaloids in Saccharomyces cerevisiae. Nat Chem Biol 4:564–573. https://doi.org/10.1038/nchembio.105
Huang P, Xu M, Xia L, Qing Z, Tang Q, Liu W, Zeng J (2017) Establishment of an efficient Agrobacterium-mediated genetic transformation method in Macleaya cordata. Sci Hortic 226:302–306. https://doi.org/10.1016/j.scienta.2017.09.004
Huang P, Zhang Y, Xiao K, Jiang F, Wang H, Tang D, Liu D, Liu B, Liu Y, He X, Liu H, Liu X, Qing Z, Liu C, Huang J, Ren Y, Long Y, Yin L, Lin Q, Zeng C, Su X, Yuan J, Lin L, Hu N, Cao H, Huang S, Guo Y, Fan W, Zeng J (2018a) The chicken gut metagenome and the modulatory effects of plant-derived benzylisoquinoline alkaloids. Microbiome 211:1–17. https://doi.org/10.1186/s40168-018-0590-5
Huang P, Liu W, Xu M, Jiang R, Xia L, Wang P, Li H, Tang Z, Zheng Q, Zeng J (2018b) Modulation of benzylisoquinoline alkaloid biosynthesis by overexpression berberine bridge enzyme in Macleaya cordata. Sci Rep 8:17988. https://doi.org/10.1038/s41598-018-36211-8
Ikezawa N, Iwasa K, Sato F (2009) CYP719A subfamily of cytochrome P450 oxygenases and isoquinoline alkaloid biosynthesis in Eschscholzia californica. Plant Cell Rep 28:123–133. https://doi.org/10.1007/s00299-008-0624-8
Inui T, Kawano N, Shitan N, Yazaki K, Kiuchi F, Kawahara N, Sato F, Yoshimatsu K (2012) Improvement of benzylisoquinoline alkaloid productivity by overexpression of 3’-hydroxy-N-methylcoclaurine 4’-O-methyltransferase in transgenic Coptis japonica plants. Biol Pharm Bull 35:650–659. https://doi.org/10.1248/bpb.35.650
Inui T, Tamura K, Fujii N, Morishige T, Sato F (2007) Overexpression of Coptis japonica norcoclaurine 6-O-methyltransferase overcomes the rate-limiting step in Benzylisoquinoline alkaloid biosynthesis in cultured Eschscholzia californica. Plant Cell Physiol 48:252–262. https://doi.org/10.1093/pcp/pcl062
Jia Y, Bai Z, Pei T, Ding K, Liang Z, Gong Y (2017) The Protein Kinase SmSnRK26 Positively Regulates Phenolic Acid Biosynthesis in Salvia miltiorrhiza by Interacting with SmAREB1. Front Plant Sci 8:1384. https://doi.org/10.3389/fpls.2017.01384
Kim YJ, Lee OR, Oh JY, Jang MG, Yang DC (2014) Functional analysis of 3-hydroxy-3-methylglutaryl coenzyme a reductase encoding genes in triterpene saponin-producing ginseng. Plant Physiol 165:373–387. https://doi.org/10.1104/pp.113.222596
Kosina P, Gregorova J, Gruz J, Vacek J, Kolar M, Vogel M, Roos W, Naumann K, Simanek V, Ulrichova J (2010) Phytochemical and antimicrobial characterization of Macleaya cordata herb. Fitoterapia 81:1006–1012. https://doi.org/10.1016/j.fitote.2010.06.020
Liu X, Liu Y, Huang P, Ma Y, Qing Z, Tang Q, Cao H, Cheng P, Zheng Y, Yuan Z, Zhou Y, Liu J, Tang Z, Zhuo Y, Zhang Y, Yu L, Huang J, Yang P, Peng Q, Zhang J, Jiang W, Zhang Z, Lin K, Ro DK, Chen X, Xiong X, Shang Y, Huang S, Zeng J (2017) The Genome of Medicinal Plant Macleaya cordata Provides New Insights into Benzylisoquinoline Alkaloids Metabolism. Mol Plant 10:975–989. https://doi.org/10.1016/j.molp.2017.05.007
Liu ZY, Wang XL, Ou SQ, Hou DX, He JH (2020) Sanguinarine modulate gut microbiome and intestinal morphology to enhance growth performance in broilers. PLoS One 15:e0234920. https://doi.org/10.1371/journal.pone.0234920
Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2–∆∆CT Method. Methods 25:402–408. https://doi.org/10.1006/meth.2001.1262
Matías-Hernández L, Jiang W, Yang K, Tang K, Brodelius PE, Pelaz S (2017) AaMYB1 and its orthologue AtMYB61 affect terpene metabolism and trichome development in Artemisia annua and Arabidopsis thaliana. Plant J 90:520–534. https://doi.org/10.1111/tpj.13509
Park SU, Yu M, Facchini PJ (2003) Modulation of berberine bridge enzyme levels in transgenic root cultures of California poppy alters the accumulation of benzophenanthridine alkaloids. Plant Mol Biol 51:153–164. https://doi.org/10.1023/A:1021199311049
Takemura T, Ikezawa N, Iwasa K, Sato F (2013) Molecular cloning and characterization of a cytochrome P450 in sanguinarine biosynthesis from Eschscholzia californica cells. Phytochemistry 91:100–108. https://doi.org/10.1016/j.phytochem.2012.02.013
Winkler A, Puhl M, Weber H, Kutchan TM, Gruber K, Macheroux P (2009) Berberine bridge enzyme catalyzes the six electron oxidation of (S)-reticuline to dehydroscoulerine. Phytochemistry 70:1092–1097. https://doi.org/10.1016/j.phytochem.2009.06.005
Zhang F, Xie J, Wang G, Zhang G, Yang H (2018) Anti-osteoporosis activity of Sanguinarine in preosteoblast MC3T3-E1 cells and an ovariectomized rat model. J Cell Physiol 233:4626–4633. https://doi.org/10.1002/jcp.26187
Zhang J, Zhou L, Zheng X, Zhang J, Yang L, Tan R, Zhao S (2017) Overexpression of SmMYB9b enhances tanshinone concentration in Salvia miltiorrhiza hairy roots. Plant Cell Rep 36:1297–1309. https://doi.org/10.1007/s00299-017-2154-8
Funding
This work was supported by the “Hunan Provincial Natural Science Foundation of China (2021JJ20032)”; “Hunan Provincial Key Research and Development Project (2020NK2031)”; “Hunan Provincial Natural Science Foundation of China (2020JJ4353)”.
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MS, PH and JZ conceived and designed the study. MS and PH wrote the manuscript. MS, ZX and YW conducted the experimental work. XL ,ZL and WL participated in the results interpretation. All authors read and approved the final manuscript.
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Communicated by Konstantin V. Kiselev.
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Sun, M., Liu, X., Liu, W. et al. The effects of protopine 6-hydroxylase (P6H) overexpression on benzylisoquinoline alkaloids in Macleaya cordata. Plant Cell Tiss Organ Cult 148, 429–437 (2022). https://doi.org/10.1007/s11240-021-02183-3
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DOI: https://doi.org/10.1007/s11240-021-02183-3