Spatial regulation of benzylisoquinoline alkaloid biosynthesis in lotus (Nelumbo nucifera) is controlled coordinately through the NnMYC2-NnMYB14-NnCYP80 modules

Abstract

Plant benzylisoquinoline alkaloids (BIAs) are a group of plant-specialized metabolites with significant pharmacological properties. In lotus (Nelumbo nucifera), BIAs accumulate primarily in the leaf blade and plumule organs. The two organs, however, accumulate quite different types of BIAs, within the former primarily aporphine-type BIAs, while the latter predominantly bis-BIAs. Herein, we demonstrate that the spatial regulation of BIA biosynthesis in lotus is coordinately controlled through the NnMYC2-NnMYB14-NnCYP80 modules. Genome-wide screening of lotus CYP80 genes discovered two tandemly arrayed yet tissue-specific NnCYP80s that are identical to the previously reported NnCYP80G and NnCYP80A, respectively. NnCYP80G is expressed primarily in the lotus laminae, while NnCYP80A is expressed particularly in the plumules. Our enzyme assays confirmed the proaporphine synthase activity of NnCYP80G and the bis-BIA synthase activity of NnCYP80A, and revealed the aporphine synthase activity of NnCYP80G by efficiently converting the (R)-reticuline substrate into corytuberine. In addition, we characterized an R2R3 MYB transcription factor (TF) NnMYB14, which binds directly to the NnCYP80G and NnCYP80A promoters and positively regulates their expression. NnMYC2, the core regulator in the JA signaling pathway, acts very upstream of NnMYB14, by binding directly to the NnMYB14 promoter and inducing its expression. Our results resolved that the organ-specific accumulation of BIAs in lotus is attributed to the tissue-specially expressed NnCYP80G and NnCYP80A genes, and the NnMYC2-NnMYB14 TF module could positively regulate the NnCYP80G and NnCYP80A expression and the lotus BIA biosynthesis.

Acknowledgements

This project was financially supported by the funds received from the National Natural Science Foundation of China (32070336 and 32370428), Hubei Provincial Natural Science Foundation of China (JCZRJQ202400159, and the Natural Science Foundation of Shandong Province (ZR2021MC163).

Authors contributions

X.D. and M.Y. (Conceptualization, Methodology), M.Z., Z.X., X.D., Z.Y., Hy.S., J.X., and H.Y. (Investigation), X.D. and M.Z. (Writing—original draft), and H.S., J.L., D.Y., Y.L., J.L., and M.Y. (Writing—review & editing)

Data availability

All data needed to evaluate the conclusions in the paper are present in the paper and/or the Supplementary materials.

Conflicts of interest statement

The authors declare that they have no competing interests.

Supplementary material

Supplementary material is available at Horticulture Research online.

References

[1] Bourgaud F, Gravot A, Milesi F. et al. Production of plant secondary metabolites: a historical perspective. Plant Sci. 2001; 161:839-51

[2] Ramakrishna A, Ravishankar GA. Influence of abiotic stress signals on secondary metabolites in plants. Plant Signal Behav. 2011; 6:1720-31

[3] Dudareva N, Pichersky E. Biochemical and molecular genetic aspects of floral scents. Plant Physiol. 2000; 122:627-34

[4] Mol J, Grotewold E, Koes R. How genes paint flowers and seeds. Trends Plant Sci. 1998; 3:212-7

[5] Pichersky E. Biochemistry and genetics of floral scent: a his-torical perspective. Plant J. 2023; 115:18-36

[6] Pichersky E, Gang DR. Genetics and biochemistry of sec-ondary metabolites in plants: an evolutionary perspective. Trends Plant Sci. 2000; 5:439-45

[7] Facchini PJ. ALKALOID BIOSYNTHESIS IN PLANTS: biochem-istry, cell biology, molecular regulation, and metabolic engi-neering applications. Annu Rev Plant Physiol Plant Mol Biol. 2001; 52:29-66

[8] Liscombe DK, Facchini PJ. Evolutionary and cellular webs in benzylisoquinoline alkaloid biosynthesis. Curr Opin Biotech-nol. 2008; 19:173-80

[9] Fan H, He ST, Han P. et al. Cepharanthine: a promising old drug against SARS-CoV-2. Adv Biol (Weinh). 2022; 6:e2200148

[10] Hijikata A, Shionyu-Mitsuyama C, Nakae S. et al. Evaluating cepharanthine analogues as natural drugs against SARS-CoV-2. FEBS Open Bio. 2022; 12:285-94

[11] Wang K, Guo H, Yin Y. AP2/ERF transcription factors and their functions in Arabidopsis responses to abiotic stresses. Environ Exp Bot. 2024; 222:105763

[12] Wiffen PJ, Wee B, Derry S. et al. Opioids for cancer pain - an overview of Cochrane reviews. Cochrane Database Syst Rev. 2017;7:CD012592

[13] Hagel JM, Facchini PJ. Benzylisoquinoline alkaloid metabolism: a century of discovery and a brave new world. Plant Cell Physiol. 2013; 54:647-72

[14] Liscombe DK, Macleod BP, Loukanina N. et al. Evidence for the monophyletic evolution of benzylisoquinoline alka-loid biosynthesis in angiosperms. Phytochemistry. 2005; 66: 1374-93

[15] Pauli HH, Kutchan TM. Molecular cloning and functional heterologous expression of two alleles encoding (S)-N-methylcoclaurine 3’-hydroxylase (CYP80B1), a new methyl jasmonate-inducible cytochrome P-450-dependent mono-oxygenase of benzylisoquinoline alkaloid biosynthesis. Plant J. 1998; 13:793-801

[16] Ikezawa N, Iwasa K, Sato F. Molecular cloning and character-ization of methylenedioxy bridge-forming enzymes involved in stylopine biosynthesis in Eschscholzia californica. FEBS J. 2007; 274:1019-35

[17] Ikezawa N, Iwasa K, Sato F. CYP719A subfamily of cytochrome P450 oxygenases and isoquinoline alkaloid biosynthesis in Eschscholzia californica. Plant Cell Rep. 2009; 28:123-33

[18] Mizutani M, Sato F. Unusual P450 reactions in plant secondary metabolism. Arch Biochem Biophys. 2011; 507:194-203

[19] Kraus PFX, Molecular cloning and heterologous expression of a cDNA encoding berbamunne synthase, a C-O phenol-coupling cytochrome P450 from the higher plant Berberis stolonifera. Proc Natl Acad Sci USA. 1995;92:2071-5

[20] An Z, Gao R, Chen S. et al. Lineage-specific CYP80 expansion and benzylisoquinoline alkaloid diversity in early-diverging eudicots. Adv Sci (Weinh). 2024; 11:e2309990

[21] Ikezawa N, Iwasa K, Sato F. Molecular cloning and charac-terization of CYP80G2, a cytochrome P450 that catalyzes an intramolecular C-C phenol coupling of (S)-reticuline in mag-noflorine biosynthesis, from cultured Coptis japonica cells. J Biol Chem. 2008; 283:8810-21

[22] Meng F, Zhang S, Su J. et al. Characterization of two CYP80 enzymes provides insights into aporphine alkaloid skele-ton formation in Aristolochia contorta. Plant J. 2024; 118: 1439-54

[23] Sun H, Xin J, Song H. et al. Harnessing genomic and molecular biology resources for genetic improvement of lotus: current achievements and future directions. Hortic Adv. 2025; 3:1

[24] Deng X, Zhu L, Fang T. et al. Analysis of isoquinoline alkaloid composition and wound-induced variation in Nelumbo using HPLC-MS/MS. J Agric Food Chem. 2016b; 64:1130-6

[25] Menendez-Perdomo IM, Facchini PJ. Benzylisoquinoline alka-loids biosynthesis in sacred lotus. Molecules. 2018; 23:2899

[26] Sharma BR, Gautam LN, Adhikari D. et al. A comprehensive review on chemical profiling of Nelumbo nucifera: potential for drug development. Phytother Res. 2016; 31:3-26

[27] Wei X, Zhang M, Yang M. et al. Lotus (Nelumbo nucifera) benzylisoquinoline alkaloids: advances in chemical profiling, extraction methods, pharmacological activities, and biosyn-thetic elucidation. Vegetable Res. 2024; 4:1-18

[28] Hao C, Yang W, Dong G. et al. Visualization and identification of benzylisoquinoline alkaloids in various Nelumbo nucifera tissues. Heliyon. 2023; 9:e16138

[29] Liu J, Shi K, Shi J. et al. A simple strategy to monitor the temporal and spatial distribution of alkaloids in sacred lotus leaves. Biosci Biotechnol Biochem. 2021; 85:1332-40

[30] Deng X, Zhao L, Fang T. et al. Investigation of benzylisoquino-line alkaloid biosynthetic pathway and its transcriptional reg-ulation in lotus. Hortic Res. 2018; 5:29

[31] Menendez-Perdomo IM, Facchini PJ. Elucidation of the (R)-enantiospecific benzylisoquinoline alkaloid biosynthetic pathways in sacred lotus (Nelumbo nucifera). Sci Rep. 2023; 13: 2955

[32] Nelson DR, Schuler MA. Cytochrome P450 genes from the sacred lotus genome. Trop Plant Biol. 2013; 6:138-51

[33] Hao C, Yu Y, Liu Y. et al. The CYP80A and CYP80G are involved in the biosynthesis of benzylisoquinoline alkaloids in the sacred lotus (Nelumbo nucifera). Int J Mol Sci. 2024; 25:702

[34] Pyne ME, Gold ND, Martin VJJ. Pathway elucidation and micro-bial synthesis of proaporphine and bis-benzylisoquinoline alkaloids from sacred lotus (Nelumbo nucifera). Metab Eng. 2023; 77:162-73

[35] Li J, Li Y, Dang M. et al. Jasmonate-responsive transcrip-tion factors NnWRKY70a and NnWRKY70b positively regulate benzylisoquinoline alkaloid biosynthesis in lotus (Nelumbo nucifera). Front Plant Sci. 2022; 13:862915

[36] Li J, Xiong Y, Li Y. et al. Comprehensive analysis and functional studies of WRKY transcription factors in Nelumbo nucifera. Int J Mol Sci. 2019; 20:5006

[37] Hagel JM, Morris JS, Lee EJ. et al. Transcriptome analysis of 20 taxonomically related benzylisoquinoline alkaloid-producing plants. BMC Plant Biol. 2015; 15:227

[38] Ishrat N, Gupta A, Khan MF. et al. Phytoconstituents of Nymphaea rubra flowers and their anti-diabetic metabolic targets. Fitoterapia. 2024; 176:106014

[39] Yogendra KN, Dhokane D, Kushalappa AC. et al. StWRKY8 tran-scription factor regulates benzylisoquinoline alkaloid path-way in potato conferring resistance to late blight. Plant Sci. 2017; 256:208-16

[40] Zhang J. Evolution by gene duplication: an update. Trends Ecol Evol. 2003; 18:292-8

[41] Sun H, Song H, Deng X. et al. Transcriptome-wide charac-terization of alkaloids and chlorophyll biosynthesis in lotus plumule. Front Plant Sci. 2022; 13:885503

[42] Poulos TL, Finzel BC, Gunsalus IC. et al. The 2.6-A crystal structure of Pseudomonas putida cytochrome P-450. J Biol Chem. 1985; 260:16122-30

[43] Kazan K, Manners JM. MYC2: the master in action. Mol Plant. 2013; 6:686-703

[44] Deng J, Li M, Huang L. et al. Genome-wide analysis of the R2R3 MYB subfamily genes in lotus (Nelumbo nucifera). Plant Mol Biol Report. 2016a; 34:1016-26

[45] Fessele S, Maier H, Zischek C. et al. Regulatory context is a crucial part of gene function. Trends Genet. 2002; 18:60-3

[46] Tjian R, Manlatis T. Transcriptional activation: a complex puz-zle with few easy pieces. Cell. 1994; 77:5-8

[47] Lau W, Sattely ES. Six enzymes from mayapple that complete the biosynthetic pathway to the etoposide aglycone. Science. 2015; 349:1224-8

[48] Chini A, Boter M, Solano R. Plant oxylipins: COI1/JAZs/MYC2 as the core jasmonic acid-signalling module. FEBS J. 2009; 276: 4682-92

[49] Luo L, Wang Y, Qiu L. et al. MYC2: a master switch for plant physiological processes and specialized metabolite synthe-sis. Int J Mol Sci. 2023; 24:3511

[50] Mu T, Luo S, Li L. et al. A review of the interaction mech-anisms between jasmonic acid (JA) and various plant hor-mones, as well as the core regulatory role of MYC2. Plant Sci. 2025; 353:112407

[51] Chen Y, Chen Z, Kang J. et al. AtMYB14 regulates cold tolerance in Arabidopsis. Plant Mol Biol Report. 2013; 31:87-97

[52] Chezem WR, Memon A, Li FS. et al. SG2-Type R2R3-MYB transcription factor MYB15 controls defense-induced lig-nification and basal immunity in Arabidopsis. Plant Cell. 2017; 29:1907-26

[53] Qian C, Chen Z, Liu Q. et al. Coordinated transcriptional reg-ulation by the UV-B photoreceptor and multiple transcrip-tion factors for plant UV-B responses. Mol Plant. 2020; 13: 777-92

[54] Edgar RC. MUSCLE: a multiple sequence alignment method with reduced time and space complexity. BMC Bioinformatics. 2004; 5:113

[55] Kumar S, Stecher G, Tamura K. MEGA7: molecular evolution-ary genetics analysis version 7.0 for bigger datasets. Mol Biol Evol. 2016; 33:1870-4

[56] Amborella Genome Project. The Amborella genome and the evolution of flowering plants. Science. 2013; 342:1241089

[57] Chen C, Chen H, Zhang Y. et al. TBtools: an integrative toolkit developed for interactive analyses of big biological data. Mol Plant. 2020; 13:1194-202

[58] Zhang M, Song H, Deng X. et al. Genome-wide identifica-tion of CYP90 family and functional analysis of NnCYP90B1 on rhizome enlargement in lotus. Vegetable Res. 2024; 4: e035

[59] Pfaffl MW. A new mathematical model for relative quantifica-tion in real-time RT-PCR. Nucleic Acids Res. 2001; 29:e45

[60] Liu J, Wang Y, Deng X. et al. Transcription factor NnMYB5 controls petal color by regulating GLUTATHIONE S-TRANSFERASE2 in Nelumbo nucifera. Plant Physiol. 2023; 193:1213-26

[61] Gouy M, Tannier E, Comte N. et al. Seaview version 5: a mul-tiplatform software for multiple sequence alignment, molec-ular phylogenetic analyses, and tree reconciliation. Methods Mol Biol. 2021; 2231:20

[62] Lescot M, Déhais P, Thijs G. et al. PlantCARE, a database of plant cis-acting regulatory elements and a portal to tools for in silico analysis of promoter sequences. Nucleic Acids Res. 2002; 30:325-7