Transcriptome-based identification of MYB transcription factors associated with flavonoid biosynthesis under LED light in Astragalus membranaceus (Fisch.) Bunge

Ji W. SEO; Hong J. CHOI; Jiu PARK; Won H. CHOI; Eun S. SEONG

doi:10.15835/nbha53314625

Authors

Ji W. SEO Kangwon National University, Interdisciplinary Program in Smart Science, Chuncheon, 24341 (KR)
Hong J. CHOI Kangwon National University, Interdisciplinary Program in Smart Science, Chuncheon, 24341 (KR)
Jiu PARK Kangwon National University, Interdisciplinary Program in Smart Science, Chuncheon, 24341 (KR)
Won H. CHOI Kangwon National University, Interdisciplinary Program in Smart Science, Chuncheon, 24341 (KR)
Eun S. SEONG Kangwon National University, Department pf Applied of Plant Sciences, Division of Bioresource Sciences, Chuncheon, 24341 (KR)

DOI:

https://doi.org/10.15835/nbha53314625

Keywords:

Astragalus membranaceus, flavonoid biosynthesis, LED light, MYB transcription factors, transcriptome data

Abstract

Astragalus membranaceus, valued for its health benefits due to flavonoids, saponins, and polysaccharides, has been studied mainly for its roots. However, the regulatory role of MYB transcription factors (TFs) in flavonoid biosynthesis under different light conditions remains largely unexplored. In this study, sterile plants were cultured for six weeks under white, red, and blue light. Transcriptome analysis identified MYB genes, which were further characterized by sequence alignment, conserved domain searches, and phylogenetic analysis with AtMYB and GmMYB176. Gene structures, conserved motifs, and cis-regulatory elements were examined using bioinformatics tools. Expression levels of selected genes were validated by real-time PCR using the 2^−ΔΔCt method. Two candidate genes, one R2R3-MYB and one R1-MYB, were found to be involved in flavonoid biosynthesis. Both showed high expression under white light, with AmMYB12 exhibiting nearly 20-fold higher expression compared with other conditions. Motif and promoter analyses indicated the presence of multiple MYB-binding sites, suggesting strong regulatory potential. Together, these results indicate that AmMYB12 may play a critical role in light-regulated flavonoid biosynthesis. This study provides essential data for functional analysis of MYB TFs and enhances understanding of molecular mechanisms underlying flavonoid accumulation in A. membranaceus in response to artificial light.

References

Abubakar AS, Feng X, Gao G, Yu C, Chen J, Chen K, … Cehn P (2022). Genome wide characterization of R2R3 MYB transcription factor from Apocynum venetum revealed potential stress tolerance and flavonoid biosynthesis genes. Genomics 114:110275. https://doi.org/10.1016/j.ygeno.2022.110275

Albert NW, Thrimawithana AH, McGhie TK, Clayton WA, Deroles SC, Schwinn KE, … Davies KM (2018). Genetic analysis of the liverwort Marchantia polymorpha reveals that R2R3 MYB activation of flavonoid production in response to abiotic stress is an ancient character in land plants. New Phytologist 218:554-566. https://doi.org/10.1111/nph.15002

Akbari F, Azadbakht M, Bagheri A, Vahedi L (2022). In vitro and in vivo wound healing activity of Astragalus Floccosus Boiss. (Fabaceae). Advances in Pharmacological and Pharmaceutical Sciences 2022:7865015. https://doi.org/10.1155/2022/7865015

Cao Y, Jia H, Xing M, Jin R, Grierson D, Gao Z, … Li X (2021). Genome-wide analysis of MYB gene family in Chinese bayberry (Morella rubra) and identification of members regulating flavonoid biosynthesis. Frontiers in Plant Science 12:691384. https://doi.org/10.3389/fpls.2021.691384

Chai G, Wang Z, Tang X, Yu L, Qi G, Wang D, … Zhou G (2014). R2R3-MYB gene pairs in Populus: evolution and contribution to secondary wall formation and flowering time. Journal of Experimental Botany 65:4255-4269. https://doi.org/10.1093/jxb/eru196

Chen C, Chen H, Zhang Y, Thomas HR, Frank MH, He Y, Xia R (2020). TBtools: an integrative toolkit developed for interactive analyses of big biological data. Molecular Plant 13:1194-1202. https://doi.org/10.1016/j.molp.2020.06.009

Chen F, Luo X, Gao W, Liu X, Zheng Y (2007). Induction and characterization of tetraploid Astragalus membranaceus plants. Plant Cell Reports 26:641-648.

Chen G, Jiang N, Zheng J, Hu H, Yang H, Lin A, … Liu H (2023). Structural characterization and anti-inflammatory activity of polysaccharides from Astragalus membranaceus. International Journal of Biological Macromolecules 241:124386. https://doi.org/10.1016/j.ijbiomac.2023.124386

Chen X, Wang P, Gu M, Lin X, Hou B, Zheng Y, … Ye N (2021). R2R3-MYB transcription factor family in tea plant (Camellia sinensis): Genome-wide characterization, phylogeny, chromosome location, structure and expression patterns. Genomics 113:1565-1578. https://doi.org/10.1016/j.ygeno.2021.03.033

Chen Y, Fang T, Su H, Duan S, Ma R, Wang P, … Zhao M (2023). A reference-grade genome assembly for Astragalus mongholicus and insights into the biosynthesis and high accumulation of triterpenoids and flavonoids in its roots. Plant Communications 4:100469. https://doi.org/10.1016/j.xplc.2022.100469

Cui L, Ma Z, Wang D, Niu Y (2022). Ultrasound-assisted extraction, optimization, isolation, and antioxidant activity analysis of flavonoids from Astragalus membranaceus stems and leaves. Ultrasonics Sonochemistry 90:106190. https://doi.org/10.1016/j.ultsonch.2022.106190

da Cristina Bungala LT, Park SU, Van Nguyen B, Lim J, Kim K, Kim JK, … Yeo HJ (2024). Effect of LED Lights on secondary metabolites and antioxidant activities in red pakchoi baby leaves. ACS omega 9:23420. https://doi.org/10.1021/acsomega.3c10261

D’Avino D, Cerqua I, Ullah H, Spinelli M, Di Matteo R, Granato E, … Daglia M (2023). Beneficial effects of Astragalus membranaceus (Fisch.) bunge extract in controlling inflammatory response and preventing asthma features. International Journal of Molecular Sciences 24:10954. https://doi.org/10.3390/ijms241310954

Del‐Castillo‐Alonso MÁ, Monforte L, Tomás‐Las‐Heras R, Ranieri A, Castagna A, Martínez‐Abaigar J, Núñez-Olivera E (2021). Secondary metabolites and related genes in Vitis vinifera L. cv. Tempranillo grapes as influenced by ultraviolet radiation and berry development. Physiologia Plantarum 173:709-724. https://doi.org/10.1111/ppl.13483

DeBonis A, Crone E (2011). Pollination ecology of Astragalus filipes (Fabaceae). Plant Species Biology 26:215-224.

Dong X, Liu X, Cheng L, Li R, Ge S, Wang S, … Jiang CZ (2024). SlBEL11 regulates flavonoid biosynthesis, thus fine‐tuning auxin efflux to prevent premature fruit drop in tomato. Journal of Integrative Plant Biology 66:749-770. https://doi.org/10.1111/jipb.13627

Dong J, Wang L, Zhang Y, Liu X, Chen M, Zhao H, Li Q (2024). Advances in cytogenetic and genomic resources of Astragalus membranaceus. Frontiers in Plant Science 15:1420512.

Du H, Liang Z, Zhao S, Nan M-G, Tran L-SP, Lu K, … Li JN (2015). The evolutionary history of R2R3-MYB proteins across 50 eukaryotes: new insights into subfamily classification and expansion. Scientific Reports 5:11037. https://doi.org/10.1038/srep11037

Durazzo A, Nazhand A, Lucarini M, Silva AM, Souto SB, Guerra F, … Santini A (2021). Astragalus (Astragalus membranaceus Bunge): botanical, geographical, and historical aspects to pharmaceutical components and beneficial role. Rendiconti Lincei. Scienze Fisiche e Naturali 32:625-642. https://doi.org/10.1007/s12210-021-01003-2

Du Y, Wan H, Huang P, Yang J, He Y (2022). A critical review of Astragalus polysaccharides: from therapeutic mechanisms to pharmaceutics. Biomedicine and Pharmacotherapy 147:112654. https://doi.org/10.1016/j.biopha.2022.112654

Gai Q-Y, Feng X, Jiao J, Xu X-J, Fu J-X, He X-J, Fu YJ (2023). Blue LED light promoting the growth, accumulation of high-value isoflavonoids and astragalosides, antioxidant response, and biosynthesis gene expression in Astragalus membranaceus (Fisch.) Bunge hairy root cultures. Plant Cell Tissue and Organ Culture 153:511-523. https://doi.org/10.1007/s11240-023-02486-7

Gangappa SN, Botto JF (2016). The multifaceted toles of HY5 in plant growth and development. Molecular Plant 9:1353-1365. http://dx.doi.org/10.1016/j.molp.2016.07.002

Ghasemian Yadegari J, Khudair Khalaf A, Darabi R (2022). Antiparasitic effects and cellular mechanism of Astragalus maximus chloroform extract against clinical isolates of Giardia lamblia. Research Journal of Pharmacognosy 9:5-13. https://www.rjpharmacognosy.ir/article_148769.html

Gong G, Yu H, Zheng Y, Qi B, He H, Yin T, … Tsim KWK (2021). Astragaloside IV, a saponin from Astragalus membranaceus var. mongholicus, induces expressions of heme recycle proteins via signaling of Nrf2/ARE in cultured macrophages. Journal of Ethnopharmacology 265:113389. https://doi.org/10.1016/j.jep.2020.113389

Guo L, Sun Y, Ping X, Liu J, Wang X, Qin N (2022). Chemical composition and antibacterial activity of ethyl acetate extract of Astragalus membranaceus aerial parts. Jounal of Food Safety 42:e12947. https://doi.org/10.1111/jfs.12947

Hu Y, Zhai W, Tan D, Chen H, Zhang G, Tan X, … Wu J (2023) Uncovering the effects and molecular mechanism of Astragalus membranaceus (Fisch.) Bunge and its bioactive ingredients formononetin and calycosin against colon cancer: An integrated approach based on network pharmacology analysis coupled with experimental validation and molecular docking. Frontiers in Pharmacology 14:1111912. https://doi.org/10.3389/fphar.2023.1111912

Jiao Y, Lau OS, Deng XW (2007). Light-regulated transcriptional networks in higher plants. Nature Reviews Genetics 8:217-230. https://doi.org/10.1038/nrg2049

Kochetova GV, Avercheva OV, Bassarskaya EM, Kushunina MA, Zhigalova TV (2023). Effects of red and blue LED light on the growth and photosynthesis of barley (Hordeum vulgare L.) seedlings. Journal of Plant Growth Regulation 42:1804-1820. https://doi.org/10.1007/s00344-022-10661-x

Konichenko EA, Selyutina IY, Selyutina GG (2014). Chromosome numbers of Fabaceae in Eurasia. Caryologia 67:1-11.

Le AT, Yu JK, Han GD, Do TK, Chung YS (2022). Potential use of colored LED lights to increase the production of bioactive metabolites Hedyotis corymbosa (L.) Lam. Plants 11:225. https://doi.org/10.3390/plants11020225

Lee SB, Lee JS, Moon SO, Lee HD, Yoon YS, Son CG (2021). A standardized herbal combination of Astragalus membranaceus and Paeonia japonica, protects against muscle atrophy in a C26 colon cancer cachexia mouse model. Journal of Ethnopharmacology 267:113470. https://doi.org/10.1016/j.jep.2020.113470

Li J, Xu S, Mei Y, Gu Y, Sun M, Zhang W, Wang J (2023). Genomic-wide identification and expression analysis of R2R3-MYB transcription factors related to flavonol biosynthesis in Morinda officinalis. BMC Plant Biology 23:381. https://doi.org/10.1186/s12870-023-04394-6

Li S, Sun Y, Huang J, Wang B, Gong Y, Fang Y, … Wang H (2020). Anti-tumor effects and mechanisms of Astragalus membranaceus (AM) and its specific immunopotentiation: Status and prospect. Journal of Ethnopharmacology 258:112797. https://doi.org/10.1016/j.jep.2020.112797

Muhammad N, Luo Z, Zhao X, Yang M, Liu Z, Liu M (2023). Transcriptome-wide expression analysis of MYB gene family leads to functional characterization of flavonoid biosynthesis in fruit coloration of Ziziphus Mill. Frontiers in Plant Science 14:1171288. https://doi.org/10.3389/fpls.2023.1171288

Pant P, Pandey S, Dall'Acqua S (2021). The influence of environmental conditions on secondary metabolites in medicinal plants: A literature review. Chemistry & Biodiversity 18:e2100345. https://doi.org/10.1002/cbdv.202100345

Paradiso R, Proietti S (2022). Light-quality manipulation to control plant growth and photomorphogenesis in greenhouse horticulture: The state of the art and the opportunities of modern LED systems. Journal of Plant Growth Regulation 41:742-780. https://doi.org/10.1007/s00344-021-10337-y

Pennisi G, Pistillo A, Orsini F, Cellini A, Spinelli F, Nicola S, … Marcelis LFM (2020). Optimal light intensity for sustainable water and energy use in indoor cultivation of lettuce and basil under red and blue LEDs. Scientia Horticulturae 272:109508. https://doi.org/10.1016/j.scienta.2020.109508

Qader M, Xu J, Yang Y, Wu X, Liu Y, Cao S (2021). Chemistry behind the immunomodulatory activity of Astragalus membranaceus. Chinese Medicine and Culture 4:201-210. https://doi.org/10.4103/cmac.cmac_40_21

Qiu YX, Chen XL, Fu CX (2001). Reproductive biology of Astragalus membranaceus var. mongholicus. Acta Botanica Boreali-Occidentalia Sinica 21:493-498.

Salehi B, Carneiro JNP, Rocha JE, Coutinho HDM, Morais Braga MFB, Sharifi‐Rad J, … de Zarate Machado V (2021). Astragalus species: Insights on its chemical composition toward pharmacological applications. Phytotherapy Research 35:2445-2476. https://doi.org/10.1002/ptr.6974

Samuel AO, Huang BT, Chen Y, Guo FX, Yang DD, Jin JQ (2021). Antioxidant and antibacterial insights into the leaves, leaf tea and medicinal roots from Astragalus membranaceus (Fisch.) Bge. Scientific Reports 11:19625. https://doi.org/10.1038/s41598-021-97109-6

Seo JW, Ham DY, Lee JG, Kim MJ, Yu CY, Seong ES (2024). The effect of different LED wavelengths on the components and biosynthesis of isoflavonoid in sprout Astragalus membranaceus. Protoplasma 261:103-110. https://doi.org/10.1007/s00709-023-01883-1

Seo JW, Lee JG, Yoo JH, Lim JD, Kim MJ, Seong ES (2022). Growth characteristics and biological activities in Astragalus membranaceus seedlings of exposed to different types of artificial light. Korean Journal of Medicinal Crop Science 30:339-346. https://doi.org/10.7783/kjmcs.2022.30.5.339

Song G, Yan Y, Guo C, Chen J, Wang Y, Wang Y, … Pia X (2024). Identification and expression analysis of R2R3-MYB transcription factors associated with flavonoid biosynthesis in Panax quinquefolius. International Journal of Molecular Sciences 25:3709. https://doi.org/10.3390/ijms25073709

Stracke R, Ishihara H, Huep G, Barsch A, Mehrtens F, Niehaus K, Weisshaar B (2007). Differential regulation of closely related R2R3‐MYB transcription factors controls flavonol accumulation in different parts of the Arabidopsis thaliana seedling. The Plant Journal 50:660-677. https://doi.org/10.1111/j.1365-313X.2007.03078.x

Stracke R, Werber M, Weisshaar B (2001). The R2R3-MYB gene family in Arabidopsis thaliana. Current Opinion in Plant Biology 4:447-456. https://doi.org/10.1016/S1369-5266(00)00199-0

Tamura K, Stecher G, Kumar S (2021). MEGA11: molecular evolutionary genetics analysis version 11. Molecular Biology and Evolution 38:3022-3027. https://doi.org/10.1093/molbev/msab120

Thakur S, Vasudev PG (2022). MYB transcription factors and their role in Medicinal plants. Molecular Biology Reports 49:10995-11008. https://doi.org/10.1007/s11033-022-07825-z

Ullah MA, Hassan A, Hamza A (2023). Clinical treatments of various diseases with the root of Astragalus Membranaceus. Journal of Clinical Research and Reports 13:299. https://doi.org/10.31579/2690-1919/299

Veremeichik G, Grigorchuk V, Makhazen D, Subbotin E, Kholin A, Subbotina N, … Bulgakov VP (2023). High production of flavonols and anthocyanins in Eruca sativa (Mill) Thell plants at high artificial LED light intensities. Food Chemistry 408:135216. https://doi.org/10.1016/j.foodchem.2022.135216

Wang Y, Zhou L-J, Song A, Wang Y, Geng Z, Zhao K, … Chen F (2023). Comparative transcriptome analysis and flavonoid profiling of floral mutants reveals CmMYB11 regulating flavonoid biosynthesis in chrysanthemum. Plant Science 336:111837. https://doi.org/10.1016/j.plantsci.2023.111837

Weerawanich K, Halbwirth H, Sirikantaramas S (2024). A novel MYB transcription factor from durian (Durio zibethinus), DzMYB1, regulates flavonoid biosynthesis in fruit pulp. Scientia Horticulturae 333:113246. https://doi.org/10.1016/j.scienta.2024.113246

Wei X, Xin J, Chen W, Wang J, Lv Y, Wei Y, … Xu X (2023). Astragalus polysaccharide ameliorated complex factor-induced chronic fatigue syndrome by modulating the gut microbiota and metabolites in mice. Biomedicine & Pharmacotherapy 163:114862. https://doi.org/10.1016/j.biopha.2023.114862

Wong DCJ, Schlechter R, Vannozzi A, Höll J, Hmmam I, Bogs J, … Matus JT (2016). A systems-oriented analysis of the grapevine R2R3-MYB transcription factor family uncovers new insights into the regulation of stilbene accumulation. DNA Research 23:451-466. https://doi.org/10.1093/dnares/dsw028

Wu BS, Hitti Y, MacPherson S, Orsat V, Lefsrud MG (2020). Comparison and perspective of conventional and LED lighting for photobiology and industry applications. Environmental and Experimental Botany 171:103953. https://doi.org/10.1016/j.envexpbot.2019.103953

Wu Y, Wen J, Xia Y, Zhang L, Du H (2022). Evolution and functional diversification of R2R3-MYB transcription factors in plants. Horticulture Research 9:uhac058. https://doi.org/10.1093/hr/uhac058

Xie D, Chen L, Zhou C, Tarin MWK, Yang D, Ren K, … Zheng Y (2020). Transcriptomic and metabolomic profiling reveals the effect of LED light quality on morphological traits, and phenylpropanoid-derived compounds accumulation in Sarcandra glabra seedlings. BMC Plant Biology 20:1-18. https://doi.org/10.1186/s12870-020-02685-w

Xing H, Yin Z (1984). Karyomorphological studies on medicinal plants in China (III). Cytologia 49:701-707.

Xue A, Liang W, Wen S, Gao Y, Huang X, Tong Y, … Luo L (2021). Metabolomic analysis based on EESI-MS indicate blue LED light promotes aliphatic-glucosinolates biosynthesis in broccoli sprouts. Journal of Food Composition and Analysis 97:103777. https://doi.org/10.1016/j.jfca.2020.103777

Yang Q, Pan J, Shen G, Guo B (2019). Yellow light promotes the growth and accumulation of bioactive flavonoids in Epimedium pseudowushanense. Journal of Photochemistry and Photobiology B: Biology 197:111550. https://doi.org/10.1016/j.jphotobiol.2019.111550

Yanhui C, Xiaoyuan Y, Kun H, Meihua L, Jigang L, Zhaofeng G, … Xiaoming Q (2006). The MYB transcription factor superfamily of Arabidopsis: expression analysis and phylogenetic comparison with the rice MYB family. Plant molecular biology 60:107-124. https://doi.org/10.1007/s11103-005-2910-y

Yavari N, Tripathi R, Wu B-S, MacPherson S, Singh J, Lefsrud M (2021). The effect of light quality on plant physiology, photosynthetic, and stress response in Arabidopsis thaliana leaves. PloS One 16:e0247380. https://doi.org/10.1371/journal.pone.0247380

Yi J, Derynck MR, Li X, Telmer P, Marsolais F, Dhaubhadel S (2010). A single‐repeat MYB transcription factor, GmMYB176, regulates CHS8 gene expression and affects isoflavonoid biosynthesis in soybean. The Plant Journal 62:1019-1034. https://doi.org/10.1111/j.1365-313X.2010.04214.x

Zhang H, Li X, Wang J, Sun R, Zhao L (2024). Genome-wide identification of secondary metabolite genes in tetraploid Astragalus membranaceus. Plants 13:1123.

Zhang X, Bian Z, Yuan X, Chen X, Lu C (2020). A review on the effects of light-emitting diode (LED) light on the nutrients of sprouts and microgreens. Trends in Food Science & Technology 99:203-216. https://doi.org/10.1016/j.tifs.2020.02.031

Zhang X, He Y, Li L, Liu H, Hong G (2021). Involvement of the R2R3-MYB transcription factor MYB21 and its homologs in regulating flavonol accumulation in Arabidopsis stamen. Journal of Experimental Botany 72:4319-4332. https://doi.org/10.1093/jxb/erab156

Zhao Y, Zhang G, Tang Q, Song W, Gao Q, Xiang G, … Li X (2022). EbMYBP1, a R2R3-MYB transcription factor, promotes flavonoid biosynthesis in Erigeron breviscapus. Frontiers in Plant Science 13:946827. https://doi.org/10.3389/fpls.2022.946827

Zhu L, Guan Y, Zhang Z, Song A, Chen S, Jiang J, Chen F (2020). CmMYB8 encodes an R2R3 MYB transcription factor which represses lignin and flavonoid synthesis in chrysanthemum. Plant Physiology and Biochemistry 149:217-224. https://doi.org/10.1016/j.plaphy.2020.02.010