Abstract
Black rice is a rare type of rice germplasm with various health benefits that are largely attributed to anthocyanin pigment accumulation in the pericarps. The anthocyanin biosynthesis in plant tissues is activated mainly by the MBW complexes, consisting of three types of transcription factors R2R3-MYB, bHLH, and WDR. In black rice, the bHLH and WDR components regulating anthocyanin biosynthesis in pericarps have been characterized, while the R2R3-MYB factor remains unknown. By examining the expression correlation between all putative rice MYB genes and anthocyanin biosynthesis–related genes based on transcriptome data of pericarps in combination with further molecular and genetic analysis, we proved that OsMYB3 (LOC_Os03g29614) was the determinant R2R3-MYB gene for anthocyanin biosynthesis in rice pericarps. The expression level of OsMYB3 in pericarps of black rice was significantly higher than that of white rice. The knockout of OsMYB3 in a black rice variety caused significant downregulation of 19 anthocyanin metabolites and many other flavonoids in grains. Our research deepens the understanding of regulatory system for anthocyanin biosynthesis in rice pericarps and provides implications for breeding black rice varieties with high anthocyanin level.
Similar content being viewed by others
Data availability
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
Code availability.
Not applicable.
References
Albert NW, Davies KM, Lewis DH, Zhang H, Montefiori M, Brendolise C, Boase MR, Ngo H, Jameson PE, Schwinn KE (2014) A conserved network of transcriptional activators and repressors regulates anthocyanin pigmentation in eudicots. Plant Cell 26(3):962–980. https://doi.org/10.1105/tpc.113.122069
Al-Kanhal MA, Al-Mohizea IS, Al-Othaimeen AI, Khan MA (1999) Nutritive value of various rice based dishes in Saudi Arabia. Ecol Food Nutr 38(3):223–235. https://doi.org/10.1080/03670244.1999.9991579
Ban Y, Honda C, Hatsuyama Y, Igarashi M, Bessho H, Moriguchi T (2007) Isolation and functional analysis of a MYB transcription factor gene that is a key regulator for the development of red coloration in apple skin. Plant Cell Physiol 48(7):958–970. https://doi.org/10.1093/pcp/pcm066
Baudry A, Heim MA, Dubreucq B, Caboche M, Weisshaar B, Lepiniec L (2004) TT2, TT8, and TTG1 synergistically specify the expression of BANYULS and proanthocyanidin biosynthesis in Arabidopsis thaliana. Plant J 39(3):366–380. https://doi.org/10.1111/j.1365-313X.2004.02138.x
Cassidy A (2018) Berry anthocyanin intake and cardiovascular health. Mol Aspects Med 61:76–82. https://doi.org/10.1016/j.mam.2017.05.002
Druka A, Kudrna D, Rostoks N, Brueggeman R, von Wettstein D, Kleinhofs A (2003) Chalcone isomerase gene from rice (Oryza sativa) and barley (Hordeum vulgare): physical, genetic and mutation mapping. Gene 302(1–2):171–178. https://doi.org/10.1016/S0378-1119(02)01105-8
Espley RV, Hellens RP, Putterill J, Stevenson DE, Kutty-Amma S, Allan AC (2007) Red colouration in apple fruit is due to the activity of the MYB transcription factor, MdMYB10. Plant J 49(3):414–427. https://doi.org/10.1111/j.1365-313X.2006.02964.x
Feller A, Machemer K, Braun EL, Grotewold E (2011) Evolutionary and comparative analysis of MYB and bHLH plant transcription factors. Plant J 66(1):94–116. https://doi.org/10.1111/j.1365-313X.2010.04459.x
Furukawa T, Maekawa M, Oki T, Suda I, Iida S, Shimada H, Takamure I, Kadowaki K (2007) The Rc and Rd genes are involved in proanthocyanidin synthesis in rice pericarp. Plant J 49(1):91–102. https://doi.org/10.1111/j.1365-313X.2006.02958.x
Gonzalez A, Zhao M, Leavitt JM, Lloyd AM (2008) Regulation of the anthocyanin biosynthetic pathway by the TTG1/bHLH/Myb transcriptional complex in Arabidopsis seedlings. Plant J 53(5):814–827. https://doi.org/10.1111/j.1365-313X.2007.03373.x
Grotewold E, Drummond BJ, Bowen B, Peterson T (1994) The myb-homologous P gene controls phlobaphene pigmentation in maize floral organs by directly activating a flavonoid biosynthetic gene subset. Cell 76(3):543–553. https://doi.org/10.1016/0092-8674(94)90117-1
Guo H, Ling W, Wang Q, Liu C, Hu Y, Xia M, Feng X, Xia X (2007) Effect of anthocyanin-rich extract from black rice (Oryza sativa L. indica) on hyperlipidemia and insulin resistance in fructose-fed rats. Plant Foods Hum Nutr 62(1):1–6. https://doi.org/10.1007/s11130-006-0031-7
Hui C, Bin Y, Xiaoping Y, Long Y, Chunye C, Mantian M, Wenhua L (2010) Anticancer activities of an anthocyanin-rich extract from black rice against breast cancer cells in vitro and in vivo. Nutr Cancer 62(8):1128–1136. https://doi.org/10.1080/01635581.2010.494821
Kim JH, Lee YJ, Kim BG, Lim Y, Ahn JH (2008) Flavanone 3beta-hydroxylases from rice: key enzymes for favonol and anthocyanin biosynthesis. Mol Cells 25(2):312–316
Kranz HD, Denekamp M, Greco R, Jin H, Leyva A, Meissner RC, Petroni K, Urzainqui A, Bevan M, Martin C, Smeekens S, Tonelli C, Paz-Ares J, Weisshaar B (1998) Towards functional characterisation of the members of the R2R3-MYB gene family from Arabidopsis thaliana. Plant J 16(2):263–276. https://doi.org/10.1046/j.1365-313x.1998.00278.x
Kushwaha UKS (2016) Black rice: research, history and development. Springer International Publishing, Berlin
Li G, Zhu Y, Zhang Y, Lang J, Chen Y, Ling W (2013) Estimated daily flavonoid and stilbene intake from fruits, vegetables, and nuts and associations with lipid profiles in Chinese adults. J Acad Nutr Diet 113(6):786–794. https://doi.org/10.1016/j.jand.2013
Li Q, Lu L, Liu H, Bai X, Zhou X, Wu B, Yuan M, Yang L, Xing Y (2020) A minor QTL, SG3, encoding an R2R3-MYB protein, negatively controls grain length in rice. Theor Appl Genet 133(8):2387–2399. https://doi.org/10.1007/s00122-020-03606-z
Lin YJ, Chen H, Cao YL, Wu CY, Wen J, Li YF, Hua HX (2002) Establishment of high-efficiency Agrobacterium-mediated genetic transformation system of Mudanjiang 8. Acta Agron Sin 28(3):294–300
Lin-Wang K, Bolitho K, Grafton K, Kortstee A, Karunairetnam S, McGhie TK, Espley RV, Hellens RP, Allan AC (2010) An R2R3 MYB transcription factor associated with regulation of the anthocyanin biosynthetic pathway in Rosaceae. BMC Plant Biol 10:50–66. https://doi.org/10.1186/1471-2229-10-50
Lu X, Zhou Y, Wu T, Hao L (2014) Ameliorative effect of black rice anthocyanin on senescent mice induced by D-galactose. Food Funct 5(11):2892–2897. https://doi.org/10.1039/c4fo00391h
Ma X, Zhang Q, Zhu Q, Liu W, Chen Y, Qiu R, Wang B, Yang Z, Li H, Lin Y, Xie Y, Shen R, Chen S, Wang Z, Chen Y, Guo J, Chen L, Zhao X, Dong Z, Liu YG (2015) A robust CRISPR/Cas9 system for convenien, t high-efficiency multiplex genome editing in monocot and dicotplants. Mol Plant 8(8):1274–1284. https://doi.org/10.1016/j.molp.2015.04.007
Maeda H, Yamaguchi T, Omoteno M, Takarada T, Fujita K, Murata K, Iyama Y, Kojima Y, Morikawa M, Ozaki H, Mukaino N, Kidani Y, Ebitani T (2014) Genetic dissection of black grain rice by the development of a near isogenic line. Breed Sci 64(2):134–141. https://doi.org/10.1270/jsbbs.64.134
Medina-Puche L, Cumplido-Laso G, Amil-Ruiz F, Hoffmann T, Ring L, Rodriguez-Franco A, Caballero JL, Schwab W, Munoz-Blanco J, Blanco-Portales R (2014) MYB10 plays a major role in the regulation of flavonoid/phenylpropanoid metabolism during ripening of Fragaria × ananassa fruits. J Exp Bot 65(2):401–417. https://doi.org/10.1093/jxb/ert377
Meng L, Qi C, Wang C, Wang S, Zhou C, Ren Y, Cheng Z, Zhang X, Guo X, Zhao Z, Wang J, Lin Q, Zhu S, Wang H, Wang Z, Lei C, Wan J (2021) Determinant factors and regulatory systems for anthocyanin biosynthesis in rice apiculi and stigmas. Rice 14(1):1–18. https://doi.org/10.1186/s12284-021-00480-1
Miyake S, Takahashi N, Sasaki M, Kobayashi S, Tsubota K, Ozawa Y (2011) Vision preservation during retinal inflammation by anthocyanin-rich bilberry extract: cellular and molecular mechanism. Lab Invest 92(1):102–109. https://doi.org/10.1038/labinvest.2011.132
Oikawa T, Maeda H, Oguchi T, Yamaguchi T, Tanabe N, Ebana K, Yano M, Ebitani T, Izawa T (2015) The birth of a black rice gene and its local spread by introgression. Plant Cell 27(9):2401–2414. https://doi.org/10.1105/tpc.15.00310
Peiffer DS, Wang LS, Zimmerman NP, Ransom BW, Carmella SG, Kuo CT, Chen JH, Oshima K, Huang YW, Hecht SS, Stoner GD (2016) Dietary consumption of black raspberries or their anthocyanin constituents alters innate immune cell trafficking in esophageal cancer. Cancer Immunol Res 4(1):72–82. https://doi.org/10.1158/2326-6066.CIR-15-0091
Petroni K, Pilu R, Tonelli C (2014) Anthocyanins in corn: a wealth of genes for human health. Planta 240(5):901–911. https://doi.org/10.1007/s00425-014-2131-1
Qiao W, Wang Y, Xu R, Yang Z, Sun Y, Su L, Zhang L, Wang J, Huang J, Zheng X, Liu S, Tian Y, Chen L, Liu X, Lan J, Yang Q (2021) A functional chromogen gene C from wild rice is involved in a different anthocyanin biosynthesis pathway in indica and japonica. Theor Appl Genet 134:1531–1543. https://doi.org/10.1007/s00122-021-03787-1
Reddy AR, Scheffler B, Madhuri G, Srivastava MN, Kumar A, Sathyanarayanan PV, Nair S, Mohan M (1996) Chalcone synthase in rice (Oryza sativa L.): Detection of the CHS protein in seedlings and molecular mapping of the chs locus. Plant Mol Biol 32(4):735–743. https://doi.org/10.1007/BF00020214
Reddy AM, Reddy VS, Scheffler BE, Wienand U, Reddy AR (2007) Novel transgenic rice overexpressing anthocyanidin synthase accumulates a mixture of flavonoids leading to an increased antioxidant potential. Metab Eng 9(1):95–111. https://doi.org/10.1016/j.ymben.2006.09.003
Shih CH, Chu H, Tang LK, Sakamoto W, Maekawa M, Chu IK, Wang M, Lo C (2008) Functional characterization of key structural genes in rice flavonoid biosynthesis. Planta 228(6):1043–1054. https://doi.org/10.1007/s00425-008-0806-1
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. Plant J 50(4):660–677. https://doi.org/10.1111/j.1365-313X.2007.03078.x
Strathearn KE, Yousef GG, Grace MH, Roy SL, Tambe MA, Ferruzzi MG, Wu QL, Simon JE, Lila MA, Rochet JC (2014) Neuroprotective effects of anthocyanin- and proanthocyanidin-rich extracts in cellular models of Parkinson’s disease. Brain Res 1555:60–77. https://doi.org/10.1016/j.brainres.2014.01.047
Sun X, Zhang Z, Chen C, Wu W, Ren N, Jiang C, Yu J, Zhao Y, Zheng X, Yang Q, Zhang H, Li J, Li Z (2018) The C-S-A gene system regulates hull pigmentation and reveals evolution of anthocyanin biosynthesis pathway in rice. J Exp Bot 69(7):1485–1498. https://doi.org/10.1093/jxb/ery001
Takos AM, Jaffe FW, Jacob SR, Bogs J, Robinson SP, Walker AR (2006) Light-induced expression of a MYB gene regulates anthocyanin biosynthesis in red apples. Plant Physiol 142(3):1216–1232. https://doi.org/10.1104/pp.106.088104
Tanaka Y, Sasaki N, Ohmiya A (2008) Biosynthesis of plant pigments: anthocyanins, betalains and carotenoids. Plant J 54(4):733–749. https://doi.org/10.1111/j.1365-313X.2008.03447.x
Wang W, Mauleon R, Hu Z, Chebotarov D, Tai S, Wu Z, Li M, Zheng T, Fuentes RR, Zhang F et al (2018) Genomic variation in 3,010 diverse accessions of Asian cultivated rice. Nature 557:43–49. https://doi.org/10.1038/s41586-018-0063-9
Xu W, Dubos C, Lepiniec L (2015) Transcriptional control of flavonoid biosynthesis by MYB-bHLH-WDR complexes. Trends Plant Sci 20(3):176–185. https://doi.org/10.1016/j.tplants.2014.12.001
Yang J, Song B, Li Y, Liu J (2006) A simple and efficient method for rna extraction from potato tuber. J Agric Sci 14(2):297–298
Yang X, Wang J, Xia X, Zhang Z, He J, Nong B, Luo T, Feng R, Wu Y, Pan Y, Xiong F, Zeng Y, Chen C, Guo H, Xu Z, Li D, Deng G (2021) OsTTG1, a WD40 repeat gene, regulates anthocyanin biosynthesis in rice. Plant J. https://doi.org/10.1111/tpj.15285
Yao SL, Xu Y, Zhang YY, Lu YH (2013) Black rice and anthocyanins induce inhibition of cholesterol absorption in vitro. Food Funct 4(11):1602–1608. https://doi.org/10.1039/c3fo60196j
Zhang Q (2021) Purple tomatoes, black rice and food security. Nat Rev Genet 22(7):414. https://doi.org/10.1038/s41576-021-00359-3
Zhang Z, Shi Y, Ma Y, Yang X, Yin X, Zhang Y, Xiao Y, Liu W, Li Y, Li S, Liu X, Grierson D, Allan AC, Jiang G, Chen K (2020) The strawberry transcription factor FaRAV1 positively regulates anthocyanin accumulation by activation of FaMYB10 and anthocyanin pathway genes. Plant Biotechnol J 18(11):2267–2279. https://doi.org/10.1111/pbi.13382
Zhao H, Yao W, Ouyang Y, Yang W, Wang G, Lian X, Xing Y, Chen L, Xie W (2015) RiceVarMap: a comprehensive database of rice genomic variations. Nucleic Acids Res 43(D1):D1018–D1022. https://doi.org/10.1093/nar/gku894
Zheng J, Wu H, Zhu H, Huang C, Liu C, Chang Y, Kong Z, Zhou Z, Wang G, Lin Y, Chen H (2019) Determining factors, regulation system, and domestication of anthocyanin biosynthesis in rice leaves. New Phytol 223(2):705–721. https://doi.org/10.1111/nph.15807
Zhu F, Cai Y-Z, Bao J, Corke H (2010) Effect of γ-irradiation on phenolic compounds in rice grain. Food Chem 120(1):74–77. https://doi.org/10.1016/j.foodchem.2009.09.072
Zong W, Tang N, Yang J, Peng L, Ma S, Xu Y, Li G, Xiong L (2016) Feedback regulation of ABA signaling and biosynthesis by a bZIP transcription factor targets drought resistance related genes. Plant Physiol 171(4):2810–2825. https://doi.org/10.1104/pp.16.00469
Funding
This research was supported by National Natural Science Foundation of China (No. 32000233), Youth Innovation Talents Project of Department of Education of Guangdong Province (No. 2019KQNCX138), and the open funds of the National Key Laboratory of Crop Genetic Improvement (No. ZK201913).
Author information
Authors and Affiliations
Contributions
Jie Zheng performed all experiments, generated the figures and tables, wrote the original draft; Hao Wu performed all bioinformatics analyses and generated figures and tables; Mingchao Zhao performed the anthocyanin content measurement, PCR, and qRT-PCR; Zenan Yang performed rice pericarp sampling and RNA extraction; Zaihui Zhou performed rice transformation and grew the rice plants; Yongmei Guo collected rice germplasms; Yongjun Lin designed the experiments; and Hao Chen designed the experiments, reviewed, and edited the paper.
Corresponding author
Ethics declarations
Ethics approval
Not applicable.
Consent to participate.
Not applicable.
Consent for publication.
Not applicable.
Competing interests
The authors declare no competing interests.
Additional declarations for articles in life science journals that report the results of studies involving humans and/or animals.
Not applicable.
Additional information
Publisher's Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Supplementary Information
Below is the link to the electronic supplementary material.
Rights and permissions
About this article
Cite this article
Zheng, J., Wu, H., Zhao, M. et al. OsMYB3 is a R2R3-MYB gene responsible for anthocyanin biosynthesis in black rice. Mol Breeding 41, 51 (2021). https://doi.org/10.1007/s11032-021-01244-x
Received:
Accepted:
Published:
DOI: https://doi.org/10.1007/s11032-021-01244-x