Identification and Spatiotemporal Expression Analysis of R2R3-MYB Transcription Factor in Crocus sativus
WU Rui1,2, LIN Ding1,2, LUO Dong1,2, ZHOU Yi-Ru1,2, ZHANG Ling-Yi1,2, SHAO Qing-Song1,2, XING Bing-Cong1,2,*
1 Zhejiang Provincial Key Laboratory of Traditional Chinese Medicine Resources Protection and Innovative Utilization, Hangzhou 311300, China; 2 College of Food and Health, Zhejiang Agriculture and Forestry University, Hangzhou 311300, China
Abstract：R2R3-MYB transcription factor is one of the largest transcription factor family in plants, and it is widely involved in the regulation of plant secondary metabolism. Based on the sequencing data of the full-length transcriptome of Crocus sativus, explored the temporal and spatial expression characteristics of R2R3-MYB transcription factors and screend the CsR2R3-MYB transcription factors related to the secondary metabolism of C. sativus, bioinformatics methods were used to identify and systematically analyzed the CsR2R3-MYB transcription factor, and the temporal and spatial expression pattern of the CsR2R3-MYB transcription factor was explored by qRT-PCR. Totally 42 R2R3-MYB transcription factors were screened from the C. sativus transcriptome. Physical and chemical properties analysis of the proteins showed that the CsR2R3-MYB proteins were all unstable hydrophilic proteins. Subcellular localization prediction showed that most of the CsR2R3-MYB were present in the nucleus. The phylogenetic tree was constructed together with Arabidopsis thaliana R2R3-MYB, and the results showed that the CsR2R3-MYB transcription factors could be divided into 26 subclasses. According to the function conserved properties of homologous proteins, CsMYB9, CsMYB16, CsMYB17, CsMYB18, CsMYB20, CsMYB21, which were homologous to the S7, S19, and S20 subclasses, were predicted to participate in the secondary metabolism regulation of active substances in C. sativus by responding to jasmonic acid (JA) signals. The results of temporal and spatial expression analysis showed that CsMYB5, CsMYB6, CsMYB7, CsMYB9, CsMYB10, CsMYB13, CsMYB17, CsMYB19, CsMYB21, CsMYB22, CsMYB23, CsMYB26, CsMYB28, CsMYB30, CsMYB31 and CsMYB32 showed relatively high expression levels in the stigma tissues and responds to JA signals. The previous studies of this research group showed that JA could promote flavonoids biosynthesis. According to the prediction results of the phylogenetic tree and the temporal and spatial expression characteristics, screened CsMYB9, CsMYB17, and CsMYB21 regulate the secondary metabolism of C. sativus, by responding to JA signals. This research provides a reference for the further exploration of the functions of CsR2R3-MYB. And it also provides ideas for the study of transcription regulation of C. sativus secondary metabolism.
[1] 樊宝莲, 王晓云. 2021. 转录因子调控植物类胡萝卜素合成途径的研究进展[J]. 分子植物育种, 19(13): 4401-4408. (Fan B L, Wang X Y.2021. Research progress of transcription factors regulating carotenoid synthesis pathway in plant[J]. Molecular Plant Breeding, 19(13): 4401-4408.) [2] 李戌彦, 杨忠义, 纪薇, 等. 2021. 基于不同颜色山楂花MYB家族的挖掘与比较分析[J]. 核农学报, 35(01): 49-59. (Li X Y, Yang Z Y, Ji W, et al.2021. Excavation and comparative analysis of MYB family based on hawthorn flowers with different colors[J]. Journal of Nuclear Agricultural Sciences, 35(01): 49-59.) [3] 刘江弟, 欧阳臻, 杨滨. 2017. 西红花品质评价研究进展[J]. 中国中药杂志, 42(03): 405-412. (Liu J D, Ouyang Z, Yang B.2017. Research progress on quality evaluation of saffron[J]. China Journal of Chinese Materia Medica, 42(03): 405-412.) [4] 刘生财, 潘君飞, 王晓, 等. 2019. MeJA对苋菜悬浮细胞类黄酮和类胡萝卜素累积及其代谢相关基因表达的影响[J]. 应用与环境生物学报, 25(05): 1168-1175. (Liu S C, Pan J F, Wang X, et al.2019. Effects of methyl jasmonate on the contents and related metabolic genes of flavonoids and carotenoids in suspension cells of Amaranthus tricolor L.[J]. Chinese Journal of Applied & Environmental Biology, 25(05): 1168-1175.) [5] 马杰, 胡文忠, 毕阳, 等. 2013. 茉莉酸甲酯处理对鲜切莴苣和甘蓝苯丙烷代谢的影响[J]. 食品工业科技, 34(07): 333-339. (Ma J, Hu W Z, Bi Y, et al.2013. Effect of MeJA treatments on benzene propane metabolism in tissues of fresh-cut lettuce and cabbage[J]. Science and Technology of Food Industry, 34(07): 333-339.) [6] 苏文炳, 蒋园园, 白昀鹭, 等. 2019. 转录因子调控植物萜类化合物生物合成研究进展[J]. 农业生物技术学报, 27(05): 919-926. (Su W B, Jiang Y Y, Bai Y L, et al.2019. Advances in transcription factors regulation on plant terpene biosynthesis[J]. Journal of Agricultural Biotechnology, 27(05): 919-926.) [7] 孙镇, 袁丽红, 吴频梅. 2013. 诱导子对藏红花悬浮培养细胞生产藏红花色素的影响[J]. 生物加工过程, 11(03): 18-23. (Sun Z, Yuan L H, Wu P M.2013. Effects of elicitors on saffron pigment production in cell suspension cultures of Crocus sativus L.[J]. Chinese Journal of Bioprocess Engineering, 11(03): 18-23. [8] 王平, 童应鹏, 陶露霞, 等. 2014. 西红花的化学成分和药理活性研究进展[J]. 中草药, 45(20): 3015-3028. (Wang P, Tong Y P, Tao L X, et al.2014. Research progress on chemical constituents of Crocus sativus and their pharmacological activities[J]. Chinese Traditional and Herbal Drugs, 45(20): 3015-3028.) [9] 张驰, 王艳芳, 陈静, 等. 2020. 植物MYB转录因子调控次生代谢的研究进展[J]. 基因组学与应用生物学, 39(09): 4171-4177. (Zhang C, Wang Y F, Chen J, et al.2020. Research advances on the regulation of secondary metabolism by plant MYB transcription factors[J]. Genomics and Applied Biology Instructions for Manuscript Preparation, 39(09): 4171-4177.) [10] Agarwal P, Mitra M, Banerjee S, et al.2020. MYB4 transcription factor, a member of R2R3-subfamily of MYB domain protein, regulates cadmium tolerance via enhanced protection against oxidative damage and increases expression of PCS1 and MT1C in Arabidopsis[J]. Plant Science, 297: 110501. [11] Albert N W, Thrimawithana A H, McGhie T K, et al.2018. Genetic analysis of the liverwort Marchantia polymorpha reveals that R2R3MYB activation of flavonoid production in response to abiotic stress is an ancient character in land plants[J]. New Phytologist, 218(2): 554-566. [12] Anwar M, Wang G Q, Wu J C, et al.2018. Ectopic overexpression of a novel R2R3-MYB, NtMYB2 from Chinese Narcissus represses anthocyanin biosynthesis in tobacco[J]. Molecules, 23(4): 781. [13] Bailey T L, Elkan C.1994. Fitting a mixture model by expectation maximization to discover motifs in biopolymers[J]. Proceedings of the Second International Conference on Intelligent Systems for Molecular Biology, 2: 28-36. [14] Bedon F, Bomal C, Caron S, et al.2010. Subgroup 4 R2R3-MYBs in conifer trees: Gene family expansion and contribution to the isoprenoid- and flavonoid-oriented responses[J]. Journal of Experimental Botany, 61(14): 3847-3864. [15] Cardone L, Castronuovo D, Perniola M, et al.2020. Saffron (Crocus sativus L.), the king of spices: An overview[J]. Scientia Horticulturae, 272: 109560. [16] Cheng H, Song S, Xiao T, et al.2009. Gibberellin acts through jasmonate to control the expression of MYB21, MYB24, and MYB57 to promote stamen filament growth in Arabidopsis[J]. PLOS Genetics, 5(3), e1000440. [17] Deléage G.2017. Alignsec: Viewing protein secondary structure predictions within large multiple sequence alignments[J]. Bioinformatics (Oxford England), 33(24): 3991-3992. [18] Deng M W, Li D, Zhang Y C, et al.2018. Protective effect of crocin on ultraviolet B-induced dermal fibroblast photoaging[J]. Molecular Medicine Reports, 18(2): 1439-1446. [19] Du M, Zhao J, Tzeng D T W, et al.2017. MYC2 orchestrates a hierarchical transcriptional cascade that regulates jasmonate-mediated plant immunity in tomato[J]. Plant Cell, 29(8): 1883-1906. [20] Dubos C, Stracke R, Grotewold E, et al.2010. MYB transcription factors in Arabidopsis[J]. Trends in Plant Science, 15(10): 573-581. [21] Gezici S.2019. Comparative anticancer activity analysis of saffron extracts and a principle component, crocetin for prevention and treatment of human malignancies[J]. Journal of Food Science and Technology, 56(12): 5435-5443. [22] Haga N, Kato K, Murase M, Araki S, et al.2007. R1R2R3 myb proteins positively regulate cytokinesis through activation of KNOLLE transcription in Arabidopsis thaliana[J]. Development, 134(6): 1101-1110. [23] Han Y, Mhamdi A, Chaouch S, et al.2013. Regulation of basal and oxidative stress-triggered jasmonic acid related gene expression by glutathione[J]. Plant, Cell & Environment, 36(6): 1135-1146. [24] Huo D, Liu X K, Zhang Y, et al.2020. A novel R2R3-MYB transcription factor PqMYB4 inhibited anthocyanin biosynthesis in Paeonia qiui[J]. International Journal of Molecular Sciences, 21(16): 5878. [25] Jain M, Srivastava P L, Verma M, et al.2016. De novo transcriptome assembly and comprehensive expression profiling in Crocus sativus to gain insights into apocarotenoid biosynthesis[J]. Scientific Reports, 6: 22456. [26] Jung C, Seo J S, Han S W, et al.2008. Overexpression of AtMYB44 enhances stomatal closure to confer abiotic stress tolerance in transgenic Arabidopsis[J]. Plant Physiology, 146(2): 623-635. [27] Kumar S, Stecher G, Li M, et al.2018. MEGA X: Molecular evolutionary genetics analysis across computing platforms[J]. Molecular Biology and Evolution, 35(6): 1547-1549. [28] Lai L B, Nadeau J A, Lucas J, et al.2005. The Arabidopsis R2R3 MYB proteins FOUR LIPS and MYB88 restrict divisions late in the stomatal cell lineage[J]. Plant Cell, 17(10): 2754-2767. [29] Li Y L, Chen X L, Wang J Q, et al.2020. Two responses to MeJA induction of R2R3-MYB transcription factors regulate flavonoid accumulation in Glycyrrhiza uralensis Fisch[J]. PLOS ONE, 15(7): e0236565. [30] Lin W K, Bolitho K, Grafton K, et al.2010. An R2R3 MYB transcription factor associated with regulation of the anthocyanin biosynthetic pathway in Rosaceae[J]. BMC Plant Biology, 10: 50. [31] Luo Q, Liu R, Zeng L, et al.2020. Isolation and molecular characterization of NtMYB4a, a putative transcription activation factor involved in anthocyanin synthesis in tobacco[J]. Gene, 760: 144990. [32] Mahjoub A, Hernould M, Joubès J, et al.2009. Over expression of a grapevine R2R3-MYB factor in tomato affects vegetative development, flower morphology and flavonoid and terpenoid metabolism[J]. Plant Physiology and Biochemistry, 47(7): 551-561. [33] Mandaokar A, Browse J.2009. MYB108 acts together with MYB24 to regulate jasmonate-mediated stamen maturation in Arabidopsis[J]. Plant Physiol, 149(2), 851-862. [34] Mandaokar A, Thines B, Shin B, et al.2006. Transcriptional regulators of stamen development in Arabidopsis identified by transcriptional profiling[J]. The Plant Journal. 46(6):984-1008. [35] Meng X, Yang D, Li X, et al.2015. Physiological changes in fruit ripening caused by overexpression of tomato SlAN2, an R2R3-MYB factor[J]. Plant Physiology and Biochemistry, 89: 24-30. [36] Millar A A, Gubler F.2005. The Arabidopsis GAMYB-like genes, MYB33 and MYB65, are microRNA-regulated genes that redundantly facilitate anther development[J]. The Plant Cell, 17(3): 705-721. [37] Nguyen N H, Lee H.2016. MYB-related transcription factors function as regulators of the circadian clock and anthocyanin biosynthesis in Arabidopsis[J]. Plant Signaling & Behavior, 11(3): e1139278. [38] Pei T, Ma P, Ding K, et al.2018. SmJAZ8 acts as a core repressor regulating JA-induced biosynthesis of salvianolic acids and tanshinones in Salvia miltiorrhiza hairy roots[J]. Journal of Experimental Botany, 69(7): 1663-1678. [39] Rajaei Z, Hadjzadeh M A, Nemati H, et al.2013. Antihyperglycemic and antioxidant activity of crocin in streptozotocin-induced diabetic rats[J]. Journal of Medicinal Food, 16(3): 206-210. [40] Reddy V A, Wang Q, Dhar N, et al.2017. Spearmint R2R3-MYB transcription factor MsMYB negatively regulates monoterpene production and suppresses the expression of geranyl diphosphate synthase large subunit (MsGPPS.LSU)[J]. Plant Biotechnology Journal, 15(9): 1105-1119. [41] Shi M, Wang Y, Wang X R, et al.2020. Simultaneous promotion of tanshinone and phenolic acid biosynthesis in Salvia miltiorrhiza hairy roots by overexpressing Arabidopsis MYC2[J]. Industrial Crops and Products, 155: 112826. [42] Song Y, Yang W, Fan H, et al.2020. TaMYB86B encodes a R2R3-type MYB transcription factor and enhances salt tolerance in wheat[J]. Plant Science, 300: 110624. [43] Stracke R, Ishihara H, Huep G, et al.2007. Differential regulation of closely related R2R3-MYB transcription factors controls flavonol accumulation in different parts of the Arabidopsis thaliana seedling[J]. The Plant Journal, 50(4): 660-677. [44] Stracke R, Jahns O, Keck M, et al.2010. Analysis of production of flavonol glycosides-dependent flavonol glycoside accumulation in Arabidopsis thaliana plants reveals MYB11-, MYB12- and MYB111-independent flavonol glycoside accumulation[J]. New Phytologist, 188(4): 985-1000. [45] Stracke R, Werber M, Weisshaar B.2001. The R2R3-MYB gene family in Arabidopsis thaliana[J]. Current Opinion in Plant Biology, 4(5): 447-456. [46] Szklarczyk D, Gable A L, Nastou K C, et al.2021. The STRING database in 2021: Customizable protein-protein networks, and functional characterization of user-uploaded gene/measurement sets[J]. Nucleic Acids Research, 49(D1): D605-D612. [47] Tian J, Zhang J, Han Z Y, et al.2017. McMYB12 transcription factors co-regulate proanthocyanidin and anthocyanin biosynthesis in Malus crabapple[J]. Scientific Reports, 7: 43715. [48] Tiwari P, Indoliya Y, Chauhan A S, et al.2020. Over-expression of rice R1-type MYB transcription factor confers different abiotic stress tolerance in transgenic Arabidopsis[J]. Ecotoxicology and Environmental Safety, 206: 111361. [49] Wang L, Huang Q, Zhang L, et al.2020. Genome-wide characterization and comparative analysis of MYB transcription factors in Ganoderma species[J]. G3 (Bethesda, Md.), 10(8): 2653-2660. [50] Waterhouse A, Bertoni M, Bienert S, et al.2018. SWISS-MODEL: Homology modelling of protein structures and complexes[J]. Nucleic Acids Research, 46(W1): W296-W303. [51] Weaver T M, Liu J, Connelly K E, et al.2019. The EZH2 SANT1 domain is a histone reader providing sensitivity to the modification state of the H4 tail[J]. Scientific Reports, 9(1): 987. [52] Wilkins M R, Gasteiger E, Bairoch A, et al.1999. Protein identification and analysis tools in the ExPASy server[J]. Methods in Molecular Biology (Clifton, N.J.), 112: 531-552. [53] Wong K H, Xie Y, Huang X, et al.2020. Delivering crocetin across the blood-brain barrier by using γ-cyclodextrin to treat alzheimer's disease[J]. Scientific Reports, 10(1): 3654. [54] Xiao Q, Xiong Z, Yu C, et al.2019. Antidepressant activity of crocin-I is associated with amelioration of neuroinflammation and attenuates oxidative damage induced by corticosterone in mice[J]. Physiology & Behavior, 212: 112699. [55] Xiong E H, Zheng C Y, Wu X L, et al.2016. Protein subcellular location: The gap between prediction and experimentation[J]. Plant Molecular Biology Reporter, 34(1): 52-61. [56] Yao L, Wang J, Sun J, et al.2020. A WRKY transcription factor, PgWRKY4X, positively regulates ginsenoside biosynthesis by activating squalene epoxidase transcription in Panax ginseng[J]. Industrial Crops and Products, 154: 112671. [57] Zhao K, Cheng Z H, Guo Q, et al.2020. Characterization of the poplar R2R3-MYB gene family and over-expression of PsnMYB108 confers salt tolerance in transgenic tobacco[J]. Frontiers in Plant Science, 11: 571881. [58] Zheng X W, Liu C, Qiao L, et al.2020. The MYB transcription factor TaPHR3-A1 is involved in phosphate signaling and governs yield-related traits in bread wheat (Triticum aestivum L.)[J]. Journal of Experimental Botany, 71(19): 5808-5822.
