Screening and Identification of Upstream Transcription Factors of OfLCYB Gene from Osmanthus fragrans
XU Meng-Han1,*, QING Hong-Sheng2,*, QIAN Jie-Yu1, ZHANG Chao1,**, FU Jian-Xin1,**
1 College of Landscape Architecture/Zhejiang Provincial Key Laboratory of Germplasm Innovation and Utilization for Garden Plants, Zhejiang A&F University, Hangzhou 311300, China; 2 National Key Laboratory for Development and Utilization of Forest Food Resources, Zhejiang A&F University, Hangzhou 311300, China;
Abstract：Carotenoids are important pigments that determine the coloration of Osmanthus fragrans petals, and lycopene β-cyclase (LCYB) is a key enzyme in the carotenoid synthesis pathway of O. fragrans. In this study, O. fragrans cultivar 'Yanhong Gui' was used as material, the bait vector of OfLCYB promoter and the yeast one-hybrid cDNA library of O. fragrans petals were constructed. The upstream transcription factor OfMYB308 interacting with OfLCYB promoter was screened by yeast one-hybrid. OfMYB308 had a typical EAR repressor motif and belonged to S4 subgroup of R2R3-MYB transcription factors. The expression level of OfLCYB was positively correlated with that of OfMYB308 gene, and both of them increased with flower opening. The results of yeast one-hybrid point-to-point verification and dual luciferase assay showed that OfMYB308 could bind to the OfLCYB promoter and significantly inhibit its transcriptional activity. This study provides theoretical basis for revealing the regulatory network of carotenoid synthesis in O. fragrans.
[1] 李莉, 庞天虹, 付建新, 等. 2025. 桂花番茄红素β-环化酶基因LCYB上游B2亚组ERF转录因子的筛选和鉴定[J]. 浙江农林大学学报, 42(1): 86-93. (Li L, Pang T H, Fu J X, et al.2025. Screening and identification of ERF transcription factors of B2 subgroup involved in regulating lycopene β-cyclase gene LCYB in Osmanthus fragrans[J]. Journal of Zhejiang A&F University, 42(1): 86-93.) [2] 李杰, 罗江宏, 杨萍. 2021. 沉默辣椒Lcyb和BZR1基因对其叶片类胡萝卜素积累的影响[J]. 农业生物技术学报, 29(01): 47-57. (Li J, Luo J H, Yang P.2021. Effects of Silencing Lcyb and BZR1 genes on carotenoid accumulation in leaves of pepper (Capsicum annuum)[J]. Journal of Agricultural Biotechnology, 29(01): 47-57.) [3] 彭琳, 王艺光, 董彬, 等. 2025. 桂花OfLCYB提高转基因烟草类胡萝卜素含量和低温胁迫的抗性[J]. 农业生物技术学报, 33(1): 1-14. (Peng L, Wang Y G, Dong B, et al.2025. Osmanthus fragrans OfLCYB increased carotenoid content and resistance to low temperature stress in transgenic tobacco (Nicotiana tabacum)[J]. Journal of Agricultural Biotechnology, 33(1): 1-14.) [4] 沈子又, 张超, 董彬, 等. 2018. 桂花OfLCYB和OfLCYE启动子的克隆和活性分析[J]. 生物技术通报, 34(1): 137-143. (Shen Z Y, Zhang C, Dong B, et al.2018. Cloning and expression analysis of the promoters of OfLCYB and OfLCYE in Osmanthus fragrans[J]. Biotechnology Bulletin, 34(1): 137-143.) [5] 向其柏, 刘玉莲. 2008. 中国桂花品种图志[M]. 杭州: 浙江科学技术出版社, pp. 86-88. (Xiang Q B, Liu Y L.2008. An illustrated monograph of the sweet osmanthus variety in China[M]. Zhejiang Science & Technology Press, Hangzhou, China, pp. 86-88.). [6] Britton G.2020. Carotenoid research: History and new perspectives for chemistry in biological systems[J]. Biochimica Et Biophysica Acta-Molecular and Cell Biology of Lipids, 1865(11): 158699. [7] Chen L H, Hu B, Qin Y H, et al.2019. Advance of the negative regulation of anthocyanin biosynthesis by MYB transcription factors[J]. Plant Physiology and Biochemistry, 136: 178-187. [8] Cunningham F X, Gantt E.2001. One ring or two? Determination of ring number in carotenoids by lycopene epsilon-cyclases[J]. Proceedings of the National Academy of Sciences of the USA, 98(5): 2905-2910. [9] He G R, Zhang R, Jiang S H, et al.2023. The MYB transcription factor RcMYB1 plays a central role in rose anthocyanin biosynthesis[J]. Horticulture Research, 10(6): uhad080. [10] Hermanns A S, Zhou X S, Xu Q, et al.2020. Carotenoid pigment accumulation in horticultural plants[J]. Horticultural Plant Journal, 6(6): 343-360. [11] Hu X M, Liang Z H, Sun T X, et al.2024. The R2R3-MYB transcriptional repressor TgMYB4 negatively regulates anthocyanin biosynthesis in tulips (Tulipa gesneriana L.)[J]. International Journal of Molecular Sciences, 25: 563. [12] Huang Y, Makkumrai W, Fu J L, et al.2025. Genomic analysis provides insights into the origin and divergence of fruit flavor and flesh color of pummelo[J]. New Phytologist, 245: 378-391. [13] Jiang L, Chen J, Qian J, et al.2024. The R2R3-MYB transcription factor ZeMYB32 negatively regulates anthocyanin biosynthesis in Zinnia elegans[J]. Plant Molecular Biology, 114:48. [14] Jin H L, Cominelli E, Bailey P, et al.2000. Transcriptional repression by AtMYB4 controls production of UV-protecting sunscreens in Arabidopsis[J]. The EMBO Journal, 10: 6150-6161. [15] Kishimoto S, Maoka T, Nakayama M, et al.2004. Carotenoid composition in petals of chrysanthemum (Dendranthema grandiflorum (Ramat.) Kitamura)[J]. Phytochemistry, 65(20): 2781-2787. [16] Li R H, Zeng Q Y, Zhang X X, et al.2023. Xanthophyll esterases in association with fibrillins control the stable storage of carotenoids in yellow flowers of rapeseed (Brassica juncea)[J]. New Phytologist, 240(1): 285-301. [17] Lu S, Li L.2008. Carotenoid metabolism: biosynthesis, regulation, and beyond[J]. Journal of Integrative Plant Biology, 50(7): 778-785. [18] Moreno J C, Pizarro L, Fuentes P, et al.2013. Levels of lycopene β-cyclase 1 modulate carotenoid gene expression and accumulation in Daucus carota[J]. PLOS One, 8(3): e58144. [19] Qing H S, Chen J H, Jiang L L, et al.2022. Functional characterization of two lycopene cyclases from sweet osmanthus (Osmanthus fragrans)[J]. Scientia Horticulturae, 299: 111062. [20] Rosas-Saavedra C, Quiroz L F, Parra S, et al.2023. Putative Daucus carota capsanthin-capsorubin synthase (DcCCS) possesses lycopene β-cyclase activity, boosts carotenoid levels, and increases salt tolerance in heterologous plants[J]. Plants-Basel, 12(15): 2788. [21] Sun T H, Rao S M, Zhou X S, et al.2022. Plant carotenoids: Recent advances and future perspectives[J]. Molecular Horticulture, 2(1): 3. [22] Sun W J, Gao Z Y, Wang J, et al.2019. Cotton fiber elongation requires the transcription factor GhMYB212 to regulate sucrose transportation into expanding fibers[J]. New Phytologist, 222: 864-881. [23] Tanaka Y, Sasaki N, Ohmiya A2008. Biosynthesis of plant pigments: Anthocyanins, betalains and carotenoids[J]. The Plant Journal, 54(4): 733-749. [24] Wan S Z, Li C F, Ma X D, et al.2017. PtrMYB57 contributes to the negative regulation of anthocyanin and proanthocyanidin biosynthesis in poplar[J]. Plant Cell Report, 36(8): 1263-1276. [25] Wang Y G, Zhang C, Dong B, et al.2018. Carotenoid accumulation and its contribution to flower coloration of Osmanthus fragrans[J]. Frontiers in Plant Science, 9: 1499. [26] Xu H F, Wang N, Liu J X, et al.2017. The molecular mechanism underlying anthocyanin metabolism in apple using the MdMYB16 and MdbHLH33 genes[J]. Plant Molecular Biology, 94: 149-165. [27] Yan J W, Zeng H, Chen W J, et al.2023a. New insights into the carotenoid biosynthesis in Torreya grandis kernels[J]. Horticultural Plant Journal, 9(6): 1108-1118. [28] Yan Y H, Zhao J X, Lin S N, et al.2023b. Light-mediated anthocyanin biosynthesis in rose petals involves a balanced regulatory module comprising transcription factors RhHY5, RhMYB114a, and RhMYB3b[J]. Journal of Experimental Botany, 74: 5783-5804. [29] Yang L C, Liu H H, Xu J Y, et al.2024. LtMYB305 transcription factor regulates the expression of LtLCYB gene to mediate carotenoids synthesis in Liriodendron petals[J]. Industrial Crops and Products, 217: 118868. [30] Yu H, Cui N, Guo K, et al.2023. Epigenetic changes in the regulation of carotenoid metabolism during honeysuckle flower development[J]. Horticultural Plant Journal, 9(3): 577-588. [31] Zhang C, Wang Y G, Fu J X, et al.2016. Transcriptomic analysis and carotenogenic gene expression related to petal coloration in Osmanthus fragrans 'Yanhong Gui'[J]. Trees-Structure and Function, 30(4): 1207-1223. [32] Zhou L J, Liu S H, Wang Y G, et al.2024. CmMYB3-like negatively regulates anthocyanin biosynthesis and flower color formation during the post-flowering stage in Chrysanthemum morifolium[J]. Horticultural Plant Journal, 10: 194-204.
