Cloning and Functional Analysis of LiMYB4 in Regulating Terpenoid Biosynthesis in Lilium‘Siberia’

doi:10.16420/j.issn.0513-353x.2025-0946

Acta Horticulturae Sinica ›› 2026, Vol. 53 ›› Issue (2): 525-537.doi: 10.16420/j.issn.0513-353x.2025-0946

• Research Papers • Previous Articles Next Articles

Cloning and Functional Analysis of LiMYB4 in Regulating Terpenoid Biosynthesis in Lilium‘Siberia’

SHEN Yan, XIA Ziyi, LENG Pingsheng, MA Bo(), HU Zenghui()

Beijing Laboratory for Urban and Rural Ecological Environment，Key Laboratory of Ancient and Famous Trees Protection，National Forestry and Grassland Administration，College of Landscape Architecture，Beijing University of Agriculture，Beijing 102206，China

Received:2025-11-25 Revised:2026-01-12 Online:2026-02-25 Published:2026-02-12

Abstract

Abstract:

Terpenoids constitute the primary volatile components of lily floral scent，and MYB transcription factors are crucial regulators of terpenoid biosynthesis. In this study，Lilium‘Siberia’was used as the experimental material for cloning and characterizing LiMYB4，including analysis of spatiotemporal expression patterns，protein sequence homology，and phylogenetic relationships. In addition，transient silencing and overexpression assays were employed in the petals to investigate the role of LiMYB4 in terpenoid biosynthesis. The results showed that LiMYB4 contains a 738 bp ORF encoding 245 amino acids. The predicted LiMYB4 possesses two conserved MYB domain repeats and was thus classified as a member of the R2R3-MYB subfamily. The phylogenetic analysis showed the highest similarity（71.26%）with AeMYB4 from Amana edulis. The spatial expression profiling demonstrated significantly higher transcript level of LiMYB4 in perianths compared with roots，stems，or leaves. During floral development LiMYB4 expression first decreased and then increased，which was inversely correlated with terpenoid emission dynamics. The functional validation showed that transient silencing of LiMYB4 increased the emission of linalool and ocimene in petals. Conversely，LiMYB4 overexpression markedly reduced the emission of these volatiles. Further analysis of the terpene backbone biosynthesis genes revealed that the transcript levels of LiDXR，LiGPPS，LiOcS，and LiLiS were significantly upregulated in LiMYB4-silenced petals，but downregulated in LiMYB4-overexpressing petals. Collectively，these results demonstrate that LiMYB4 acts as a negative regulator of terpenoid biosynthesis in Lilium‘Siberia’by directly or indirectly suppressing the expression of key genes in the terpenoid biosynthesis pathway.

Key words: Lilium, floral scent, terpenoids, LiMYB4, function

SHEN Yan, XIA Ziyi, LENG Pingsheng, MA Bo, HU Zenghui. Cloning and Functional Analysis of LiMYB4 in Regulating Terpenoid Biosynthesis in Lilium‘Siberia’[J]. Acta Horticulturae Sinica, 2026, 53(2): 525-537.

Figures/Tables 10

Fig. 1 Different floral development stages（A），organs and tissues（B）in full flowering stage of Lilium‘Siberia’

Table 1 Primers used in this study

引物用途 Primer function	引物名称 Primer name	序列（5′-3′） Primer sequence
基因克隆 Gene clone	LiMYB4-QCF	ATGGTGAGAGCTCCTTGCTGTGA
基因克隆 Gene clone	LiMYB4-QCR	TCAGATTGTGGGCAAGTCCTGCA
亚细胞定位 Subcellular localization	LiMYB4-F	CAGTGGTCTCACAACATGGTGAGAGCTCCTTGCTG
亚细胞定位 Subcellular localization	LiMYB4-R	CAGTGGTCTCATACAGATTGTGGGCAAGTCCTGCA
实时荧光定量PCR qRT-PCR	qLiMYB4-F	AAATCTGAAGCACCCCACATT
实时荧光定量PCR qRT-PCR	qLiMYB4-R	CATCCACCATGCGAGTATGAT
基因沉默 Gene silencing	pTRV2-LiMYB4-EcoR Ⅰ-F	TTAGATTCTGTGAGTAAGGTTACCGATGGTGAGAGCTCCTTGCTG
基因沉默 Gene silencing	pTRV2-LiMYB4-Xho Ⅰ-R	AATGTCTTCGGGACATGCCCGGGCCACTATAGATGATTCGGTGGCCG
基因过表达 Gene overexpression	35S-LiMYB4-Sfi I-F	GTCCAGTCCTGGCCTCGTCGGCCATGGTGAGAGCTCCTTGCTG
基因过表达 Gene overexpression	35S-LiMYB4-Sfi I-R	GACCACAAGTGGCCAGACTGGCCGATTGTGGGCAAGTCCTGCAG

Fig. 2 Cloning of LiMYB4 gene A：Agarose gel electrophoresis detection of total RNA in Lilium‘Siberia’；M1：Trans2K Plus DNA marker；B：Agarose gel electrophoresis of LiMYB4 gene；M2：DL2000 DNA marker

Fig. 3 Homology alignment analysis of LiMYB4 with MYB4 from other plants Lr：Lilium regale；Ae：Amana edulis；Cn：Cocos nucifera；Tl：Typha latifolia. The blue，yellow，and green parts represent homology = 100%，≥ 75%，≥ 50%，respectively. Black horizontal lines mark two MYB conserved domains

Fig. 4 Phylogenetic tree of LiMYB4 and MYB4 from other species

Fig. 5 The expression patterns of LiMYB4 at different floral developmental stages（A）and in different organs and tissues（B）of Lilium‘Siberia’ Different lowercase letters indicate significant differences at the P < 0.05 level

Fig. 6 Subcellular localization of LiMYB4 A：Arabidopsis protoplast，with red fluorescence representing chloroplast autofluorescence；B：Nicotiana benthamiana leaf，with red fluorescence indicating nuclear marker

Fig. 7 Effect of silencing LiMYB4 on terpenoid emission in Lilium‘Siberia’ A：Gene silencing efficiency；B：The release amount of the two main monoterpenoids；C：Representative total ion chromatograms of the floral scent compounds from TRV and TRV-LiMYB4. t-test，* P < 0.05，****P < 0.0001

Fig. 8 Effect of overexpression LiMYB4 on terpenoid emission in Lilium‘Siberia’ A：Gene overexpression efficiency；B：The release amount of the two main monoterpenoids；C：Representative total ion chromatograms of the floral scent compounds from 35S and 35S-LiMYB4. t-test，**P < 0.01，****P < 0.0001

Fig. 9 Effects of transient silencing（A）and overexpression（B）of LiMYB4 on the expression of terpenoid biosynthesis pathway genes LiDXR，LiGPPS，LiOcS，and LiLiS t-test，*P < 0.05，**P < 0.01，****P < 0.0001

References 24

[1]	Chen Q, Wang M, Gao L, Lou Q, Gan Y, Li X, Li Y, Xin T, Xu W, Song J. 2025. A pivotal switch in β-domain determines the substrate selectivity of terpene synthases involved in Gardenia jasminoides floral scent synthesis. International Journal of Biological Macromolecules, 288:138333.
[2]	Guo Y, Guo Z, Zhong J, Liang Y, Feng Y, Zhang P, Zhang Q, Sun M. 2023. Positive regulatory role of R2R3 MYBs in terpene biosynthesis in Lilium‘Siberia’. Horticultural Plant Journal, 9 (5):1024-1038. doi: 10.1016/j.hpj.2023.05.004 URL
[3]	Han J, Li T, Wang X, Zhang X, Bai X, Shao H, Wang S, Hu Z, Wu J, Leng P. 2022. AmMYB24 regulates floral terpenoid biosynthesis induced by blue light in snapdragon flowers. Frontiers in Plant Science, 13:885168.
[4]	Han Y, Wang H, Wang X, Li K, Dong M, Li Y, Zhu Q, Shang F. 2019. Mechanism of floral scent production in Osmanthus fragrans and the production and regulation of its key floral constituents,beta-ionone and linalool. Horticulture Research, 6:106. doi: 10.1038/s41438-019-0189-4
[5]	Ke Y, Abbas F, Zhou Y, Yu R, Fan Y. 2021. Auxin-responsive R2R3-MYB transcription factors HcMYB1 and HcMYB2 activate volatile biosynthesis in Hedychium coronarium flowers. Frontiers in Plant Science, 12:710826.
[6]	Lan Kaer, Liu Yuyang, Wu Shiyi, Yin Xiaopu, Wei Qiuhui. 2023. Research progress of MYB transcription factors regulating terpenoid biosynthesis in plants. Plant Physiology Journal, 59 (9):1709-1719. (in Chinese)
	蓝卡尔, 刘宇阳, 吴诗怡, 殷晓浦, 卫秋慧. 2023. MYB转录因子调控植物萜类生物合成的研究进展. 植物生理学报, 59 (9):1709-1719.
[7]	Lei G, Gao G, Zhou M, Guo J, Chen Y. 2024. Water soluble essential oil components of flowers of Paeonia × suffruticosa cultivars and in silico analysis with antidepressant targets. Natural Product Research, 38 (10):1776-1779. doi: 10.1080/14786419.2023.2217706 URL
[8]	Liu L, Chao N, Yidilisi K, Kang X, Cao X. 2022. Comprehensive analysis of the MYB transcription factor gene family in Morus alba. BMC Plant Biology, 22:362.
[9]	Ma B, Wu J, Zou J R, Wang J X, Hu Z H, Jia L M, Leng P S. 2024. Genome-wide identification and functional characterization of trans-IDS genes give new insights into terpene biosynthesis in Syringa oblata. Industrial Crops & Products, 213:118413.
[10]	Ma Bo, Yang Jun, Li Yingying, Wang Haonan, Hu Zenghui, Leng Pingsheng. 2020. Cloning and expression characteristic analysis of LiMCT gene in Lilium‘Siberia’. Molecular Plant Breeding, 18 (18):5933-5942. (in Chinese)
	马波, 杨珺, 李迎迎, 王浩楠, 胡增辉, 冷平生. 2020. ‘西伯利亚’百合LiMCT基因的克隆及表达特性分析. 分子植物育种, 18 (18):5933-5942.
[11]	Muhlemann J K, Klempien A, Dudareva N. 2014. Floral volatiles:from biosynthesis to function. Plant Cell & Environment, 37 (8):1936-1949. doi: 10.1111/pce.2014.37.issue-8 URL
[12]	Ramya M, Park P H, Chuang Y, Kwon O K, An H R, Park P M, Baek Y S, Kang B, Tsai W, Chen H. 2019. RNA sequencing analysis of Cymbidium goeringii identifies floral scent biosynthesis related genes. BMC Plant Biology, 19 (1):337. doi: 10.1186/s12870-019-1940-6
[13]	Reddy V A, Wang Q, Dhar N, Kumar N, Venkatesh P N, Rajan C, Panicker D, Sridhar V, Mao H Z, Sarojam R. 2017. Spearmint R2R3-MYB transcription factor MsMYB negatively regulates monoterpene production and suppresses the expression of geranyl diphosphate synthase large subunit(MsGPPS.LSU). Plant Biotechnology Journal, 15 (9):1105-1119. doi: 10.1111/pbi.2017.15.issue-9 URL
[14]	Sun Lichao, Li Shuying, Wang Fengzhong, Xin Fengjiao. 2017. Research progresses in the synthetic biology of terpenoids. Biotechnology Bulletin, 33 (1):64-75. (in Chinese) doi: 10.13560/j.cnki.biotech.bull.1985.2017.01.007
	孙丽超, 李淑英, 王凤忠, 辛凤姣. 2017. 萜类化合物的合成生物学研究进展. 生物技术通报, 33 (1):64-75. doi: 10.13560/j.cnki.biotech.bull.1985.2017.01.007
[15]	Xu M, Gao Q, Jiang M, Wang W, Hu J, Chang X, Liu D, Liang Y, Jiang Y, Chen F, Li C, Huang H, Chen F, Li F, Trigiano R N, Wang J, Jiao C, Zhou X, Zhang L. 2023. A novel genome sequence of Jasminum sambac helps uncover the molecular mechanism underlying the accumulation of jasmonates. Journal of Experimental Botany, 74 (4):1275-1290. doi: 10.1093/jxb/erac464 URL
[16]	Yang Xue, Luo Jun, Yang Caixia, Wang Zhenheng, Xia Qi, Wang Yinquan. 2018. Cloning and expression analysis of transcription factor MYB 4 gene from Angelica sinensis. Journal of Henan Agricultural Sciences, 47 (12):48-56. (in Chinese)
	杨雪, 雒军, 杨彩霞, 王振恒, 夏琦, 王引权. 2018. 当归MYB4转录因子基因的克隆及表达分析. 河南农业科学, 47 (12):48-56.
[17]	Yang Y, Liu X, Hu Z, Zhang Y, Yu H, Yang L, Leng P. 2024. Negative regulatory role of LiSRM1 in monoterpene biosynthesis in Lilium‘Siberia’. Plant Physiology and Biochemistry, 216:109167.
[18]	Yang Y, Zhang X, Han M, Hu Z, Wu J, Ma N, Leng P, Zhou X-F. 2023. LiMYB 108 is involved in floral monoterpene biosynthesis induced by light intensity in Lilium‘Siberia’. Plant Cell Reports, 42:763-773. doi: 10.1007/s00299-023-02995-x
[19]	Yang Yunyao, Zhang Yongchun, Chen Minmin, Han Xin, Yang LiuYan. 2024. Research progress on the synthesis and regulation mechanism of Lilium fragrance substances. Journal of Plant Genetic Resources, 33 (1):1-20. (in Chinese)
	杨云尧, 张永春, 陈敏敏, 韩炘, 杨柳燕. 2024. 百合花香物质合成与调控机制研究进展. 植物遗传资源学报, 33 (1):1-20.
[20]	Yang Y Y, Ma B, Li Y Y, Han M Z, Wu J, Zhou X F, Hu Z H. 2022. Transcriptome analysis identifies key gene LiMYB305 involved in monoterpene biosynthesis in Lilium‘Siberia’. Frontiers in Plant Science, 13:1021576.
[21]	Yang Z, Li Y, Gao F, Jin W, Li S, Kimani S, Yang S, Bao T, Gao X, Wang L. 2020. MYB 21 interacts with MYC2 to control the expression of terpene synthase genes in flowers of Freesia hybrida and Arabidopsis thaliana. Journal of Experimental Botany, 71 (14):4140-4158. doi: 10.1093/jxb/eraa184 URL
[22]	Yue Yuanzheng, Hu Hongmin, Liu Jiawei, Shen Huimin, Shi Tingting, Yang Xiulian, Wang Lianggui. 2023. Functional analysis of OfMYB1R 47 transcription factor in Osmanthus fragrans during the formation of aromatic volatiles. Journal of Zhejiang A & F University, 40 (3):465-474. (in Chinese)
	岳远征, 胡宏敏, 刘家伟, 申慧敏, 施婷婷, 杨秀莲, 王良桂. 2023. 桂花OfMYB1R47转录因子在芳香挥发物形成过程中的功能分析. 浙江农林大学学报, 40 (3):465-474.
[23]	Zhang Huixiu, Hu Zenghui, Leng Pingsheng, Wang Wenhe, Xu Fang, Zhao Jing. 2013a. Qualitative and quantitative analysis of floral volatile components from different varieties of Lilium spp. Scientia Agricultura Sinica, 46 (4):790-799. (in Chinese)
	张辉秀, 胡增辉, 冷平生, 王文和, 徐芳, 赵静. 2013a. 不同品种百合花挥发性成分定性与定量分析. 中国农业科学, 46 (4):790-799.
[24]	Zhang Huixiu, Leng Pingsheng, Hu Zenghui, Zhao Jing, Wang Wenhe, Xu Fang. 2013b. The floral scent emitted from Lilium‘Siberia’at different flowering stages and diurnal variation. Acta Horticulturae Sinica, 40 (4):693-702. (in Chinese)
	张辉秀, 冷平生, 胡增辉, 赵静, 王文和, 徐芳. 2013b. ‘西伯利亚’百合花香随开花进程变化及日变化规律. 园艺学报, 40 (4):693-702.

Cloning and Functional Analysis of LiMYB4 in Regulating Terpenoid Biosynthesis in Lilium‘Siberia’

RichHTML

PDF

Knowledge

Abstract

Cite this article

share this article

Figures/Tables 10

References 24

Related Articles 15

Recommended Articles

Metrics

Comments

[1]	WANG Fang, FAN Yanping. Molecular Insights into the Formation and Regulation of Floral Scent Traits in Ornamental Plants [J]. Acta Horticulturae Sinica, 2026, 53(2): 359-385.
[2]	XIE Yuying, SHI Tingting, YANG Xiulian, WANG Lianggui, YUE Yuanzheng. Research Progress on the Biological Functions and Biosynthetic Mechanisms of Floral Scent [J]. Acta Horticulturae Sinica, 2026, 53(2): 386-396.
[3]	ZHAO Yuqing, TANG Feihong, ZHENG Shijie, GUAN Zeen, WU Jiankai, LIU Zhongjian, PENG Donghui, LAN Siren, ZHAO Kai, ZHOU Yuzhen. Research Progress on Volatile Substances in Orchidaceae：From Synthetic Mechanisms to Evolutionary Drivers [J]. Acta Horticulturae Sinica, 2026, 53(2): 412-436.
[4]	FU Qi, SHANG Junzhong, PEI Wenwen, LIU Guoliang, JING Weikun, ZHANG Hao, JIAN Hongying, QIU Xianqin, TANG Kaixue, YAN Huijun. Advances in the Biosynthetic Genes and Evolutionary Mechanisms of Floral Volatile Organic Compounds in Rosa hybrida [J]. Acta Horticulturae Sinica, 2026, 53(2): 481-495.
[5]	HUANG Yawen, HUANG Hongfeng, WANG Zhiling, CHEN Siyu, ZHANG Shiqi, HONG Bo, GU Zhaoyu. Identification of Aroma Components and Analysis of Expression Characteristics of Terpene Synthase Genes in Chrysanthemum Floral Scent Mutants [J]. Acta Horticulturae Sinica, 2026, 53(2): 513-524.
[6]	WEI Yonglu, YE Guangying, GAO Jie, JIN Jianpeng, LU Chuqiao, LI Jie, XIE Qie, GOU Yajun, ZHU Genfa, YANG Fengxi. Integrated Metabolome and Transcriptome Analysis of Floral Scent in Maxillaria tenuifolia [J]. Acta Horticulturae Sinica, 2026, 53(2): 538-556.
[7]	LI Chonghui, HONG Xiaoyu, LU Shunjiao, LIAO Yi, YIN Junmei. Elucidation of Floral Scent Formation in Phalaenopsis-Type Dendrobium Hybrids by Integrated Metabolome and Transcriptome Analysis [J]. Acta Horticulturae Sinica, 2026, 53(2): 557-573.
[8]	XU Yong, YANG Yang, WANG Ruotong, MAO Yiru, FENG Liguo. Effects of Exogenous Melatonin on Rose Fragrance [J]. Acta Horticulturae Sinica, 2026, 53(2): 574-584.
[9]	CHEN Yiquan, FAN Ronghui, LIN Bing, CHEN Yan, WU Jianshe, ZHONG Huaiqin. Screening and Validation of Reference Genes for qRT-PCR Analysis of Genes Related to Floral Scent Biosynthesis in Camellia japonica [J]. Acta Horticulturae Sinica, 2026, 53(2): 598-612.
[10]	LI Jian, WANG Juntao, SHI Gensheng, HAO Huazheng, JI Zhongrui, XU Taoyu, WANG Jing, CHEN Lin. Comprehensive Evaluation on Cold Tolerance of Eleven Walnut Cultivars [J]. Acta Horticulturae Sinica, 2026, 53(1): 174-184.
[11]	ZHAO Xiaoyong, OU Ruohan, MEI Yuyang, CUI Tonghao, LIU Yilong, XIE Xiaodong, CAO Peijian, LI Xian. Research Progress on Key Enzymes Involved in Protein N-Glycosylation and Their Biological Functions in Plants [J]. Acta Horticulturae Sinica, 2026, 53(1): 285-302.
[12]	LI Jialong, LUAN Xiyun, TANG Zhanghu, HAN Aizhi, GUO Ling. Drought Resistance Evaluation of Apricot Germplasm Resources Based on Leaf Microstructure [J]. Acta Horticulturae Sinica, 2025, 52(9): 2300-2316.
[13]	LI Maofu, YANG Yuan, WANG Hua, SUN Pei, KANG Yanhui, SUN Xiangyi, LUO Jie, LIU Xin, JIN Min, JIN Wanmei. Cloning and Preliminary Functional Exploration of the Transposon-Derived Gene RcHTD in Rose [J]. Acta Horticulturae Sinica, 2025, 52(9): 2377-2386.
[14]	LI Xiao, YANG Qinrong, XIA Yuelin, WANG Chenhao, JIN Rongrong, CAO Hong, BO Yongming, ZHANG Mingfang, and LÜ Xiaolong. Progress in Functional Gene Research on Important Traits of Melons [J]. Acta Horticulturae Sinica, 2025, 52(8): 2208-2232.
[15]	FENG Lingjia, LIU Yujie, HE Lintong, WANG Xinchao, and YE Meng. Research Progress on the Regulation Mechanism of Linalool Synthesis and Its Ecological Functions [J]. Acta Horticulturae Sinica, 2025, 52(8): 2270-2290.