Functional Characterization of Carotenoid Cleavage Dioxygenases 4 Gene（RcCCD4）Involved in Biosynthetic Pathway of Floral Scent in Rosa chinensis‘Old Blush’

doi:10.16420/j.issn.0513-353x.2024-0382

Acta Horticulturae Sinica ›› 2025, Vol. 52 ›› Issue (3): 623-634.doi: 10.16420/j.issn.0513-353x.2024-0382

• Genetic & Breeding·Germplasm Resources·Molecular Biology • Previous Articles Next Articles

Functional Characterization of Carotenoid Cleavage Dioxygenases 4 Gene（RcCCD4）Involved in Biosynthetic Pathway of Floral Scent in Rosa chinensis‘Old Blush’

FU Qi¹^,², WANG Dan¹^,², JING Weikun¹, ZHANG Hao¹, WANG Huichun¹, JIAN Hongying¹, QIU Xianqin¹, WANG Qigang¹, TANG Kaixue¹, YAN Huijun¹^,^*()

¹ Flower Research Institute of Yunnan Academy of Agricultural Sciences，Kunming 650051，China
² Institute of Resource Plants，Yunnan University，Kunming 650500，China

Received:2025-01-02 Revised:2025-02-20 Online:2025-03-25 Published:2025-03-25
Contact: YAN Huijun

Abstract

Abstract:

To reveal the role of carotenoid cleavage dioxygenases（CCDs）in biosynthetic pathway of floral scent，RcCCD4 gene were cloned from the petals of Rosa chinensis‘Old Blush’and functionally characterized. RT-qPCR showed that RcCCD4 was mainly expressed in petals，with the highest expression level at the fully opening stage and lowest at the bud stage. Subcellular localization assays showed that RcCCD4 were located in the plastid. Moreover，a prokaryotic expression protein vector for RcCCD4 were constructed and found that the RcCCD4 enzyme can specifically cleave β-carotene substrates in vitro enzymatic activity assay. Virus-induced gene silencing（VIGS）further suggested that silencing of RcCCD4 significantly reduced the content of dihydro-β-ionone in petals，and RcCCD4 overexpression led to increase in dihydro-β-ionone content. These results indicated that RcCCD4 can specifically cleave β-carotene substrates，thereby synthesizing dihydro-β-ionone.

Key words: rose, floral scent, carotenoid cleavage dioxygenase（CCD）, prokaryotic expression, virus induced gene silencing（VIGS）

FU Qi, WANG Dan, JING Weikun, ZHANG Hao, WANG Huichun, JIAN Hongying, QIU Xianqin, WANG Qigang, TANG Kaixue, YAN Huijun. Functional Characterization of Carotenoid Cleavage Dioxygenases 4 Gene（RcCCD4）Involved in Biosynthetic Pathway of Floral Scent in Rosa chinensis‘Old Blush’[J]. Acta Horticulturae Sinica, 2025, 52(3): 623-634.

Figures/Tables 9

Table 1 Primers used in this study

用途 Function	引物名称 Primer name	引物序列（5′-3′）Primer sequence
克隆基因	RcCCD4-F	ATGGATGCCTTGTCTTCTTCT
Gene clone	RcCCD4-R	CAACTTCTTCAGGTCACTCTCC
病毒诱导的基因沉默	pTRV1-F	TTACAGGTTATTTGGGCTAG
Virus induced gene	pTRV1-R	CCGGGTTCAATTCCTTATC
silencing	pTRV2-F	TGGGAGATGATACGCTGTT
	pTRV2-R	CCTAAAACTTCAGACACG
	pTRV2-RcCCD4-F	GAGTAAGGTTACCGAATTCAGCTCGACGTGTCGG
	pTRV2-RcCCD4-R	CTCGAGACGCGTGAGCTCGGTACCATACAACAGTCACAACGTTTGTG
原核表达	Pet28a（+）-RcCCD4-F	CGCGGCAGCCATATGATGGATGCCTTGTCTTCTTCTT
Prokaryotic expression	Pet28a（+）-RcCCD4-R	GTGGTGGTGGTGCTCGAGCAACTTCTTCAGGTCACTCTCC
过表达	pSurper-1300-RcCCD4-F	AAGCTTCTGCAGGGGCCCGGGATGGATGCCTTGTCTTCTTCT
Overexpression	pSurper-1300-RcCCD4-R	TCCTCGCCCTTGCTCACCATGGTACCCAACTTCTTCAGGTCACTCTCC
实时荧光定量PCR qRT-PCR	qRT-RcCCD4-F	AGCTCGACGTGTCGGG
	qRT-RcCCD4-R	CCTCCGGATTCTCCGGATCC
	qRT-RcCCD1-F	GTTGATGTACGACAGTTCTCAGCCT
	qRT-RcCCD1-R	ACAAACTCTCCATTCAAGCAATCAGG
	qRT-RcCCD7-F	AGTGGAACAAGTCATCAGATTTTCCAGT
	qRT-RcCCD7-R	TAGTTGAAGCATTCAGCTTCACCAC
	qRT-RcCCD8-F	TGTTTTAATGACATGCCGCCTTGAG
	qRT-RcCCD8-R	ATAATTTCTTCTGTGTTGCTAAACCGGT
	qRT-RcZAS-F	GAGGGTGAAGCGATTGCAAAAATT
	qRT-RcZAS-R	TGACACGGAGAAAATTCATGGTTACAG
	qRT-RcZAS-like-F	TGCCAACCTCATCGAACACCTAA
	qRT-RcZAS-like-R	ATGGTCAACTACACACTTGTATACGACT
	qRT-RcNCED1-F	CAATCTCACTGAACCCACTTGCTT
	qRT-RcNCED1-R	CACTACTACTTTAACTCAAAGCCTGCTCC
	qRT-RcNCED6-F	AGAAGACGTGGAAGTCGGAGC
	qRT-RcNCED6-R	AGCTACTGCTATGCCTGGTTTTTCA

Fig. 1 Phylogenetic trees of rose RcCCD4 and other species

Fig. 2 Subcellular localization of rose RcCCD4 in leaf cells of Nicotiana benthamiana

Fig. 3 Expression of CCD family genes in different organs of Rosa chinensis‘Old Blush’ Compared with leaf as a control，t-test，* represents a significance level of α = 0.05；** represents a significance level of α = 0.01；ns：No significant

Fig. 4 Expression of RcCCD4 in the petals during different developmental stages of Rosa chinensis‘Old Blush’ Compared with S1 stage in the bud stage as a control，t-test，* represents a significance level of α = 0.05；** represents a significance level of α = 0.01；ns：No significant

Fig. 5 Conditions optimization（A）and Western-Blot analysis（B）of RcCCD4 recombination protein M：Marker. The arrows indicate the protein of interest；1-4：IPTG-induced samples at different temperatures and concentrations；5：Uninduced sample；6，8，10，12：Precipitated sample；7，9，11，13：Supernatant samples

Fig. 6 Expression analysis of RcCCD4 in Escherichia coli（A）and HPLC detection of β-carotenoids（B）

Fig. 7 Expression of RcCCD4 in infected plants by qRT-PCR and relative contents of dihydro-β-ionone in Rosa hybrida‘Luoshen’ t-test，* represents a significance level of α = 0.05；** represents a significance level of α = 0.01；ns：No significant；NT：No-treatment sample；Mock：TRV empty sample. The same below

Fig. 8 Expression of RcCCD4 in overexpression plants by qRT-PCR and relative contents of dihydro-β-ionone in Rosa chinensis‘Old Blush’and Rosa hybrida‘Luoshen’

References 36

[1]	Auldridge M E, McCarty D R, Klee H J. 2006. Plant carotenoid cleavage oxygenases and their apocarotenoid products. Current Opinion in Plant Biology, 9 (3):315-321. doi: 10.1016/j.pbi.2006.03.005 pmid: 16616608
[2]	Baldermann S, Kato M, Kurosawa M, Kurobayashi Y, Fujita A, Fleischmann P, Watanabe N. 2010. Functional characterization of a carotenoid cleavage dioxygenase 1 and its relation to the carotenoid accumulation and volatile emission during the floral development of Osmanthus fragrans Lour. Journal of Experimental Botany, 61 (11):2967-2977. doi: 10.1093/jxb/erq123 pmid: 20478967
[3]	Brandi F, Bar E, Mourgues F, Horváth G, Turcsi E, Giuliano G, Liverani A, Tartarini S, Lewinsohn E, Rosati C. 2011. Study of‘Redhaven’peach and its white-fleshed mutant suggests a key role of CCD 4 carotenoid dioxygenase in carotenoid and norisoprenoid volatile metabolism. BMC Plant Biology, 11 (1):24.
[4]	Bruno M, Beyer P, Al-Babili S. 2015. The potato carotenoid cleavage dioxygenase 4 catalyzes asingle cleavage of β-ionone ring-containing carotenes and non-epoxidated xanthophylls. Archives of Biochemistry and Biophysics, 572:126-133. doi: S0003-9861(15)00073-9 pmid: 25703194
[5]	Fang Qiang. 2020. Cloning and functional characterization of carotenoid cleavage dioxygenases from Freesia hybrida[Ph. D. Dissertation]. Changhcun: Northeast Normal University. (in Chinese)
	房强. 2020. 香雪兰类胡萝卜素裂解双加氧酶(FhCCDs)基因克隆与功能鉴定[博士论文]. 长春: 东北师范大学.
[6]	González-Verdejo CI, Obrero Á, Román B, Gómez P. 2015. Expression profile of carotenoid cleavage dioxygenase genes in summer squash (Cucurbita pepo L.). Plant Foods for Human Nutrition, 70 (2):200-206. doi: 10.1007/s11130-015-0482-9 pmid: 25861766
[7]	Guterman I, Shalit M, Menda N, Piestun D, Dafny-Yelin M, Shalev G, Bar E, Davydov O, Ovadis M, Emanuel M, Wang J, Adam Z, Pichersky E, Lewinsohn E, Zamir D, Vainstein A, Weiss D. 2002. Rose scent:genomics approach to discovering novel floral fragrance-related genes. Plant Cell, 14 (10):2325-2338. pmid: 12368489
[8]	Han Y, Lu M, Yue S, Li K, Dong M, Liu L, Wang H, Shang F. 2022. Comparative methylomics and chromatin accessibility analysis in Osmanthus fragrans uncovers regulation of genic transcription and mechanisms of key floral scent production. Horticulture Research, 9:uhac096.
[9]	Hou Yixuan, Zhang Ling, Lü Mengwen, Wu Yaoxing, Wang Liangsheng, Zhang Xiuqing, Li Shanshan. 2023. Analysis of volatile components from the petal of intersectional hybrids of Paeonia. Acta Horticulturae Sinica, 50 (4):842-852. (in Chinese) doi: 10.16420/j.issn.0513-353x.2021-1221 URL
	侯异璇, 张玲, 吕梦雯, 武耀星, 王亮生, 张秀卿, 李珊珊. 2023. 芍药属组间杂种花香成分分析. 园艺学报, 50 (4):842-852. doi: 10.16420/j.issn.0513-353x.2021-1221 URL
[10]	Huang F C, Horváth G, Molnár P, Turcsi E, Deli J, Schrader J, Sandmann G, Schmidt H, Schwab W. 2009a. Substrate promiscuity of RdCCD1, a carotenoid cleavage oxygenase from Rosa damascena. Phytochemistry, 70 (4):457-464.
[11]	Huang F C, Molnár P, Schwab W. 2009b. Cloning and functional characterization of carotenoid cleavage dioxygenase 4 genes. J Exp Bot, 60 (11):3011-3022.
[12]	Li Y Q, Bao T T, Zhang J, Li H J, Shan X T, Yan H J, Kimani S, Zhang L S, Gao X. 2025. The coordinated interaction or regulation between floral pigments and volatile organic compounds. Horticultural Plant Journal, 11 (2):463-485.
[13]	Liu J R, Chen B Q, Yang B F, Dong H W, Sun C H, Wang Q, Song G, Song Y Q. 2004. Apoptosis of human gastric adenocarcinoma cells induced by β-ionone. World Journal of Gastroenterology, 10 (3):348-351.
[14]	Liu Jiaren, Yang Yanmei, Dong Hongwei, Sun Xiangrong, Yu Jia, Zhao Shuyuan, Chen Bingqing. 2005. Effect of β-ionone in human mammary cancer cells(Er-)by MAPK pathway. Journal of Hygiene Research, 34 (6):4. (in Chinese)
	刘家仁, 杨艳梅, 董宏伟, 孙向荣, 于佳, 赵淑媛, 陈炳卿. 2005. β-紫罗兰酮对人乳腺癌细胞(Er-)MAPK途径的影响. 卫生研究, 34 (6):4.
[15]	Liu Q, Dong H W, Sun W G, Liu M, Ibla J C, Liu L X, Parry J W, Han X H, Li M S, Liu J R. 2013. Apoptosis initiation of β-ionone in SGC-7901 gastric carcinoma cancer cells via a PI3K-AKT pathway. Archives of Toxicology, 87 (3):481-490. doi: 10.1007/s00204-012-0962-8 pmid: 23100158
[16]	Ma N, Ma C, Liu Y, Shahid M O, Wang C, Gao J. 2018. Petal senescence:a hormone view. Journal of Experimental Botany, 69 (4):719-732.
[17]	Mir J I, Ahmed N, Wafai A H, Qadri R A. 2012. Relative expression of CsZCD gene and apocarotenoid biosynthesis during stigma development in Crocus sativus L. Physiology & Molecular Biology of Plants, 18 (4):371-375.
[18]	Nawade B, Shaltiel-Harpaz L, Yahyaa M, Bosamia T C, Kabaha A, Kedoshim R, Zohar M, Isaacson T, Ibdah M. 2020. Analysis of apocarotenoid volatiles during the development of Ficus carica fruits and characterization of carotenoid cleavage dioxygenase genes. Plant Science, 290:110292.
[19]	Ohmiya A, Kishimoto S, Aida R, Yoshioka S, Sumitomo K. 2006. Carotenoid cleavage dioxygenase(CmCCD4a)contributes to white color formation in chrysanthemum petals. Plant Physiology, 142 (3):1193-1201. pmid: 16980560
[20]	Pino, Jorge, Antonio, Quijano, Clara, Elizabeth. 2012. Study of the volatile compounds from plum(Prunus domestica L. cv. Horvin)and estimation of their contribution to the fruit aroma. Ciência E Tecnologia De Alimentos, 32 (1):76-83
[21]	Rubio A, Rambla J L, Santaella M, Gómez M D, Orzaez D, Granell A, Gómez-Gómez L. 2008. Cytosolic and plastoglobule-targeted carotenoid dioxygenases from Crocus sativus are both involved in β-ionone release. Journal of Biological Chemistry, 283 (36):24816-24825.
[22]	Schwab W, Huang F C, Péter Molnár. 2013. Carotenoid cleavage dioxygenase genes from fruit. ACS Symposium Series, 1134 (10):11-19.
[23]	Scognamiglio J, Jones L, Letizia C S, Api A M. 2012. Fragrance material review on β,β,3-trimethyl-benzenepropanol. Food Chem Toxicol,Suppl 2:S263-S268.
[24]	Simkin A J, Schwartz S H, Auldridge M, Taylor M G, Klee H J. 2010. The tomato carotenoid cleavage dioxygenase 1 genes contribute to the formation of the flavor volatiles β-ionone,pseudoionone,and geranylacetone. Plant Journal, 40 (6):882-892.
[25]	Simkin A J, Underwood B A, Auldridge M, Loucas H M, Shibuya K, Schmelz E, Clark D G, Klee H J. 2004. Circadian regulation of the PhCCD 1 carotenoid cleavage dioxygenase controls emission of β-ionone,a fragrance volatile of petunia flowers. Plant Physiol, 136 (3):3504-3514.
[26]	Sorefan K, Booker J, Haurogné K, Goussot M, Bainbridge K, Foo E, Chatfield S, Ward S, Beveridge C, Rameau C, Leyser O. 2003. MAX4 and RMS1 are orthologous dioxygenase-like genes that regulate shoot branching in Arabidopsis and pea. Genes & Development, 17 (12):1469-1474.
[27]	Tan B C, Joseph L M, Deng W T, Liu L, Li Q B, Cline K, McCarty D R. 2003. Molecular characterization of the Arabidopsis 9-cis epoxycarotenoid dioxygenase gene family. The Plant Journal, 35 (1):44-56.
[28]	Walter M H, Strack D. 2011. Carotenoids and their cleavage products:biosynthesis and functions. Natural Product Reports, 28 (4):663-692.
[29]	Wang J, Wu B, Zhang N, Zhao M, Jing T, Wu Y, Hu Y, Yu F, Wan X, Schwab W, Song C. 2020. Dehydration-induced carotenoid cleavage dioxygenase 1 reveals a novel route for β-ionone formation during tea(Camellia sinensis)withering. Journal of Agricultural and Food Chemistry, 68 (39):10815-10821.
[30]	Wang J M, Zhao M Y, Gao T, Feng Y Y, Wang F Q, Pan Y T, Jin J Y, Jing T T, Lu M Q, Zhang M T, Guo D Y, Wan X C, Schwab W, Song C K. 2023. Promoter and coding sequence diversity of CsCCD1may contribute to the differential accumulation of floral β-ionone in fresh tea leaves. Horticultural Plant Journal, 9 (4):837-847.
[31]	Watanabe K, Oda-Yamamizo C, Sage-Ono K, Ohmiya A, Ono M. 2017. Alteration of flower colour in Ipomoea nil through CRISPR/Cas9-mediated mutagenesis of carotenoid cleavage dioxygenase 4. Transgenic Research, 27 (1):25-38.
[32]	Wu L, Ma N, Jia Y, Zhang Y, Feng M, Jiang C Z, Ma C, Gao J. 2016. An ethylene-induced regulatory module delays flower senescence by regulating cytokinin content. Plant Physiology, 173 (1):853-862.
[33]	Ye Yunfang, Tian Qingyin, Shi Tingting, Wang Liang, Yue Yuanzheng, Yang Xiulian, Wang Lianggui. 2023. Research progress in the biosynthesis and regulation of β-ionone in plants. Biotechnology Bulletin, 39 (8):91-105. (in Chinese) doi: 10.13560/j.cnki.biotech.bull.1985.2023-0208
	叶云芳, 田清尹, 施婷婷, 王亮, 岳远征, 杨秀莲, 王良桂. 2023. 植物中β-紫罗兰酮生物合成及调控研究进展. 生物技术通报, 39 (8):91-105. doi: 10.13560/j.cnki.biotech.bull.1985.2023-0208
[34]	Yi Xing. 2014. Embryonic callus induction and genetic transformation of basic study in Rosa chinensis‘Yueyuehong’[M. D. Dissertation]. Changsha: Hunan Agricultural University. (in Chinese)
	易星. 2014. ‘月月红’月季胚性愈伤组织诱导及遗传转化基础研究[硕士论文]. 长沙: 湖南农业大学.
[35]	Zhang B, Liu C, Wang Y, Yao X, Wang F, Wu J, King G J, Liu K. 2015. Disruption of a CAROTENOID CLEAVAGE DIOXYGENASE 4 gene converts flower colour from white to yellow in Brassica species. New Phytologist, 206 (4):1513-1526. doi: 10.1111/nph.13335 pmid: 25690717
[36]	Zhao Jing, Hu Zenghui, Leng Pingsheng, Yang Xiaohong. 2013. Anakysus on fragrance composition of two snaodragon cultivars. Journal of Beijing University of agriculture, 28 (3):5. (in Chinese)
	赵静, 胡增辉, 冷平生, 杨晓红. 2013. 两个金鱼草品种香气成分分析. 北京农学院学报, 28 (3):5.

Functional Characterization of Carotenoid Cleavage Dioxygenases 4 Gene（RcCCD4）Involved in Biosynthetic Pathway of Floral Scent in Rosa chinensis‘Old Blush’

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