Cloning and functional characterization of a carotenoid cleavage dioxygenase 2 gene in safranal and crocin biosynthesis from Freesia hybrida.

Qiang Fang, Yueqing Li, Baofeng Liu, Xiangyu Meng, Zhongzhou Yang, Song Yang, Tingting Bao, Shadrack Kimani, Xiang Gao, Li Wang

Journal: Plant physiology and biochemistry : PPB 2020;154():439-450

PMID: 32912484

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Abstract

Safranal and crocin, commonly derived from the oxidative cleavage reaction of zeaxanthin in plants, are two kinds of apocarotenoids with versatile functions, which were only found in limited number of plant species. In this study, both metabolites were detected and varied concomitantly with the expression of carotenoid cleavage dioxygenase (CCD) genes in Freesia hybrida, Red River® and Ambiance cultivars. The newly isolated CCD, denoted here as FhCCD2, was phylogenetically clustered with other reported saffron CCD2s. Besides, ten introns were also observed in the genomic DNA sequence of FhCCD2 and the presence of N-terminal transporter peptide suggested its plastidial sub-localization. Biochemical analysis showed that the FhCCD2 cleaved zeaxanthin at the 7, 8 and 7', 8' double bonds to generate intermediates prerequisite for the biosynthesis of safranal and crocin. Further, gene transient expression analysis showed that the promoter of FhCCD2 was functional in Ambiance as well as Red River® cultivars, even with slight variation in their promoter sequence. At present, CCD2 proteins have only been found in Freesia and Crocus genus of Iridaceae family. Phylogenetic and intron position analysis infer that CCD2 perhaps emerged after the intron loss during evolutionary process of CCD1 or their shared ancestry.

Address: Key Laboratory of Molecular Epigenetics of MOE and Institute of Genetics & Cytology, Northeast Normal University, Changchun, China; School of Chemistry and Life Science, Changchun University of Technology, Changchun, China.; Key Laboratory of Molecular Epigenetics of MOE and Institute of Genetics & Cytology, Northeast Normal University, Changchun, China. Electronic address: [email protected].; Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun, China.; Key Laboratory of Molecular Epigenetics of MOE and Institute of Genetics & Cytology, Northeast Normal University, Changchun, China.; Key Laboratory of Molecular Epigenetics of MOE and Institute of Genetics & Cytology, Northeast Normal University, Changchun, China; School of Pure and Applied Sciences, Karatina University, Karatina, Kenya.; Key Laboratory of Molecular Epigenetics of MOE and Institute of Genetics & Cytology, Northeast Normal University, Changchun, China; National Demonstration Center for Experimental Biology Education, Northeast Normal University, Changchun, China.; Key Laboratory of Molecular Epigenetics of MOE and Institute of Genetics & Cytology, Northeast Normal University, Changchun, China. Electronic address: [email protected].

DOI: 10.1016/j.plaphy.2020.06.035

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MeSH Terms: Carotenoids, Cloning, Molecular, Cyclohexenes, Dioxygenases, Iridaceae, Phylogeny, Plant Proteins, Terpenes