Characterization of Carotenoid Cleavage Oxygenase Genes in Cerasus humilis and Functional Analysis of ChCCD1

Abstract

Carotenoid cleavage oxygenases (CCOs) are key enzymes that function in degrading carotenoids into a variety of apocarotenoids and some other compounds. In this study, we performed genome-wide identification and characterization analysis of CCO genes in Cerasus humilis. Totally, nine CCO genes could be classified into six subfamilies, including carotenoid cleavage dioxygenase 1 (CCD1), CCD4, CCD7, CCD8, CCD-like and nine-cis-epoxycarotenoid dioxygenase (NCED), were identified. Results of gene expression analysis showed that ChCCOs exhibited diverse expression patterns in different organs and in fruits at different ripening stages. To investigate the roles of ChCCOs in carotenoids degradation, enzyme assays of the ChCCD1 and ChCCD4 were performed in Escerichia coli BL21(DE3) that can accumulate lycopene, β-carotene and zeaxanthin. The prokaryotic expressed ChCCD1 resulted in obvious degradation of lycopene, β-carotene and zeaxanthin, but ChCCD4 did not show similar functions. To further determine the cleaved volatile apocarotenoids of these two proteins, headspace gas chromatography/mass spectrometer analysis was performed. Results showed that ChCCD1 could cleave lycopene at 5, 6 and 5', 6' positions to produce 6-methy-5-hepten-2-one and could catalyze β-carotene at 9, 10 and 9', 10' positions to generate β-ionone. Our study will be helpful for clarifying the roles of CCO genes especially ChCCD1 in regulating carotenoid degradation and apocarotenoid production in C. humilis.

Keywords: Cerasus humilis; apocarotenoids; carotenoid cleavage oxygenase; carotenoids; functional analysis.

Figure 1

Phylogenetic analysis results (A) of CCO proteins from Cerasus humilis (Ch), P. persica (Pp), Fragaria vesca (Fv), Solanum Lycopersicum (Sl), Oryza sativa (Os) and Arabidopsis thaliana (At), and nucleotides (B) and proteins (C) similarity analysis results of ChCCOs. CCD1, CCD4, CCD-like, CCD8, CCD7 and NCED are six subfamilies of CCOs. Red stars in A represent C. humilis CCD members. In B and C: the redder the color, the higher the similarity; the greener the color, the lower the similarity.

Figure 2

Protein–protein interaction network for ChCCOs based on the Prunus persica protein database.

Figure 3

Heatmap for the transcriptome data-based expression analysis of ChCCOs in the fruit, leaf, kernel, rhizome and root of C. humilis. For heatmap drawing, log₂(FPKM + 1) values of ChCCO genes were used. The redder the color, the higher the gene’s expression, and white represents no expression.

Figure 4

Quantitative real-time PCR analysis results of ChCCOs in fruits at four different ripening stages. (A–F) represents expression analysis result for ChCCD-like-a, ChCCD1, ChCCD4, ChCCD8, ChNCED1 and ChNCED5, respectively. DAF: days after flowering. The different letters above the columns represent significant differences at p < 0.05 level.

Figure 5

Functional analysis results of ChCCD1 and ChCCD4 proteins. (A) The influences of ChCCD1 and ChCCD4 expression on the color changes of E. coli strains that can accumulate lycopene (carrying pACCRT-EIB vector), β-carotene (carrying pACCAR16ΔcrtX vector) and zeaxanthin (carrying pACCAR25ΔcrtX vector); CK: control bacteria with no IPTG addition. (B) ChCCD1 can cleave lycopene into 6-methyl-5-heptene-2-one. (C) ChCCD1 can cleave β-carotene into β-ionone.

Figure 6

GC-MS detection results of the ChCCD1 cleaved volatile products of lycopene and β-carotene in E. coli. (A,B) for the cleaved volatile products of lycopene and β-carotene, respectively; (C,D) fragments pattern for 6-methy-5-hepten-2-one and β-ionone, respectively. Blue circles in (A) and (B) represent starting and ending time points of peak.