Genomic analysis and gene structure of the plant carotenoid dioxygenase 4 family: A deeper study in Crocus sativus and its allies

The plastoglobule-targeted enzyme carotenoid cleavage dioxygenase (CCD4) mediates the formation of volatile C13 ketones, such as β-ionone, by cleaving the C9-C10 and C9'-C10' double bonds of cyclic carotenoids. Here, we report the isolation and analysis of CCD4 genomic DNA regions in Crocus sativus. Different CCD4 alleles have been identified: CsCCD4a which is found with and without an intron and CsCCD4b that showed the presence of a unique intron. The presence of different CCD4 alleles was also observed in other Crocus species. Furthermore, comparison of the locations of CCD4 introns within the coding region with CCD4 genes from other plant species suggests that independent gain/losses have occurred. The comparison of the promoter region of CsCCD4a and CsCCD4b with available CCD4 gene promoters from other plant species highlighted the conservation of cis-elements involved in light response, heat stress, as well as the absence and unique presence of cis-elements involved in circadian regulation and low temperature responses, respectively. Functional characterization of the Crocus sativus CCD4a promoter using Arabidopsis plants stably transformed with a DNA fragment of 1400 base pairs (P-CsCCD4a) fused to the β-glucuronidase (GUS) reporter gene showed that this sequence was sufficient to drive GUS expression in the flower, in particular high levels were detected in pollen.