Characterization of the rice carotenoid cleavage dioxygenase 1 reveals a novel route for geranial biosynthesis

Carotenoid cleavage products - apocarotenoids - include biologically active compounds, such as hormones, pigments and volatiles. Their biosynthesis is initiated by the oxidative cleavage of C-C double bonds in carotenoid backbones, leading to aldehydes and/or ketones. This step is catalyzed by carotenoid oxygenases, which constitute an ubiquitous enzyme family, including the group of plant carotenoid cleavage dioxygenases 1 (CCD1s), which mediates the formation of volatile C₁₃ ketones, such as β-ionone, by cleaving the C9-C10 and C9′-C10′ double bonds of cyclic and acyclic carotenoids. Recently, it was reported that plant CCD1s also act on the C5-C6/C5′- C6′ double bonds of acyclic carotenes, leading to the volatile C ₈ ketone 6-methyl-5-hepten-2-one. Using in vitro and in vivo assays, we show here that rice CCD1 converts lycopene into the three different volatiles, pseudoionone, 6-methyl-5-hepten-2-one, and geranial (C₁₀), suggesting that the C7-C8/C7′-C8′ double bonds of acyclic carotenoid ends constitute a novel cleavage site for the CCD1 plant subfamily. The results were confirmed by HPLC, LC-MS and GC-MS analyses, and further substantiated by in vitro incubations with the monocyclic carotenoid 3-OH-γ-carotene and with linear synthetic substrates. Bicyclic carotenoids were cleaved, as reported for other plant CCD1s, at the C9-C10 and C9′-C10′ double bonds. Our study reveals a novel source for the widely occurring plant volatile geranial, which is the cleavage of noncyclic ends of carotenoids.