Abstract
In many organisms, various enzymes mediate site-specific carotenoid cleavage to generate biologically active apocarotenoids. These carotenoid-derived products include provitamin A, hormones, and flavor and fragrance molecules. In plants, the CCD1 enzyme cleaves carotenoids at 9,10 (9',10') bonds to generate multiple apocarotenoid products. Here we systematically analyzed volatile apocarotenoids generated by maize CCD1 (ZmCCD1) from multiple carotenoid substrates. ZmCCD1 did not cleave geranylgeranyl diphosphate or phytoene but did cleave other linear and cyclic carotenoids, producing volatiles derived from 9,10 (9',10') bond cleavage. Additionally the Arabidopsis, maize, and tomato CCD1 enzymes all cleaved lycopene to generate 6-methyl-5-hepten-2-one. 6-Methyl-5-hepten-2-one, an important flavor volatile in tomato, was produced by cleavage of the 5,6 or 5',6' bond positions of lycopene but not geranylgeranyl diphosphate, zeta-carotene, or phytoene. In vitro, ZmCCD1 cleaved linear and cyclic carotenoids with equal efficiency. Based on the pattern of apocarotenoid volatiles produced, we propose that CCD1 recognizes its cleavage site based on the saturation status between carbons 7 and 8 (7' and 8') and carbons 11 and 12 (11' and 12') as well as the methyl groups on carbons 5, 9, and 13 (5', 9', and 13').
Highlights
Apocarotenoids are widely distributed in nature and serve important biological functions
The Arabidopsis thaliana genome contains nine genes similar to VP14, five of which appear to be involved in abscisic acid biosynthesis [16, 17]
ZmCCD1 Cleaves Multiple Carotenoids to Generate Apocarotenoid Volatiles Derived from 9,10 and 5,6 Double Bond Cleavage—To determine the substrate specificities and bond cleavage preferences of ZmCCD1, we undertook a systematic analysis of the volatile products generated by the enzyme in various carotenoid-accumulating strains of E. coli
Summary
Cloning of ZmCCD1—A near full-length ZmCCD1 cDNA was amplified by reverse transcription-PCR from developing White cap (Wc) kernels of maize (18 days after pollination) with forward primer 5Ј-CCCTTCGCTACAAGCCTACA-3Ј and reverse primer 5Ј-TTCGAATACACGTCCTGCAA-3Ј. The PCR product was cloned into the pCR4-TOPO vector (Invitrogen) and sequenced. To create a fusion of ZmCCD1 to glutathione S-transferase (GST), the ZmCCD1 coding sequence was first amplified with forward primer 5Ј-GGATCCATGGGGACGGAGGCGGAGCAGCCG-3Ј and reverse primer 5Ј-ATGCCCTGCAATAATTTCCA-3Ј using Pfu DNA polymerase and cloned into the pCR-blunt-TOPO vector (Invitrogen). Volatile Analysis from E. coli Cultures—pGEX2T-ZmCCD1, pGEX2T empty vector, pDEST15-AtCCD1, pDEST15LeCCD1A, pDEST15-LeCCD1B, or pDEST15-GUS [23, 30] was transformed into chemically competent BL21-AI (Invitrogen) cells harboring plasmids encoding carotenoid biosynthetic genes (36 –39). Three individual colonies for each carotenoid strain were used to inoculate three different 3-ml cultures of LB containing 100 g/ml carbenicillin and 34 g/ml chloramphenicol (Sigma). The following day, 1 ml of overnight culture was used to inoculate 100 ml of LB containing 100 g/ml carbenicillin and 34 g/ml chloramphenicol in a 250-ml baffle flask. Any background present in control reactions was subtracted, and values
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