Cloning And Characterization Of Carotenoid Isomerase And Lycopene Epsilon Cyclase Genes From Maize (Zea Mays L.)

Carotenoids have fundamental roles in human nutrition as antioxidants and vitamin A precursors, and a high dietary intake of carotenoids is associated with a reduced risk for a range of diseases[1]. Animals are unable to synthesize carotenoids directly and must obtain them from their diets. Cereal grains are generally deficient in these compounds, leading to deficiency diseases in countries where cereals are the staple diet. For these reasons there is much interest in studying the carotenoid biosynthetic pathway in plants and in modifying crop plants to enhance their carotenoid content. The limited data concerning endogenous regulation of carotenogenic genes has made the precise engineering of crop plants to enhance carotenoid content and composition difficult despite recent progress in cereal crops, particularly in corn[2-4]. In order to gain further insight into the partly-characterized carotenoid biosynthetic pathway in corn (Zea mays L.), we cloned cDNAs encoding the enzymes carotenoid isomerase (CRTISO) and lycopeneε-cyclase (LYCE) using endosperm mRNA isolated from inbred corn line B73. For CRTISO enzyme, two different cDNAs (Zmcrtiso1 and Zmcrtiso2), encoding full-length carotenoid isomerase (CRTISO) enzymes, were amplified from 25 DAP (days after pollination) B73 corn endosperm mRNA. The full-length Zmcrtiso1 cDNA encoded a 587-amino-acid protein with a molecular weight of 63.7 kDa, a pI of 8.24, and a putative 43-residue transit peptide for chloroplast targeting. The B73 Zmcrtiso2 cDNA contained a C-to-A transversion at position 143 (the putative translation start codon of the Zmcrtiso2 cDNA is referred to as 1) which was not present in homologous EST and cDNA sequences from white maize (M37W), yellow maize (EP42 and A632), and sequences from other cultivars (accession nos. FL133727 and EU957482). The resulting nonsense mutation prematurely terminates protein synthesis and yields a truncated 127-amino-acid protein. Zmcrtiso1 and Zmcrtiso2 were identified mapping to unlinked genes each containing 12 introns, a feature conserved among all crtiso genes studied thus far. ZmCRTISO1 was able to convert tetra-cis prolycopene to all-trans lycopene but could not isomerize the 15-cis double bond of 9,15,9′‐tri‐cis-ζ-carotene. ZmCRTISO2 is inactivated by the premature termination codon in B73 corn as described above, but importantly the mutation is absent in other corn cultivars and the active enzyme showed the same activity as ZmCRTISO1.ZmLYCE from maize B73 was predicted to be 59.7 kDa (537 residues) and a pI of 6.33. The full-length amino acid residues of Zmlyce have high similarities (72.1% to 73.8%) and identities (63.2% to 63.9%) with functional LYCE proteins from Arabidopsis, tomato and gentian. However, by heterologous complementation experiment in Escherichia coli, ZmLYCE can add twoε-ionone rings at the both ends of lycopene to generate the bicyclicε,ε-carotene through mono-cyclicδ-carotene, which is significant different from that from Arabidopsis, tomato and gentian where LYCE can add only oneε-ionone ring at one end of lycopene to give rise to mono-cyclicδ-carotene. Furthermore, in order to understand how carotenoid accumulation is regulated in maize endosperm development, we investigated the correlation between carotenoid accumulation in endosperm and mRNA expression of Zmcrtiso1 and Zmcrtiso2. Both of Zmcrtiso1 and Zmcrtiso2 genes were expressed during endosperm development, with mRNA levels rising in line with carotenoid accumulation (especiallyβ-carotene, zeaxanthin and lutein) until 25 DAP. Thereafter, expression declined for Zmcrtiso1 gene, whereas Zmcrtiso2 mRNA levels maintained by 30 DAP. We discuss the impact of paralogs with different expression profiles and functions on the regulation of carotenoid synthesis in corn.