Cloning and functional characterization of a gene for capsanthin-capsorubin synthase from tiger lily (Lilium lancifolium Thunb. 'Splendens')

• Published in: Plant and cell physiology 2012-11, Vol.53 (11), p.1899-1912
• Main Authors: Jeknić, Zoran, Morré, Jeffrey T, Jeknić, Stevan, Jevremović, Sladana, Subotić, Angelina, Chen, Tony H H
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Chromatography, High Pressure Liquid; Cloning, Molecular; Color; DNA, Complementary - genetics; DNA, Complementary - metabolism; Flowers - enzymology; Flowers - genetics; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Plant; Genes, Plant; Intramolecular Lyases - genetics; Intramolecular Lyases - metabolism; Iris germanica; Iris Plant - genetics; Iris Plant - metabolism; Lilium; Lilium - enzymology; Lilium - genetics; Lolium; Molecular Sequence Data; Open Reading Frames; Oxidoreductases - genetics; Oxidoreductases - metabolism; Phylogeny; Plant Proteins - genetics; Plant Proteins - metabolism; Plants, Genetically Modified - enzymology; Plants, Genetically Modified - genetics; Plants, Genetically Modified - metabolism; Regular Papers; Reverse Transcriptase Polymerase Chain Reaction; Sequence Homology, Amino Acid; Tandem Mass Spectrometry - methods; Xanthophylls - biosynthesis
• ISSN: 0032-0781; 1471-9053
• DOI: 10.1093/pcp/pcs128

The orange color of tiger lily (Lolium lancifolium 'Splendens') flowers is due, primarily, to the accumulation of two κ-xanthophylls, capsanthin and capsorubin. An enzyme, known as capsanthin-capsorubin synthase (CCS), catalyzes the conversion of antheraxanthin and violaxanthin into capsanthin and capsorubin, respectively. We cloned the gene for capsanthin-capsorubin synthase (Llccs) from flower tepals of L. lancifolium by the rapid amplification of cDNA ends (RACE) with a heterologous non-degenerate primer that was based on the sequence of a gene for lycopene β-cyclase (lcyB). The full-length cDNA of Llccs was 1,785 bp long and contained an open reading frame of 1,425 bp that encoded a polypeptide of 474 amino acids with a predicted N-terminal plastid-targeting sequence. Analysis by reverse transcription-PCR (RT-PCR) revealed that expression of Llccs was spatially and temporally regulated, with expression in flower buds and flowers of L. lancifolium but not in vegetative tissues. Stable overexpression of the Llccs gene in callus tissue of Iris germanica, which accumulates several xanthophylls including violaxanthin, the precursor of capsorubin, resulted in transgenic callus whose color had changed from its normal yellow to red-orange. This novel red-orange coloration was due to the accumulation of two non-native κ-xanthophylls, capsanthin and capsorubin, as confirmed by HPLC and ultraperformance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) analysis with authentic standards. Cloning of the Llccs gene should advance our understanding of the molecular and genetic mechanisms of the biosynthesis of κ-carotenoids in general and in the genus Lilium in particular, and will facilitate transgenic alterations of the colors of flowers and fruits of many plant species.