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.

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Phytohormones are important regulators of numerous developmental and physiological processes in plants. Spontaneous morphogenesis of the common centaury (Centaurium erythraea Rafn.) is possible on nutrition medium without addition of any plant growth regulator depending solely on endogenous phytohormone levels. Thus, this plant species represents a very good model system for the investigation of numerous physiological processes under phytohormonal control in vitro. We analysed the total amount of endogenous cytokinins (CKs) including the contents of their individual groups in shoots and roots of C. erythraea plants grown in vitro. The total amount of endogenous CKs was 1.4 times higher in shoots than in roots. Inactive or weakly active N-glucosides found to predominate in both organs of centaury plants, whereas free bases and O-glucosides represented only a small portion of the total CK pool. Consequently, centaury roots showed higher IAA content as well as IAA/ free CK base ratios compared to shoots. Centaury tissues also showed increased levels of ''stress hormones''. In contrast to SA, considerably higher levels of ABA were found in centaury shoots than in roots. Our results could serve as a basis for understanding and elucidating spontaneous de novo shoot organogenesis and further plant regeneration of C. erythraea in vitro. Abbreviations for CKs Adopted and Modified According to Kamínek and Others (2000) ADPAdenosine diphosphate AMP Adenosine monophosphate CK Cytokinin cisZ cis-zeatin cisZ7G cis-zeatin 7-glucoside cisZ9G cis-zeatin 9-glucoside cisZOG cis-zeatin O-glucoside cisZR cis-zeatin 9-riboside cisZRMP cis-zeatin 9-riboside-5 0 -monophosphate cisZROG cis-zeatin 9-riboside O-glucoside DHZ Dihydrozeatin DHZ7GDihydrozeatin 7-glucoside DHZ9GDihydrozeatin 9-glucoside DHZOG Dihydrozeatin O-glucoside DHZR Dihydrozeatin 9-riboside DHZRMP Dihydrozeatin 9-riboside-5 0 -monophosphate DHZROG Dihydrozeatin 9-riboside O-glucoside iP N 6 -(D 2 -isopentenyl)adenine iP7G N 6 -(D 2 -isopentenyl)adenine 7-glucoside iP9G N 6 -(D 2 -isopentenyl)adenine 9-glucoside iPR N 6 -(D 2 -isopentenyl)adenine 9-riboside Milana Trifunović-Momčilov and Václav Motyka have contributed equally to this work. Electronic supplementary materialThe online version of this article (iPRMP N 6 -(D 2 -isopentenyl)adenine 9-riboside-5 0 -monophosphate transZ trans-zeatin transZ7Gtrans-zeatin 7-glucoside transZ9G trans-zeatin 9-glucoside transZOG trans-zeatin O-glucoside transZR trans-zeatin 9-riboside transZRMP trans-zeatin 9-riboside-5 0 -monophosphate transZROG trans-zeatin 9-riboside O-glucoside