Physiologia Plantarum 141: 1–10. 2011 Copyright © Physiologia Plantarum 2010, ISSN 0031-9317 Expression and phylogenetic analysis of two new lycopene β -cyclases from Citrus paradisi Amanda F. S. Mendes a , Chunxian Chen b , Fred G. Gmitter Jr b,c , Gloria A. Moore c,d and Marcio G. C. Costa a,∗ a Center for Biotechnology and Genetics, Biological Sciences Department, State University of Santa Cruz, Ilh ´ eus, BA 45662-000, Brazil b Citrus Research & Education Center, University of Florida, Lake Alfred, FL 33850, USA c Plant Molecular and Cellular Biology Program, University of Florida, Gainesville, FL, USA d Horticultural Sciences Department, University of Florida, Gainesville, FL 32611, USA Correspondence *Corresponding author, e-mail: mcosta@labbi.uesc.br Received 24 August 2010 doi:10.1111/j.1399-3054.2010.01415.x Two new lycopene β -cyclases (LCYBs) were cloned and characterized from grapefruit (Citrus paradisi Macf.). During fruit ripening, CpLCYB1 expression did not show significant differences between ‘Flame’ (red flesh) and ‘Marsh’ (white flesh), and was much lower than CpLCYB2 and nearly constant; however, CpLCYB2 expression dramatically changed in a similar tendency in the pulp of both grapefruit cultivars, but the relative abundance of mRNA in ‘Flame’ was significantly lower than in ‘Marsh’. Phylogenetically and structurally, CpLCYB1 was a chloroplast-specific member and CpLCYB2 a chromoplast-specific member, the two subfamilies of all the LCYB genes. An intron was found in the 5 ′ -untranslated region of CpLCYB1 and in two other Citrus LCYB1 genes (CcLCYB1 and CsLCYB1-2), resulting in an extra 20 amino acids, compared with all the other LCYB1s. It suggested that a different genomic event, in addition to gene duplication, has contributed to the evolution of these LCYB genes, and likewise, the change of their functions. Introduction Lycopene cyclases (LCYs) are a large group of enzymes that catalyze the cyclization of the linear carotenoid trans-lycopene to produce the cyclic carotenoids, including α-, β -, γ - and ε-carotene, in the carotenoid biosynthetic pathway in plants, bacteria and fungi (Bouvier et al. 2005). Cyclic carotenoids are essential components of the photosynthetic membranes in all plants, algae and cyanobacteria, providing functions of photoprotection and light harvesting, and additionally furnishing plant flowers and fruits with distinct colors to attract pollinators and seed dispersers (Cunningham and Gantt 1998). β -carotenes are essential precursors Abbreviations – bp, base pairs; CCS, capsanthin-capsorubin synthase; IME, intron-mediated enhancement; LCY, lycopene cyclase; LCYB, lycopene β -cyclase; LCYE, lycopene ε-cyclase; NCBI, National Center for Biotechnology Information; NSY, neoxanthin synthase; PCR, polymerase chain reaction; qPCR, quantitative PCR; RACE, rapid amplification of cDNA ends; RT, reverse transcription; UTR, untranslated region. for vitamin A in human and animal diets and have other health benefits, particularly reducing cardiovascular disease and certain cancers (Fraser and Bramley 2004). In higher plants, the formation of β - and ε-ionone rings is catalyzed by two enzymes, lycopene β -cyclase (LCYB) and lycopene ε-cyclase (LCYE), which share high similarity in their amino acid sequences and likely evolved from the same ancestor (Sandmann 2002). They are also related to two other carotenoid cyclase enzymes: the capsanthin-capsorubin synthase (CCS) of pepper (Cunningham et al. 1996) and the neoxanthin synthase (NSY) of tomato (Bouvier et al. 2000) and potato (Al-Babili et al. 2000). CCS catalyzes the formation of the unusual five-carbon κ ring (Bouvier Physiol. Plant. 141, 2011 1