Expression and Functional Analysis of Two Lycopene Β-cyclases from Citrus

23 In the present study, two LCYb genes (CitLCYb1 and CitLCYb2) were 24 isolated from Satsuma mandarin (Citrus unshiu Marc.), Valencia orange (Citrus 25 sinensis Osbeck) and Lisbon lemon (Citrus limon Burm.f.) and their functions 26 were analyzed by the color complementation assay in lycopene-accumulating E. 27 coli cells. The results showed that CitLCYb1 and CitLCYb2 shared high identity 28 at the amino acid level among the three citrus varieties. The N-terminal region 29 of the two proteins encoded by CitLCYb1 and CitLCYb2 was predicted to 30 contain a 51-residue chloroplastic transit peptide, which shared low similarity. 31 In Satsuma mandarin, the secondary structures of the CitLCYb1 and CitLCYb2 32 encoding proteins without the transit peptide were quite similar. Moreover, 33 functional analysis showed that both enzymes of CitLCYb1 and CitLCYb2 34 participated in the formation of β-carotene, and when they were co-expressed 35 with CitLCYe, α-carotene could be produced from lycopene in E. coli cells. 36 However, although CitLCYb2 could convert lycopene to α-carotene in E. coli 37 cells, its extremely low level of expression indicated that CitLCYb2 did not 38 participate in the formation of α-carotene during the green stage in the flavedo. 39 In addition, the high expression levels of CitLCYb1 and CitLCYb2 during the 40 orange stage played an important role in the accumulation of β,β-xanthophylls in 41 citrus fruits. The results presented in this study might contribute to elucidate the 42 mechanism of carotenoid accumulation in citrus fruits.