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Expression and functional analysis of two lycopene β-cyclases from citrus fruits

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Abstract

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

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Abbreviations

ABA:

Abscisic acid

GGPP:

Geranylgeranyl diphosphate

HYb:

β-Ring hydroxylase

HYe:

ε-Ring hydroxylase

LCY:

Lycopene cyclase

LCYb:

Lycopene β-cyclase

LCYe:

Lycopene ε-cyclase

PDS:

Phytoene desaturase

PSY:

Phytoene synthase

ZDS:

ζ-Carotene desaturase

ZEP:

Zeaxanthin epoxidase

α-Car:

α-Carotene

β-Car:

β-Carotene

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Acknowledgments

We thank Professor Norihiko Misawa for providing the pACCRT-EIB plasmid (Research Institute for Bioresources and Biotechnology, Ishikawa Prefectural University, Nonoichi Ishikawa, Japan). This work was supported by Grant-in-Aid for Young Scientists (22780020) and JSPS Postdoctoral Fellowships for Research Abroad.

Author information

Correspondence to Masaya Kato.

Additional information

L. Zhang and G. Ma contributed equally to this article.

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Zhang, L., Ma, G., Shirai, Y. et al. Expression and functional analysis of two lycopene β-cyclases from citrus fruits. Planta 236, 1315–1325 (2012). https://doi.org/10.1007/s00425-012-1690-2

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Keywords

  • Carotenoid
  • Citrus
  • Lycopene β-cyclase
  • α-Carotene
  • β-Carotene