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


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.

This is a preview of subscription content, access via your institution.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6



Abscisic acid


Geranylgeranyl diphosphate


β-Ring hydroxylase


ε-Ring hydroxylase


Lycopene cyclase


Lycopene β-cyclase


Lycopene ε-cyclase


Phytoene desaturase


Phytoene synthase


ζ-Carotene desaturase


Zeaxanthin epoxidase






  1. Alquézar B, Zacarías L, Rodrigo MJ (2009) Molecular and functional characterization of a novel chromoplast-specific lycopene β-cyclase from Citrus and its relation to lycopene accumulation. J Exp Bot 60:1783–1797

    PubMed  Article  Google Scholar 

  2. Altucci L, Gronemeyer H (2001) The promise of retinoids to fight against cancer. Nat Rev Cancer 1:181–193

    PubMed  Article  CAS  Google Scholar 

  3. Ampomah-Dwamena C, McGhie T, Wibisono R, Montefiori M, Hellens RP, Allan AC (2009) The kiwifruit lycopene beta-cyclase plays a significant role in carotenoid accumulation in fruit. J Exp Bot 60:3765–3779

    PubMed  Article  CAS  Google Scholar 

  4. Bai L, Kim EH, DellaPenna D, Brutnell TP (2009) Novel lycopene epsilon cyclase activities in maize revealed through perturbation of carotenoid biosynthesis. Plant J 59:588–599

    PubMed  Article  CAS  Google Scholar 

  5. Cunningham FX, Gantt E (1998) Genes and enzymes of carotenoid biosynthesis in plants. Annu Rev Plant Physiol Plant Mol Biol 49:557–583

    PubMed  Article  CAS  Google Scholar 

  6. Cunningham FX, Pogson B, Sun Z, McDonald KA, DellaPenna D, Gantt E (1996) Functional analysis of the β and ε lycopene cyclase enzymes of Arabidopsis reveals a mechanism for control of cyclic carotenoid formation. Plant Cell 8:1613–1626

    PubMed  CAS  Google Scholar 

  7. Dalal M, Chinnusamy V, Bansal KC (2010) Isolation and functional characterization of Lycopene β-cyclase (CYC-B) promoter from Solanum habrochaites. BMC Plant Biol 10:61–76

    PubMed  Article  Google Scholar 

  8. Devitt LC, Fanning K, Dietzgen RG, Holton TA (2010) Isolation and functional characterization of a lycopene beta-cyclase gene that controls fruit colour of papaya (Carica papaya L.). J Exp Bot 61:33–39

    PubMed  Article  CAS  Google Scholar 

  9. Fu HF, Xie BJ, Fan G, Ma SJ, Zhu XR, Pan SY (2010) Effect of esterification with fatty acid of β-cryptoxanthin on its thermal stability and antioxidant activity by chemiluminescence method. Food Chem 122:602–609

    Article  CAS  Google Scholar 

  10. Giovannucci E (1999) Tomatoes, tomato-based products, lycopene, and cancer: review of the epidemiologic literature. J Natl Cancer Inst 91:317–331

    PubMed  Article  CAS  Google Scholar 

  11. Goodner KL, Rouseff RL, Hofsommer HJ (2001) Orange, mandarin, and hybrid classification using multivariate statistics based on carotenoid profiles. J Agric Food Chem 49:1146–1150

    PubMed  Article  CAS  Google Scholar 

  12. Harvaux M, Kloppstech K (2001) The protective functions of carotenoid and flavonoid pigments against excess visible radiation at chilling temperature investigated in Arabidopsis npq and tt mutants. Planta 213:953–966

    PubMed  Article  CAS  Google Scholar 

  13. Havaux M (1998) Carotenoids as membrane stabilizers in chloroplasts. Trends Plant Sci 3:147–151

    Article  Google Scholar 

  14. Hugueney P, Badillo A, Chen HC, Klein A, Hirschberg J, Camara B, Kuntz M (1995) Metabolism of cyclic carotenoids: a model for the alteration of this biosynthetic pathway in Capsicum annuum chromoplasts. Plant J 8:417–424

    PubMed  Article  CAS  Google Scholar 

  15. Ikoma Y, Yano M, Ogawa K, Yoshioka T, Xu ZC, Hisada S, Omura M, Moriguchi T (1996) Isolation and evaluation of RNA from polysaccharide-rich tissues in fruit for quality by cDNA library construction and RT-PCR. J Jpn Soc Hortic Sci 64:809–814

    Article  CAS  Google Scholar 

  16. Ikoma Y, Komatsu A, Kita M, Ogawa K, Omura M, Yano M, Moriguchi T (2001) Expression of a phytoene synthase gene and characteristic carotenoid accumulation during citrus fruit development. Physiol Plant 111:232–238

    Article  CAS  Google Scholar 

  17. Inoue K, Furbee KJ, Uratsu S, Kato M, Dandekar AM, Ikoma Y (2006) Catalytic activities and chloroplast import of carotenogenic enzymes from citrus. Physiol Plant 127:561–570

    Article  CAS  Google Scholar 

  18. Kato M, Ikoma Y, Matsumoto H, Sugiura M, Hyodo H, Yano M (2004) Accumulation of carotenoids and expression of carotenoid biosynthetic genes during maturation in citrus fruit. Plant Physiol 134:824–837

    PubMed  Article  CAS  Google Scholar 

  19. Kato M, Matsumoto H, Ikoma Y, Okuda H, Yano M (2006) The role of carotenoid cleavage dioxygenases in the regulation of carotenoid profiles during maturation in citrus fruit. J Exp Bot 57:2153–2164

    PubMed  Article  CAS  Google Scholar 

  20. Kato M, Matsumoto H, Ikoma Y, Kuniga T, Nakajima N, Yoshida T, Yano M (2007) Accumulation of carotenoids and expression of carotenoid biosynthetic genes and carotenoid cleavage dioxygenase genes during fruit maturation in the juice sacs of ‘Tamami’, ‘Kiyomi’ tangor, and ‘Wilking’ mandarin. J Jpn Soc Hortic Sci 76:103–111

    Article  CAS  Google Scholar 

  21. Krinsky NI, Landrum JT, Bone RA (2003) Biologic mechanisms of the protective role of lutein and zeaxanthin in the eye. Annu Rev Nutr 23:171–201

    PubMed  Article  CAS  Google Scholar 

  22. Ledford HK, Niyogi KK (2005) Singlet oxygen and photo-oxidative stress management in plants and algae. Plant Cell Environ 28:1037–1045

    Article  CAS  Google Scholar 

  23. Lee HS, Castle WS (2001) Seasonal changes of carotenoid pigments and color in Hamlin, Earlygold, and Budd Blood orange juices. J Agric Food Chem 49:877–882

    PubMed  Article  CAS  Google Scholar 

  24. Liang C, Zhao F, Wei W, Wen Z, Qin S (2006) Carotenoid biosynthesis in cyanobacteria: structural and evolutionary scenarios based on comparative genomics. Int J Biol Sci 2:197–207

    PubMed  Article  CAS  Google Scholar 

  25. Matsumoto A, Mizukami H, Mizuno S, Umegaki K, Nishikawa J, Shudo K, Kagechika H, Inoue M (2007) β-Cryptoxanthin, a novel natural RAR ligand, induces ATP-binding cassette transporters in macrophages. Biochem Pharmacol 74:256–264

    PubMed  Article  CAS  Google Scholar 

  26. Mendes AF, Chen C, Gmitter FG Jr, Moore GA, Costa MG (2011) Expression and phylogenetic analysis of two new lycopene β-cyclases from Citrus paradisi. Physiol Plant 141:1–10

    PubMed  Article  CAS  Google Scholar 

  27. Mialoundama AS, Heintz D, Jadid N, Nkeng P, Rahier A, Deli J, Camara B, Bouvier F (2010) Characterization of plant carotenoid cyclases as members of the flavoprotein family functioning with no net redox change. Plant Physiol 153:970–979

    PubMed  Article  CAS  Google Scholar 

  28. Misawa N, Shimada H (1997) Metabolic engineering for the production of carotenoids in non-carotenogenic bacteria and yeasts. J Biotechnol 59:169–181

    PubMed  Article  CAS  Google Scholar 

  29. Molnár P, Szabolcs J (1980) β-Citraurin epoxide, a new carotenoid from Valencia orange peel. Phytochemistry 19:633–637

    Article  Google Scholar 

  30. Pecker I, Gabbay R, Cunningham FX Jr, Hirschberg J (1996) Cloning and characterization of the cDNA for lycopene beta-cyclase from tomato reveals decrease in its expression during fruit ripening. Plant Mol Biol 30:807–819

    PubMed  Article  CAS  Google Scholar 

  31. Ronen G, Carmel-Goren L, Zamir D, Hirschberg J (2000) An alternative pathway to β-carotene formation in plant chromoplasts discovered by map-based cloning of Beta and old-gold color mutations in tomato. Proc Natl Acad Sci USA 26:11102–11107

    Article  Google Scholar 

  32. Sandmann G (2002) Combinatorial biosynthesis of carotenoids in a heterologous host: a powerful approach for the biosynthesis of novel structures. ChemBioChem 3:629–635

    PubMed  Article  CAS  Google Scholar 

  33. Schwartz SH, Tan BC, Gage DA, Zeevaart JA, McCarty DR (1997) Specific oxidative cleavage of carotenoids by VP14 of maize. Science 276:1872–1874

    PubMed  Article  CAS  Google Scholar 

  34. Schweiggert RM, Steingass CB, Heller A, Esquivel P, Carle R (2011) Characterization of chromoplasts and carotenoids of red- and yellow-fleshed papaya (Carica papaya L.). Planta 234:1031–1044

    PubMed  Article  CAS  Google Scholar 

  35. Tadmor Y, King S, Levi A, Davis A, Wasserman B, Hirschberg J, Lewinsohn E (2005) Comparative fruit colouration in watermelon and tomato. Food Res Int 38:837–841

    Article  CAS  Google Scholar 

  36. Zhang LC, Ma G, Kato M, Yamawaki K, Takagi T, Kiriiwa Y, Ikoma Y, Matsumoto H, Nesumi H, Yoshioka T (2012) Regulation of carotenoid accumulation and the expression of carotenoid metabolic genes in citrus juice sacs in vitro. J Exp Bot 63:871–886

    PubMed  Article  CAS  Google Scholar 

Download references


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



Corresponding author

Correspondence to Masaya Kato.

Additional information

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

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary material (DOC 1784 kb)

Rights and permissions

Reprints and Permissions

About this article

Cite this article

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).

Download citation


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