SURE: Shizuoka University REpositorySURE: Shizuoka University REpository
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Expression and functional analysis of two lycopene β -cyclases
from citrus fruits
Zhang, Lancui; Ma, Gang; Shirai, Yuki; Kato, Masaya;
Yamawaki, Kazuki; Ikoma, Yoshinori; Matsumoto, Hikaru
© Springer, Part of Springer Science+Business Media. The
original publication is available at www.springerlink.com
Expression and functional analysis of two lycopene β-cyclases from citrus
Lancui Zhang1,*, Gang Ma1,*, Yuki Shirai1,§, Masaya Kato1,†, Kazuki Yamawaki1,
Yoshinori Ikoma2, Hikaru Matsumoto2
1Department of Biological and Environmental Sciences, Faculty of Agriculture, Shizuoka
University, 836 Ohya, Suruga, Shizuoka 422-8529, Japan
2 Department of Citrus Research, National Institute of Fruit Tree Science, Okitsunakacho,
Shimizu, Shizuoka 424-0292, Japan
* These authors contributed equally to this article.
§ Present address: Fruit Tree Research Center, Shizuoka Prefectual Research Institute of 13
Agriculture and Forestry, 2-12-10 Komagoe-nishi, Shimizu, Shizuoka 424-0905, Japan 14
† Corresponding author: Masaya Kato
Telephone: 81-54-238-4830 Fax: 81-54-238-4830
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.
Keywords Carotenoid · Citrus · Lycopene β-cyclase · α-Carotene · β-Carotene
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 expoxidase
Carotenoids are important natural isoprenoid pigments, which provide
distinct yellow, red and orange colors to flowers and fruits (Ronen et al. 2000;
Schweiggert et al. 2011). In addition, carotenoids fulfill a variety of other
critical functions in plants, such as the stabilization of lipid membranes, light
harvesting for photosynthesis, as well as protecting the photosystem from
photo-oxidation (Havaux 1998; Havaux and Kloppstech 2001; Ledford and
Niyogi 2005). Carotenoids are also the precursors of the plant hormone abscisic
acid (ABA) (Schwartz et al. 1997; Cunningham and Gantt 1998). Carotenoids
are not only important to the plants themselves, but also beneficial to human
health. Some carotenoids with a β-ring are the precursors of vitamin A, which is
a fundamental nutrient for humans (Giovannucci 1999; Krinsky et al. 2003).
Recent studies have identified that the benefits from carotenoids might be due to
β-cryptoxanthin, which is one of the major carotenoids in human blood (Fu et al.
2010). Altucci and Gronemeyer (2001) reported that β-cryptoxanthin played an
important role in the prevention of some diseases, especially cancers, because of
its antioxidant activity. Additionally, β-cryptoxanthin served as a retinoic acid
receptor (RAR) ligand and exerted beneficial effects on atherogenesis through
RAR activation (Matsumoto et al. 2007).
The pathway of carotenoid biosynthesis is a series of desaturation,
cyclization, hydroxylation, and epoxidation steps (Cunningham and Gantt 1998;