Biotechnology of Fruit Quality - Fruit Ripening: Physiology, Signalling and Genomics - page 273

E -carotene by lycopene E -cyclase (CRTL-B),
a precursor of vitamin A, or D -carotene by
lycopene D -cyclase (CRTL_E). Lutein, a
major xanthophyll involved in light harvest-
ing and preventing macular degeneration of
the eyes in older people, is synthesized
from D -carotene (Ronen et al. , 1999).
Transgenic approaches have been
widely used to enhance levels of caroten-
oids in many crop species by expressing
various genes of the carotenoid pathways
(Table 16.1). Ectopic expression of a
bacterial PSY ( crtB ) under the control of a
fruit-specifi c promoter increased phytoene
(2.4-fold), lycopene (1.8-fold), E -carotene
(2.2-fold) and lutein levels in tomato fruit
(Fraser et al. , 2002). The constitutive
expression of citrus lycopene E -cyclase
( CRTL-B ) increased E -carotene levels 4.1-
fold with a 30% increase in total
carotenoids while suppressing fl uxes
downstream into the E -carotene pathway
and the concomitant increase in D -carotene
(Guo et al. , 2012). A mutation in lycopene
H -cyclase ( CRTL-E ) caused accumulation of
G -carotene at the expense of lycopene in
Delta ( Del ), a fruit-colour mutant (Ronen et
al. , 1999). Two other genes, CYP97A29 and
CYP97C11 , have been functionally char-
acterized by expressing them in tomato
under the control of the CaMV 35S
promoter. CYP97A29 and CYP97C11
encode P450 carotenoid E -hydroxylase
( CRTR-B ) and carotenoid H -hydroxylase
( CRTR-E ), respectively. CRTR-E converts
D -carotene into lutein, and CRTR-B
converts E -carotene into zeaxanthin and
D -carotene into lutein (Stigliani et al. ,
2011). Zeaxanthin is further converted to
violaxanthin by zeaxanthin epoxidase
( ZEP1 ) . A mutation in zep1 caused ABA-
defi ciency in tomato plants with a
concomitant accumulation of 30% more
carotenoids in mature red tomato fruit
(Galpaz et al. , 2008). RNAi-mediated
fruit-specifi c suppression of 9- cis -
epoxycarotenoid dioxygenase 3 ( NCED3 ),
an enzyme that catalyses the fi rst step of
ABA biosynthesis converting 9- cis -
violaxanthin to 2- cis ,4- trans -xanthoxin, not
only suppressed ABA synthesis but also

stimulated accumulation of upstream
compounds such as E -carotene and lyco-
pene in transgenic tomato fruits (Sun et al .,
2012).
Characterization of several tomato
mutants that accumulate higher levels of
carotenoids than wild-type fruits has
helped to discover factors regulating fl ux
through the carotenoid pathways. UV-
DAMAGED DNA BINDING PROTEIN 1
( DDB1 ) and DE-ETIOLATED 1 ( DET1 ) are
transcription factors that negatively
regulate photomorphogenic responses
(Azari et al. , 2010a). Mutations in DDB1
and DET1 exhibit recessive high-pigment 1
( hp-1 ) and hp-2 phenotypes with severe
developmental defects (Mustilli et al. ,
1999; Levin et al. , 2003; Davuluri et al. ,
2004; Azari et al. , 2010b). However, organ-
specifi c silencing of DET1 by RNAi under a
fruit-specifi c promoter resulted in a
twofold increase in lycopene, a fourfold
increase in E -carotene and up to a 3.5-fold
increase in fl avonoids without signifi cant
changes in fruit weight and total soluble
solids in red-ripe fruit (Davuluri et al. ,
2005). The transgenic overexpression of
cryptochrome 2 ( 35S:CRY2 ) resulted in a
1.7-fold increase in carotenoids and a 2.9-
fold increase in fl avonoids (Giliberto et al. ,
2005). RNAi-mediated repression of
ELONGATED HYPOCOTYL 5 HY5 )
reduced carotenoid accumulation and
repression of CONSTITUTIVELY PHOTO-
MORPHOGENIC 1 ( COP1 )-like showed an
elevation in tomato fruit carotenoids
suggesting involvement of light-signalling
factors in carotenoid biosynthesis (Liu et
al. , 2004).
The biogenic amines spermidine and
spermine, which belong to the group of
ubiquitous polycations called polyamines,
have also been implicated in delaying fruit
ripening and increasing carotenoid content
in tomato (Mehta et al. , 2002; Nambeesan et
al. , 2010). Constitutive overexpression
of Saccharomyces cerevisiae spermidine
synthase or fruit-specifi c overexpression of
yeast S -adenosylmethionine decarboxylase
( E8:ySAMdc ) in tomato led to a 40% or
200-300% increase in lycopene content,