(II) enzymes, the carotenoid cleavage dioxygenases (CCDs) that
catalyze the oxidative cleavage of carbon-carbon double bonds in
carotenoid backbones, generating products with aldehyde or
ketone end groups [19]. Plants CCDs enzymes are classified into
two big subfamilies [20]. The nine-cis-epoxy-carotenoid-dioxygenases (NCED) subfamily represented by the first characterized
CCD, the maize VP14 [1], that catalyzes the synthesis of the ABA
precursor xanthoxin by cleaving the C11,C12 double bond in
9-cis-violaxanthin and 9
0 -cis-neoxanthin. NCEDs are represented
in practically all plant species by a multigene family [21]. The
second subfamily is generally referred to as CCD, regardless the
type of carotenoid substrates and the cleavage site, and includes all
other members of the CCD family, which are not involved in ABA
biosynthesis. Indeed, plant CCDs cleave a variety of carotenoid and
apocarotenoid substrates at different positions [22–28]. The plant
CCD subfamily includes CCD1, CCD2, CCD4, CCD7, and
CCD8 enzymes [20]. Large scale genomic sequencing projects
on plant species has allowed the identification of many putative
CCDs that have expanded this subfamily. However, biochemical
studies have only been reported for a limited number of CCDs
[20, 29].
Two main strategies, in vitro and/or in vivo studies, have been
followed to characterize the activity of CCD enzymes. These studies have been performed with CCDs from many plant species,
including Arabidopsis [30], saffron [31], rose [32], petunia [33],
tomato [34, 35], potato [22], rice [36], melon [37], maize [38],
Vitis [39, 40], citrus [26, 41], and other plant species [42]. The
in vivo studies have been done by expressing CCDs in carotenoid
accumulating E. coli strains containing specific plasmids that
allowed the accumulation of different carotenoids substrates
(Table 1). The in vitro approach allows testing of much more
substrates, including apocarotenoids themselves, and enables comprehensive and quantitative characterization of investigated
enzymes. In both cases, products formed have been detected and
identified through thin-layer chromatography (TLC), HPLC,
LC-MS, or GC-MS.
2 Materials
2.1 In Vivo Assays
1. Bacterial strains. Most common E. coli strains, such as JM109,
XL1-Blue, and TOPO-10, used for cloning are utilized to
accumulate carotenoids.
2. Plasmids. They are listed in (Table 1) and can be purchased
from Addgene (https://www.addgene.org).
3. Luria Bertani (LB) medium. Combine 10 g tryptone, 10 g
NaCl, and 5 g yeast extract in 900 mL ddH 2 O and shake
64
Lourdes Go ´ mez-Go ´ mez et al.
catalyze the oxidative cleavage of carbon-carbon double bonds in
carotenoid backbones, generating products with aldehyde or
ketone end groups [19]. Plants CCDs enzymes are classified into
two big subfamilies [20]. The nine-cis-epoxy-carotenoid-dioxygenases (NCED) subfamily represented by the first characterized
CCD, the maize VP14 [1], that catalyzes the synthesis of the ABA
precursor xanthoxin by cleaving the C11,C12 double bond in
9-cis-violaxanthin and 9
0 -cis-neoxanthin. NCEDs are represented
in practically all plant species by a multigene family [21]. The
second subfamily is generally referred to as CCD, regardless the
type of carotenoid substrates and the cleavage site, and includes all
other members of the CCD family, which are not involved in ABA
biosynthesis. Indeed, plant CCDs cleave a variety of carotenoid and
apocarotenoid substrates at different positions [22–28]. The plant
CCD subfamily includes CCD1, CCD2, CCD4, CCD7, and
CCD8 enzymes [20]. Large scale genomic sequencing projects
on plant species has allowed the identification of many putative
CCDs that have expanded this subfamily. However, biochemical
studies have only been reported for a limited number of CCDs
[20, 29].
Two main strategies, in vitro and/or in vivo studies, have been
followed to characterize the activity of CCD enzymes. These studies have been performed with CCDs from many plant species,
including Arabidopsis [30], saffron [31], rose [32], petunia [33],
tomato [34, 35], potato [22], rice [36], melon [37], maize [38],
Vitis [39, 40], citrus [26, 41], and other plant species [42]. The
in vivo studies have been done by expressing CCDs in carotenoid
accumulating E. coli strains containing specific plasmids that
allowed the accumulation of different carotenoids substrates
(Table 1). The in vitro approach allows testing of much more
substrates, including apocarotenoids themselves, and enables comprehensive and quantitative characterization of investigated
enzymes. In both cases, products formed have been detected and
identified through thin-layer chromatography (TLC), HPLC,
LC-MS, or GC-MS.
2 Materials
2.1 In Vivo Assays
1. Bacterial strains. Most common E. coli strains, such as JM109,
XL1-Blue, and TOPO-10, used for cloning are utilized to
accumulate carotenoids.
2. Plasmids. They are listed in (Table 1) and can be purchased
from Addgene (https://www.addgene.org).
3. Luria Bertani (LB) medium. Combine 10 g tryptone, 10 g
NaCl, and 5 g yeast extract in 900 mL ddH 2 O and shake
64
Lourdes Go ´ mez-Go ´ mez et al.