Characterization of the rice carotenoid cleavage
dioxygenase 1 reveals a novel route for geranial
biosynthesis
Andrea Ilg, Peter Beyer and Salim Al-Babili
Faculty of Biology, Institute of Biology II, Albert-Ludwigs University of Freiburg, Germany
Carotenoids are isoprenoid pigments synthesized by all
photosynthetic organisms and some nonphotosynthetic
bacteria and fungi. In plants, carotenoids are essential
in protecting the photosynthetic apparatus from
photo-oxidation, and represent essential constituents of
the light-harvesting and of the reaction center com-
plexes [1–4]. Carotenoids are also the source of apoca-
rotenoids [5–7], which are physiologically active
compounds, including the ubiquitous chromophore
retinal, the chordate morphogen retinoic acid and the
phytohormone abscisic acid as the best-known exam-
ples. Further carotenoid-derived signaling molecules
are represented by strigolactones, a group of C
15
apoc-
arotenoids attracting both symbiotic arbuscular mycor-
rhizal fungi and parasitic plants [8,9] and, as recently
shown, exerting functions as novel plant hormones reg-
ulating shoot branching [10,11]. In addition, the devel-
opment of arbuscular mycorrhiza is also accompanied
by accumulation of cyclohexenone (C
13
) and mycor-
radicin (C
14
) derivatives [12], all of which are apoca-

ketones, such as b-ionone, by
cleaving the C9–C10 and C9¢–C10¢ double bonds of cyclic and acyclic
carotenoids. Recently, it was reported that plant CCD1s also act on the
C5–C6 ⁄ C5¢–C6¢ double bonds of acyclic carotenes, leading to the volatile
C
8
ketone 6-methyl-5-hepten-2-one. Using in vitro and in vivo assays,
we show here that rice CCD1 converts lycopene into the three different
volatiles, pseudoionone, 6-methyl-5-hepten-2-one, and geranial (C
10
),
suggesting that the C7–C8 ⁄ C7¢–C8¢ double bonds of acyclic carotenoid
ends constitute a novel cleavage site for the CCD1 plant subfamily. The
results were confirmed by HPLC, LC-MS and GC-MS analyses, and
further substantiated by in vitro incubations with the monocyclic caroten-
oid 3-OH-c-carotene and with linear synthetic substrates. Bicyclic carote-
noids were cleaved, as reported for other plant CCD1s, at the C9–C10 and
C9¢–C10¢ double bonds. Our study reveals a novel source for the widely
occurring plant volatile geranial, which is the cleavage of noncyclic ends of
carotenoids.
Abbreviations
CCD, carotenoid cleavage dioxygenase; GST, glutathione S-transferase; NIST, National Institute of Standards and Technology; OsCCD1,
Oryza sativa carotenoid cleavage dioxygenase 1; SPME, solid-phase microextraction.
736 FEBS Journal 276 (2009) 736–747 Journal compilation ª 2008 FEBS. No claim to original German government works
backbones, generally catalyzed by carotenoid oxygen-
ases, nonheme iron enzymes that are common in all
taxa [5–7,16]. VP14 (viviparous14) from maize, which
catalyzes the formation of the abscisic acid precursor
xanthoxin by cleaving 9-cis-epoxy carotenoids [17], is
the first identified member of this enzyme class. On the

13
compounds, including b-ionophores, a-ionones,
pseudoionone and geranylacetone. The first member of
the CCD1 subfamily was identified from Arabidopsis
thaliana [26], and was later shown to act as a
dioxygenase [27]. Sequence homology then allowed the
identification and charcterization of orthologs from
several plant species, such as crocus, tomato, grape,
melon, petunia and maize [15,28–32].
The biological function of CCD1s was confirmed by
loss-of-function experiments in tomato fruits and petu-
nia flowers, leading to decreased emission of b-ionone
[27,31]. Moreover, recent studies on the CCD1 from
maize indicated its involvement in the formation of
cyclohexenone and mycorradicin in mycorrhizal roots
[33]. Underscoring a role of CCD1 in carotenoid
catabolism, seeds of Arabidopsis ccd1 mutants con-
tained elevated carotenoid levels [34]. This suggested
that the modification of CCD1 expression is instru-
mental for altering volatile production contributing to
taste, or in increasing the carotenoid content in crop
plant tissues where elevated provitamin A carotenoid
levels are aimed for, such as high-b-carotene tomato
[35,36], canola [37], golden rice [38] or golden potato
[39]. Hence, the identification of substrates and cleav-
age sites of CCD1s from crop plants is considered to
be a worthwhile approach.
It has recently been discovered that plant CCD1s
exert additional activity at the C5–C6 and ⁄ or the C5¢–
C6¢ double bonds of acyclic carotenoids, leading to the

), forming b-ionone
(C
13
) and the C
17
dialdehyde apo-8,10¢-carotendial
[27,32]. To test the cleavage activity of OsCCD1,
in vitro assays were performed with this substrate,
using purified enzyme. Subsequent HPLC analyses
(data not shown) revealed a cleavage pattern identical
to that of the plant CCD1s mentioned above. To
determine the impact of the chain length and of the io-
none ring on the cleavage pattern, purified OsCCD1
was incubated with b-apo-10¢-carotenal (C
27
), which is
shorter than b-apo-8¢-carotenal (C
30
), and the acyclic
substrates apo-10¢-lycopenal (C
27
) and apo-8¢-lycopenal
(C
30
). HPLC analyses of the incubation with the cyclic
b-apo-10¢-carotenal (Fig. 1; substrate I) revealed an
almost complete conversion of this substrate
(Table S1) and the formation of the C
14
dialdehyde

on the basis of the fine structure of the corresponding
UV–visible spectra. The three dialdehydes differed in
their chain lengths, as indicated by their retention
times and absorption maxima. Products 1 and 5 were
supposed to represent a C
14
and a C
19
dialdehyde,
respectively. These are expected to arise upon cleavage
at the known plant CCD1 sites (C9–C10 and C5–C6).
The retention time of product 4 indicated a chain
length between C
14
and C
19
. This pointed to the recog-
nition of a novel cleavage site, at the C7–C8 double
bond, between the above mentioned C9–C10 and
C5–C6 positions, yielding a C
17
dialdehyde. To con-
firm their nature, the dialdehyde products, 1, 4 and 5,
were purified and analyzed by LC-MS. In order to sta-
bilize the C
14
dialdehyde (product 1), it was derivatized
with O-methyl-hydroxylamine-hydrochloride prior to
LC-MS analyses. As shown in Fig. 2, derivatized
A

14
dialdehyde); 2, b-ionone (C
13
); 3,
pseudoionone (C
13
); 4, apo-8,10¢-carotendial (C
17
dialdehyde); 5,
apo-6,10¢-carotendial (C
19
dialdehyde); 6, apo-8,8¢-carotendial (C
20
dialdehyde); 7, apo-6,8¢-carotendial (C
22
dialdehyde).
A novel route for geranial formation A. Ilg et al.
738 FEBS Journal 276 (2009) 736–747 Journal compilation ª 2008 FEBS. No claim to original German government works
product 1 exhibited an [M + (NCH
3
)
2
+H]
+
molec-
ular ion of mass 275.17 (substrate I), consistent with
the C
14
dialdehyde structure, and the molecular ions
for products 4 and 5 (Fig. 2; substrates II and III)

6-methyl-5-hepten-2-one (C
8
) (for structures, see
Fig. 4). Pseudoionone was found by HPLC analysis
(Fig. 1; product 3) and its presence was further demon-
strated by GC-MS analyses, which also showed the
formation of geranial and and 6-methyl-5-hepten-2-one
(data not shown).
OsCCD1 mediates double cleavage of
different site combinations in 3-OH-c-carotene
and lycopene
To determine the cleavage sites in natural substrates,
purified OsCCD1 was incubated with the bicyclic
zeaxanthin, the monocyclic 3-OH-c-carotene and the
acyclic lycopene. As shown in Fig. 3, OsCCD1 con-
verted zeaxanthin (substrate I) into the two products
3-OH-b-ionone (C
13
; product 1) and rosafluene-dialde-
hyde (C
14
; product 2), as confirmed by LC-MS and
GC-MS analyses, respectively (data not shown). This
suggested that OsCCD1 cleaves the C9–C10 and
C9¢–C10¢ double bonds of bicyclic carotenoids, like
other plant orthologs.
Although it occurred at lower conversion rates than
with the synthetic substrates (Table S1), we clearly
observed the accumulation of the C
17

The three dialdehydes formed from apo-10¢-lycopenal in vitro
(Fig. 1; products 1, 4 and 5 ) represent a C
14
,aC
17
and a C
19
dialde-
hyde, respectively, as suggested by their molecular ions of mass
275.17 (I, [M + (NCH
3
)
2
+H]
+
; product 1), 257.13 (II,[M+H]
+
;
product 4) and 283.14 (III,[M+H]
+
; product 5).
A. Ilg et al. A novel route for geranial formation
FEBS Journal 276 (2009) 736–747 Journal compilation ª 2008 FEBS. No claim to original German government works 739
geranial (Fig. 4; substrate IV), as indicated by the
detection of the expected [M]
+
molecular ion of mass
152.3 and the fragmentation pattern (Fig. 4;
substrate IV), which was correctly recognized by the
National Institute of Standards and Technology

bond in vivo, OsCCD1 was expressed in lycopene-
A
B
C
Fig. 3. (A) HPLC analyses of OsCCD1 in vitro incubations with
three natural carotenoids. UV–visible spectra of the products are
shown in the insets. The bicyclic zeaxanthin (I) was converted into
3-OH-b-ionone (1) and apo-10,10¢-carotendial (C
14
, 2). The products
3-OH-b-ionone (C
13
, 1), apo-10,10¢-carotendial (C
14
dialdehyde, 2),
pseudoionone (C
13
, 3), apo-8,10¢-carotendial (C
17
dialdehyde, 4) and
apo-6,10¢-carotendial (C
19
dialdehyde, 5) were obtained from 3-OH-
c-carotene (II). The cleavage of lycopene (III) led to pseudoionone
(C
13
, 3), apo-8,10¢-carotendial (C
17
dialdehyde, 4) and apo-6,10¢-caro-
tendial (C

B
Fig. 5. Determination of the relative amounts of dialdehyde prod-
ucts. (A) Incubations with apo-8¢-lycopenal (C
30
). The peak areas of
the three dialdehydes (C
17
,C
20
and C
22
) formed from apo-8¢-lyco-
penal were calculated at a max. plot of 350–550 nm. The values
represent the proportion of each dialdehyde in the sum of the three
peak areas. (B) Incubations with apo-10¢-lycopenal (C
27
), 3-OH-c-car-
otene and lycopene. The values represent ratios of the C
17
and C
19
dialdehydes in the sum of their peak areas calculated by integrating
each peak at its individual k
max
. Data represent the average of six
independent incubations.
Fig. 4. GC-MS analyses of volatile OsCCD1 products. Volatile com-
pounds produced in lycopene-accumulating and OsCCD1-expres-
sing cells were collected using SPME and subjected to GC-MS
(I–III). As suggested by the [M]

22
dialdehyde products
formed upon incubation with apo-8¢-lycopenal (C
30
),
corresponding to C9–C10, C7–C8 and C5–C6 double
bond recognition, respectively (Fig. 1B; substrate III).
The enzyme exhibited by far the highest preference for
the C9–C10 double bond in vitro, as suggested by the
predominance of the C
17
dialdehyde, which accounted
for about 90% of the total dialdehyde products
(Fig. 5A). The relative amount (about 7%) of the C
20
dialdehyde was much higher than of the C
22
dialde-
hyde (about 2%), indicating a higher affinity for the
novel C7–C8 double bond than for the C5–C6 double
bond. The instability of the C
14
dialdehyde arising
from the double cleavage at the C9–C10 ⁄ C9¢–C10¢
double bonds in lycopene and 3-OH-c-carotene ham-
pered determination of the preference for these sites,
and allowed only a comparison of the C9–C10 ⁄
C7¢–C8¢ and C9–C10 ⁄ C5¢–C6¢ cleavage products corre-
sponding to the C
17

that plant CCD1s may also be able to cleave the
C7–C8 ⁄ C7¢–C8¢ double bonds. Here, we demonstrate
that the rice enzyme OsCCD1 cleaves linear ends of
carotenoids at three different double bond positions,
including the C7–C8 ⁄ C7¢–C8¢ double bonds, leading to
geranial.
To avoid further metabolization of products that
can occur in vivo, we relied first on in vitro incubations
using purified enzyme, which allowed clear identifica-
tion of the products formed. In a first approach, we
checked the site specificities using synthetic apocarote-
nals packed in octyl-b-glucoside micelles. This enabled
us to observe the cleavage of the C7–C8 double bond.
However, the confirmation of this novel activity with
the highly lipophilic lycopene and c-carotene required
the use of different detergents. The best activities were
obtained with micelles produced with a Triton X mix-
ture, following the protocol of Prado-Cabrero et al.
[41]. The accumulation of the C
17
dialdehyde in the
lycopene assays confirmed the cleavage at the
C9–C10 ⁄ C7¢–C8¢ double bonds. However, the activities
determined were still low in comparison to the incuba-
tions with zeaxanthin, and did not allow clear identifi-
cation of geranial. Furthermore, we did not detect
significant conversion of lycopene in the 30 min incu-
bations used to determine the substrate preferences of
the enzyme (Table S1). This weak activity is probably
due to the use of the Triton X mixture, which was nec-

22
dial-
dehydes in the lycopene incubation, which would be
expected if the cleavage reactions occurred only at the
C7–C8 ⁄ C7¢–C8¢ and ⁄ or C5 ⁄ C6 ⁄ C5¢–C6¢ double bonds.
A further conclusion is that the first cleavage site plays
a role in the determination of the second one in acyclic
A novel route for geranial formation A. Ilg et al.
742 FEBS Journal 276 (2009) 736–747 Journal compilation ª 2008 FEBS. No claim to original German government works
substrates. This is shown by the different preferences
for the C7–C8 and C5–C6 double bonds (Fig. 5) in
apo-10¢-lycopenal (C
27
) and apo-8¢-lycopenal (C
30
),
which mimic a lycopene molecule cleaved at the C9¢–
C10¢ and C7¢–C8¢ double bonds, respectively. More-
over, comparison of the relative amounts of the C
19
and C
17
dialdehydes accumulated in the incubations
with the natural substrates lycopene and 3-OH-c-caro-
tene indicates that the preference of OsCCD1 for the
C5¢–C6¢ and C7¢–C8¢ double bonds depends on the
nature of the substrate.
The cleavage of a sole double bond in the cyclic moi-
ety of 3-OH-c-carotene and of monocyclic b-apocarote-
nals with different chain lengths indicates that the

monocyclic and acyclic carotenoids into geranial repre-
sents a novel biosynthetic route, and may provide an
explanation for the impact of lycopene accumulation
on the emission of geranial, as observed in the fruits of
several tomato and watermelon varieties [46], as well
as in transgenic tomato fruits, where elevated caroten-
oid levels were accompanied by an increased emission
of citral [50]. The possible synthesis of geranial by
tomato CCD1s is now under investigation.
Experimental procedures
Cloning procedures
Five micrograms of total RNA, isolated from 14-day-old
seedlings (O. sativa var. japonica cv. TP309), was used for
cDNA synthesis using SuperScript III RnaseH
)
(Invitrogen,
Paisley, UK), according to the instructions of the manufac-
turer. Two microliters of cDNA was then applied for the
amplification of OsCCD1 (accession no. AK066766,
encoded by Os12g0640600), using the primers CCD-1 (5¢-
ATGGGAGGCGGCGATGGCGATGAG-3¢) and CCD-2
(5¢-TCACGCTGATTGTTTTGCCAGTTG-3¢). The PCR
reaction was performed with 100 ng of each primer, 200 lm
dNTPs and 1 lL of Advantage cDNA Polymerase Mix
(BD Biosciences, San Jose, CA, USA) in the buffer pro-
vided, as follows: 2 min of initial denaturation at 94 °C,
followed by 32 cycles of 30 s at 94 °C, 30 s at 58 °C, and
2 min at 68 °C, and 10 min of final polymerization at
68 °C. The obtained PCR product was purified using GFX
PCR DNA and a Gel Band Purification Kit (Amersham

temperature for 30 min. After centrifugation for 10 min at
12 000 g, the fusion protein was purified using glutathione–
Sepharose 4B (Amersham Biosciences), according to the
manufacturer’s instructions. OsCCD1 was then released by
overnight treatment with the protease factor Xa in NaCl ⁄ P
i
(pH 7.3) at room temperature, according to the manu-
facturer’s instructions (Amersham Biosciences). The protein
eluate contained approximately 50% purified OsCCD1.
Purification steps and protein expression were monitored by
SDS ⁄ PAGE. The control strain expressed only GST.
A. Ilg et al. A novel route for geranial formation
FEBS Journal 276 (2009) 736–747 Journal compilation ª 2008 FEBS. No claim to original German government works 743
Enzyme assays
Synthetic substrates were kindly provided by BASF (Lud-
wigshafen, Germany). Lycopene was obtained from Roth
(Karlsruhe, Germany). Zeaxanthin and 3-OH-c-carotene
were isolated from E. coli cells transformed with carotenoid
biosynthetic genes (unpublished data). The substrates were
purified using TLC, and quantified spectrophotometrically
at their individual k
max
values, using extinction coefficients
calculated from E1% [51]. Protein concentrations were
determined using the BioRad protein assay kit (BioRad,
Hercules, CA, USA).
In vitro assays contained 40 lg of purified enzyme eluate
at substrate concentrations of 80 lm (lycopene and 3-OH-
c-carotene) or 40 lm (zeaxanthin and synthetic substrates).
For the production of lycopene micelles, dried substrate

performed using 20 lL of the extracts. For GC-MS analy-
ses, volatile compounds were collected with solid-phase
microextraction (SPME) fibers (100 lm polydimethylsil-
oxane; Sigma-Aldrich) for 30 min.
Conversion rates were determined in 30 min incubation
assays using 30 lg of purified enzyme eluate. For quantifi-
cation, 200 lL of an acetonic solution of a-tocopherole
acetate (1 mgÆmL
)1
) was added as internal standard to each
assay prior to extraction. The conversion rates were
determined by calculating the decrease of substrate peak
areas measured at their individual k
max
values using the
max plot function of the software empower pro (Waters,
Eschborn, Germany). Peak areas were normalized relative
to the peak area of the internal standard, which was quan-
tified at its absorption maximum of 285 nm.
In vivo test using lycopene-accumulating
E. coli cells
Lycopene-accumulating XL1-Blue E. coli cells (unpublished
data), harboring the corresponding biosynthetic genes from
Erwinia herbicola , were transformed with pBAD–OsCCD1
or with pBAD–TOPO as a negative control. Overnight cul-
tures were used to inoculate 50 mL of LB medium. Bacteria
were grown at 28 °CtoaD
600 nm
of 0.5, and induced with
0.08% (w ⁄ v) arabinose. Cells were harvested after 6 h, and

17
and C
19
dialdehydes was performed as described in [40]. The oxime
of the C
14
dialdehyde was produced by adding 50 lLof
O-methyl-hydroxylamine-hydrochloride (15 mgÆmL
)1
)toan
MeOH solution of the HPLC-purified compound, and then
incubating for 20 min at 50 °C. The product was then par-
titioned against petroleum ether ⁄ diethyl ether 1 : 4 (v ⁄ v).
The identification of the oxime was carried out according
to [40].
GC-MS analyses were carried out with a Finnigan Trace
DSQ mass spectrometer coupled to a Trace GC gas chro-
matograph equipped with a 30 m Zebron ZB 5 column
(5% phenylpolysilanoxane ⁄ 95% dimethylpolysilanoxane,
0.25 mm internal diameter, and 0.25 lm film thickness;
Phenomenex, Aschaffenburg, Germany). The temperature
program used was as follows: 50 °C held isocratically
for 5 min, followed by a ramp of 25 °CÆmin
)1
to a final
A novel route for geranial formation A. Ilg et al.
744 FEBS Journal 276 (2009) 736–747 Journal compilation ª 2008 FEBS. No claim to original German government works
temperature of 340 °C, which was maintained for an addi-
tional 5 min. The He carrier gas flow was maintained at
1mLÆmin

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