The synthesis of apocarotenoids is initiated by the oxidative cleavage of double bonds in carotenoid Keywords apocarotenoids; carotenoid cleavage; carotenoid dioxygenase; geranial; lycop
Trang 1dioxygenase 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 C15
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 (C13) and mycor-radicin (C14) derivatives [12], all of which are apoca-rotenoids conferring a yellow pigmentation to infected roots [13] Apocarotenoids, such as bixin in Bixa orell-ana [14] and saffron in Crocus sativus [15], are plant pigments of economic value
The synthesis of apocarotenoids is initiated by the oxidative cleavage of double bonds in carotenoid
Keywords
apocarotenoids; carotenoid cleavage;
carotenoid dioxygenase; geranial; lycopene
cleavage
Correspondence
S Al-Babili, Institute for Biology II, Cell
Biology, Albert-Ludwigs University of
Freiburg, Schaenzlestr 1, D-79104 Freiburg,
Germany
Fax: +49 761 203 2675
Tel: +49 761 203 8454
E-mail: salim.albabili@biologie.uni-freiburg.de
(Received 19 September 2008, revised 25
November 2008, accepted 26 November
2008)
doi:10.1111/j.1742-4658.2008.06820.x
Carotenoid cleavage products – apocarotenoids – include biologically active compounds, such as hormones, pigments and volatiles Their biosynthesis
is initiated by the oxidative cleavage of C–C double bonds in carotenoid backbones, leading to aldehydes and⁄ or ketones This step is catalyzed by carotenoid oxygenases, which constitute an ubiquitous enzyme family, including the group of plant carotenoid cleavage dioxygenases 1 (CCD1s), which mediates the formation of volatile C13ketones, 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
C8 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 (C10), 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.
Trang 2backbones, 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
basis of their substrate specificity, VP14 and its
ortho-logs have been termed 9-cis-epoxy-carotenoid
dioxy-genases Plants possess a second group of carotenoid
oxygenases, carotenoid cleavage dioxygenases (CCDs),
which act on different carotenoid substrates The
CCDs of higher plants contribute to diverse
physio-logical processes, including the regulation of the
out-growth of lateral shoot buds [18–20] and plastid
development [21]
Plants release volatile apocarotenoids, including C13
ketones such as b-ionone and damascone [22], which
constitute an essential aroma note in tea, grapes, roses,
tobacco and wine [23] Such compounds may arise by
unspecific oxidative degradation or lipid co-oxidation
processes, involving lipoxygenases [24] Alternatively,
they are produced by double bond-specific cleavage
reactions mediated by peroxidases [25] or by CCDs
such as members of the plant CCD1 subfamily Plant
CCD1s cleave numerous cyclic and linear
all-trans-car-otenoids at the C9–C10 and C9¢–C10¢ double bonds
into C14 dialdehydes, which are common to all
carot-enoid substrates, and two variable end-group-derived
C13 ketones [6,7,16] The wide substrate specificity of
plant CCD1s allows the production of divergent
vola-tile C13compounds, 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 formation of the C8 ketone 6-methyl-5-hepten-2-one [32] In this study, we investigated the enzymatic activi-ties of the sole CCD1 [Oryza sativa CCD1 (OsCCD1)] occurring in rice Our study revealed the C7¢–C8¢ dou-ble bond of linear and monocyclic carotenoids to be
an additional novel cleavage site of OsCCD1, leading
to geranial and indicating a novel plant route for the formation of this widespread volatile compound
Results
Purified OsCCD1 cleaves acyclic apolycopenals at the C7–C8 double bond
To investigate the activity of OsCCD1, the correspond-ing cDNA was cloned and expressed as a glutathione S-transferase (GST)-fusion protein in Escherichia coli cells However, the insolubility of the fusion protein, which could not be improved by modulating the expression conditions, hampered its purification Therefore, the GST–OsCCD1 fusion was expressed in BL21(DE3) E coli cells harboring the vector pGro7, which encodes the chaperones groES–groEL, enhanc-ing correct foldenhanc-ing This resulted in a strikenhanc-ing improve-ment of the GST–OsCCD1 solubility, allowing protein purification (Fig S1)
It has been shown that CCD1s from Arabidopsis and maize maintain their regional specificity in cleav-ing the C9–C10 double bond with the synthetic apoca-rotenoid b-apo-8¢-carotenal (C30), forming b-ionone (C13) and the C17 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 (C27), which is shorter than b-apo-8¢-carotenal (C30), and the acyclic substrates apo-10¢-lycopenal (C27) and apo-8¢-lycopenal (C30) 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 C14 dialdehyde apo-10,10¢-carotendial (rosafluene dialdehyde; prod-uct 1) and b-ionone (C13; product 2), as confirmed by
Trang 3LC-MS and GC-MS analyses, respectively (data not shown) The relatively low amount of the C14 dialde-hyde is probably a result of instability The formation
of b-ionone from b-apo-8¢-carotenal (C30) and b-apo-10¢-carotenal (C27) suggested that the cleavage of the C9–C10 double bond occurs independently of the chain length of monocyclic apocarotenals, pointing to the ionone ring as a determinant governing the reac-tion site
The two acyclic substrates were cleaved almost com-pletely within 30 min (Table S1), proving them to be
as suitable as the monocyclic apocarotenals However,
as shown in the HPLC analyses (Fig 1), the cleavage
of apo-10¢-lycopenal (C27; substrate II) led to a more complex mixture of products, including three com-pounds (products 1, 4 and 5) identified as dialdehydes
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 C14 and a C19 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 C14and C19 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 C17 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 C14dialdehyde (product 1), it was derivatized with O-methyl-hydroxylamine-hydrochloride prior to LC-MS analyses As shown in Fig 2, derivatized
A
B
C
Fig 1 (A) HPLC analyses of OsCCD1 in vitro assays with synthetic apocarotenoids The cyclic b-apo-10¢-carotenal (C 27 , I) was cleaved into apo-10,10¢-carotendial (C 14 , 1) and b-ionone (C13, 2); the lower amount of the former is probably a result of its instability The acy-clic substrates apo-10¢-lycopenal (C 27 , II) and apo-8¢-lycopenal (C 30 , III) were converted into pseudoionone (3) and three dialdehydes of different chain lengths (1, 4 and 5 from II; 4, 6 and 7 from III), as indicated by their UV ⁄ visible-spectra (shown in the insets) and retention times (B) Structures of the synthetic substrates showing the cleavage I, b-apo-10¢-carotenal; II, apo-10¢-lycopenal; III, apo-8¢-lycopenal The substrates were cleaved at three different double bonds, including the novel site (b, shaded) Cleavage of the C9–C10 double bond (a) results in products 1 and 2 from I, products 1 and
3 from II, and products 3 and 4 from III The cleavage of (b) leads
to product 4 from II, and product 6 from III The cleavage of the C5–C6 double bond (c) leads to product 5 from II, and prod-uct 7 from III (C) Strprod-uctures of the cleavage prodprod-ucts detected: 1, apo-10,10¢-carotendial (C 14 dialdehyde); 2, b-ionone (C 13 ); 3, pseudoionone (C13); 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).
Trang 4product 1 exhibited an [M + (NCH3)2 + H]+
molec-ular ion of mass 275.17 (substrate I), consistent with
the C14 dialdehyde structure, and the molecular ions
for products 4 and 5 (Fig 2; substrates II and III)
proved their identities as C17 and C19 dialdehydes,
respectively
HPLC analyses of the incubation with apo-8¢-lyco-penal (C30; substrate III in Fig 1) confirmed novel cleavage of the C7–C8 double bond As shown in Fig 1, the reaction led to an equivalent series of C17,
C20and C22dialdehydes (products 4, 6 and 7), corre-sponding to the cleavage of the C9–C10, C7–C8 and C5–C6 double bonds, respectively The nature of these dialdehyde products was confirmed by LC-MS anal-yses (data not shown)
Cleavage of the three double bonds described above must also lead to the three different mono-oxygenated products pseudoionone (C13), geranial (C10) and 6-methyl-5-hepten-2-one (C8) (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 (C13; product 1) and rosafluene-dialde-hyde (C14; 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 C17 and C19 dialde-hydes (Fig 3; products 4 and 5, respectively) from 3-OH-c-carotene (Fig 3; substrate II) Owing to its instability, the third dialdehyde (C14), expected from the cleavage of the C9–C10 and C9¢–C10¢ double bonds, occurred at low levels only (Fig 3; product 2) The formation of the C14, C17 and C19 dialdehydes (for structures, see Fig 3C) suggested a single cut at the C9–C10 double bond of the ring-bearing side of 3-OH-c-carotene in combination with several cleavage options in the linear half of the molecule, namely at the C9¢–C10¢, C7¢–C8¢ and C5¢–C6¢ double bond The occurrence of the C17 dialdehyde confirmed the novel site at the C7¢–C8¢ double bond observed with apoly-copenals and implied the formation of geranial Accordingly, the GC-MS analyses of the in vitro assays pointed to the conversion of 3-OH-c-carotene into
Fig 2 LC-MS identification of the dialdehydes produced in vitro.
The three dialdehydes formed from apo-10¢-lycopenal in vitro
(Fig 1; products 1, 4 and 5) represent a C14, a C17and a C19
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).
Trang 5geranial (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 (NIST) library (mass spectral search program Ver-sion 2.0) In addition, these GC-MS analyses (data not shown) revealed the formation of the known plant CCD1 mono-oxo products 6-methyl-5-hepten-2-one, 3-OH-b-ionone and pseudoionone, the latter two of which had already been detected in the HPLC analyses (Fig 3; products 1 and 3)
The high lipophilicity of lycopene did not allow the use of octyl-b-glucoside as detergent in the correspond-ing in vitro assays Therefore, lycopene micelles were produced with a Triton X mixture The products formed from this substrate (Fig 3; substrate III, prod-ucts 3, 4 and 5) suggested the cleavage of the double bond combinations C9–C10⁄ C7¢–C8¢, C9–C10 ⁄ C5¢–C6¢ and their symmetrical counterparts The C14 dialde-hyde formed by cleavage of the C9–C10⁄ C9¢–C10¢ double bond combination was only detectable in longer incubations (data not shown) GC-MS analyses
of the lycopene incubations demonstrated the forma-tion of pseudoionone and 6-methyl-5-hepten-2-one However, geranial could not be detected, although the formation of the C17 dialdehyde confirmed the cleav-age of lycopene at the novel C7–C8 or C7¢–C8¢ site
OsCCD1 catalyzes the formation of three different volatiles from lycopene in vivo
To confirm cleavage at the C7–C8 or C7¢–C8¢ double 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 (C13, 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 (C13, 3), apo-8,10¢-carotendial (C 17 dialdehyde, 4) and apo-6,10¢-caro-tendial (C19 dialdehyde, 5) Longer incubations with lycopene also resulted in the accumulation of the C 14 dialdehyde apo-10,10¢-caro-tendial corresponding to compound 2 (not shown) (B) Structures of the substrates showing the cleavage sites, including the novel C7–C8 ⁄ C7¢–C8¢ double bonds (b, b¢, shaded) I, zeaxanthin; II, 3-OH-c-carotene; III, lycopene OsCCD1 cleaves zeaxanthin at the C9–C10 ⁄ C9¢–C10¢ double bonds (a ⁄ a¢) 3-OH-c-Carotene (II) is cleaved at the C9–C10 double bond (a) in combination with the C9¢–C10¢ double bond (a¢), the C7¢–C8¢ double bond or the C5¢–C6¢ (c¢) double bond Lycopene (III) is cleaved either at the C9–C10 dou-ble bond (a) in combination with one of the three doudou-ble bonds C9¢–C10¢ (a¢), C7¢–C8¢ (b¢) or C5¢–C6¢ (c¢), or at the C9¢–C10¢ double bond (a¢) in combination with one of the a, b or c sites (C) Structures of the cleavage products detected.
Trang 6accumulating E coli cells, and volatile compounds were collected from the medium and analyzed by
GC-MS As shown in Fig 4, the activity of the enzyme resulted in the accumulation of 6-methyl-5-hepten-2-one (I), the reduced form of geranial, geraniol (II), and pseudoionone (III) The three compounds, undetect-able in the controls, were identified by their correct [M]+molecular ions and by comparing the mass
spec-A
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 (C17, C20 and C22) 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 kmax 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 uOsCCD1-expres-sing SPME and subjected to GC-MS (I–III) As suggested by the [M] + molecular ions (bold) and compari-son of mass spectra with the NIST library, the in vivo activity of OsCCD1 led to the volatiles 6-methyl-5-hepten-2-one (C8, I), gera-niol (C10, II) and pseudoionone (C13, III) IV represents the detection
of geranial (C 10 ) produced from 3-OH-c-carotene in vitro.
Trang 7tra with the NIST library HPLC analyses of the
corre-sponding cell pellets revealed the accumulation of a
complex mixture of compounds, tentatively identified
as dialcohols corresponding to three dialdehydes
described above (data not shown)
OsCCD1 exhibits different preferences for the
three cleavage sites
To gain insights into the preference of OsCCD1 for
the three cleavage sites, we determined the relative
amounts of the C17, C20 and C22 dialdehyde products
formed upon incubation with apo-8¢-lycopenal (C30),
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 C17 dialdehyde, which accounted
for about 90% of the total dialdehyde products
(Fig 5A) The relative amount (about 7%) of the C20
dialdehyde was much higher than of the C22
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 C14 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 C17 and C19 dialdehydes, respectively
The higher relative amount of the C17dialdehyde
indi-cated a higher preference for the C7–C8 than for the
C5–C6 double bond in 3-OH-c-carotene, whereas the
opposite tendency was observed with lycopene and
apo-10¢-lycopenal (Fig 5B)
Discussion
Plant CCD1s are known to catalyze the cleavage of
the C9–C10 and the C9¢–C10¢ double bonds of several
carotenoids Recently, it was shown that these enzymes
can also cleave at the C5–C6 and⁄ or the C5¢–C6¢
dou-ble bonds in lycopene [32] This was deduced from
GC-MS analyses of lycopene-accumulating E coli cells
expressing different plant CCD1s and from in vitro
incubations with a CCD1 from maize (ZmCCD1),
showing in both cases the formation of
6-methyl-5-hepten-2-one (C8), besides pseudoionone Geranial
(C10) was not detected, and cleavage of the C7–
C8⁄ C7¢–C8¢ double bonds was therefore excluded [32]
On the basis of our recent work on a Nostoc
caroten-oid oxygenase producing geranial and derivatives
thereof from monocyclic carotenoids [40], we assumed
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 C17 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-essary to soulibilize lycopene, but led to an overall reduction of enzyme activity (Table S1) Therefore, we used the more polar 3-OH-c-carotene, which could be incorporated into octyl-b-glucoside micelles and which was readily cleaved at the double bond combinations C9–C10⁄ C9¢–C10¢, C9–C10⁄ C7¢–C8¢ and C9–C10⁄ C5¢–C6¢
The formation of multiple dialdehyde products allows some conclusions to be drawn on the site pref-erences of OsCCD1 For instance, the C14, C17 and
C19 dialdehydes formed from lycopene and 3-OH-c-carotene arise from cleavage of the C9–C10 double bond, which is combined with the C9¢–C10¢, C7¢–C8¢
or C5¢–C6¢ double bonds The C9–C10 ⁄ C9¢–C10¢ dou-ble bonds constitute the main site, as suggested by the determination of the relative amounts of the dialdehy-des produced from apo-8¢-lycopenal; the C9–C10 ⁄ C9¢– C10¢ double bonds also constitute the sole cleavage site
in ring-bearing moieties of substrates This preference may explain the absence of the C20, C24 and C22 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
Trang 8substrates This is shown by the different preferences
for the C7–C8 and C5–C6 double bonds (Fig 5) in
apo-10¢-lycopenal (C27) and apo-8¢-lycopenal (C30),
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 C19
and C17 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
b-ionone end-group may determine the reaction site in
the polyene chain This may provide an explanation for
the ‘wobbling’ of the enzyme on linear substrate
moieties, where three different double bonds are being
recognized Similar results were obtained with the
cyanobacterial enzyme SynACO, representing up to
now the only carotenoid oxygenase with a known
crys-tal structure [42] SynACO cleaved b-apocarotenoids
with different chain lengths at a sole site, the C15–C15¢
double bond, leading to retinal and derivatives thereof
[43] In contrast, apolycopenals were cleaved at multiple
positions, including the C15–C15¢ double bond,
and indicating ‘wobbling’ of the enzyme (S Ruch,
S Al-Babili and P Beyer; unpublished data)
Owing to the high sequence similarity of plant
CCD1s, it appears likely that the cleavage of the
C7–C8⁄ C7¢–C8¢ double bonds of linear substrates is
not unique to OsCCD1 Apart from the formation of
geranial, the OsCCD1 cleavage reactions were identical
to those of other plant CCD1s, as supported by the
formation of pseudoionone, 6-methyl-5-hepten-2-one
and b-ionones Geranial is biologically active and
known to exert antifungal and antimicrobial activities
[44,45]; it represents a major volatile of tomato fruits
[46] and roses [47] Moreover, citral, the mixture of
geranial and its cis-isomer neral, is a major component
of the aroma of lemon grass and other lemon-scented
plants Geranial is synthesized from geranyl
diphos-phate by the enzymes geraniol synthase [48] and
gera-niol dehydrogenase [49] The enzymatic cleavage of
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 94C, followed by 32 cycles of 30 s at 94C, 30 s at 58 C, and
2 min at 68C, and 10 min of final polymerization at
68C The obtained PCR product was purified using GFX PCR DNA and a Gel Band Purification Kit (Amersham Biosciences, Piscataway, NJ, USA), and cloned into the pCR2.1–TOPO vector and pBAD⁄ TOPO (Invitrogen) to yield pCR–OsCCD1 and pBAD–OsCCD1, respectively The nature of the cDNA was verified by sequencing To express OsCCD1 as a GST-fusion protein, the correspond-ing cDNA was excised as an EcoRI fragment from pCR–OsCCD1, and then ligated into accordingly digested and alkaline phosphatase-treated pGEX–5X-1 (Amersham Biosciences) to yield pGEX–OsCCD1
Protein expression and purification pGEX–OsCCD1 was transformed into BL21(DE3) E coli cells harboring the plasmid pGro7 (Takara Bio Inc.; Mobitec, Go¨ttingen, Germany), which encodes the groES–groEL chaperone system under the control of an arabinose-inducible promoter Overnight cultures (2.5 mL) were inoculated into
50 mL of 2· YT medium containing 0.2% (w ⁄ v) arabinose, grown at 28C to a D600 nmof 0.7, and induced with 0.2 mm isopropyl thio-b-d-galactoside for 4 h Cells were harvested
by centrifugation (10 min, 6000 g), resuspended in 4 mL of NaCl⁄ Pi(pH 7.3), and lysed using a French press Six millili-ters of NaCl⁄ Pi (pH 7.3) containing 1% Triton X-100 was then added, and the suspension was incubated at room 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⁄ Pi (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
Trang 9Enzyme 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 kmaxvalues, using extinction coefficients
calculated from E1% [51] Protein concentrations were
determined using the BioRad protein assay kit (BioRad,
Hercules, CA, USA)
In vitroassays 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
was resuspended in 200 lL of benzene and mixed with
150 lL of an ethanolic detergent mixture consisting of
0.7% (v⁄ v) Triton X-100 and 1.6% (v ⁄ v) Triton X-405
The mixture was then dried using a vacuum centrifuge to
produce a carotenoid-containing gel The gel was
resus-pended in 110 lL of 2· incubation buffer containing
2 mm tris(2-carboxyethyl)phosphine, 0.6 mm FeSO4 and
2 mgÆmL)1 catalase (Sigma, Deisenhofen, Germany) in
200 mm Hepes⁄ NaOH (pH 7.8) One hundred microliters
of this lycopene suspension was then used in the in vitro
assay, which was started by adding water and purified
Os-CCD1 to obtain a final volume of 200 lL
3-OH-c-Caro-tene, zeaxanthin and apocarotenoids were solubilized using
octyl-b-glucoside at a final concentration of 1% (v⁄ v) For
this purpose, substrates were mixed with 50 lL of a 4%
octyl-b-glucoside ethanolic solution, dried using a vacuum
centrifuge, and resuspended in 100 lL of the 2·
incuba-tion buffer menincuba-tioned above Water and purified OsCCD1
were then added to obtain the final volume of 200 lL
Depending on the substrates, the incubations were
per-formed at 28C for 4 h (lycopene and 3-OH-c-carotene),
2 h (zeaxanthin) or 30 min (synthetic substrates)
Reac-tions were stopped by adding two volumes of acetone
Lipophilic compounds were partitioned against petroleum
ether⁄ diethyl ether 1 : 4 (v ⁄ v), vacuum-dried, and
dis-solved in 40 lL of chloroform HPLC analyses were then
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 kmax 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 28C to a D600 nm of 0.5, and induced with 0.08% (w⁄ v) arabinose Cells were harvested after 6 h, and volatile compounds were collected by introducing the SPME fiber into the cell-free medium for 30 min For HPLC analyses, cells were harvested after 4 h, and carote-noids were extracted and processed as described above
Analytical methods For HPLC analyses, a Waters system equipped with a photodiode array detector (model 996) was used A
C30-reversed phase column (YMC Europe, Schermbeck, Germany) was developed with solvent system B [MeOH⁄ t-butylmethyl ether⁄ water (60 : 2 : 20, v ⁄ v ⁄ v)] and solvent system A [MeOH⁄ t-butylmethyl ether (1 : 1, v ⁄ v)] at a flow rate of 1 mLÆmin)1, using a linear gradient from 100% solvent B to 43% solvent B within 45 min, and then to 0% solvent B within 1 min The final conditions were main-tained for 26 min at a flow rate of 2 mLÆmin)1, and this was followed by re-equilibration
LC-MS analyses of compounds collected from HPLC were performed using a Thermo Finnigan LTQ mass spec-trometer coupled to a Surveyor HPLC system consisting of
a Surveyor Pump Plus, Surveyor PDA Plus and Surveyor Autosampler Plus (Thermo Electron, Waltham, MA, USA) Separations were carried out using a YMC-Pack C30-reversed phase column (150· 3 mm internal diameter,
3 lm) Separation and identification of the C17 and C19 dialdehydes was performed as described in [40] The oxime
of the C14 dialdehyde was produced by adding 50 lL of O-methyl-hydroxylamine-hydrochloride (15 mgÆmL)1) to an MeOH solution of the HPLC-purified compound, and then incubating for 20 min at 50C 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: 50C held isocratically for 5 min, followed by a ramp of 25CÆmin)1 to a final
Trang 10temperature of 340C, which was maintained for an
addi-tional 5 min The He carrier gas flow was maintained at
1 mLÆmin)1 using a split flow of 1 : 20 The splitless time
was 3 min, and the injector oven temperature was set at
220C Standard electrospray ionization was used at an
ion source potential of 70 eV and with an ion source
tem-perature of 200C Identification of compounds was done
by comparing the mass spectra with the NIST database
Acknowledgements
This work was supported by the HarvestPlus
pro-gramme (http://www.harvestplus.org) and by the
Deut-sche Forschungsgemeinschaft (DFG), Grant 892⁄ 1-3
We are indebted to J Mayer for valuable discussions
We thank H Ernst for providing the synthetic
sub-strates and E Scheffer for skilful technical assistance
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