The Plant Cell, Vol. 26: 2223–2233, May 2014, www.plantcell.org ã 2014 American Society of Plant Biologists.

All rights reserved.

Carotene Hydroxylase Activity Determines the Levels of Both

a-Carotene and Total Carotenoids in Orange Carrots W

Jacobo Arango,a,1 Matthieu Jourdan,b Emmanuel Geoffriau,b Peter Beyer,a and Ralf Welscha,2
a University of Freiburg, Faculty of Biology II, Freiburg, Germany
b Agrocampus Ouest, Unité Mixte de Recherche 1345 Institut de Recherche en Horticulture et Semences, Angers, France
ORCID IDs: 0000-0003-3039-818X (M.J.); 0000-0003-4384-3107 (E.G.); 0000-0002-2865-2743 (R.W.)

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The typically intense carotenoid accumulation in cultivated orange-rooted carrots (Daucus carota) is determined by a high protein
abundance of the rate-limiting enzyme for carotenoid biosynthesis, phytoene synthase (PSY), as compared with white-rooted
cultivars. However, in contrast to other carotenoid accumulating systems, orange carrots are characterized by unusually high levels
of a-carotene in addition to b-carotene. We found similarly increased a-carotene levels in leaves of orange carrots compared with
white-rooted cultivars. This has also been observed in the Arabidopsis thaliana lut5 mutant carrying a defective carotene
hydroxylase CYP97A3 gene. In fact, overexpression of CYP97A3 in orange carrots restored leaf carotenoid patterns almost to those
found in white-rooted cultivars and strongly reduced a-carotene levels in the roots. Unexpectedly, this was accompanied by a 30 to
50% reduction in total root carotenoids and correlated with reduced PSY protein levels while PSY expression was unchanged. This
suggests a negative feedback emerging from carotenoid metabolites determining PSY protein levels and, thus, total carotenoid flux.
Furthermore, we identified a deficient CYP97A3 allele containing a frame-shift insertion in orange carrots. Association mapping
analysis using a large carrot population revealed a significant association of this polymorphism with both a-carotene content and
the a-/b-carotene ratio and explained a large proportion of the observed variation in carrots.

INTRODUCTION The first carotenoid-specific reaction of plastid prenyllipid bio-

Carrot (Daucus carota) roots exhibit a panoply of colors ranging
from yellow to orange and red (Surles et al., 2004; Arscott and
Tanumihardjo, 2010). This color spectrum is mostly determined
by the abundance of carotenoids, with the exception of purple
carrots, which also accumulate anthocyanins (Simon et al., 2008;
Montilla et al., 2011). The earliest cultivated carrots were yellow or
purple and emerged in the Afghanistan region before the 900s, while
orange and white cultivars were bred in the 16th century (Banga,
1957; Soufflet-Freslon et al., 2013). Despite significant progress in
the identification of genetic markers associated with root color in
recent years, knowledge of the molecular basis of diverse color
phenotypes is sparse (Santos and Simon, 2006; Just et al., 2009;
Fuentes et al., 2012; Rodriguez-Concepcion and Stange, 2013).
In carrot roots, carotenoids accumulate as crystals within chro-
moplasts (Maass et al., 2009; Kim et al., 2010; Wang et al., 2013). In
leaf chloroplasts, these pigments are mostly protein-bound, forming
essential constituents of light-harvesting complexes and pho-
tosynthetic reaction centers. Furthermore, carotenoids represent
ultimate precursors for at least two classes of plant hormones,
abscisic acid (ABA) and the strigolactones (Walter and Strack,
2011; Alder et al., 2012).
1 Current
address: International Center for Tropical Agriculture, Tropical
Forages, AA 6713 Cali, Colombia.
2 Address correspondence to ralf.welsch@biologie.uni-freiburg.de.
The author responsible for distribution of materials integral to the findings
presented in this article in accordance with the policy described in the
Instructions for Authors (www.plantcell.org) is: Ralf Welsch (ralf.welsch@
biologie.uni-freiburg.de).
W
Online version contains Web-only data.
www.plantcell.org/cgi/doi/10.1105/tpc.113.122127
synthesis is catalyzed by the enzyme phytoene synthase (PSY;
Figure 1) and yields the C40 hydrocarbon phytoene (for a review
on carotenoid biosynthesis, see Cazzonelli, 2011). Following
desaturation and isomerization reactions, which are catalyzed
by four enzymes in plants (phytoene desaturase, z-carotene
desaturase, z-carotene isomerase, and carotenoid isomerase),
the red-colored all-trans-lycopene is formed (Isaacson et al.,
2002; Park et al., 2002; Li et al., 2007). Two different cyclases
introduce e-ionone (e-cyclase, LCYe) and/or b-ionone rings
(b-cyclase, LCYb), yielding a-carotene (e-b-carotene) or b-carotene
(b-b-carotene), respectively.
The synthesis of the oxygen-containing xanthophylls from
either a- or b-carotene requires ring-specific hydroxylation reactions.
In Arabidopsis thaliana, these reactions are catalyzed by a set of
four enzymes (Kim et al., 2009). Two non-heme di-iron enzymes
(b-carotene hydroxylase 1 [BCH1] and BCH2) are primarily re-
sponsible for b-ring hydroxylation of b-carotene and produce
zeaxanthin, while two heme-containing cytochrome P450 enzymes
(CYP97A3 and CYP97C1) preferentially hydroxylate the e- and
b-ionone rings of a-carotene, yielding lutein. Zeaxanthin is further
epoxidized by the enzyme zeaxanthin epoxidase, leading to
antheraxanthin, violaxanthin, and neoxanthin, a reversible reaction
representing the xanthophyll cycle (Niyogi et al., 1998). The analysis
of leaf carotenoid patterns from hydroxylase-deficient Arabidopsis
mutants revealed partially overlapping substrate specificities of the
hydroxylases involved and reflect the requirement for the complete
set of hydroxylases to generate a balanced pigment composition
(Kim et al., 2009).
High carotenoid levels in non-green plant tissues have frequently
been found to be dependent on PSY expression, indicating this
enzyme to be rate-limiting in carotenogenesis. The overexpression
of PSY, e.g., in tomato fruits (Solanum lycopersicum), cassava
2224 The Plant Cell
Figure 1. The Carotenoid Biosynthesis Pathway in Plants.
Enzymes catalyzing specific reactions are indicated in boxes. Carotene
hydroxylases exhibiting main activity for the reaction are shown in bold,
while those with minor activities are shown in parentheses. The central
part covering C9 to C9’ is replaced with “R” in cyclic carotenoids (adapted
from Kim et al., 2009).
roots (Manihot esculenta), canola seeds (Brassica napus), and
rice endosperm (Oryza sativa), effectively increased the flux
through the biosynthetic pathway (Shewmaker et al., 1999; Ye
et al., 2000; Fraser et al., 2007; Welsch et al., 2010). Fluxes can
be driven at levels where even b-carotene crystals form. This
was observed in transgenic white-rooted carrots overexpressing
the bacterial PSY (crtB; Maass et al., 2009). Accordingly, high
PSY protein levels can be revealed by immunoblotting in or-
ange-rooted cultivars while being undetectable in white-rooted
cultivars. However, these large differences are not equivalently
reflected in PSY mRNA levels, suggesting the involvement of
additional regulatory mechanisms. Identification of the remain-
ing genes involved in carotenoid biosynthesis permitted analysis
of their expression during root development of various color
cultivars (Clotault et al., 2008). However, the results obtained
can only partially explain the different carotenoid patterns and
fail to account for the wide range of carotenoid levels found.
Compared with other carotenoid accumulating tissues, one
remarkable feature of orange carrots is the high abundance of
a-carotene, in addition to b-carotene. We found that unusually
high a-carotene levels are also present in leaves of orange-
rooted carrots. This is a distinctive characteristic of plants with
a deficiency in one cytochrome P450-type carotene hydroxylase,
as concluded from investigations of Arabidopsis mutants (Kim
and DellaPenna, 2006). In this work, we took advantage of this
knowledge and investigated whether the high a-carotene phe-
notype in carrots is attributed to such a defect by overexpressing
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the corresponding Arabidopsis carotene hydroxylase gene in
orange-rooted carrots. Our data show that this assumption is
correct inasmuch as the carotenoid pattern is concerned and
identified a feedback mechanism on total carotenoid content
that might be involved in carotenoid differences observed in
various carrot varieties.
RESULTS
a-Carotene Levels in Carrot Cultivars
Most cultivated orange carrots exhibit remarkably high a-carotene
levels, between 15 and 30% of the total carotenoid content, and
an accordingly high a-/b-carotene ratio of 0.2 up to 0.5 (Surles
et al., 2004; Clotault et al., 2008; Simon et al., 2008; Arscott and
Tanumihardjo, 2010). For instance, tomato fruits accumulate ;4%
a-carotene with an a-/b-carotene ratio of 0.02 (Stigliani et al.,
2011), and Arabidopsis leaves contain <1% a-carotene with an
a-/b-carotene ratio of ;0.03 (Kim et al., 2009). Furthermore,
root-specific overexpression of PSY in a white-rooted carrot
background (cultivar Queen Anne’s Lace [QAL]) increases the
total carotenoid amounts, but this is almost exclusively due to
elevated b-carotene while a-carotene remains at low levels. Simi-
larly, PSY overexpression in Arabidopsis roots results in significant
carotenoid accumulations with only 3% a-carotene and low
a-/b-carotene ratios (Maass et al., 2009).
We compared the carotenoid profiles in leaves from two
orange-rooted cultivars (Chantenay Red Cored [CRC] and
Nantaise [NAN]) with two white-rooted cultivars (QAL and Küttinger
[KUT]), which accumulate high and low PSY protein levels, re-
spectively (Maass et al., 2009). Table 1 shows that irrespective
of root color and PSY abundance, total leaf carotenoid levels
and the carotenoid/chlorophyll ratios were very similar and so
were the levels of the major xanthophyll lutein, an a-carotene
derivative. The difference to be noted is that the proportion of
a-carotene is strongly elevated, up to 10% of the total carotenoids,
in leaves of orange-rooted cultivars, compared with those of white-
rooted cultivars where only ;1% a-carotene was found. Based on
previous results where a-carotene levels were unchanged upon
overexpression of PSY (lines At12 and At22 in Table 1; Maass
et al., 2009), we conclude that high levels of a-carotene in leaves,
and supposedly also in roots of orange carrots, involve additional
mechanisms that are present in orange but absent from white-
rooted cultivars.
Alteration in e-ring hydroxylation pattern affecting a-carotene
levels are known from Arabidopsis mutants with reduced carotene
hydroxylase capacity (Kim et al., 2009). Among the cytochrome
 a-Carotene in Orange Carrots 2225

Table 1. Carotenoids in Arabidopsis and Carrot Leaves

Line Tot. Car Lutein a-Crypt a-Carotene b-Carotene VAZN chla/b

QAL 431.4 6 2 206.6 6 0 (47.9) ND 2.6 6 0 (0.6) 84.0 6 1 (19.5) 138.2 6 1 (32.0) 3.6 6 0.1
KUT 458.1 6 36 213.4 6 3 (46.8) ND 3.4 6 1 (0.7) 81.5 6 17 (17.6) 159.8 6 15 (34.8) 3.4 6 0.4
CRC 438.0 6 8 213.4 6 3 (48.7) 1.4 6 0.3 (0.3) 26.1 6 1 (6.0) 64.9 6 4 (14.8) 132.3 6 0 (30.2) 3.6 6 0.1
NAN 458.3 6 2 222.8 6 5 (48.6) 6.9 6 1.1 (1.5) 45.7 6 5 (10.0) 59.6 6 8 (13.0) 123.2 6 5 (26.9) 3.4 6 0.1
At-Wt 521.7 6 94 233.6 6 30 (45.0) ND 3.9 6 1 (0.7) 114.8 6 19 (22.0) 169.5 6 44 (32.3) 3.1 6 0.5
At12 486.2 6 31 227.0 6 14 (46.7) ND 3.1 6 1 (0.6) 105.8 6 10 (21.7) 150.3 6 9 (30.9) 3.2 6 0.1
At22 534.0 6 2 248.6 6 8 (46.6) ND 2.4 6 2 (0.4) 107.6 6 15 (20.2) 175.4 6 24 (32.9) 2.9 6 0.1
lut5-1 503.8 6 18 248.1 6 6 (49.3) 8.9 6 0.9 (1.8) 78.9 6 4 (15.7) 45.5 6 7 (9.0) 122.4 6 8 (24.3) 3.1 6 0.1

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Carotenoids were quantified by HPLC from leaves of white-rooted (QAL and KUT) and orange-rooted carrot cultivars (CRC and NAN). For comparison,
leaf carotenoids are shown from two CaMV35Spro:At-PSY Arabidopsis lines (At12 and At22), the corresponding wild type (At-Wt), and from the lut5-1 mutant
carrying a T-DNA integration within CYP97A3. Carotenoids are expressed in mmol pigment mol21 chlorophyll. The relative percentage of each carotenoid is
given in parentheses. Tot. Car, total carotenoids; a-Crypt, a-cryptoxanthin; VAZN, sum of violaxanthin, antheraxanthin, zeaxanthin, and neoxanthin; chla/b,
ratio between chlorophyll a and b; ND, not detectable. Data represent the mean 6 SD of four (carrot) and three (Arabidopsis) replicates, respectively.

P450 (CYP97C1 and CYP97A3) and non-heme hydroxylases
(BCH1 and BCH2) involved, only the CYP97A3 mutant showed
a high proportion of a-carotene (between 15 and 20%), exhibiting
the lut5 phenotype (Kim and DellaPenna, 2006). The increase of
a-carotene in lut5 is compensated for by an almost equivalent re-
duction in b-carotene, resulting in wild-type total carotenoid levels
(Table 1; HPLC chromatograms are provided in Supplemental
Figure 1). This was also found in leaves of orange-rooted carrot
cultivars (;14% b-carotene in CRC and NAN compared with
;18% in QAL and KUT; Table 1). Furthermore, the (e-ring) mono-
hydroxylated intermediate a-cryptoxanthin, which accumulates
due to the CYP97A3 deficiency in Arabidopsis lut5, was also de-
tected in CRC and NAN, while being absent in QAL and KUT.
Expression of Arabidopsis CYP97A3 in Orange Carrots
Rescues the Leaf Phenotype
The similarity in the leaf carotenoid patterns between Arabidopsis
lut5 and orange-rooted carrots suggests the presence of reduced
CYP97A3 activity in orange cultivars. Aiming at rescuing the low
a-carotene “chemotype,” we transformed the orange carrot cultivar
CRC with a construct harboring the CYP97A3 cDNA from Arabi-
dopsis (At-CYP97A3; Kim and DellaPenna, 2006) under control of
the DJ3S promoter from yams. This promoter is highly active in
carrot roots and ;100-fold less active in leaves (Arango et al., 2010).
In leaves of all three transgenic DJ3Spro:AtCYP97A3 events ge-
nerated, a-carotene was found to be strongly reduced, resulting
in an accordingly decreased a-/b-carotene ratio (Figure 2; see
Supplemental Table 1 for detailed carotenoid data). The changes
in different DJ3Spro:AtCYP97A3 lines largely corresponded with
transgene expression levels (Supplemental Figure 2). Thus, leaf
carotenoid patterns in the transgenic CRC gradually approached
those from white-rooted carrot leaves upon only the small increases
in At-CYP97A3 expression allowed by the promoter characteristics.
Root-Specific Overexpression of Arabidopsis CYP97A3 in
Orange Carrots Strongly Reduces a-Carotene Amounts
and Causes a Reduction in Total Carotenoid Levels
DJ3Spro:AtCYP97A3 CRC lines and wild-type CRC carrots were
grown in soil and their roots harvested after 8, 12, and 16 weeks.
Growth of transgenic lines was indistinguishable from non-
transformed CRC and roots developed similar to the wild type
(Supplemental Table 2). At-CYP97A3 mRNA was strongly expressed
throughout all selected stages (Figure 3A). Furthermore, immunoblot
analysis performed with roots from two transgenic DJ3Spro:AtCYP97A3
lines, Arabidopsis wild-type and lut5 leaves, confirmed the presence
of the mature At-CYP97A3 protein and mirrored overall transgene
expression differences between these lines (Figure 3C).
As with the leaves, a-carotene was found strongly reduced in
these roots throughout all stages, comprising only ;2% of total
carotenoids compared with 20% in untransformed CRC control
plants (Figure 3B; Supplemental Figure 3). Accordingly, the
a-/b-carotene ratio decreased from 0.5 in nontransformed CRC
to 0.1 and 0.04 in average in lines Dc#1 and Dc#25, respectively;
this decrease was ;3-fold during root growth (Figure 3E). Young
roots of transgenic lines also accumulated CYP97A3-catalyzed
monohydroxylated intermediates, such as zeinoxanthin and
b-cryptoxanthin, which were absent in the controls (Supplemental
Table 2). These intermediates disappeared in older transgenic
roots (12 and 16 weeks), suggesting a limited activity of other
carotene hydroxylases, e.g., the e-ring specific CYP97C1, during
early root development. Surprisingly, the decrease in a-carotene
from ;300 µg in untransformed CRC to 10 µg in line Dc#25 did not
entail an equivalent increase in CYP97A3-derived xanthophylls.
These remained at only small total amounts in both young roots
(100 µg in Dc#25 versus 71 µg in CRC) as well as older roots
(150 µg in Dc#25 versus 100 µg in CRC).
Moreover, the conceivable decrease in a-carotene was accom-
panied by an unexpected 30 to 50% reduction in total carotenoid
levels in transgenic DJ3Spro:AtCYP97A3 relative to untransformed
control roots of the same age (Figures 3B and 3D). This decrease
affected not only b-carotene, but all other carotenoids, including
desaturation intermediates such as phytoene, phytofluene, and
z-carotene. For instance, b-carotene was reduced by 77%, while
noncolored carotenes were reduced by 32% in 8-week-old roots
from line Dc#25 compared with the control. Expression of the
putative carrot homolog of the ABA-responsive gene 9-cis-
epoxycarotenoid dioxygenase 3 (NCED3) was largely unchanged
in the transgenic lines (Supplemental Figure 4; Auldridge et al.,
2006; Just et al., 2007). This suggests that the altered carotenoid
levels did not entail changes in ABA synthesis.
2226 The Plant Cell
Figure 2. a-Carotene and a-/b-Carotene Ratio in Leaves from DJ3Spro:AtCYP97A3 Carrots.
Orange-rooted carrots (CRC) were transformed with At-CYP97A3 under control of the yam DJ3S promoter (DJ3Spro). Both leaf a-carotene levels (A) as
well as a-/b-carotene ratios (B) are strongly reduced in DJ3Spro:AtCYP97A3 lines (Dc#1, Dc#25, and Dc#7) and approached levels determined in leaves
of white-rooted carrots (QAL). a-Carotene content is given in mmol mol21 chlorophyll. Data are mean of four (controls) and three (transgenic lines)
biological replicates + SD, respectively.
Since PSY has frequently been reported as the rate-limiting
step in non-green plant tissues (Fraser et al., 2002; Paine et al.,
2005; Rodríguez-Villalón et al., 2009; Welsch et al., 2010), reduced
phytoene and total carotenoid levels may suggest an attenuated
phytoene synthesis capacity as a consequence of At-CYP97A3
overexpression. However, transcript levels of both carrot PSY
genes were quite similar in all lines throughout all stages com-
pared with nontransformed CRC carrots (Figure 4A). We therefore
investigated PSY protein levels in roots from DJ3Spro:AtCYP97A3
lines and nontransformed CRC carrots. Protein gel blot analysis
revealed that, in fact, PSY protein amounts were very strongly
reduced, e.g., by ;50% in line Dc#25 (Figures 4B and 4C). Levels
of a putative PSY degradation product did not increase, which
suggests that PSY translation rather than its turnover rate is
affected. Furthermore, Arabidopsis wild-type and lut5 roots ac-
cumulate barely detectable PSY protein levels, which appear
somewhat higher in the mutant (Supplemental Figure 5). This
might indicate that the observed dependence of PSY protein
levels on hydroxylation capacity is not only a distinct feature of
carrots. In conclusion, the altered carotene hydroxylation upon
At-CYP97A3 overexpression in orange carrots negatively affected
PSY protein levels, explaining the reduced total carotenoid ac-
cumulation observed.
Cloning of CYP97A3 from White Carrots
High a-carotene levels in CRC wild type and their strong re-
duction upon Arabidopsis CYP97A3 overexpression suggest a de-
ficient CYP97A3 activity in CRC. Since sequence information for
carrot cytochrome P450 hydroxylases is available for the e-ring-
specific CYP97A1 (Just et al., 2007), but not for CYP97A3, we first
cloned CYP97A3 from QAL, assuming its intactness in white-rooted
carrots. Based on sequence conservation of CYP97A3 homologs,
we amplified a 1.3-kb fragment and completed the contiguous
coding region by rapid amplification of cDNA ends (39end) and
genome walking (59end). A phylogenetic analysis with the de-
duced amino acid sequence (616 amino acids) confirmed its
identity as carrot CYP97A3 (Figure 5A). This was concluded
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from the high sequence identity to several cytochrome P450
enzymes, which were functionally confirmed as b-ring-specific
a-carotene hydroxylases, e.g., from tomato (Sl-CYP97A29, 76%
identity; Stigliani et al., 2011), rice (Os-CYP97A4, 69% identity;
Quinlan et al., 2012), and Arabidopsis CYP97A3 (71% identity;
alignment in Supplemental Figure 6). In contrast, the sequence
exhibited only 41% identity to e-ring-specific carotene hydrox-
ylases, including carrot CYP97C1, and branched-off separately
in phylogenetic analysis, excluding that the sequence obtained
represented an additional CYP97C1 copy (Figure 5A). DNA gel
blot analysis with genomic DNA from QAL and CRC revealed the
presence of carrot CYP97A3 as single-copy gene in both culti-
vars. Quantitative RT-PCR (qRT-PCR) conducted on root RNA
from QAL and CRC revealed no large differences in expression
levels (Supplemental Figure 7), so that the possibility of muta-
tions was considered.
An Insertion Present in a CYP97A3 Allele in Orange Carrots
Total leaf RNA was isolated from CRC and NAN plants and used to
amplify CYP97A3 with the same primer combination used to amplify
CYP97A3 from QAL. Remarkably, CYP97A3 cDNAs from both or-
ange-rooted varieties were 8 nucleotides longer than from QAL,
which was caused by a single insertion at position #1074 (Figure 5B;
the electrophoretic mobility shift is shown in Supplemental Figure 7).
The insertion results in a premature translational stop, encoding
a truncated protein with only 382 instead of 616 amino acids
(CYP97A3Ins, Figure 5C). The truncated protein lacks a C-terminal
cysteine residue that is essential for the assembly of the heme
cofactor; it is therefore nonfunctional.
In order to confirm the involvement of the CYP97A3 gene in
a-carotene accumulation in carrot roots, the identified insertion/
deletion was genotyped in a broad unstructured population of
380 individuals, and an association mapping strategy was con-
ducted on carotenoid content. Three associations were found to be
significant with the insertion/deletion tested. The a-carotene
content was significantly associated with this polymorphism,
as expressed in dry (P value = 3.53 3 10 25 ) or fresh weight
 a-Carotene in Orange Carrots 2227

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Figure 3. Characterization of Roots from DJ3Spro:AtCYP97A3 CRC Lines.

(A) At-CYP97A3 expression in roots from 8-, 12-, and 16-week-old DJ3Spro:AtCYP97A3 carrots (cultivar CRC). Transcript levels determined by qRT-
PCR, normalized to 18S rRNA levels, and expressed relative to one selected sample from line Dc#1 (8 weeks).
(B) Carotenoids in roots from DJ3Spro:AtCYP97A3 lines and nontransformed CRC.
(C) Immunoblot analysis using 60 µg leaf protein from Arabidopsis wild type and lut5-1 (right) and 40 µg carrot root protein from wild-type CRC and
DJ3Spro:AtCYP97A3 lines (left). Antiserum directed against At-CYP97A3 was used; actin signals are shown as loading control.
(D) Transverse section of 8-week-old roots from DJS3pro:AtCYP97A3 CRC line Dc#25 (right) and nontransformed CRC as control (left).
(E) Ratio of a-carotene to b-carotene.
Data represent the mean 6 SD of three biological replicates, except Dc#1/16 weeks (one sample; two technical replicates were used in [A]). For detailed
carotenoid data, see Supplemental Table 2.

(P value = 5 3 1025) and explained 12 and 14% of the observed
variation, respectively (Figure 5D; Supplemental Figure 8). Finally, this
insertion was significantly associated with the a-/b-carotene ratio
(P value = 3.72 3 1024), but explained a small proportion of the
observed variation (r2 = 0.04). Associations with any other carotenoid
were not significant. These results confirm the involvement of
CYP97A3 in a-carotene accumulation and explained a relatively high
proportion of the variability of a-carotene content in carrot roots.
DISCUSSION
Cytochrome P450 Carotene Hydroxylase Deficiency
in Orange Carrots
a-Carotene is the second most abundant carotene present in
orange carrot cultivars. This is not limited to roots; a-carotene
accumulates to unusually high amounts in leaves and is increased
up to 10-fold compared with leaves of white-rooted cultivars. In
this work, we addressed this widespread feature of orange-rooted
carrots that is absent from white-rooted cultivars.
Leaf a-carotene content varies between different plant species
and is also increased in response toward low light intensities
(Matsubara et al., 2009). However, the constitutively high a-carotene
levels in orange carrots point to a genetic alteration like in
the Arabidopsis lut5 mutant, carrying a disruption within the
cytochrome P450 carotene hydroxylase CYP97A3 (Kim and
DellaPenna, 2006). In fact, the expression of the Arabidopsis
CYP97A3 gene in CRC largely rescued leaf carotenoid patterns,
approaching those of white-rooted carrots, regarding their low
a-carotene content, low a-/b-carotene ratio, and the complete
absence of a-cryptoxanthin. Similarly, roots of CYP97A3-
overexpressing CRC lines revealed carotenoid patterns matching
overexpressing a bacterial phytoene synthase (Maass et al.,
2009) as well as of PSY-overexpressing tissues from other
crops, such as potato tubers (Solanum tuberosum) or cas-
sava roots (Ducreux et al., 2005; Welsch et al., 2010). These
observations corroborate that a-carotene abundance in or-
ange carrots is not a concomitant of increased pathway flux
by high PSY levels, but rather represents a particular prop-
erty of orange carrots.
2228 The Plant Cell

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Figure 4. PSY Expression in Roots of DJ3Spro:AtCYP97A3 Lines.
(A) Transcript levels of both putative carrot PSY paralogs (Dc-PSY1 and Dc-PSY2) were determined by qRT-PCR, normalized to 18S rRNA, and are
expressed relative to 8-week-old CRC roots.
(B) PSY protein levels were determined in roots from CRC and DJ3Spro:AtCYP97A3 line Dc#1 and Dc#25 by immunoblot analysis using antibodies directed
against Arabidopsis PSY. Along with the imported form (imp, 42 kD) a putative degradation product (deg, 27 kD) is detected. An immunoblot using
Arabidopsis leaf protein (At-lvs) is shown for comparison. Actin levels were used as loading control. The plant age given refers to weeks after transfer to soil.
(C) PSY protein levels were normalized to actin of the corresponding sample and expressed relative to the ratio detected in 8-week-old CRC roots.
Data represent the mean 6 SD of three biological replicates, except Dc#1/16 weeks (two technical replicates).

These findings are in agreement with our identification of a
CYP97A3 allele in various orange carrot cultivars that carries an 8-
nucleotide insertion and encodes a truncated protein that is most
likely dysfunctional. An association study conducted on a large
unstructured carrot population revealed that this polymorphism is
associated with both a-carotene content and a-/b-carotene ratio in
roots. More specifically, 88% of the individuals homozygous for
CYP97A3Ins contained high a-carotene levels (above 2 mg g21
fresh weight), while this applied for only 47% of those individuals
carrying the wild-type allele. Therefore, the CYP97A3 polymorphism
identified explains a large proportion of the variation observed,
even though there are other currently unknown factors affecting
a-carotene levels in orange carrots. This is in agreement with
a quantitative trait loci study, which estimated four genes as the
minimum number of genes involved in a-carotene inheritance
(Santos and Simon, 2006). Apparently, the CYP97A3Ins allele rep-
resents one of these genes, and it remains to be determined whether
other candidate genes cause similar CYP97A3 deficiencies through
alternative mechanisms, e.g., by altered CYP97A3 expression.
Considering the remarkably high a-carotene levels coinciding
with high b-carotene levels in various carrot cultivars, we
questioned whether CYP97A3 activity also modulates the level
of total carotenoids in roots. On one side, this is supported by
our experimental data: The defective enzyme present in the
orange-rooted varieties investigated correlated with high total
carotenoids, while the overexpression of the intact version brings
them down again, apparently by downregulation of PSY protein.
However, this notion is not corroborated in the association analysis,
as the deficient CYP97A3 allele was associated with a-carotene
content, while an association with total carotenoid levels was not
found. It must be concluded that this is because the association
panel does not provide the situation observed with the over-
expression of a functional CYP97A3. One can expect one half or
one dosage of functional CYP97A3 but not more. The transgenic
carrots are therefore thought to reveal a phenomenon that cannot
be discovered in a biparental cross.
However, the principle discovered is that downstream carotenoids
can regulate the total pathway flux, which might contribute to
the explanation of several observations. For instance, a recent
quantitative trait loci study identified two major interacting loci that
determine much of the variability of carotenoid amounts in carrots
(Just et al., 2009). Markers for the e-ring specific a-carotene
hydroxylase CYP97C1 were found in close proximity to one of
the two loci (Just et al., 2007). While it was difficult to explain
reductions in total carotene content by a deficiency of an enzyme
acting downstream of b-carotene synthesis, our findings open the
way for possible explanations.
Feedback Regulation of Carotenoid Biosynthesis
Similar to other nuclear-encoded plastid-localized pathways, the
regulation of carotenogenic enzymes requires retrograde sig-
naling to coordinate (at the level of gene expression) metabolic
flux with product requirements. Molecules from various path-
ways are currently considered as likely candidates for plastid to
nucleus communication, e.g., the chlorophyll intermediate Mg
protoporphyrin IX for photosynthesis-related or methylerythritol
cyclodiphosphate (MEP pathway) for stress-responsive gene
expression (Chi et al., 2013). It is currently unknown how these
molecules shuttle between compartments traversing the plastid
envelope membranes. Carotenoids, especially, appear very unsuit-
able to function as immediate signaling metabolites in the regulation
of carotenogenic gene expression because of their lipophilicity.
Nevertheless, there are several published examples documenting
an impact of altered xanthophyll patterns on total carotenoid
amounts. For instance, silencing of zeaxanthin epoxidase and
BCH in potatoes resulted in 6- and 4-fold higher total carotenoid
amounts, respectively (Römer et al., 2002; Diretto et al., 2007).
 a-Carotene in Orange Carrots 2229

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Figure 5. The CYP97A3 Gene from Carrots.

(A) Phylogenetic tree of cytochrome P450 subfamily 97A and C proteins. At, A. thaliana, Dc, D. carota, Os, O. sativa, Rc, R. communis, Sl, S. lycopersicum,
Mt, M. truncatula. Arabidopsis CYP86A1 was included as the outgroup sequence. The identified carrot CYP97A3 sequence (framed) groups with other b-ring
carotene hydroxylases. Bootstrap values are reported next to the branches (only bootstraps above 50% are shown).
(B) Sections of sequencing chromatograms of RT-PCR fragments from Dc-CYP97A3 (top) and Dc-CYP97A3Ins (bottom); the 8-nucleotide insertion is marked with a box.
(C) Schematic map of proteins encoded by Dc-CYP97A3 and Dc-CYP97A3Ins. The insertion results in a premature translational termination prior to the
sequence encoding the heme binding cysteine residue (P450 cys). Primers used for RT-PCR in (B) are indicated by arrows.
(D) Allelic effect of the insertion/deletion polymorphism in CYP97A3 on a-carotene content (in mg 100 g21 FW). The box plots show the quartile division
with the median indicated by lines within the boxes. The two whiskers correspond to the first and third quartile.

Likewise, both LCYb-deficient maize mutants as well as LCYe-
silenced potato tubers accumulated higher total carotenoid
levels (Diretto et al., 2006; Bai et al., 2009). Furthermore, enhanced
lycopene accumulation in the tomato ogc mutant is considered to
be due to increased activity of earlier enzymes of the pathway in
addition to reduced lycopene cyclization caused by a deficient
fruit-specific lycopene cyclase (Ronen et al., 2000; Bramley,
2002). The induced changes in metabolite abundance entailed
altered expression of carotenogenic enzymes, which in most
cases included elevated PSY transcript levels. Furthermore, recent
findings suggest the involvement of cis-carotene intermediates in the
regulation of tomato fruit-specific PSY1 expression (Kachanovsky
et al., 2012).
PSY levels/activity are key to carotenoid accumulation in non-
green plant tissues (Ye et al., 2000; Lindgren et al., 2003; Ducreux
et al., 2005; Welsch et al., 2010), including carrot roots (Santos
et al., 2005; Maass et al., 2009). The context described here be-
tween CYP97A3 overexpression leading to reduced PSY protein
levels suggests the presence of a negative feedback regulation,
stemming from carotenoids downstream of a-carotene or metabo-
lites thereof, acting on PSY levels. The difference with respect to the
examples given above is that the expression of carrot PSY
genes remained unchanged upon At-CYP97A3 overexpression
in our study, while PSY protein levels were reduced. We there-
fore present an example of the modulation of PSY protein levels
by carotenoid metabolites through mechanisms acting beyond
transcription. Whether the feedback regulation at this level af-
fects additional enzymes of the pathway cannot be conclusively
answered as yet due to the lack of suitable antibodies. However,
there are several examples in non-green tissues in which varying
the rate of phytoene synthesis affects the total carotenoid con-
tent strongly while the carotenoid pattern is hardly affected (Paine
et al., 2005; Maass et al., 2009). It is therefore conceivable that
feedback-inhibited phytoene synthesis is sufficient to explain the
reduced carotene amounts in At-CYP97A3–overexpressing carrots.
Metabolite-Induced Feedback Regulation of PSY
Protein Abundance
In contrast to carotenoids, apocarotenoids are known to function as
signaling molecules. In fact, it is reasonable to assume that strongly
reduced a-carotene levels in At-CYP97A3 overexpressing CRC
roots are caused by enhanced metabolization. However, this is
contrasted by the lack of accordingly elevated b-ring hydroxylated
a-carotene derivatives, i.e., zeinoxanthin or lutein, especially
in older roots. The levels of these xanthophylls increased only
2230 The Plant Cell
slightly, and total xanthophyll levels remained at as low levels as in
nontransformed CRC control plants. This suggests increased
xanthophyll turnover through their enhanced cleavage by caro-
tenoid cleavage oxygenases (Simkin et al., 2004; Rodrigo et al.,
2013). In fact, several carotenoid-derived cleavage products are
important signaling molecules involved in developmental pro-
cesses (strigolactones) and mediate responses toward abiotic
stress (ABA; Walter and Strack, 2011; Alder et al., 2012). However,
the expression of the ABA-responsive carrot NCED3 was un-
changed upon carotenoid changes in the transgenics. It therefore
appears unlikely that ABA is involved in the observed response.
A putative function of other metabolites might be to trigger
total carotenoid flux via the regulation of the rate-limiting steps
of the pathway, most notably PSY. A dose dependence of PSY
protein levels from carotenoid-derived metabolites might in fact
be concluded from our data. a-Carotene levels in line Dc#25 are
much lower than in line Dc#1, which might correlate with higher
levels of cleavage products derived from e-ring xanthophylls.
Interestingly, this correlates with a more pronounced negative
feedback of Dc#25 over Dc#1 since PSY protein levels are lower
in Dc#25, corresponding with lower total carotenoid levels.
Similarly, an involvement of metabolites derived from e-ring hy-
droxylated xanthophylls might also be concluded from transgenic
tomato plants fruit-specifically overexpressing the bacterial desa-
turase crtI, which show an unexpected strong reduction in total
carotenoid levels including phytoene (Römer et al., 2000). While
a-carotene is almost absent in control fruits, crtI-overexpressing
fruits accumulate small amounts of a-carotene and e-ring xantho-
phylls, which correlate with PSY enzyme activity reduced by half.
As shown previously, high abundance of carotenes results in their
crystallization, a mode of sequestration that reduces their accessi-
bility toward degradation, either enzymatically or nonenzymatically
(Maass et al., 2009; Nogueira et al., 2013). Therefore, the sug-
gested negative feedback inhibition might also be part of a regu-
latory network that prevents critically high carotene concentrations
in order to maintain carotenoid homeostasis.
It is currently unclear which of the two putative carrot PSY
paralogs is affected by the negative feedback, as the antibody
used cannot differentiate between these variants. Whether re-
duced PSY translation or increased protein turnover results in
decreased steady state PSY protein levels cannot be conclu-
sively answered yet. However, lower PSY protein levels in At-
CYP97A3–overexpressing carrots do not entail higher levels of
putative PSY breakdown products. Furthermore, PSY protein
can be very strongly expressed in Arabidopsis (Maass et al.,
2009), which may point to protein turnover being less relevant
for flux adjustments. Metabolite-dependent regulation of trans-
lation required for the fine-tuning of biosynthetic efficiency ap-
pears more likely. Investigations to further discriminate between
these possibilities are currently underway.
METHODS
Plant Material and Growth
Arabidopsis thaliana (ecotype Wassilewskija) seeds were grown asepti-
cally on Murashige and Skoog medium under long-day conditions for
14 d. Seedlings were ground in liquid nitrogen immediately after harvest
and stored at 270°C for further analysis. Arabidopsis roots were harvested
from seedlings grown according to Hétu et al. (2005). Seeds from carrots
(Daucus carota) were obtained as follows: Queen Anne’s Lace, Richters Herbs;
Küttiger, Dreschflegel Saatgut; Nantaise, Freya; Chantenay Red Cored, B&T
World Seeds. Carrot plants were grown in soil under long-day conditions.
Roots of 8-, 12-, and 16-week-old carrot plants were removed from the soil,
ground in liquid nitrogen, and stored at 270°C for further analysis.
Carrot Transformation
The At-CYP97A3 coding region was amplified by PCR with primers 59-
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GTGGCGTTCCATGGCTATGGCCTTTCCTCTTTCTTATACT-39 and 59-
AAAGATGAACACGTGAGAAAGAGCAGATGAAACTTCATCC-39 from the
RIKEN full-length cDNA clone RAFL09-10-L12, thereby introducing NcoI
and PmlI restriction sites, respectively. The fragment was digested with
NcoI and PmlI and ligated into the vector backbone obtained from the
accordingly digested vector pCAMBIA-DJ3S-GUS (Arango et al., 2010),
revealing pCAMBIA-DJ3S-AtCYP97A3. This vector was used to generate
transgenic CRC lines as described by Maass et al. (2009), with the exception
that 10 mg mL21 hygromycin was used for selection of transgenic calli.
Cloning of Dc-CYP97A3
Sequence alignments with CYP97A3 sequences from various taxa were
performed to identify conserved regions. Degenerated oligonucleotides
with wobble nucleotides were deduced: forward, ATT GCT/G TCT/C
GGT/A/G/C GA/C G/T/ATT CAC T/C/GGT, and reverse, TGA AAG TT/CT
AA/CC GT/A C/G A/G A/T CCT CGA/T GG. Total QAL leaf RNA was
reverse transcribed using the reverse primer and the verso cDNA kit
(Thermo), followed by a PCR using both primers and Phusion polymerase
(Thermo). Specific primers were deduced from the partial CYP97A3 se-
quence obtained and the 39 end of the mRNA was retrieved using the 59/39
RACE kit (Roche Diagnostics) and the forward primer 59-ACATCGC-
TGTCCTCAAAGGTGGGA-39 according to the manufacturer’s instructions.
Sequence information of the 59 mRNA end was retrieved by genome walking
(GenomeWalker Universal Kit; Clontech) followed by exon predictions using
the FGENESH algorithm available in Softberry (www.softberry.com). Full-
length CYP97A3 coding regions were amplified using the primers forward
59-CCCAGCCAACGTCTCCAAATGATAGCC-39 and reverse 59-GAACA-
TTGCTGCACATACAAAGAT-39 and cloned in the vector pCR2.1 (Invitrogen
Life Technologies). For CYP97A3 RT-PCR, 200 ng total RNA was reverse
transcribed with primer reverse 59-CATCTCCAGATGCCAACAAAAAAT-39
followed by PCR upon the addition of primer forward 59-ATTCCCATT-
TGGAAAGACATTTCG-39 (1 min 93°C, 1 min 60°C, 1 min 72°C, 28 cycles).
Amplification products were separated by agarose gel electrophoresis.
DNA Gel Blot Analysis
Genomic DNA isolation and DNA gel blot analysis was performed as de-
scribed by Arango et al. (2010) using the DIG DNA labeling and detection kit
(Roche Diagnostics). A probe covering 676 bp of the Dc-CYP97A3 cDNA
was used (positions 318 to 994; accession number JQ655297).
Phylogenetic Analysis
Sequences used for phylogenetic analysis were selected according to
Stigliani et al. (2011). Sequences were aligned with T-Coffee (Notredame
et al., 2000), and the phylogenetic tree was reconstructed using the
neighbor-joining method in MEGA5 (Tamura et al., 2011). The evolutionary
distances were computed using the Poisson correction method and
bootstrap test was selected with 1000 replicates. A member of family 86 of
the cytochrome superfamily, the fatty acid hydroxylase At-CYP86A1, was
chosen as an outgroup. For GenBank accession numbers in Figure 5A.

TaqMan Real-Time RT-PCR Assay
Total RNA was isolated using the plant RNA purification reagent (Invi-
trogen Life Technologies). RNA purification, DNaseI digestion, and real-
time RT-PCR assays were performed as described (Welsch et al., 2008).
Carrot PSY1, PSY2, CYP97A3, and nos 39UTR (present in the At-CYP973
transgene mRNA) expression was detected with 6FAM-labeled probes,
while SYBR green was used for NCED3 expression analysis. For primers
and probes, see Supplemental Table 3. For 18S rRNA quantification, the
eukaryotic 18S rRNA endogenous control kit (Life Technologies) was
used. The relative quantity of the transcripts was calculated using the
comparative threshold cycle method (Livak and Schmittgen, 2001). Data
were normalized first to the corresponding 18S rRNA levels and then
expressed relative to a selected sample indicated.
Carotenoid Extraction and Quantification
Carotenoids were extracted using lyophilized plant materials (5 mg for
leaves; 20 mg for carrot roots) and analyzed by HPLC as described
(Welsch et al., 2008).
Immunoblot Analysis
Generation and affinity purification of antibodies directed against Arab-
idopsis PSY is described by Maass et al. (2009). Serum containing anti-
At-CYP97A3 antibodies was used in 1:5000 dilutions. Proteins were
extracted with phenol as described (Welsch et al., 2007). After SDS-
PAGE, blotting onto polyvinylidene fluoride membranes (Carl Roth), and
treatment with blocking solution (TBS containing 5% [w/v] milk powder),
membranes were incubated with antibodies in PBS containing 0.1% (v/v)
Tween 20 and 1% (w/v) milk powder. For detection, the ECL system (GE
Healthcare) was used. Protein gel blots were stripped and reprobed with
anti-actin antibodies (Sigma-Aldrich). Quantification of band intensities
was performed with ImageJ (Abramoff et al., 2004).
Validation through Association Mapping Analysis
Polymorphism in CYP97A3 was tested in association mapping analysis in
a broad unstructured population obtained by three intercrossing gen-
erations of a diversified panel of 67 carrot cultivars. A total of 380 in-
dividuals were randomly chosen at the third generation and were grown in
field (Agrocampus Ouest, Angers, France) following standard agronomic
practices. Carotenoid contents were quantified by HPLC with a modified
procedure adapted from Clotault et al. (2008). CYP97A3 polymorphism
was genotyped by KASPar Assay technology (LGC Genomics), along with
92 single nucleotide polymorphisms (SNPs) spread all over the genome.
Raw data on carotenoid content and CYP97A3 polymorphism are given
in Supplemental Data Set 1. Association analysis (Hall et al., 2010) was
performed using the TASSEL software (Bradbury et al., 2007). As re-
latedness between individuals can lead to false positive detection, the
association tests were based on a mixed linear model in which a kinship
matrix, estimated by identity by states calculated on SNP data set, was
added as a covariable (Kang et al., 2008).
Accession Numbers
Sequence data from this article can be found in the GenBank/EMBL data
libraries under the following accession numbers: JQ655297 (Dc-CYP97A3
mRNA), JQ655298 (Dc-CYP97A3 gene, partial), DQ192196 (Dc-CYP97C1),
EEC67393.1 (Os-CYP97C2), EEC74248.1 (Os-CYP97A4), NP_190881
(At-CYP97C1), At1g31800 (At-CYP97A3), ABC59096 (Mt-CYP97C10),
ABD28565.1 (Mt-CYP97A10), ACJ25968 (Sl-CYP97C11), ACJ25969
(Sl-CYP97A29), At5g58860 (At-CYP86A1), XP_002512609.1 (Rc-CYP97A),
XP_002519427.1 (Rc-CYP97C), and DQ192202 (Dc-NCED3).
a-Carotene in Orange Carrots 2231
Supplemental Data
The following materials are available in the online version of this article.
Supplemental Figure 1. HPLC Chromatograms from Carrot and
Arabidopsis Leaves.
Supplemental Figure 2. At-CYP97A3 Expression in Transgenic CRC.
Supplemental Figure 3. Carotenoid Patterns of DJ3Spro:AtCYP97A3
CRC Lines.
Supplemental Figure 4. Dc-NCED3 Expression in Roots of DJ3Spro:
AtCYP97A3 Lines.
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Supplemental Figure 5. Carotenoid and PSY Protein Levels in
Arabidopsis and Carrot Roots.
Supplemental Figure 6. Alignment of Carrot CYP97A3 with Other
Cytochrome p450 Proteins.
Supplemental Figure 7. Gene Copy Number and Expression of Carrot
CYP97A3.
Supplemental Figure 8. Allelic Effects of CYP97A3 Polymorphism on
a-Carotene Content and a-/b-Carotene Ratio.
Supplemental Table 1. Carotenoids in Leaves from DJ3Spro:AtCYP97A3
Carrots.
Supplemental Table 2. Detailed Carotenoid Amounts in Roots from
At-CYP97A3-Overexpressing CRC Lines.
Supplemental Table 3. Primers and Probes Used for qRT-PCR.
Supplemental Data Set 1. Carotenoid Contents and CYP97A3
Genotype.
ACKNOWLEDGMENTS
This work was supported by the HarvestPlus research consortium and
a Deutsche Forschungsgemeinschaft Research Grant to R.W. (Grant
WE4731/2-1). We thank Dean DellaPenna (Michigan State University) for
providing lut5 seeds and the At-CYP97A3 cDNA. We also thank Stefano
Cazzaniga and Roberto Bassi (Università di Verona, Italy) for providing
antibodies against At-CYP97A3. We thank Jérémy Clotault (Université
d’Angers, France) for initiating the collaboration between R.W. and E.G.
M.J. was supported by the French Ministry of Research. We are indebted
to Carmen Schubert for her skillful contribution to carrot transformation
and analysis.
AUTHOR CONTRIBUTIONS
R.W., P.B., and E.G. conceived this project and designed all experi-
ments. R.W., J.A., and M.J. performed experiments. R.W., P.B., J.A.,
E.G., and M.J. analyzed data. R.W., P.B., and E.G. wrote the article.
Received December 19, 2013; revised March 14, 2014; accepted April
16, 2014; published May 23, 2014.
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