Biosynthesis, regulation, and domestication of bitterness in cucumber

Article abstract of DOI:10.1126/science.1259215

Cucurbitacins are triterpenoids that confer a bitter taste in cucurbits such as cucumber, melon, watermelon, squash, and pumpkin. These compounds discourage most pests on the plant and have also been shown to have antitumor properties. With genomics and biochemistry, we identified nine cucumber genes in the pathway for biosynthesis of cucurbitacin C and elucidated four catalytic steps.We discovered transcription factors Bl ( Bitter leaf) and Bt (Bitter fruit) that regulate this pathway in leaves and fruits, respectively. Traces in genomic signatures indicated that selection imposed on Bt during domestication led to derivation of nonbitter cucurbits from their bitter ancestors.

Full text of DOI:10.1126/science.1259215

Biosynthesis, regulation, and domestication of bitterness in
cucumber
Yi Shang et al.
Science 346, 1084 (2014);
DOI: 10.1126/science.1259215
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RESEARCH
| RESEARCH ARTICLES
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PLANT SCIENCE
Biosynthesis, regulation, and
domestication of bitterness
in cucumber
Yi Shang,1,2* Yongshuo Ma,1,3* Yuan Zhou,1,4* Huimin Zhang,1,3* Lixin Duan,5
Huiming Chen,6 Jianguo Zeng,4 Qian Zhou,1 Shenhao Wang,1 Wenjia Gu,1,7
Min Liu,1,3 Jinwei Ren,8 Xingfang Gu,1 Shengping Zhang,1 Ye Wang,1
Ken Yasukawa,9 Harro J. Bouwmeester,10 Xiaoquan Qi,5 Zhonghua Zhang,1
William J. Lucas,11 Sanwen Huang1,2
†
Cucurbitacins are triterpenoids that confer a bitter taste in cucurbits such as cucumber,
melon, watermelon, squash, and pumpkin. These compounds discourage most pests on
the plant and have also been shown to have antitumor properties. With genomics and
biochemistry, we identified nine cucumber genes in the pathway for biosynthesis of
cucurbitacin C and elucidated four catalytic steps. We discovered transcription factors
Bl (Bitter leaf) and Bt (Bitter fruit) that regulate this pathway in leaves and fruits,
respectively. Traces in genomic signatures indicated that selection imposed on Bt during
domestication led to derivation of nonbitter cucurbits from their bitter ancestors.
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lant specialized metabolites play essential
roles in mediating interactions between
the plant and its environment and con-
stitute a valuable resource in discovery
of economically important molecules. In
hepatoprotective activities, in the form of tradi-
tional herbal medicines (4, 5). Bitter fruits and
leaves of wild cucurbit plants have been used
as a purgative and emetic in India (6). The bitter
fruit stem of melon (in Chinese, “gua di”) is pre-
scribed as a traditional hepatoprotective medi-
cine whose effect and usage were well documented
in Ben Cao Gang Mu, the Chinese Encyclopedia
of Botany and Medicines composed by the Ming
Dynasty physician Li Shi-Zhen in 1590 CE. Recent
studies revealed that cucurbitacins can cause cell-
cycle arrest, apoptosis, and growth suppression of
cancer cells through the specific inhibition of
the Janus kinase–signal transducers and activa-
tors of transcription (JAK-STAT) pathway (7, 8).
At present, their low concentrations in plants
and nonspecific cytotoxicity limit their phar-
maceutical applications.
To date, plant metabolic diversification studies
(9, 10), as well as recently reported gene clusters
in plants [reviewed in (11)], indicate that clustering
of functionally-related genes for the biosynthesis
of secondary metabolites may well be a common
feature of plant genomes. In cucumber, two inter-
acting Mendelian loci were reported to control the
bitterness, conferred predominantly by cucurbita-
cin C (CuC) (3, 12). The Bi gene (1) confers bitterness
to the entire plant and is genetically associated with
an operon-like gene cluster (13), similar to the gene
cluster involved in thalianol biosynthesis in Ara-
bidopsis (14). Fruit bitterness requires both Bi and
the dominant Bt (Bitter fruit) gene. Nonbitterness
of cultivated cucumber fruit is conferred by bt, an
allele selected during domestication as indicated
by population genomics (15). Exploiting these
genetic clues, here we report the discovery of 11
genes involved in the biosynthesis, regulation,
and domestication of cucumber bitterness.
P
the plant family Cucurbitaceae, a group of highly
oxygenated tetracyclic and bitter triterpenes,
the cucurbitacins, mediated the coevolution be-
tween cucurbits and herbivores. They serve ei-
ther as protectants against generalists or feeding
attractants to specialists (1–3). Widely consumed
as vegetables and fruits, cucurbits were domes-
ticated from their wild ancestors that had ex-
tremely bitter fruits. Drought and temperature
stress can increase the bitterness in certain do-
mesticated cultivars, which can affect fruit qual-
ity and marketability. Molecular insights into the
occurrence and domestication of bitterness in
cucurbits remain largely unknown.
ACKNOWLEDGMENTS
We thank the Instituto de Astrofisica de Canarias for the excellent
working conditions at the Observatorio del Roque de los
Muchachos in La Palma. The support of the German BMBF and
MPG, the Italian INFN, the Swiss National Fund SNF, and the
Spanish MICINN is gratefully acknowledged. This work was also
supported by the CPAN CSD2007-00042 and MultiDark CSD2009-
00064 projects of the Spanish Consolider-Ingenio 2010 program,
by grant 127740 of the Academy of Finland, by the DFG Cluster of
Excellence “Origin and Structure of the Universe”, by the Croatian
Science Foundation (HrZZ) Projects 09/176, by the University of
Rijeka Project 13.12.1.3.02, by the DFG Collaborative Research
Centers SFB823/C4 and SFB876/C3, and by the Polish MNiSzW
grant 745/N-HESS-MAGIC/2010/0. We thank also the support by
DFG WI 1860/10-1. J. S. was supported by ERDF and the Spanish
MINECO through FPA2012-39502 and JCI-2011-10019 grants.
E. R. was partially supported by the Spanish MINECO projects
AYA2009-13036-C02-02 and AYA2012-38491-C02-01 and by the
Generalitat Valenciana project PROMETEO/2009/104, as well as
by the COST MP0905 action ’Black Holes in a Violent Universe.’
The European VLBI Network is a joint facility of European, Chinese,
South African and other radio astronomy institutes funded by their
national research councils. The research leading to these results
has received funding from the European Commission Seventh
Framework Programme (FP/2007-2013) under grant agreement
No. 283393 (RadioNet3). The MAGIC data are archived in the data
center at the Port dÍnformació Cientfica (PIC) in Barcelona. The
EVN data are available at the Data Archive at the Joint Institute for
VLBI in Europe (JIVE).
Despite their presence in fruits as a negative
agricultural taste, cucurbitacins have for cen-
turies been exploited for anti-inflammatory and
1Institute of Vegetables and Flowers, Chinese Academy of
Agricultural Sciences, Key Laboratory of Biology and Genetic
Improvement of Horticultural Crops of the Ministry of
Agriculture, Sino-Dutch Joint Laboratory of Horticultural
Genomics, Beijing 100081, China. 2Agricultural Genomic
Institute at Shenzhen, Chinese Academy of Agricultural
Sciences, Shenzhen 518124, China. 3College of Life Sciences,
Nanjing Agricultural University, Nanjing 210095, China.
4Horticulture and Landscape College, Hunan Agricultural
University, National Chinese Medicinal Herbs Technology
Center, Changsha 410128, China. 5Institute of Botany,
Chinese Academy of Sciences, Beijing 100093, China.
6Hunan Vegetable Research Institute, Hunan Academy of
Agricultural Sciences, Changsha 410125, China. 7College of
Life Sciences, Wuhan University, Wuhan 430072, China.
8Institute of Microbiology, Chinese Academy of Sciences,
Beijing 100190, China. 9School of Pharmacy, Nihon
University, Tokyo 101-8308, Japan. 10Laboratory of Plant
Physiology, Wageningen University, Wageningen 6700,
Netherlands. 11Department of Plant Biology, College of
Biological Sciences, University of California, Davis, CA
95616, USA.
SUPPLEMENTARY MATERIALS
www.sciencemag.org/content/346/6213/1080/suppl/DC1
Materials and Methods
Figs. S1 to S5
Tables S1 and S2
References (42–69)
First committed step in CuC biosynthesis
To identify genetic variants associated with Bi, a
genome-wide association study was performed
16 May 2014; accepted 23 October 2014
10.1126/science.1256183
*These authors contributed equally to this work. †Corresponding
author. E-mail: huangsanwen@caas.cn
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based on the variation map (15) of 115 diverse
cucumber lines (Fig. 1A and table S1). The most
significant single-nucleotide polymorphism (SNP)
was located within the region where Bi had
been mapped and resulted in a nonsynonymous
change from cysteine (C) to tyrosine (Y) at residue
393 (C393Y) of the cucumber gene Csa6G088690
(Fig. 1B). In the 115 lines, this SNP explained the
phenotype in all but one line, CG7744. In-depth
analysis of the variation map identified a 1–base
pair (bp) deletion at Csa6G088690 in CG7744
that resulted in a frameshift mutation at the
760th amino acid residue (FS760) (Fig. 1B). Ge-
netic analysis pinpointed that Csa6G088690 de-
fines the Mendelian Bi gene (fig. S1A).
Bi is a member of the oxidosqualene cyclase
(OSC) gene family. Phylogenetic analysis showed
that Bi is the ortholog of cucurbitadienol syn-
thase gene CPQ in squash (Cucurbita pepo) (16)
(fig. S1B). We next used yeast to express Bi, as
well as its two mutant alleles, C393Y and FS760,
to test its biochemical function. As revealed by
gas chromatography–mass spectrometry (GC-MS)
analysis, formation of cucurbitadienol occurred
only in the yeast strain expressing the wild-type
gene (Fig. 1C and fig. S1, C and D). Thus, in cu-
cumber, Bi encodes a cucurbitadienol synthase
that catalyzes the cyclization of 2,3-oxidosqualene
into the tetracyclic cucurbitane skeleton, the first
committed step of CuC biosynthesis (fig. S1E).
EIC 498
TIC
X105
X106
5
2
pYES2
0
5
0
2
Bi
0
5
0
2
bi C393Y
0
2
0
5
1
2
3
4
5
6
7
Chromosome
bi FS760
0
2
0
5
Csa6G088690
Cucurbitadienol
0
0
A leaf-specific regulator of Bi
9930:
9110Gt:
CG7744:
393 760
18
20
22
18
20
22
To investigate the molecular mechanism in
regulating CuC biosynthesis, we searched for
naturally occurring mutants and screened an
ethylmethane sulfonate–induced cucumber mutant
library and subsequently identified two non-
bitter mutants (XY-3 and E3-231). The foliage
expression level of Bi in the natural mutant XY-3
Time (min)
Time (min)
783
Fig. 1. The Bi gene. (A) Genome-wide association study for the bitter foliage trait. Red arrow, most sig-
nificant association. Scale, –log10 of P value of SNPs. (B) Amino acid alignment between wild Csa6G088690
and two mutant alleles. (C) GC-MS analysis of extracts prepared from yeast INVSc1 that harbored Bi, two
mutant alleles (C393Y and FS760), empty vector, or an authentic standard. TIC, total ion chromatograms;
EIC 498, extracted ion chromatograms at a mass/charge ratio (m/z) of 498 [M+TMS (trimethysilyl)].
Fig. 2. The Bl gene. (A) Expression of Bl
(Csa5G156220) and Bi in nonbitter mutant XY-3
200
XY-3
XY-2
Bl
Exon 1
Exon 2
Intron
and bitter XY-2 cucumber lines (means T SEM, n =
3). (B) Sequence alignment between wild Bl and
two mutated alleles. (C and D) Transient expres-
sion of Bl in cotyledons complemented the non-
bitter phenotype of XY-3. (C) Expression of Bl and
Bi determined 7 days after agroinfiltration (means T
SEM, n = 6).Value obtained from control (CK) was
set to 1 and used to obtain relative values for the
test sample. INF, sample infiltrated with Bl; CK,
sample infiltrated with empty vector. (D) Presence
or absence of CuC detected by high-performance
liquid chromatography (HPLC) analysis of extracts
prepared from Bl or control infiltrated cotyledons.
mAU, milli–arbitrary units. (E) Schematic of the Bi
promoter region (2000 bp upstream of the start
codon). Black vertical lines indicate locations of
E-box motifs, and red horizontal lines indicate
regions amplified in ChIP assays or used in EMSA.
Localization of mutated E-box used in EMSA is in-
dicated in red. (F) ChIP analysis of Bl recruitment
to the Bi promoter region by PCR. ChIP assays
conducted with or without (+/−) Myc antibody.
INF, sample infiltrated with Bl; CK, sample infil-
trated with empty vector. (G) qPCR analysis of Bl
recruitment to the indicated Bi promoter region
(means T SEM, n = 3). (H) EMSA showing that Bl-
His specifically binds, in vitro, to the E-box region
within the Bi promoter. Lane identified by a red
triangle indicates that the E-box element within the
probe has been mutated from CANNTG to GANNTG.
Comp, competitor (unlabeled probe); His, His-tag;
+/−, presence or absence of protein or competitor;
closed triangle, increasing amount of protein or
competitor.
100
20
10
0
XY-2 (bitter):
XY-3 (non-bitter):
E3-231 (non-bitter):
basic domain splice site
E2
Bi Bl (Csa5G156220)
10
8
CK
INF
PBi
E7
E6 E5
E4
E3
E1
6
-1
-2000
4
PCR 2
PCR 1
& probe
2
0
Bi
Bl
INF 1
CK
Bl INF
40
30
15
10
Input
Input
-
-
+
+
Bl INF
CK
20
5
0
15
10
0
INF 2
PCR 1 PCR 2
10
5
PCR 1 PCR 2
0
15
INF 3
CK
10
-
-
-
-
-
-
-
-
Comp
Bl-His
His
5
0
+ -
-
+
-
+
-
+
-
+
15
10
shifted
probe
5
0
300
CuC
200
100
0
free
probe
6.6 7.0
7.4 7.8
Time (min)
SCIENCE sciencemag.org
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was reduced to less than 1% of that in the bitter
isogenic line XY-2 (Fig. 2A), which indicated that
cucurbitacin biosynthesis is disrupted in XY-3.
Genomes of XY-2 and XY-3 were resequenced
and compared to identify possible mutations.
A SNP in the cucumber gene Csa5G156220 caught
our attention, as it encodes a putative basic helix-
loop-helix (bHLH) transcription factor (TF) ex-
pressed specifically in leaves (table S2). The
mutation resides at the splicing site of the pre-
dicted intron that likely disrupts proper gene
transcription (Fig. 2, A and B).
Resequencing of E3-231 revealed another SNP
located within Csa5G156220 that caused a change
from arginine (R) to lysine (K) at the 85th amino
acid residue (R85K), which is located inside the
basic domain (Fig. 2B). This mutation may af-
fect regulatory ability, as the basic domain is es-
sential for DNA binding ability for bHLH TFs
(17). Genetic analyses showed that the mutations
in XY-3 and E3-231 are actually two recessive
alleles of the same gene (fig. S2A). Increased
expression of both Bi and Csa5G156220 was also
observed in cucumber plants either exposed to
drought stress or treated with the phytohor-
mone ABA (fig. S2, B and C), which indicated
that abiotic stress may stimulate the bitterness
biosynthesis in cucumber by up-regulation of
Csa5G156220.
considered Csa5G157230 to be a candidate for
Bt, given that it is specifically expressed in the
fruit of the wild line, PI 183967, consistent with
the distribution of bitterness in these plants
(Fig. 3A and table S2). In addition, positive cor-
relations were observed between expression lev-
els of Csa5G157230 and Bi, and between fruit
bitterness and gene expression in various cucum-
ber lines, especially in those five extremely bitter
wild lines (Fig. 3B). These studies established a
correlation between Csa5G157230 expression and
accumulation of bitterness in the fruit.
Next, we performed a local association analy-
sis within the 442-kb region to further identify
genetic variants associated with the extremely
bitter phenotype. This led to finding 11 signals at
the regulatory region of Csa5G157230, including
10 SNPs and one structural variant, a 699-bp
insertion 2195 bp upstream of the Csa5G157230
start codon (SV-2195). Another variant was also
identified at the regulatory region of Csa5G157230,
a SNP at the 1601 bp upstream of the start codon
(SNP-1601), which cosegregated with the Bt locus
in a large F2 population (n = 1822). In the 115
lines, 22 carrying a homozygous “A” at SNP-1601
all bear nonbitter fruits (table S3). These analyses
indicated that selection at the regulatory region
of Csa5G157230 may down-regulate Csa5G157230
expression in cultivated lines, which results in re-
duced fruit bitterness.
In some cucumber lines, fruits become bitter
under stress conditions. For instance, the fruits
of the cucumber line HAN become bitter when
plants were grown at a low temperature (18°C
ancestral
domesticated
SNPBt5-25
SNPBt5-27
Bt
Relative
CuC content
2
3
4
5
6
7
8
Chr 5 (Mb)
0
4
8
2
1
0
WF
WF
CF
WL
CL
CF
WL
CL
Bl
Csa5G157220 Bt (Csa5G157230)
A cucumber cotyledon transient agro-infiltration
expression system was developed to further con-
firm the in vivo function of Csa5G156220 (18).
Increasing expression of Csa5G156220 in XY-3
cotyledons up-regulated expression of Bi, which
in turn functionally complemented the nonbitter
phenotype (Fig. 2, C and D, and fig. S3, A and B).
Thus Csa5G156220 regulates the bitterness bio-
synthesis in cucumber leaves, and hence, this
gene was named Bl (Bitter leaf ).
Next, we investigated how Bl regulates Bi. Analy-
sis of the Bi promoter region revealed the occur-
rence of seven E-box (CANNTG) sequences (Fig.
2E), a cis-element to which bHLH TFs could po-
tentially bind (17). Yeast one-hybrid (Y1H) assay
and a tobacco transient reporter (luciferase) acti-
vation system showed that Bl indeed could bind
to this promoter (fig. S2, D and E). Chromatin im-
munoprecipitation (ChIP) assays were performed
by using formaldehyde-fixed cotyledons of XY-3
that were transiently expressing a Bl-Myc fusion
protein. As revealed by the polymerase chain re-
action (PCR) products and quantitative real-time
PCR (qPCR), Bl was selectively recruited to the Bi
promoter region containing E-box elements (Fig.
2, F and G). Electrophoretic mobility-shift assays
(EMSAs) also confirmed selective binding of Bl to
the E-box elements within the Bi promoter (Fig.
2H). Thus, Bl regulates cucurbitacin biosynthesis by
activating transcription of Bi in cucumber leaves.
40
30
20
10
0
Bt
Bi
60
50
CK
Bt
han
INF
40
F1
40
20
30
HAN
20
10
0
900
500
100
25
0
Bi
Bt
1200
han
F1
HAN
Bi
20
10
800
400
15
10
5
INF 1
INF 2
5
0
0
5
0
1000
800
CuC
0
5
0
5
0
300
200
han
F1
HAN
INF 3
CK
16
12
8
10
8
CuC
6
4
0
4
CuC
100
0
2
0
7.2 7.6 8.0 8.4
normal cold
+ + + + + + + + + + + + + + + + - - - - -
SV-2195:
Time (min)
condition stress
SNP-1601: A AAAAGGUGUUGGGYGGGGGG
Fig. 3. The Bt gene. (A) The Bt-mapped region on chromosome 5 overlaps with a large domestication
sweep region showing almost zero nucleotide diversity in the domesticated population (top). Differential
expression profiles of genes predicted within the Bt region illustrated by a gradient in red (bottom).
Numeric expression values of predicted genes are shown in table S2. Candidate Bt gene is indicated in
red. CuC content of wild and cultivated cucumber was compared (means T SEM, n = 3).WF, wild fruit; CF,
cultivated fruit; WL, wild leaf; CL, cultivated leaf. (B) High consistency observed between expression of
Bt, Bi, and the CuC content in 21 cucumber lines, including five extremely bitter lines (means T SEM, n = 3,
indicated in red). Presence or absence of SV-2195 indicated by +/−. Genotype of SNP-1601 (Y: A or G, U:
unknown). (C) High consistency among cold-stress treatment: expression of Bt, Bi, and CuC content in
fruit of HAN, han, and F1 individual plants (means T SEM, n = 3). (D and E) Transient expression of Bt in fruit
complemented the nonbitter phenotype of cucumber line XinTaiMiCi-2. (D) Expression of Bt and Bi
determined 15 days after agroinfiltration (means T SEM, n = 3).Value obtained from control (CK) was set to
1 and used to obtain relative values for the test sample. INF, sample infiltrated with Bt; CK, sample
infiltrated with empty vector. (E) Presence or absence of CuC detected by HPLC analysis of extracts
prepared from Bt or control infiltrated fruits 15 days after agroinfiltration. mAU, milli–arbitrary units.
A cucumber domestication gene
Bt was previously mapped to a 442-kilobase (kb)
region on chromosome 5 that harbors 67 predicted
genes (15). Bl and its two homologs (Csa5G157220
and Csa5G157230) are among these candidates
and clustered in an 8.5-kb region (Fig. 3A). As Bl
positively regulates Bi in cucumber leaves, we
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day, 12°C night), whereas, at a normal temper-
ature (30°C day, 22°C night), the fruits are not
bitter. We identified a natural HAN mutant (han),
whose fruits were nonbitter even under such low
temperature conditions. Resequencing both lines
revealed a mutation corresponding to SNP-1601
(G in HAN and A in han). Genetic analysis showed
that SNP-1601 cosegregates with the phenotype
(fig. S4A). Our qPCR analysis indicated that SNP1601
is essential for regulating Bi expression in re-
sponse to this environmental factor (Fig. 3C).
To confirm the in vivo function of Csa5G157230,
a fruit transient gene expression system was de-
veloped (18). Expression of Csa5G157230 activated
transcription of Bi and promoted biosynthesis
of CuC in the fruit (Fig. 3, D and E). In parallel
experiments, we expressed Csa5G157230 in XY-3
cotyledons, with the method described above. An
increase in CuC content in the infiltrated XY-3
tissue was also observed (fig. S3C), which in-
dicated that the TFs, Bl, and Csa5G157230 have
a similar biochemical function and that they
control CuC biosynthesis in different organs.
Next, we tested whether Csa5G157230 could di-
rectly regulate the Bi gene. Here, we expressed
the Myc-tagged protein in cotyledons of XY-3
to prepare sufficient material for ChIP assays.
Similar to Bl, Csa5G157230 could bind to the
E-box elements within the Bi promoter (fig. S4, B
to F). Taken together, these studies provide strong
support for the hypothesis that Csa5G157230 is
the Bt gene, which activates Bi and regulates CuC
biosynthesis in the fruit.
which in turn is likely to result in the nonbitter
phenotype of cucumber (E3-231). Although the
Y1H assay showed that Bt could also interact
with the promoter of Csa6G088180 (fig. S5A),
Bt cannot activate Csa6G088180’s transcription
(figs.S5B and S6C).
Nine genes in CuC biosynthetic pathway
To catalyze the formation of CuC, cucurbitadienol
has to be further modified with a series of oxi-
dation reactions and acetylation, likely catalyzed
by cytochrome P-450 enzymes (P450s) and an
acyltransferase (ACT). On chromosome 6, Bi co-
localizes with four P450 genes (Csa6G088160,
Csa6G088170, Csa6G088180, and Csa6G088710)
and one ACT gene (Csa6G088700) within a 35-kb
genomic region. Except for Csa6G088180, all
other genes shared nearly identical expression
patterns, with high expression occurring in leaves
of line 9930 as well as in fruits of wild line PI
183967 (Fig. 4, A and B). In addition, these co-
expressed genes were down-regulated in leaves
of XY-3 as compared with XY-2 and in fruits
of han as compared to HAN, and they were
up-regulated in cucumber leaves under ABA treat-
ment or drought stress (Fig. 4, C to F, and table
S4). Furthermore, our studies showed that Bl
and Bt could specifically bind to the promoters
of these coexpressed genes and could activate
their transcription (Fig. 4G, and figs. S5 and
S6). Mutation (R85K) within the basic domain
of Bl appeared to affect its binding ability to
the CuC biosynthetic genes (fig. S5, C and D),
We failed in a search for the specific P450
within the cluster responsible for oxidizing cu-
curbitadienol, which suggests there should be
other candidates located outside this 35-kb ge-
nomic region. We reasoned that other genes
would be coexpressed with the Bi cluster and
coregulated by Bl and Bt. Therefore, by applying
the integrative bioinformatics and molecular
biology approach described above, we identify
four additional P450 genes (three on chromosome
3, Csa3G698490, Csa3G903540, and Csa3G903550,
and one on chromosome 1, Csa1G044890) that
are coexpressed with the Bi cluster and are acti-
vated by Bl and Bt in leaves and fruits, respec-
tively (Fig. 4, A to F, and table S4).
The relation of CuC biosynthesis and these
candidate tailoring enzymes was probed by using
a transient RNA interference (RNAi) system acting
on the bitter cotyledon of the cucumber line 9930
(18). RNAi-mediated target-specific down-regulation
of transcripts for all these candidate genes resulted
in a decrease in CuC content in the infiltrated
cotyledons (Fig. 4H and fig. S7). Thus, Bl and Bt
regulate bitterness formation in leaves and fruits,
Fig. 4. Nine pathway genes that are
coordinately regulated. (A and B)
Identification of coexpressed candidate
enzymes by analyzing transcriptomic
data acquired from cultivar 9930 (A)
and wild line PI 183967 (B). Candidate
enzymes are indicated with different
colors according to their annotations.
Low-expressed gene Csa6G088180 is
indicated with hatched green and was
used as a negative control in the
Chr 6
Chr 3
Chr 1
CYP81Q58CYP89A140
OSC
ACT CYP87D19 CYP712D8 CYP88L2 CYP88L3 CYP87D20
6G088160 6G088170 6G088180
Bi 6G088700 6G088710 3G698490 3G903540 3G903550 1G044890
Root
Stem
Leaf
Flower
Fruit
Tendril
Root
Stem
Leaf
following analyses. (C to F) Coregulation
of candidate genes (means T SEM,
n = 3). Down-regulation of the nine
genes in XY-3 as compared with XY-2
(C) and han as compared with HAN
(D) (asterisk indicates samples
Flower
Fruit
Tendril
100%
100%
XY-3 (blbl)
XY-2 (BlBl)
prepared from plants grown under low
temperature), and up-regulation of
the nine genes in the presence of ABA
treatment (E) or drought stress (F).
(G) Summary of interaction between
candidate gene promoter and Bl or Bt.
Luc, luciferase trans-activation assay.
(H) Function of enzymes elucidated by
transient RNAi assays (means T SEM,
n = 6). RNAi sample in blue; CK in red.
Value obtained from control (CK) was
set to 1 and used to obtain relative
values for the RNAi sample. CK, sample
infiltrated with empty vector. More
information is provided in figs. S5 to S7.
han*(btbt)
HAN*(BtBt)
100%
100%
-
ABA
+
-
Drought
+
+
+
Bl
Y1H
Bt
+
+
+
+
-
+
+
+
+
+
+
+
+
+
+
+
+
+
+
Bl
+
+
-
+
+
+
+
+
+
Luc
+
+
+
Bt
+
+
+
+
+
+
-
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
Bl
EMSA
Bt
-
-
+
+
+
Bl
+
+
-
+
+
+
+
+
+
ChIP
Bt
+
+
+
-
+
+
+
+
+
+
100%
CuC content
Gene
expression
CK
RNAi
SCIENCE sciencemag.org
28 NOVEMBER 2014 • VOL 346 ISSUE 6213 1087

RESEARCH
| RESEARCH ARTICLES
respectively, by direct transactivation of nine genes
(one OSC, seven P450s, and one ACT) involved in
the CuC biosynthetic pathway.
We continued to search for downstream
P450s using this same approach. As revealed
by liquid chromatography–mass spectrometry
(LC-MS) assays, we identified an expected peak
in the yeast expressing Bi, CPR, Csa3G903540,
and Csa6G088160 (a member of CYP81 family,
located within the Bi cluster) (Fig. 5B). Tan-
dem mass spectrometry (MS/MS) and NMR
analysis revealed that a hydroxyl group was
transferred to the C-25 position of 19-hydroxy
cucurbitadienol and that the double bond be-
tween C-24,25 was shifted to the position of
C-23,24 (figs. S11 and S12). The product (compound
2) of Csa6G088160 was named 19,25-dihydroxy
cucurbitadienol.
From fresh bitter cucumber leaves, our NMR
analysis identified a deacetyl CuC (figs. S13 and
S14, compound 3). LC-MS analysis showed that
the ACT enzyme (Csa6G088700) was able to acetyl-
ate this compound to yield CuC (Fig. 5C). These
studies indicate that Csa6G088700 is the enzyme
involved in the final step in the CuC biosynthetic
pathway.
In summary, we discovered that two TFs regu-
late nine genes in the CuC biosynthetic path-
way and propose a model as to how extremely
bitter wild cucumber was domesticated into
nonbitter cultivars (fig. S15). As revealed in this
study, such regulators must contribute to the
highly coordinated and efficient transcription
of plant specialized metabolic pathways. The
new knowledge on cucurbitacin biosynthesis
will open a door for biological manufacturing
and engineering of these triterpenoids as anti-
tumor drugs, for example, in a manner similar
to the biosynthesis of artemisinic acid, the anti-
malarial drug precursor (19, 20).
Three more steps in CuC biosynthesis
To characterize the biochemical function of
these candidate P450s, we expressed each P450
in the engineered yeast (EY10) that accumu-
lates 10 times as much cucurbitadienol as its
original strain (18) (fig. S8). No expected product
was detected from yeast extract at first (Fig. 5A).
However, once an NADPH–cytochrome P450
oxidoreductase gene (CPR, Csa1G423150) was
expressed with candidate P450 in the EY10, we de-
tected a specific product catalyzed by Csa3G903540
(a member of CYP88 family, located outside the
Bi cluster) (Fig. 5A). The structure of this purified
product (compound 1) was interrogated by nuclear
magnetic resonance (NMR) spectroscopy (figs. S9
and S10), which indicated that it was a derivative of
cucurbitadienol in which the 19-CH3 was hydroxy-
lated. The product of Csa3G903540 was named
19-hydroxy cucurbitadienol.
REFERENCES AND NOTES
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2. R. L. Metcalf, R. A. Metcalf, A. M. Rhodes, Proc. Natl. Acad.
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510–513 (2014).
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1658–1661 (2012).
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91–99 (2014).
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15. J. Qi et al., Nat. Genet. 45, 1510–1515 (2013).
16. M. Shibuya, S. Adachi, Y. Ebizuka, Tetrahedron 60, 6995–7003
(2004).
X106
X104
TIC
EIC: 586
5
0
5
0
1
Bi+CPR
0
1
0
OH
Bi+Csa3G903540
Bi+CPR
HO
5
0
5
0
1
+Csa3G903540
0
1
19-hydroxy-
cucurbitadienol
Csa3G903540+CPR
0
19.5 20.5 21.5 22.5
18.5
19.5 20.5 21.5 22.5
18.5
Time (min)
Time (min)
17. G. Toledo-Ortiz, E. Huq, P. H. Quail, Plant Cell 15, 1749–1770 (2003).
18. Materials and methods are available as supplementary
material on Science Online.
19. D. K. Ro et al., Nature 440, 940–943 (2006).
20. C. J. Paddon et al., Nature 496, 528–532 (2013).
X106
X104
TIC
+ESI EIC: 459.3833
2
5
OH
OH
Bi+CPR+3G903540
0
0
5
Bi+CPR+3G903540 2
+6G088160
0
2
0
5
HO
Bi+CPR+3G903540
+6G088170
19,25-dihydroxy-
cucurbitadienol
0
2
ACKNOWLEDGMENTS
0
5
Bi+CPR+3G903540
+6G088710
We thank J. Bohlmann and D.-K. Ro for critical comments on the
manuscript and J.-J. Qi, X.-Z. Lin, T. Lin, X.-F. Xue, and X.-Y. Liu for
bioinformatic and experimental assistance. The P450s were named
according to the alignment made by D. Nelson (http://drnelson.uthsc.
edu/cytochromeP450.html). This work was funded by the National
Program on Key Basic Research Projects in China (the
0
0
5
Bi+CPR+3G903540 2
+1G044890
0
2
0
5
Bi+CPR+3G903540
+3G903550
0
2
0
5
Bi+CPR+3G903540
+3G698490
973 Program; 2012CB113900), National Science Fund for
0
0
Distinguished Young Scholars (31225025), National Natural Science
Foundation of China (31272161, 31322047, and 31101550),
Agricultural Science and Technology Innovation Program, and
National Key Technology R&D Program (the 863 Program;
2012BAI29B04). This work was also supported by the Shenzhen
Municipal and Dapeng District Governments. The Institute of Flowers
and Vegetables has three pending patent applications relating the
genes reported in this study. Supplementary materials contain
additional data. This whole-genome shotgun project has been
deposited at DNA Data Bank of Japan/European Molecular Biology
Laboratory/GenBank under the accession ACHR00000000. The
version described in this paper is version ACHR02000000. Genes
reported in the study are deposited in the National Center for
Biotechnology Information (NCBI), NIH, with accession numbers
(KM655851–KM655862, KM677686–KM677688). RNA-seq data may
be obtained from NCBI with the accession number SRA046916.
8
12
16
20
24
8
12
16
20
24
Time (min)
Time (min)
O
X107
X106
OH
TIC
+ESI EIC: 583.3241
OH
O
OH
5
HO
5
0
5
CuC
0
5
HO
deacetyl-CuC
Csa6G088700
deacetyl-CuC
Csa6G088700
Csa5G639480
0
5
0
5
O
O
0
5
0
5
OH
O
O
OH
HO
0
0
2
3
4
5
6
7
8
2
3
4
5
6
7
8
HO
CuC
Time (min)
Time (min)
Fig. 5. Three more catalytic steps. (A) GC-MS analysis of putative product (red arrow) generated by
Csa3G903540 in the engineered yeast (EY10). Partial enlarged details are shown as insets.TIC, total ion
chromatograms; EIC 586, extracted ion chromatograms at m/z of 586.The product structure (right) was
elucidated by NMR (18). (B) Ultra-performance liquid chromatography coupled with quadropole time-of-
flight mass spectrometry (UPLC-qTOF-MS) analysis of yeast extracts with electrospray ionization (ESI)
on positive mode. EIC 459.3833, extracted ion chromatograms of the accurate parent ion at m/z of
459.3833.The product (indicated by red arrow) structure was elucidated by MS/MS and NMR (18). (C)
UPLC-qTOF-MS analysis of the acetyltransferase catalytic reaction product. Deacetyl-CuC is acetylated
by Csa6G088700-His in vitro (indicated by red arrow). A leaf-specific ACT (Csa5G639480) served as a
negative control. Schematic of this biosynthetic pathway from deacetyl-CuC to CuC is shown at right.
SUPPLEMENTARY MATERIALS
www.sciencemag.org/content/346/6213/1084/suppl/DC1
Materials and Methods
Figs. S1 to S15
Tables S1 and S8
References (21–24)
25 July 2014; accepted 3 November 2014
10.1126/science.1259215
1088 28 NOVEMBER 2014 • VOL 346 ISSUE 6213
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