Herbivore-induced and floral homoterpene volatiles are biosynthesized by a single P450 enzyme (CYP82G1) in Arabidopsis

Article abstract of DOI:10.1073/pnas.1009975107

Terpene volatiles play important roles in plant-organism interactions as attractants of pollinators or as defense compounds against herbivores. Among the most common plant volatiles are homoterpenes, which are often emitted from night-scented flowers and from aerial tissues upon herbivore attack. Homoterpene volatiles released from herbivore-damaged tissue are thought to contribute to indirect plant defense by attracting natural enemies of pests. Moreover, homoterpenes have been demonstrated to induce defensive responses in plant-plant interaction. Although early steps in the biosynthesis of homoterpenes have been elucidated, the identity of the enzyme responsible for the direct formation of these volatiles has remained unknown. Here, we demonstrate that CYP82G1 (At3g25180), a cytochrome P450 monooxygenase of the Arabidopsis CYP82 family, is responsible for the breakdown of the C₂₀-precursor (E,E)-geranyllinalool to the insect-induced C₁₆-homoterpene (E,E)-4,8,12-trimethyltrideca-1,3,7,11-tetraene (TMTT). Recombinant CYP82G1 shows narrow substrate specificity for (E,E)- geranyllinalool and its C ₁₅-analog (E)-nerolidol, which is converted to the respective C ₁₁-homoterpene (E)-4,8-dimethyl-1,3,7-nonatriene (DMNT). Homology-based modeling and substrate docking support an oxidative bond cleavage of the alcohol substrate via syn-elimination of the polar head, together with an allylic C-5 hydrogen atom. CYP82G1 is constitutively expressed in Arabidopsis stems and inflorescences and shows highly coordinated herbivoreinduced expression with geranyllinalool synthase in leaves depending on the F-box protein COI-1. CYP82G1 represents a unique characterized enzyme in the plant CYP82 family with a function as a DMNT/TMTT homoterpene synthase.

Full text of DOI:10.1073/pnas.1009975107

Herbivore-induced and floral homoterpene volatiles
are biosynthesized by a single P450 enzyme
(
CYP82G1) in Arabidopsis
a b c
d
e
a,1
Sungbeom Lee , Somayesadat Badieyan , David R. Bevan , Marco Herde , Christiane Gatz , and Dorothea Tholl
a
b
c
d
Departments of Biological Sciences, Biological Systems Engineering, and Biochemistry, Virginia Tech, Blacksburg, VA 24061; Department of Biochemistry
e
and Molecular Biology, Michigan State University, East Lansing, MI 48824; and The Albrecht-von-Haller-Institute for Plant Sciences, Georg-August-University
Göttingen, D-37073 Göttingen, Germany
Edited by May R. Berenbaum, University of Illinois at Urbana–Champaign, Urbana, IL, and approved October 26, 2010 (received for review July 9, 2010)
Terpene volatiles play important roles in plant-organism interac-
tions as attractants of pollinators or as defense compounds against
herbivores. Among the most common plant volatiles are homo-
terpenes, which are often emitted from night-scented flowers and
from aerial tissues upon herbivore attack. Homoterpene volatiles
released from herbivore-damaged tissue are thought to contribute
to indirect plant defense by attracting natural enemies of pests.
Moreover, homoterpenes have been demonstrated to induce de-
fensive responses in plant–plant interaction. Although early steps
in the biosynthesis of homoterpenes have been elucidated, the
identity of the enzyme responsible for the direct formation of
these volatiles has remained unknown. Here, we demonstrate that
CYP82G1 (At3g25180), a cytochrome P450 monooxygenase of the
Arabidopsis CYP82 family, is responsible for the breakdown of the
In Arabidopsis, emission of TMTT and other volatiles is in-
duced upon leaf damage by the crucifer-specialists Pieris rapae
and Plutella xylostella (10, 11). The volatile blend was shown to
attract the parasitic wasp Cotesia rubecula, which parasitizes
P. rapae larvae and, therefore, led to increased plant fitness (10,
12). Although Arabidopsis leaves release none or negligible
amounts of DMNT under natural conditions, olfactometer
experiments with transgenic Arabidopsis plants, which were con-
stitutively emitting DMNT and its precursor (E)-nerolidol,
demonstrated the ability of the two volatile compounds to attract
P. persimilis (13).
Homoterpenes may also exert other defensive activities, such as
the direct repulsion of aphids (14). Moreover, homoterpene emis-
sion from Arabidopsis is induced upon infection by Pseudomonas
syringae DC3000 (15) and after fungal elicitor treatment (11). Fi-
nally, studies in lima bean revealed the ability of TMTT to induce
the expression of defense genes in plant–plant interactions (5).
Despite the wide occurrence of homoterpenes in floral odors
and volatile blends induced by biotic stress, knowledge of the
biosynthesis of these compounds has been fragmentary. The first
committed step in the formation of TMTT is the conversion of the
central C20-diterpene precursor geranylgeranyl diphosphate to the
tertiary alcohol (E,E)-geranyllinalool (Fig. 1). A geranyllinalool
synthase (GES) has been identified from Arabidopsis (11) and
terpene synthases catalyzing the analogous conversion of the
C -prenyldiphosphate farnesyl diphosphate to (E)-nerolidol in
C20-precursor (E,E)-geranyllinalool to the insect-induced C16-homo-
terpene (E,E)-4,8,12-trimethyltrideca-1,3,7,11-tetraene (TMTT). Re-
combinant CYP82G1 shows narrow substrate specificity for (E,E)-
geranyllinalool and its C15-analog (E)-nerolidol, which is converted
to the respective C11-homoterpene (E)-4,8-dimethyl-1,3,7-nona-
triene (DMNT). Homology-based modeling and substrate docking
support an oxidative bond cleavage of the alcohol substrate via
syn-elimination of the polar head, together with an allylic C-5 hy-
drogen atom. CYP82G1 is constitutively expressed in Arabidopsis
stems and inflorescences and shows highly coordinated herbivore-
induced expression with geranyllinalool synthase in leaves depend-
ing on the F-box protein COI-1. CYP82G1 represents a unique char-
acterized enzyme in the plant CYP82 family with a function as
a DMNT/TMTT homoterpene synthase.
1
5
DMNT formation have been characterized (3, 7). Experiments
with stable-isotope precursors suggested a subsequent oxidative
degradation of (E,E)-geranyllinalool and (E)-nerolidol to their
respective homoterpenes (Fig. 1) (16). Analogous biosynthetic
pathways involving the oxidative C-C bond cleavage of a tertiary
alcohol have been described for the dealkylation of 22-hydroxy-
cholesterol into androstenolone (17) and the formation of the
furanocoumarin psoralen from its precursor (+)-marmesin in
Apiaceae (Ammi majus) (18). Because these pathways are cata-
lyzed by one or two enzymes of the cytochrome P450 mono-
oxygenase (P450) family, it was assumed that P450-type enzymes
may catalyze the final degradation steps in homoterpene bio-
synthesis (Fig. 1).
floral scent
| herbivory | terpene biosynthesis
lants interact with the environment by producing a variety of
P
chemical compounds. In particular, volatile compounds emit-
ted from flowers and vegetative plant tissues serve as attractants for
pollinators or exert defensive activities against herbivores, thereby
contributing to plant survival and reproductive success. Among the
most common plant volatiles are the irregular C16-homoterpene
E,E)-4,8,12-trimethyltrideca-1,3,7,11-tetraene (TMTT) and its
11-analog (E)-4,8-dimethyl-1,3,7-nonatriene (DMNT), both of
which are widespread floral odor constituents contributing to the
white-floral image”ofnightscentedflowers(1). Moreover, TMTT
(
C
Here, we report that CYP82G1 (At3g25180), encoding a P450
enzyme of the Arabidopsis thaliana CYP82 family, is responsiblefor
the conversion of (E,E)-geranyllinalool to TMTT. CYP82G1 is
expressed constitutively in flowers and coexpressed locally with
“
and DMNT are released in response to herbivore attack from the
foliage of gymnosperms (2) and numerous angiosperms, both
monocots and dicots (3–7). Several studies have indicated a role of
homoterpene volatiles in the attraction of herbivore predators in
indirect plant defense. For example, de Boer et al. (8) demon-
strated that TMTT influenced the foraging behavior of predatory
mites when emitted in the presence of other induced volatiles from
lima bean leaves infested by spider mites (Tetranychus urticae). In
addition, treatment of lima bean with the terpenoid pathway in-
hibitor fosmidomycin, which severely reduced the emission of
homoterpenes, led to a reduced attraction of the predatory mite
Phytoseiulus persimilis (9).
Author contributions: S.L., S.B., D.R.B., M.H., C.G., and D.T. designed research; S.L., S.B.,
and M.H. performed research; S.L., S.B., D.R.B., M.H., and D.T. analyzed data; and S.L., S.B.,
and D.T. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
1
To whom correspondence should be addressed. E-mail: tholl@vt.edu.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
1073/pnas.1009975107/-/DCSupplemental.
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CYP82G1 Gene Knockout Plants Do Not Produce TMTT and Their
Phenotype Is Complemented by the Constitutive Expression of
CYP82G1. To determine the function of CYP82G1 in the forma-
tion of TMTT in vivo, a corresponding gene knockout line, GABI-
Kat (GK) 377A01 (21), was analyzed. GK377A01 plants carry a
T-DNA insertion in the first intron of the CYP82G1 gene (Fig. 2A).
When leaves of homozygous GK377A01 plants were treated with
alamethicin, no CYP82G1 transcript was detected by RT-PCR
OPP
OPP
GGPP
GES
FPP
NES
OH
OH
(
E,E)-Geranyllinalool
Oxidaꢀve
degradaꢀon
(E)-Nerolidol
(
Fig. 2B) compared with wild-type plants. In contrast, induction of
P450
P450
the GES transcript was found in wild-type and CYP82G1 knockout
plants, confirming a successful elicitation by alamethicin (Fig. 2B).
In agreement with these results, the CYP82G1 mutant produced
geranyllinalool but no TMTT in response to alamethicin treat-
ment (Fig. 2C and Fig. S1C). Interestingly, emission levels of gera-
nyllinalool in alamethicin-treated CYP82G1 knockout plants were
similar to those in wild-type plants and no major accumulation of
geranyllinalool was observed, suggesting feedback regulatory mech-
anisms in the formation of the TMTT precursor. We also tested
for the formation of TMTT in alamethicin-treated knockout lines of
At1g19250 (salk_026163) and two other genes (salk_114795 for
At3g55970 and salk_073705 for At5g05600), which showed no ex-
TMTT
DMNT
Fig. 1. Proposed biosynthetic pathways for the formation of volatile
homoterpenes in plants. GGPP, geranylgeranyl diphosphate; FPP, farnesyl
diphosphate; TMTT, 4,8,12-trimethyltrideca-1,3,7,11-tetraene; DMNT, 4,8-
dimethyl-1,3,7-nonatriene; GES, geranyllinalool synthase; NES, nerolidol
synthase; P450, cytochrome P450 monooxygenase.
GES at the sites of insect feeding damage. We further show that the pression in Pro35S:GES × coi1 plants (Fig. S1A and Table S1). None
recombinant CYP82G1 enzyme is able to convert (E)-nerolidol, of the mutants showed disrupted TMTT formation (Fig. S1D).
To complement the GK377A01 phenotype, a 1,548-bp CYP82G1
cDNA (GenBank NM_113423) was expressed constitutively under
the control of the CaMV 35S promoter in the GK377A01 mutant
background. Expression of CYP82G1 in the transgenic lines
the C15-analog of geranyllinalool, to the respective C11-terpene
DMNT and that substrate specificity is dependent on the position
of the hydroxyl group and configuration of the prenyl chain.
CYP82G1 is unique as a characterized enzyme in the plant CYP82
family that functions in homoterpene volatile formation.
(
Fig. 3A and Fig. S1E) restored the formation of TMTT (Fig. 3B).
In the absence of an elicitor, emission of TMTT from Pro35S
:
Results
CYP82G1 plants was higher than TMTT background emissions
from wild-type plants (Fig. 3B), indicating an increased conversion
of basal levels of geranyllinalool. Interestingly, treatment of leaves
of CYP82G1-expressing plants with alamethicin caused higher
levels of CYP82G1 transcript (and to some extent GES transcript)
compared with mock-controls, suggesting an increased mRNA
stability under these conditions (Fig. 3A). In contrast, the rise in
alamethicin-induced TMTT emission was only ≈1.5-fold over that
CYP82G1 Is Coexpressed with GES upon Elicitor Treatment. As a
strategy to identify putative P450s involved in TMTT biosynthesis,
we performed in silico screening of P450 gene candidates, which
are coexpressed with GES encoding the precursor enzyme ger-
anyllinalool synthase (Table S1). Besides P450s, putative coex-
pressed flavin-dependent monooxygenases, dioxygenases, and per-
oxidases, were considered as possible gene candidates because
their role in TMTT formation could not be entirely excluded at the
beginning of the study. A total of 16 genes with the highest coex-
pression coefficients were selected for further analysis (Table S1).
We then performed semiquantitative RT-PCR to examine
transcripts of the selected genes in detached rosette leaves treated
with alamethicin, an ion-channel–forming peptide elicitor from
the fungus Trichoderma viride (19) (Fig. S1A). Because alame-
thicin induces the expression of GES and the formation of TMTT
GABI-Kat 377A01
A
(
GK377A01) T-DNA
5
’
3’
(bp) 0
Ala
400
800 1200 1600 2000
-
+
-
+
-
+
B
(
11), enhanced mRNA levels of candidate genes coexpressed with
Col-0
GK377A01
GES were expected in comparison with basal transcription levels
in control leaves. Gene transcript analysis was also performed in
plant lines constitutively expressing GES under the control of the
cauliflower mosaic virus (CaMV) 35S promoter (Fig. S1A). These
plants were previously shown to produce geranyllinalool and
TMTT at ratios of 1:2 to 1:5 (11), indicating expression of the
genes responsible for the conversion of geranyllinalool to TMTT.
As an additional screening strategy, we tested whether the tran-
scription of candidate genes was dependent on the F-box protein
COI1, a central regulator in jasmonate-dependent responses.
Plants constitutively expressing GES (11) and crossed into the
coi1 background produced geranyllinalool but no TMTT (Fig.
S1B), demonstrating COI1 dependency of the final steps in
TMTT biosynthesis. Therefore, no expression of candidate genes
was expected in these lines. A comparison of transcripts of all 16
genes in the different plants under the described conditions
resulted in the selection of two gene candidates as best matches
for the expected transcript profile (Fig. S1A): the putative P450
gene At3g25180 (CYP82G1) and the putative flavin-dependent
monooxygenase At1g19250 (20).
CYP82G1
GES
Actin8
C
6
TMTT
Geranyllinalool
6
TMTT
Geranyllinalool
4
2
0
Col-0
4
2
0
GK377A01
Mock Ala
Mock Ala
Fig. 2. Emission of TMTT and expression of CYP82G1 in wild type and
CYP82G1 knockout plants in response to mock- and alamethicin-treatment.
(A) Schematic of the CYP82G1 gene and position of the T-DNA insertion in the
GK377A01 line. Gray boxes represent exons, and introns are shown by the
black line. The position of the T-DNA insertion in intron 1 is indicated. (B)
Semiquantitative RT-PCR analysis of CYP82G1 and GES (geranyllinalool syn-
thase) gene transcripts in rosette leaves of Col-0 and GK377A01 plants in
response to 30 h of mock- and alamethicin (Ala)-treatment. Actin8 transcripts
were analyzed as a control. (C) Quantitative analysis of geranyllinalool and
TMTT emission from wild-type and CYP82G1 mutants treated as described
in B. Numbers are means ± SEM (n = 3).
2
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Lee et al.

6
5
4
3
2
1
0
0
0
0
0
0
0
To probe the substrate specificity of CYP82G1, enzyme assays
were also conducted with the smaller C15- and C10-analogs of
geranyllinalool, (E)-nerolidol and linalool. (E)-Nerolidol [>95%
A
B
CYP82G1
GES
b
(
3S)-enantiomer] was converted into DMNT (Fig. S3B and
Table S2), although no C -degradation product (4-methyl-
6
1,3-pentadiene) was detected from racemic linalool or (R)-(−)-
linalool (Table S2).
a
b
+
a
+
a
+
Conversion of (E,E)-geranyllinalool and (E)-nerolidol to their
respective homoterpene products could also be achieved by in
vivo assays and headspace volatile analysis of the transformed
-
-
+
-
-
Ala Ala
Ala Ala
Col-0 Pro35S
:
Col-0 Pro35S:
CYP82G1
CYP82G1
yeast cultures (Table S2). A C -cleavage product (but-1-en-3-one)
4
resulting from the breakdown of (E,E)-geranyllinalool or (E)-
nerolidol was not observed, neither in vitro nor in vivo, and none of
the previously proposed ketone intermediates, C -farnesylacetone
6
5
4
3
2
1
0
1
Col-0
2 Empty vector
Pro35S:CYP82G1mut
4 Pro :CYP82G1 #1
1
8
3
and C13- geranylacetone (16), were detected in comparison with
empty vector control assays.
In addition to (E)-nerolidol and linalool, we tested eight other
putative substrates using in vitro and in vivo assays (Table S2).
With the exception of the in vivo formation of small amounts of
DMNT and TMTT in the presence of (E,E)-farnesylacetone and
3
5S
5
Pro35S:CYP82G1 #2
1
2345 12345 12345 12345
Mock Mock
Ala Ala
TMTT Geranyllinalool
(
E)-geranylacetone, none of the selected compounds was con-
verted into the homoterpene products (Table S2). The trace
amounts of TMTT and DMNT found by incubation with (E,E,E)-
geranylgeraniol and (E,E)-farnesol, respectively, can most likely
be attributed to contamination by (E,E)-geranyllinalool and (E)-
nerolidol.
Fig. 3. Complementation analysis of the CYP82G1 T-DNA insertion line
GK377A01. (A) Quantitative RT-PCR analysis of relative CYP82G1 and GES
transcript levels in Col-0 and Pro35S:CYP82G1 plants with and without ala-
methicin (Ala) treatment. Transcript levels of Col-0 mock controls were ar-
bitrarily set to 1. The average GES mRNA level in mock controls of transgenic
plants was 11.1 (not visible at the selected y-axis scale). Different letters
above bars indicate significant differences among the means calculated with
one-way analysis of variance (ANOVA) and Tukey-Kramer HSD test for
CYP82G1 (P < 0.01) and GES (P < 0.05). Values were normalized to transcript
levels of protein phosphatase 2A and are means ± SEM of three individual
wild type plants or transgenic plants of three independent lines (#1–3). (B)
Quantification of TMTT and geranyllinalool emission from wild type and
Pro35S:CYP82G1 plants of two independent lines. Lines carrying the empty
expression vector or an expression construct with a truncated CYP82G1 gene
were used as controls (Fig. S1E). Numbers are means ± SEM from at least
We further determined basic kinetic properties of CYP82G1 for
the substrates (3RS)-(E,E)-geranyllinalool and (3S)-(E)-nerolidol
(
Table 1). Apparent K
m
values were similar for both substrates and
of A. majus psoralen synthase,
comparable to the apparent K
m
which catalyzes an analogous cleavage reaction of (+)-marmesin
(18). In contrast, catalytic efficiencies for (E)-nerolidol and (E,E)-
geranyllinalool were 30- to 70-fold lower than that reported for
psoralen synthase. A more detailed comparison of kinetic param-
eters between the single (3S)- or (3R)-enantiomers of (E)-nerolidol
and (E,E)-geranyllinalool was not possible because of substrate
unavailability.
three individual plants. Differences in TMTT emission between Pro35S
CYP82G1 plants and wild-type plants were not statistically significant.
:
CYP82G1 is Expressed in Inflorescences and in Leaves upon Insect
Feeding. To allow for a more detailed analysis of the tissue-specific
expression of CYP82G1, we performed histochemical CYP82G1
promoter-beta-glucuronidase (GUS) assays. In Arabidopsis plants
stably transformed with a GUS vector construct carrying a 2.6-kb
CYP82G1 promoter fragment, GUS staining occurred in flower
peduncles, in the receptacle of developing and mature flowers,
and in the stigma of mature opening flower buds (Fig. 4 A and B).
GUS staining was also observed in stems with GUS activity
gradually increasing toward the inflorescence (Fig. 4C). Although
no GUS staining was detected in undamaged leaves of mature
plants, reporter-gene activity was induced within 24 to 48 h of
feeding damage by P. xylostella larvae (Fig. 4D). GUS staining
occurred locally in a 0.3-inch wide zone around the site of damage
(Fig. 4D). A similar response was found in leaves infested with
thrips (Fig. 4 E and F), and after 24 h of mechanical wounding
(Fig. 4G).
of induced wild-type plants (Fig. 3B), which further indicates ad-
ditional regulatory mechanisms in TMTT formation based on
substrate limitation or feedback control. Nevertheless, the higher
emission rates of TMTT in elicitor-treated CYP82G1-expressing
lines correlated with undetectable levels of geranyllinalool in con-
trast to wild-type plants (Fig. 3B), suggesting an efficient conversion
of geranyllinalool to TMTT by the constitutively expressed
CYP82G1 enzyme.
In addition to the full-length ORF of CYP82G1, a 1,121-bp
cDNA encoding a CYP82G1 protein truncated by 150 amino
acids at the carboxy-terminus was expressed in the GK377A01
mutant background (Fig. S1E). Complementation with this trun-
cated CYP82G1 protein, which lacked the heme-binding domain
(
FGSGRRSCPG) and the highly conserved PERF region (Fig.
S2A), did not result in the production of TMTT (Fig. 3B).
Induction of CYP82G1 in response to feeding by P. xylostella
larvae was confirmed at the gene transcript level using quan-
titative real-time PCR (Fig. S4A). Transcription of CYP82G1 was
also found upon infection with P. syringae DC3000, which induces
TMTT emission and expression of GES (11, 15) (Fig. S4A). Tran-
CYP82G1 Functions as a TMTT and DMNT Synthase. To verify the
biochemical function of CYP82G1 in vitro, the ORF of the
CYP82G1 cDNA encoding 515 amino acids was cloned into
the yeast expression vector YEp352 under the control of the con-
stitutive alcohol dehydrogenase 1 (ADH1) promoter (22). Micro- script levels were in both cases lower than those in alamethicin-
somes were isolated from transformed yeast WAT11 cells and treated leaves in comparison with the respective mock-controls
tested for P450 enzyme activity using (3RS)-(E,E)-geranyllinalool (Fig. S4A). In contrast to the induction of CYP82G1 transcripts by
as the substrate. As expected, TMTT was detected as the enzyme biotic stress and elicitor-treatment, only a transient accumulation of
product in the presence of the P450 cofactor NADPH (Fig. S3A). CYP82G1 mRNA similar to that of GES was found after me-
No TMTT was found in assays with microsomes extracted from chanical damage, which is in agreement with the absence of TMTT
yeast carrying the empty YEp352 vector.
emission after 24 h of mechanical wounding (11) (Fig. S4B). The
Lee et al.
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Table 1. Kinetic parameters of the CYP82G1 recombinant enzyme
Substrate (μM) max (pkat/mg)
cat (s−
1
)
kcat/K
m
(s−1/μM)
K
m
V
k
(
(
3RS)-(E,E)-geranyllinalool
3S)-(E)-nerolidol
2.68 ± 0.71
1.84 ± 0.11
16.37 ± 0.78
29.09 ± 1.39
0.11 ± 0.01
0.20 ± 0.01
0.05 ± 0.01
0.11 ± 0.01
Values shown are means ± SEM of three replicates. K
m
, Michaelis-Menten constant; Vmax, maximal velocity;
k
cat, turnover number.
observed GUS activity after mechanical wounding is a result of the
stability of the transiently induced GUS protein.
The recombinant CYP82G1 enzyme showed narrow substrate
specificity for (E,E)-geranyllinalool and (E)-nerolidol. No enzy-
matic product was found for the (Z)-stereoisomer of nerolidol, for
primary prenylalcohols, or unsaturated substrate analogs (Table
Discussion
We have identified CYP82G1 from Arabidopsis as a P450 enzyme S2), indicating a critical role for the position of the hydroxyl group
that catalyzes the final step in the biosynthesis of the common plant and the configuration and degree of saturation of the aliphatic
chain for substrate binding and conversion. The latter result is in
agreement with feeding experiments in lima bean using several
partially saturated analogs of (E)-nerolidol (30).
homoterpene volatiles TMTT and DMNT. The CYP82G1 amino
acidsequencecontainsthehighly conservedheme-bindingdomain,
as well as the P450-specific PERF motif and the proline-rich do-
main required for efficient protein folding (Fig. S2A). Phylogenetic
analysis revealed highest similarity of CYP82G1 to CYP82L2 from
Populus trichocarpa (53% amino acid sequence identity) and to
CYP82L3 from papaya (52% identity) within the CYP82 family of
the CYP71 clan (Fig. S2B). No biochemical function has been as-
To further predict the binding mode of the two substrates, the
CYP82G1 fold was modeled on structural cores and regions of
templates derived from four mammalian P450 structures (2F9Q,
3
CZH, 3E6I, 2Q9F) (SI Materials and Methods), which were se-
lected based on highest amino acid sequence similarity (∼42%) to
the CYP82G1 protein. Although these P450s catalyze primarily
cribedtothelatterproteinsandtomost oftheothermembersofthe hydroxylation reactions of different substrates (SI Materials and
CYP82 family except for tobacco CYP82E4v1 and CYP82E5v2, Methods), several of them accommodate substrates of hydro-
which catalyze an oxidative N-demethylation reaction in the con-
phobic nature in hydrophobic active-site cavities. The generated
version of nicotine to nornicotine (23, 24) and Arabidopsis CYP82G1 model was supported by the identification of 10 pu-
tative substrate-binding residues (Fig. 5), all of which are posi-
tioned in five substrate recognition sites (SRS1, -2, -3, -4, -6) pre-
viously predicted for Arabidopsis P450s (31) (Fig. S2A). Molecular
docking of (E,E)-geranyllinalool and (E)-nerolidol showed that
both substrates occupied the same position in the enzyme binding
site with the hydroxyl group at C-3, forming a strong hydrogen
bond to the carbonyl oxygen of Thr313 (Fig. 5). Positioning of
the hydrophobic chain of both substrates was supported by sev-
eral hydrophobic residues lining the active site cavity (Fig. 5). The
model further indicated that the position of the allylic hydrogen
atoms at C-5 of (E,E)-geranyllinalool and (E)-nerolidol and the
CYP82C2 and CYP82C4, which can hydroxylate the substrate
-methoxypsoralen (25). Given the common emission of homo-
terpenes among a large number of angiosperms, including tobacco
26), Medicago (6), and poplar (27, 28), it is possible that other
members of the CYP82 family (Fig. S2B) are involved in homo-
terpene biosynthesis. Interestingly, the CYP82 family is absent in
monocots (29), indicating an independent convergent evolution of
P450s with functions in homoterpene biosynthesis in monocots
and dicots.
8
(
A
B
C
Phe113
Tyr222
Phe251
Leu491
Leu308
Leu126
Ala492
Leu312
D
E
F
G
C-5
C-3
Thr313
Ile320
2.1 Å
Fig. 4. Histochemical analysis of CYP82G1 promoter activity. A 2.6-kb
fragment of the CYP82G1 promoter was cloned upstream of the GUS re-
porter gene. ProCYP82G1:GUS gene expression pattern in inflorescences (A
and B), and stems (C). GUS activity was induced locally at sites of feeding
damage by P. xylostella (D), and thrips (E, Inset F), as well as by mechanical
wounding (G). The arrow indicates the part of the shoot, which is closest to
the inflorescence. Images are representative for at least five independent
transgenic lines.
Fig. 5. Position of (3S)-(E,E)-geranyllinalool (purple) and (3S)-(E)-nerolidol
(orange) in the active site of the CYP82G1 homology model. The main
interacting residues including hydrophobic active site residues are illus-
trated. According to the best-ranked docking mode for both substrates,
a strong hydrogen bond is formed between the hydroxyl groups at C-3 and
the carbonyl oxygen of Thr313 with a distance of 2.1 Å (black dashed line). A
hydrogen at C-5 and the hydroxyl group at C-3 have equal distances of 5.3 Å
(red dashed lines) to the Fe atom of the heme group.
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Lee et al.

hydroxyl group at C-3 relative to the reactive iron-oxo heme moiety
Fig. 5) supports an oxidative-bond cleavage reaction proceeding
by a syn-elimination (β-elimination) mechanism, as previously re-
ported by Boland et al. (16). In contrast, a docked position for
linalool with the least-favorite binding energy among the com-
pounds studied made the hydrogen atoms at C-5 inaccessible to
molecular oxygen (Fig. S5A).
Volatile Collection and Analysis. Volatiles from alamethicin-treated leaves
were collected for 28 h (7-h light period I, 14-h dark, 7-h light period II) on
(
5
-mg charcoal traps using a closed-loop stripping method, as described
previously (11, 32). Volatiles eluted from the traps were analyzed by GC-MS.
For qualitative and quantitative analysis of volatiles from P450 in vitro or in
vivo enzyme assays, automated solid-phase microextraction (SPME)-GC-MS
was performed using a Shimadzu AOC-5000 autoinjector. Further details are
described in SI Materials and Methods.
Further analysis to explore structural elements possibly con-
tributing to the oxidative degradation activity of the enzyme will
be required. The tertiary alcohol substrate may be converted by
a one-step reaction into an equimolar mixture of the homo-
Transcript-Profile Analysis of CYP82G1 and Selected Candidate Genes. For
semiquantitative and quantitative RT-PCR analysis of gene transcripts, total
RNA was isolated from leaves with the TRI REAGENT (Molecular Research
Center, Inc.). First-strand cDNA was synthesized from 3 μg of DNase-treated
total RNA using an oligo(dT) primer and SuperScript II reverse transcriptase
terpene product and the C
4
-carbonyl fragment but-1-en-3-one
(Fig. S5B, Path a), which is analogous to the conversion of the
(
Invitrogen). Detailed information on semiquantitative and real-time PCR
furanocoumarin (+)-marmesin into psoralen and acetone cat-
alyzed by CYP71AJ1 from A. majus (18). Similar to results of
previous experiments applying deuterium-labeled (E)-nerolidol
to floral tissues or lima bean leaves (16, 17, 26), we did not detect
analysis is given in SI Materials and Methods.
Plant Transformation and Screening of Transformants. Stable plant trans-
formation was conducted with Agrobacterium tumefaciens strain GV3101
using the floral vacuum-infiltration method (34). Transformed seedlings
were screened according to Harrison et al. (35) on 1/2 MS medium con-
taining 0.8% sucrose and 50 μg/mL kanamycin.
a C -carbonyl cleavage product, which might be because of its
4
high chemical reactivity. In an alternative reaction, (E)-nerolidol
or (E,E)-geranyllinalool may first be degraded into a C11-geranyl-
2
or C18-farnesylacetone intermediate by removal of a C -moiety,
as suggested previously (17), before a second C-C bond cleavage
yielding the final homoterpene product and acetate (30) (Fig.
S5B, Path b). This pathway is in part analogous to steroid deal-
kylation reactions (17). In our experiments, geranylacetone and
farnesylacetone were not detected as products of CYP82G1 and
both compounds were rather inefficient substrates for conversion
into homoterpenes. Docking of both substrates suggested an
antiorientation of the carbonyl-group relative to the C-4 hydrogen
Genetic Complementation Analysis. The coding region of CYP82G1 (1,548 bp)
was amplified by RT-PCR from total RNA extracted from alamethicin-treated
leaves and transferred to the binary destination vector pK7WG2 (36). As
a negative control of the complementation experiment, a heme-domain
truncated CYP82G1 gene fragment (1,121 bp) was amplified and cloned into
the same vector. Transformation of Arabidopsis and selection of kanamycin-
resistant transgenic plants was performed as described above. Additional
details are provided in SI Materials and Methods.
(Fig. S5A), which does not allow syn-elimination and may explain
Yeast Expression and Enzyme Assay. The full-length CYP82G1 cDNA was
cloned into the modified YEp352 gateway vector under control of the
constitutive ADH1 promoter (22). Expression of CYP82G1 was performed in
the yeast WAT11 strain following the procedure described previously (22).
CYP82G1 enzyme activity was measured in vivo or in vitro by incubating
a 5-mL culture or 1-mL microsomal reaction in 20- or 10-mL PTFE/Silicon
Septa screw cap glass vials, respectively, in the presence of different putative
substrates (Table S2). Volatiles in the headspace of the vial were collected
and analyzed by automated SPME-GC-MS as described under Volatile
Analysis in SI Materials and Methods. Volatiles were sampled by SPME rather
than organic solvent extracted or trapped by small-scale closed-loop strip-
ping, as none of the latter methods proved to be successful for recovering
the lack of enzyme activity. On the other hand, it is possible that
the active site of the enzyme might undergo conformational
changes after initial binding of the alcohol substrate, which may
allow subsequent cleavage of a ketone intermediate.
The demonstrated enzymatic conversion of both (E,E)-ger-
anyllinalool and (E)-nerolidol to TMTT and DMNT, respectively,
indicates that a single P450 enzyme can produce both homo-
terpenes. Although emission of DMNT from Arabidopsis leaves is
absent or marginal because of the lack of an (E)-nerolidol syn-
thase activity, many plants produce both DMNT and TMTT as
components of insect-induced or floral volatile blends. The in-
duced expression of CYP82G1 is tightly coordinated with that of
GES in a local-wound response depending on COI1 as the central
regulator of jasmonate-mediated reactions. Interestingly, the ex-
pression of GES and CYP82G1 overlap only partially in the
peduncles and receptacles of flowers, which may be a reason why
only trace emissions of homoterpenes have been observed in
Arabidopsis flowers (32).
measurable amounts of enzymatic product. K
m
and Vmax values were cal-
culated by using the HYPER 1.01 software (J. S. Easterby, University of Liv-
erpool, United Kingdom). Additional details are described in SI Materials
and Methods.
Histochemical Assay. A CYP82G1 promoter-GUS reporter gene fusion con-
struct was generated by amplifying a 2.6-kb CYP82G1 promoter fragment
(
ProCYP82G1) upstream of the start codon from genomic DNA with primers C
and D (Table S3). The ProCYP82G1 PCR product was inserted into the TOPO-
pENTR vector (Invitrogen) and then recombined into the binary pKGWFS7.0
vector (36). Transgenic plants were generated as described above. Histo-
chemical GUS staining was performed as previously described (37).
Materials and Methods
Plant Material and Growth Conditions. A. thaliana ecotype Columbia wild-
type plants and gene-knockout mutants were grown in potting mix under
short-day conditions (10-h light/14-h dark) at 22 °C and ∼150 μmol m− ·s
2
−1
Comparative Modeling of CYP82G1 and Docking of Substrates. Four high-res-
olution mammalian P450, structures with highest sequence similarity to
CYP82G1 (PDB: 2F9Q, 3CZH, 3E6I, 2Q9F) were selected for multiple template
comparative modeling using Modeler 9v7 (38). Details of homology mod-
eling and substrate docking are described in SI Materials and Methods and
Table S4.
PAR. The GABI-377A01 At3g25180 gene-knockout mutant was purchased
from the Nottingham Arabidopsis Stock Centre (http://nasc.nott.ac.uk) and
the other gene-knockout lines were from the ABRC Stock Center (http://
www.arabidopsis.org). Pro35S:GES plants were generated as described pre-
viously (11). Additional information is given in SI Materials and Methods.
Plant Treatments. Plants used for treatments were 6- to 7-wk-old. Treatments
of leaves with the elicitor alamethicin and with larvae of P. xylostella were
performed with some modification, as described previously (11). For in-
fection with P. syringae, leaves were sprayed with a P. syringae pv. tomato
DC3000 cell suspension (OD600 = 0.01) according to Yan et al. (33). For me-
chanical wounding, leaves were wounded with a sterile needle (for GUS
assays) or standard pliers (for RT-PCR) at independent sites. For RNA iso-
lation, only the wounded area of six sites was harvested and immediately
frozen at 0 to 24 h after treatment. Further details are provided in SI
Materials and Methods.
ACKNOWLEDGMENTS. We thank Eran Pichersky for helpful comments and
Janet Webster for editing the manuscript, Wilhelm Boland (The Max Planck
Institute for Chemical Ecology, Jena, Germany) for providing standards for
DMNT and TMTT, Tony Shelton and Hilda Collins (Cornell University, Ithaca,
NY) for providing Plutella xylostella larvae, Joe Chappell (University of Ken-
tucky, Lexington, KY) for making available the modified YEp352 vectors, and
Daniele Werck-Reichhart (Centre National de la Recherche Scientifique,
Strasbourg, France) for providing yeast WAT11 cells. This work was sup-
ported by a US Department of Agriculture Cooperative State Research, Ed-
ucation, and Extension Service National Research Initiative Grant 2007-
35318-18384 and by funds from Virginia Tech (to D.T.)
Lee et al.
PNAS
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vol. 107
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no. 49
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