Identification of (Z)-3:(E)-2-Hexenal isomerases essential to the production of the leaf aldehyde in plants

Article abstract of DOI:10.1074/jbc.M116.726687

The green odor of plants is characterized by green leaf volatiles (GLVs) composed of C6 compounds. GLVs are biosynthesized from polyunsaturated fatty acids in thylakoid membranes by a series of enzymes. A representative member of GLVs (E)-2-hexenal, known as the leaf aldehyde, has been assumed to be produced by isomerization from (Z)-3-hexenal in the biosynthesis pathway; however, the enzyme has not yet been identified. In this study, we purified the (Z)-3:(E)-2-hexenal isomerase (HI) from paprika fruits and showed that various plant species have homologous HIs. Purified HI is a homotrimeric protein of 110 kDa composed of 35-kDa subunits and shows high activity at acidic and neutral pH values. Phylogenetic analysis showed that HIs belong to the cupin superfamily, and at least three catalytic amino acids (His, Lys, Tyr) are conserved in HIs of various plant species. Enzymatic isomerization of (Z)-3-hexenal in the presence of deuterium oxide resulted in the introduction of deuterium at the C4 position of (E)-2-hexenal, and a suicide substrate 3-hexyn-1-al inhibited HI irreversibly, suggesting that the catalytic mode of HI is a keto-enol tautomerism reaction mode mediated by a catalytic His residue. The gene expression of HIs in Solanaceae plants was enhanced in specific developmental stages and by wounding treatment. Transgenic tomato plants overexpressing paprika HI accumulated (E)-2-hexenal in contrast to wild-type tomato plants mainly accumulating (Z)-3-hexenal, suggesting that HI plays a key role in the production of (E)-2-hexenal in planta.

Full text of DOI:10.1074/jbc.M116.726687

JBC Papers in Press. Published on April 29, 2016 as Manuscript M116.726687
The latest version is at http://www.jbc.org/cgi/doi/10.1074/jbc.M116.726687
(Z)-3:(E)-2-Hexenal isomerases producing the leaf aldehyde
Identification of (Z)-3:(E)-2-hexenal isomerases essential to the production
of the leaf aldehyde in plants*
Mikiko Kunishima, Yasuo Yamauchi1, Masaharu Mizutani, Masaki Kuse,
Hirosato Takikawa, Yukihiro Sugimoto
From the Graduate School of Agricultural Science, Kobe University, 1-1 Rokkodai,
Nada, Kobe 657-8501, Japan
Running title: (Z)-3:(E)-2-Hexenal isomerases producing the leaf aldehyde
1To whom correspondence should be addressed. Tel.: 81-78-803-5886; Fax:
81-78-803-5886; E-mail: yamauchi@kobe-u.ac.jp
Keywords: enzyme purification, lipid peroxidation, plant biochemistry,
polyunsaturated fatty acid (PUFA)
ABSTRACT
Phylogenetic analysis showed that HIs
belong to the cupin superfamily, and at
least three catalytic amino acids (His, Lys,
Tyr) are conserved in HIs of various plant
species. Enzymatic isomerization of
(Z)-3-hexenal in the presence of
deuterium oxide resulted in the
introduction of deuterium at the C4
position of (E)-2-hexenal, and a suicide
substrate 3-hexyn-1-al inhibited HI
irreversibly, suggesting that the catalytic
mode of HI is a keto-enol tautomerism
reaction mode mediated by a catalytic
His residue. The gene expression of HIs
in Solanaceae plants was enhanced in
specific developmental stages and by
wounding treatment. Transgenic tomato
plants overexpressing paprika HI
accumulated (E)-2-hexenal in contrast to
The green odor of plants is characterized
by green leaf volatiles (GLVs) composed
of
C6
compounds.
GLVs
are
biosynthesized from polyunsaturated
fatty acids in thylakoid membranes by a
series of enzymes. A representative
member of GLVs (E)-2-hexenal, known
as the leaf aldehyde, has been assumed to
be produced by isomerization from
(Z)-3-hexenal in the biosynthesis
pathway; however, the enzyme has not
yet been identified. In this study, we
purified
the
(Z)-3:(E)-2-hexenal
isomerase (HI) from paprika fruits and
showed that various plant species have
homologous HIs. Purified HI is a
homotrimeric protein of 110 kDa
composed of 35-kDa subunits and shows
high activity at acidic and neutral pHs.
wild-type
tomato
plants
mainly
1
Copyright 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

accumulating (Z)-3-hexenal, suggesting
that HI plays a key role in the production
of (E)-2-hexenal in planta.
(HPL) to produce (Z)-3-hexenal in
chloroplasts (9, 10). Branching from
(Z)-3-hexenal to (E)-2-hexenal then
forms unsaturated GLVs in two series,
i.e., (Z)-3 and (E)-2, based on the position
of the unsaturated bond in their structures.
(Z)-3-Hexenal and (E)-2-hexenal are
subsequently reduced to alcohols by
aldehyde reductases, aldo/keto reductases
(11), and alcohol dehydrogenases (12).
Alcohol forms of GLVs are further
converted to ester forms by a BADH
acyltransferase (2). A saturated form of
GLV, n-hexanal, is produced through the
oxidation of linoleic acid mediated by
13-lipoxygenase and hydroperoxide lyase
along with the hydrogenation of the C–C
double bond in (E)-2-hexenal by alkenal
reductase (13).
Plants emit various volatile
organic compounds (VOCs), such as
terpenes, volatile plant hormones, and
fatty acid derivatives. Abiotic and/or
biotic stresses often stimulate the
emission of VOCs (1). In response to
wounding, the emission of C6
compounds has been widely observed in
many plants. This emission is a fast
bioprocess, with that of (Z)-3-hexenal
being observed within a few minutes
after wounding (2) followed by
(E)-2-hexenal (3). C6 compounds such as
n-hexanal, (Z)-3-hexenal, (E)-2-hexenal,
and the corresponding alcohol and ester
derivatives are collectively named green
leaf volatiles (GLVs) because they have a
characteristic green leaf odor. Recent
investigations of the biological effects of
GLVs suggested that GLVs contribute to
protection against the invasion of fungi
and insects due to their pathological
effects (4-6). In addition, GLVs may be
involved in abiotic stress response; a
GLV with α,β-unsaturated carbonyl
bonds (E)-2-hexenal has been shown to
be a signal chemical inducing abiotic
stress-associated gene expression (7).
Among
GLV
species,
(E)-2-hexenal was identified by Curtius
and Franzen (14) at an early stage in
GLV research history and named the leaf
aldehyde
(originally
named
Blätteraldehyd in German). Despite this
early discovery, enzymes involved in
(E)-2-hexenal production have remained
unknown, although it has been assumed
to be catalyzed by an isomerase (15, 16).
In
this
study,
we
identified
(Z)-3:(E)-2-hexenal isomerase (HI) in
various plant species and identified the
common catalytic amino acids needed for
isomerase activity by analysis of the
enzymatic mechanism of isomerization.
Overexpression of paprika HI (CaHI) in
tomato plants led to a drastic change in
hexenal composition in both leaves and
fruits, indicating that this HI is a critical
GLVs are produced from
thylakoid
membrane-bound
fatty acids in
polyunsaturated
chloroplasts by a series of enzymes (Fig.
1). Briefly, linolenic acid released from
lipase from thylakoid membranes is
peroxidized by 13-lipoxygenase (LOX, 8)
and then cleaved by hydroperoxide lyase
2

enzyme determining green odor in
planta.
the activity) was incubated in 1 ml of
reaction mixture containing 10 mM
(Z)-3-hexenal in 50 mM Hepes-NaOH,
pH 7.0 for 30 min at 25°C. The reaction
was stopped and derivatized with
2,4-dinitrophenylhydrazine (DNPH) by
addition of 25 µl of 20 mM DNPH in
acetonitrile and 20 µl of HCOOH. After
10 min, dinitrophenylhydrazone (DNP)
derivatives were extracted with 300 µl of
n-hexane. After centrifugation, 150 µl of
the hexane layer was recovered and dried
in vacuo. The residue was dissolved in 50
µl acetonitrile and filtered through a
Cosmonice Filter (Nacalai Tesque, Kyoto,
Japan), after which 10 µl aliquots were
subjected to HPLC as described
previously (17).
EXPERIMENTAL PROCEDURES
Materials- Paprika (Capsicum
annuum L.), Arabidopsis (Arabidopsis
thaliana, Columbia-0), tomato (Solanum
lycopersicum, cv. Micro-Tom), potato
(Solanum tuberosum cv. Sassy), tobacco
(Nicotiana
benthamiana),
alfalfa
(Medicago sativa), and rice (Oryza sativa,
cv Nipponbare) were sown on Jiffy-7
peat pellets (Sakata Seed Co., Yokohama,
Japan) and kept at 4°C for 3 days in the
dark. The plants were then transferred to
the conditions of a 14-h-light (80 µmol
photons m–2 s–1)/10-h-dark cycle at 23°C
(for Arabidopsis, tomato, potato, tobacco,
alfalfa, and rice) or 28°C (for paprika).
(Z)-3-Hexenal was obtained from
Bedoukian Research Inc. (Danbury, CT,
USA). Other chemicals of research grade
were purchased from Wako Pure
Chemicals (Osaka, Japan) or Nacalai
Tesque (Kyoto, Japan). (Z)-3-Nonenal
and (Z,Z)-3,6-nonadienal were obtained
by oxidation with the Dess–Martin
periodinane (Wako Pure Chemical) from
Purification of CaHI- Paprika
pericarp (640 g) was homogenized with
1.5 volumes of 50 mM Hepes-NaOH, pH
7.0. Debris was removed by filtration
through two layers of gauze, and the
filtrate was centrifuged at 10,000g for 10
min. Ammonium sulfate was added to the
supernatant to 30% saturation, followed
by centrifugation at 10,000g for 10 min.
After lipids floating on the supernatant
were removed by filtration through two
layers of gauze, the supernatant was
applied to a phenyl-sepharose column (40
ml, GE Healthcare UK, Ltd., England)
(Z)-3-nonen-1-ol
and
(Z,Z)-3,6-nonadienol (Tokyo Chemical
Industry, Tokyo, Japan), respectively.
equilibrated
with
30%
saturated
Crude extract preparation and
HI activity measurement- Plant material
was homogenized with two volumes of
50 mM Hepes-NaOH, pH 7.0. After
centrifugation at 10,000g for 10 min, the
supernatant was used as a crude extract.
Crude extract (up to 500 µl, depending on
ammonium sulfate in 50 mM potassium
phosphate buffer (K-PB), pH 7.0.
Proteins were eluted with 50 mM K-PB,
pH 7.0, and the eluent was applied
directly to a hydroxyapatite (Nacalai
Tesque) column equilibrated with 50 mM
K-PB, pH 7.0. After washing with 250
3

mM K-PB, pH 7.0, HI activity was eluted
with 250 mM K-phosphate, pH 7.0,
containing 1% Triton X-100. Nine
volumes of 20 mM Hepes-NaOH, pH 7.0,
containing 0.1% β-D-dodecyl maltoside
(DDM) was added to the fraction
containing activity, which was then
applied to a Mono Q mini column (total
volume 2 ml, GE Healthcare). After
washing with 20 mM Hepes-NaOH, pH
7.0, containing 0.1% DDM, proteins were
eluted with 20 mM Hepes-NaOH, pH 7.0,
containing 0.1% DDM and 1 M NaCl.
The enzyme solution was concentrated by
ultrafiltration using Centricon-30 (Merck
Millipore, Germany) and diluted by nine
volumes of 20 mM Hepes-NaOH, pH 7.0,
containing 0.1% DDM. The proteins
were subjected to anion exchange column
membrane, and their amino acid
sequences were determined with a protein
sequencer (Procise Model 492, Applied
Biosystems, Foster City, CA, USA).
Cloning
of
CaHI
and
quantitative RT-PCR
(qRT-PCR)-
Similarity search of internal amino acid
sequences was performed against a
Solanaceae-specific
database
(Solcyc.solgenomics.net). To confirm
that Ca08g14620, including both internal
amino acid sequences, was identical to
CaHI, the open reading frame (ORF) of
Ca08g14620 was cloned as follows. Total
RNA was isolated from paprika fruits
with an RNeasy Plant Mini Kit
(QIAGEN), and cDNA was then
synthesized with ReverTra Ace® qPCR
RT Master Mix with gDNA Remover
(TOYOBO, Osaka, Japan). To obtain the
ORF of Ca08g14620, PCR was
performed with ExTaq DNA polymerase
(Takara Bio Inc., Shiga, Japan) using
chromatography
(Mono
Q,
GE
Healthcare) and fractionated with a
gradient of 0–0.3 M NaCl. Protein
concentration was determined by the
method of Bradford (18).
primers
(5′-GGATCCATGGATTTAATATTGGC
ATCG-3′) and CaHI SalI 3′
CaHI
BamHI
5′
Internal amino acid sequencing
of purified enzyme- Purified paprika
enzyme (2 µg) dissolved in 1 ml of 70%
HCOOH was chemically fragmented
with 1 mg of BrCN at room temperature
overnight in the dark. Fragmented
peptides recovered by centrifugal
(5′-GTCGACTTAAGGTGGGGCAATG
ACTGC-3′). The PCR product was
cloned into the TA Cloning vector
pMD19 and then sequenced with a
R
ꢀ
BigDye
Terminator v3.1 Cycle
evaporation
SDS-sample buffer and then separated by
Tricine-SDS-PAGE. After
were
dissolved
in
Sequencing Kit for confirmation.
Quantitative
(qRT-PCR)
real-time
RT-PCR
with
was performed
electrophoresis, the separated peptides
were blotted onto a PVDF membrane
with a semidry blotting system (Atto
Corp., Tokyo, Japan), and peptides were
visualized by CBB R-250 staining.
Stained bands were excised from the
Thunderbird SYBR Green qPCR Mix
(TOYOBO) and a LightCycler Nano
System (Roche, Basel, Switzerland)
using template cDNA prepared as
described above. Primers used for
qRT-PCR are shown in Table S1. For
4

analysis of relative transcript levels,
internal standard mRNA (list is shown in
Table S1) was used in all qRT-PCR
experiments, and the expression levels of
genes of interest were normalized to that
of the internal standard by subtraction of
the cycle threshold (CT) value of the
internal standard from the CT value of
the gene of interest.
Electrophoretically homogenous HI
fraction was used for enzyme assays.
Point mutation of recombinant CaHI
(rCaHI) was performed with
a
PrimeSTAR Mutagenesis Basal Kit
(Takara Bio) using primers shown in
Table S4.
3-Hexyn-1-al synthesis and
measurement of its inhibitory activity
against HI- 3-Hexyn-1-al was chemically
synthesized from 3-hexyn-1-ol (Tokyo
Chemical Industry, Tokyo, Japan) by
Phylogenetic
analysis-
Alignment and phylogenetic tree analysis
were performed using the software
MEGA 5 (19). List of protein sequences
used for analysis is shown in Table S2.
oxidation
with
the
Dess–Martin
periodinane (Wako Pure Chemical)
reagent according to the method of
Wavrin and Viala (20). To confirm its
purity, synthesized 3-hexyn-1-al was
derivatized with DNPH as described
above and then analyzed by LC–MS and
NMR. Synthesized 3-hexyn-1-al was
incubated with purified rCaHI for 4 h on
ice, and free 3-hexyn-1-al was then
removed by ultrafiltration using Vivaspin
500-30K (GE Healthcare). Residual HI
activity was measured by standard assay
as described above.
Heterogeneous expression of
HIs- The ORF of HI was amplified by
PCR using the primers shown in Table S3
and then subcloned into pMD19 (Takara
Bio). After confirming DNA sequence,
each HI gene was inserted into
pColdProS2 vector (Takara Bio), and
Escherichia coli strain BL21(DE3) was
transformed with the resultant expression
plasmid. E. coli harboring the expression
vector was grown with shaking at 37°C in
LB broth with 50 µg/ml ampicillin to
midlogarithmic phase. Expression was
induced by addition of IPTG to 0.1 mM,
and cultures were grown for a further 16
h at 15°C and harvested by centrifugation.
The pellet was washed twice with
phosphate-buffered saline (PBS) and then
resuspended with 400 µl of PBS. After
disruption of the cells by sonication (40
W, 10 s, three times) on ice, recombinant
HI protein in the soluble fraction was
purified on a His SpinTrap column (GE
1H-NMR analysis- All NMR
experiments were performed on a JEOL
JNM-AL300 (300 MHz at 1H, JEOL Ltd.,
Tokyo, Japan), and chemical shifts were
assigned relative to the solvent signal.
Hexenal-DNP derivatives were dissolved
in chloroform-d2 and added to tubes
having a diameter of 5 mm.
Production
of
transgenic
healthcare)
according
to
the
tomato overexpressing CaHI- A DNA
fragment of CaHI amplified with primers
manufacturer’s
instructions.
5

CaHI
NdeI
5′
30 min (for leaf) or 60 min (for fruit) at
25°C. The fiber was inserted into the
insertion port of a GC–MS (QP-5050,
Shimadzu, Kyoto, Japan) equipped with a
0.25 µm × 30 m Stabiliwax column
(Restek, Bellefonte, PA, USA). The
column temperature was programmed as
follows: 40°C for 1 min, increasing by
15°C min−1 to 180°C for 1 min (22). The
carrier gas (He) was delivered at a flow
rate of 1 ml min−1. Splitless injection with
a sampling time of 1 min was used. The
fiber was held in the injection port for 1
min to remove all compounds fully from
the matrix. The temperatures of the
injector and interface were 200°C and
230°C, respectively. The mass detector
was operated in electron impact mode
with ionization energy of 70 eV. To
identify compounds, retention indices and
MS profiles of corresponding authentic
specimens were used.
(5′-CATATGGATTTAATATTGGCATCG
-3′) and CaHI SalI 3′ was digested with
NdeI and SalI and ligated into the
pRI101-AN vector (Takara Bio Inc.,
Shiga, Japan) to construct a 35S
promoter-driven expression plasmid.
After Agrobacterium tumefaciens (strain
C58C1Rifr)
was
transformed
by
pRI101-AN::35S::CaHI, cotyledons of
tomato plants (cv Micro-Tom) were
transformed by Agrobacterium-mediated
transformation with kanamycin resistance
as
confirmation of insertion of 35S-driven
CaHI and the neomycin
a
selectable marker (21). For
phosphotransferase II gene in regenerated
transgenic tomato plants, genome PCR
was performed using the primers 35S-F
(5’-TCGCCGTAAAGACTGGCGAACA
-3’) and CaHI SalI 3′. Candidates were
further examined by qRT-PCR and
volatile analysis to establish transgenic
lines. Finally, transgenic lines showing
stable seed production were used for
analysis.
Homology
modeling-
Three-dimensional structures of HI
proteins were deduced by homology
modeling using SWISS-MODEL (23-25).
The amino acid sequences of HIs were
used to search for templates in the
database. The templates were selected
based on the Global Model Quality
Estimation values and the QMEAN
scores (26). Templates and the sequence
identity between the HIs and their
corresponding templates are shown in the
figures. The protein structures were
analyzed with Swiss PdbViewer (27).
Volatile analysis- Harvested
tomato materials were placed in a Falcon
tube (15 ml) with three stainless steel
beads (5 mm i.d.), and the tube was then
sealed tightly with Parafilm. After
samples were frozen in liquid N2, the
tissues were completely disrupted by
vigorous vortexing. When volatiles in
intact tissues were analyzed, 1 ml
saturated CaCl2 solution was added to
inactivate enzymes. An SPME fiber
(50/30
µm
DVB/Carboxen/PDMS,
Accession number—The full
sequences of the CaHI and alfalfa HI
(MsHI) have been deposited at the DDBJ
Supelco, Bellefonte, PA, USA) was
exposed to the headspace of the vial for
6

data
bank
with
accession
be 110 kDa (Fig. 2C). Isomerization of
hexenal by purified CaHI is
numbers LC146479 and LC146718,
respectively.
unidirectional, given that CaHI did not
convert (E)-2-hexenal to (Z)-3-hexenal
(Fig. S2, A and B). (Z)-3-Nonenal and
RESULTS
(Z,Z)-3,6-nonadienal,
substrates for other fatty acid-derived
volatiles (E)-2-nonenal (29) and
which
are
Purification of CaHI from red
paprika- At the start of this study, we
screened plant materials for high HI
activity. We first assayed activity in red
bell pepper (Capsicum annuum L.) fruits
because bell pepper showed a large
change in leaf aldehyde composition
during ripening; (Z)-3-hexenal was the
predominant GLV in green bell pepper,
whereas (E)-2-hexenal became the
predominant GLV during the change in
fruit color from green to red (28). This
phenomenon suggested that abundant HI
activity occurs in ripening bell pepper. As
expected, we detected activity in red
pepper fruits, and another bell pepper
variant, red paprika, showed the most
abundant activity (Fig. S1). We detected
no isomerase activities in Arabidopsis
leaves, tobacco leaves, or tomato fruits.
We accordingly used red paprika as a
source for purification of HI.
(E,Z)-2,6-nonadienal (30), respectively,
were also isomerized (Fig. S2, C and D).
Kinetic parameters against (Z)-3-hexenal
are shown in Table 2. CaHI showed high
activity at acidic to neutral pH (Fig. S3A).
Cloning of CaHI- To clone the
cDNA encoding CaHI, we determined
the internal amino acid sequences. After
the purified protein was chemically
cleaved by BrCN, polypeptide fragments
were separated by Tricine-SDS-PAGE
and blotted onto PVDF membrane (Fig.
S4A). Among the blotted peptides, amino
acid sequences of two major polypeptides
could be determined (Fig. S4B).
Homology search of these sequences
using a Solanaceae-specific database
(Solcyc.solgenomics.net) indicated that
the amino acid sequence of an unknown
CaHI was purified from
protein
encoded
by
Ca08g14620
pericarp of red paprika fruits by
successive column chromatography steps
(Table 1). At the final step of purification,
fractions containing activity matched
elution peaks of proteins (Fig. 2A).
SDS-PAGE analysis showed that each
fraction contained a single protein of 35
kDa (Fig. 2B), suggesting that CaHI was
purified to homogeneity. Native CaHI is
a trimetric protein, given that the
contained both sequences. To confirm
that this unknown protein was identical to
CaHI, the ORF of the unknown protein
was cloned into pColdProS2 plasmid and
then produced as a recombinant protein
(Fig. S5A). The purified recombinant
protein in soluble form showed high HI
activity (Fig. S5B), indicating that
Ca08g14620 encoded CaHI. rCaHI, like
native CaHI, showed high activity at
weakly acidic and neutral pHs (Fig. S3B).
molecular
mass
of
nondenatured
isomerase was estimated by BN-PAGE to
7

HI belongs to the cupin
superfamily- BLAST search using the
amino acid sequence of CaHI indicated
that it is a member of the cupin
superfamily. This superfamily is a large
protein family containing diverse
functional proteins such as storage
proteins and various enzymes (31). A
subfamily including CaHI is located near
germin and germin-like protein family
including 11S globulin and vicilin (Fig.
3). CaHI and highly homologous proteins
in Solanaceae species such as tomato and
potato are included in a clade named
Solanaceae HI, and another clade closely
related to HI is named Solanaceae HI-like.
We produced proteins belonging to clade
HI and HI-like as recombinant proteins
and assayed their activity. Proteins
belonging to clade HI showed activity but
one representative (SlHI-like 1 from
tomato) belonging to clade HI-like did
not (Fig. S5C). To investigate the
physiological role of HIs, their gene
considered the action mode of bacterial
β-hydroxydecanoyl thiolester dehydrase
(32) in which a His residue plays a
critical role in the isomerization reaction.
Among 357 amino acid residues, 38
differed among proteins belonging to
Solanaceae HI and HI-like clades, and
only His54 was conserved in all proteins
in the HI clade but not in the HI-like
clade, suggesting this His as a candidate
catalytic amino acid in HIs. To test this
hypothesis, we produced point-mutated
rCaHI(H54A).
As
expected,
rCaHI(H54A) showed no activity (Fig.
4A), indicating that His54 plays a critical
role in enzymatic reaction. By homology
modeling to deduce other catalytic amino
acids, K60 and Y128 were found to be
candidates owing to their locations near
H54 (Fig. 4B). We accordingly produced
rCaHI(K60A) and rCaHI(Y128A) and
found that these point mutations caused
loss of isomerase activity (Fig. 4A).
These results suggested that these three
amino acid residues (named catalytic
HKY) form a catalytic site in HI.
expression
levels
at
different
developmental stages were analyzed.
Expression levels of HIs in paprika and
potato
showed
developmental
being
HIs are distributed
among various plant species- Given that
(E)-2-hexenal has been detected in
various plant species other than
Solanaceae (9), the presence of other
types of HI was expected. To identify
other HIs, we searched in other plant
species for proteins showing lower
similarity but conserving the catalytic
HKY, finding candidates in alfalfa,
cucumber, and rice sequences (alignment
is shown in Fig. S7). We produced
recombinant HIs and assayed their
activity. All recombinant proteins having
stage-specific
expression,
extremely high in ripe fruits and sprouts,
respectively (Fig. S6, A and B). In
contrast, expression levels of tomato HIs
were consistently low (Fig. S5C),
consistent with our inability to detect HI
activity in tomato leaves and fruits (Fig.
S1).
Determination of amino acid
residues essential for enzymatic activity-
To identify amino acid residues essential
for the isomerization reaction, we
8

catalytic HKY showed HI activity (Fig.
S8), confirming that HIs are widely
distributed among various plant species.
Phylogenetic tree analysis showed that
these miscellaneous HIs form a branched
clade from Solanaceae HI, and HI-like
proteins having no HKY catalytic amino
acids are present as in the case of
Solanaceae HI (Fig. 3). HIs of
monocotyledonous plants (OsHIs) are
located far from those of dicotyledonous
plants.
These results indicate that rCaHI
isomerized (Z)-3-hexenal to
(E)-2-hexenal by abstraction of H+ at the
C2 position and subsequent H+ donation
at the C4 position in a keto–enol
tautomerism reaction mode (a plausible
catalytic mechanism is shown in Fig. 6A).
To further investigate the catalytic
mechanism, we produced point-mutated
rCaHI (K60R) and rCaHI (Y120F) to
evaluate the roles of the ε-NH2 group in
the K60 and OH groups in Y128,
respectively (Fig. S9A). rCaHI (K60R)
completely lost HI activity but rCaHI
(Y128F) retained the activity (Fig. S9B),
suggesting that K60 was involved in the
catalysis mechanism.
Catalysis mode of HI-
To investigate the catalytic mechanism of
HI, (Z)-3-hexenal was incubated with
rCaHI in the presence of D2O as a solvent,
and the enzymatic product was analyzed
by 1H-NMR to identify the positions and
geometry of the C–C double bond in
3-Hexyn-1-al acts as a suicidal substrate
for HI- As suggested by studies of
bacterial isomerases (32, 33), an
analogous compound having a C–C triple
bond can behave as a suicidal substrate in
an isomerization reaction by binding
irreversibly to the catalytic His.
Accordingly, we prepared 3-hexyn-1-al, a
1
2-hexenal. H-NMR spectra of authentic
(E)-2-hexenal-dinitrophenylhydrazone
(DNP)
and
enzymatic
D-labeled
2-hexenal are shown in Fig. 5. The
signals H-2 (6.35 ppm, m, 1H) and H-3
(6.27 ppm, m, 1H) were identified as the
protons on the double bond, and the
coupling constants between H-2 and H-3
(J=15.6 Hz) were determined to be E. By
comparison of the authentic and
enzymatic products, deuterium was
introduced mainly at the C4 position
(H-4) given that the integrated area of
triple
bond-containing
analog
of
(Z)-3-hexenal, and tested its inhibitory
activity. As shown in Fig. 6B,
3-hexyn-1-al
inhibited
rCaHI
stoichiometrically. The removal of
non-bound
3-hexyn-1-al
after
by
ultracentrifugation
HI
and
H-4
hexenal-DNP was half that in authentic
2-hexenal-DNP. Incorporation of
in
enzymatically
produced
3-hexyn-1-al incubation did not restore
activity, showing that inhibition by
3-hexyn-1-al was irreversible. This
finding showed that plant HI shares a
similar enzymatic property with bacterial
isomerases that are irreversibly inhibited
by suicidal substrates (a plausible
inhibitory mechanism is shown in Fig.
deuterium in C4 position (H-4) was also
1
supported by disturbed H signals of C3
position
(H-3)
in
D-labeled
(E)-2-hexenal-DNP (Fig. 5) due to
substituted deuterium of H-4 position.
9

6C).
leaves (Fig. 8A), corresponding to the
much higher expression of CaHI
introduced exogenously (Fig. 8A, inset).
In fruits, the wild type contained
(Z)-3-hexenal as the main hexenal (85%
± 5.8%), but (E)-2-hexenal was the sole
hexenal in transgenic plants (Fig. 8B).
These results suggest that the CaHI
transgene CaHI functions effectively and
thus determines hexenal composition in
planta.
Wounding treatment induced HI gene
expression- (E)-2-Hexenal is assumed to
play a protective role in wounding
response because it shows antifungal
activity (5, 34), leading to a hypothesis
that HI genes are induced by wound
treatment. To test this hypothesis,
induction of HI gene expression in
wounded paprika and tomato leaves was
investigated. Expression of SlHI1, CaHI,
and PINII genes, known as typical
wound-inducible genes (35), was
enhanced by wounding treatment (Fig. 7),
suggesting that HIs may be regulated at
the transcriptional level in response to
DISCUSSION
In this study, we identified HIs
responsible for (E)-2-hexenal production
in various plant species and showed
evidence
that
plants
produce
wounding,
leading
to
enhanced
(E)-2-hexenal by enzymatic reaction.
Expression analysis of HIs indicated that
HI levels remained low except under
several physiological conditions such as
wounding and at specific developmental
stages (Fig. 7 and S6), suggesting that the
physiological roles of (E)-2-hexenal are
limited to certain conditions. Previous
reports suggested that (E)-2-hexenal as
(E)-2-hexenal production.
Overexpression of CaHI in
transgenic tomatoes drastically changes
the (E)-2-hexenal production in planta-
(Z)-3-Hexenal is known to be the most
abundant volatile in tomato fruits and is
thus recognized as
a
source of
tomato-like flavor (36). To assess the
biological impact of HI on hexenal
composition, we produced transgenic
tomato plants overexpressing CaHI
(oxCaHI) and assayed their hexenal
composition. In both leaves and fruits of
wild-type tomatoes, (Z)-3-hexenal was
detected as the main hexenal, as reported
by previous authors (Fig. 8), suggesting
that the very low expression of inherited
tomato HI genes does not contribute to
production of (E)-2-hexenal. In contrast,
overexpression of CaHI in transgenic
tomato plants resulted in a drastic change
in (Z)-3/(E)-2-hexenal proportion in
well
as
(Z)-3-hexenal
were
wounding-responsive volatiles, given that
they showed antibiotic and defense
gene-inducing activities (4, 5, 34). In
response to wounding, the production of
GLVs including both (Z)-3- and
(E)-2-hexenals is an early event, a
reflexive response that starts within a few
minutes after wounding and leads to the
presence of large amounts of detectable
GLVs after 10 min (3). (Z)-3- and
(E)-2-hexenal production is mediated by
a series of enzymes including lipase,
13-LOX, HPL, and HI (Fig. 1), and these
enzymes need no cofactors for their
10

reactions. This biochemical property
would facilitate the rapid hexenal burst
after wounding. At the transcriptional
level, expression of HIs was low except a
specific developmental stage (Fig. S6)
and was induced by wounding treatment
over a period of hours (Fig. 7). Given that
emission of (E)-2-hexenal continues for
hours after wounding (3), this
transcriptional regulation of HIs in
wounding response supports this
long-term emission.
(E)-2-hexenal as an insect repellent to
protect sprouts from pests (39).
The isomerization catalyzed by
HI is likely to be the keto–enol
tautomerism reaction mode (Fig. 6A),
similar to that of the keto–enol
tautomerism-mediated isomerization of
the double bond of fatty acid derivatives,
which has been well studied in bacterial
fatty acid metabolism. In the case of
β-hydroxydecanoyl thioester dehydrase,
which catalyzes the reaction of double
bond isomerization on 10-carbon
thioesters of acyl carrier protein in the
biosynthesis of unsaturated fatty acids
under anaerobic conditions (40), the
catalytic mode is also the keto–enol
tautomerism mediated by a catalytic His,
Site
of
production
of
(Z)-3-hexenal are chloroplasts because
both 13-LOX and HPL are chloroplastic
proteins whereas cytosol is likely to be a
subsequent
production
site
of
(E)-2-hexenal judged from amino acid
sequence of HI indicating HIs as
cytosolic proteins, that is, native CaHI
includes N-terminal amino acid sequence
(Fig. S4B), and computational protein
localization predictors also indicated that
no transit signature sequence was found
in C-terminal region.
and
a
specific suicidal substrate,
3-decynoyl-N-acetylcysteamine,
inactivated the enzyme by irreversible
binding to the catalytic His (32). Also in
HIs,
a
specific suicidal substrate,
3-hexyn-1-al, completely inhibited HI
activities irreversibly (Fig. 6), suggesting
that the His residue plays a critical role in
catalytic function, plausibly the migration
of H+. The importance of two other
catalytic amino acids (K60 and Y128)
was also shown by complete loss of HI
HIs showed characteristic
expression at specific developmental
stages. In the case of paprika, higher
expression of CaHI was observed in ripe
fruits (Fig. S6A). This expression might
contribute to producing (E)-2-hexenal as
an antifungus volatile as in strawberry
(37). In potato, higher expression of
StHI1 and StHI2 was observed in sprouts
(Fig. S6B). Because in transgenic potato,
depletion of HPL to decrease GLV
contents caused an increase in aphid
performance (38), higher expression of
StHIs may promote the production of
activity
of
rCaHI(K60A)
and
rCaHI(Y128A) (Fig. 4). In the case of
K60, basic amino acid substitution with
Arg (K60R) resulted in the complete loss
of activity (Fig. S9), suggesting that the
protonated ε–NH2 group in K60 under
neutral and acidic pH conditions was
essential to the keto-enol tautomerism as
a proton donor. On the other hand, Y128
might contribute in forming the
11

substrate-binding pocket, given that the
OH group in Y128 was apparently not
involved in the catalysis, as shown by the
retained activity of rCaHI (Y128F).
(E)-2-hexenal as the main green odor but
not (Z)-3-hexenal. Alonso et al. (42)
found that (E)-2-hexenal is one of the
major contributors determining sensory
differences among traditional and hybrid
tomato types, suggesting that gene
manipulation of HI is a candidate
approach with high potential in molecular
breeding for tomato flavor.
Identification of quantitative
trait loci that affect the volatile emissions
of tomato fruits has been studied, because
tomato breeders wish to combine good
flavor with high fruit firmness, long shelf
life, and high disease resistance (41).
However, conventional breeding for
sensory quality has been severely limited
In conclusion, we identified
HIs in various plant species, and our
identification of HIs allows the
completion of the scheme of GLV
biosynthesis in plants. HIs share a small
number of catalytic amino acids, and
catalytic His is plausibly responsible for
the keto–enol tautomerism involved in
isomerization of (Z)-3-hexenal to
(E)-2-hexenal. Higher expression of the
gene encoding HI in transgenic tomatoes
led to enhanced (E)-2-hexenal production,
suggesting that HI plays a key role in the
production of (E)-2-hexenal in planta.
(42).
For
this
reason,
genetic
manipulation is applied for improvement
of tomato flavor. Changes in expression
level of alcohol dehydrogenase (43), fatty
acid desaturase (44), and linalool
synthase (45) resulted in changes in
composition of volatiles. Among tomato
volatiles, (Z)-3-hexenal is the main flavor
volatile determining tomato flavor, thus
that (Z)-3-hexenal is assigned as a source
of tomato-like flavor (36). As shown in
this study (Fig. 8), both leaves and fruits
of
oxCaHI
plants
contained
Acknowledgement: Tomato seeds (Micro-Tom, TOMJPF00001) were provided by
University of Tsukuba, Gene Research Center, through the National Bio-Resource
Project (NBRP) of the Japan Agency for Research and Development (AMED), Japan.
Conflict of interest: The authors declare that they have no conflicts of interest with the
con- tents of this article.
Author contributions: Y. Yamauchi designed the study. M. Kunishima, and Y. Y.
performed research; M. Kuse and H. Takikawa analyzed chemical data; M. Mizutani
analyzed molecular biological data; Y. Sugimoto supervised research; and M.
Kunishima and Y. Y. wrote the paper.
12

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FOOTNOTES
*This study was supported by JSPS KAKENHI Grant Number 15K07325 (to Y. Y.).
The abbreviations used are: GLV, green leaf volatile; HI, (Z)-3:(E)-2-hexenal
isomerases; VOC, volatile organic compound; LOX, lipoxigenase; HPL, hydroperoxide
liase; DNP, 2,4-dinitrophenylhydrazone; DNPH, 2,4-dinitrophenylhydrazine; DDM,
β-D-dodecyl maltoside.
TABLE1
Summary of purification of CaHI from red paprika
Total
Total
Specific
activity
(nmol/min mg)
38.3
Purification Yield
protein
(mg)
activity
(nmol/min)
647,000
130,000
98,700
10,000
3,840
(fold)
(%)
Crude extracta
16,800
1.0
100
Phenyl-sepharose 866
150
3.92
16.9
328
20.1
15.3
1.56
0.59
Hydroxyapatite
Mono Q5/5
153
646
0.80b
0.012b
12,600
320,000
Mono Q1.6/5
8,350
aPrepared from 640 g of red paprika pericarp.
bProtein concentration was estimated using EA280 (0.1% protein) = 1.
TABLE 2
Kinetic parameters of purified CaHI and recombinant HIs
kcat (s−1)
Km (mM)
kcat/Km (s−1 mM−1)
CaHI
760
0.73
1,040
rCaHI
521 ± 13.0
1.78 ± 0.06
293 ± 2.60
17

rSlHI1
rStHI1
rStHI2
rMsHI
rCsHI1
rOsHI1
264 ± 18.1
27.7 ± 1.75
159 ± 13.9
308 ± 13.9
951 ± 66.2
43.5 ± 2.23
0.20 ± 0.04
0.33 ± 0.03
0.72 ± 0.10
0.32 ± 0.04
0.56 ± 0.08
1.23 ± 0.12
1349 ± 126
83.7 ± 1.08
224 ± 13.7
990 ± 79.1
1749 ± 172
35.8 ± 1.61
HI, (Z)-3:(E)-2-hexenal isomerase.
Figure legends
FIGURE 1. GLV biosynthesis pathway in higher plants. Major GLV species are
enclosed by round-shaped squares. Enzymes catalyzing each reaction are underlined.
Isomerization step catalyzed by HI is shown by white arrow. Enzymatic reactions for
alcohol- and acetate-forms formation need cofactors.
FIGURE 2. Purification of CaHI from paprika pericarp. (A) Elution profile of CaHI
in the final step of purification. HI activity in fractions containing proteins was
measured by standard assay. Activity is shown by gray bar. (B) Protein profile in each
fraction (alphabets in small letter are same as panel A; M, molecular marker) was
analyzed by SDS-PAGE. (C) Molecular mass of native HI was determined by
BN-PAGE. Arrowheads indicate bands of purified CaHI.
FIGURE 3. Phylogenic tree of HIs. Proteins having catalytic HKY and homologous
proteins but not having catalytic HKY are named as HI and HI-like, respectively. Left,
HI and HI-like clades (shown by a dotted circle) belong to germin and germin-like
protein family in the cupin superfamily. Right, Enlarged view of HI and HI-like clades.
Recombinant proteins showing HI activity or no activity are marked with “o” or “-“,
respectively. Numbers in parentheses indicate identity against CaHI. Values at the nodes
indicate percentage of bootstrap support (of 1,000 bootstrap replicates).
FIGURE 4. Determination of catalytic amino acids of HI. (A) Effect of point
mutation on HI activity. Amino acids written by large size indicate point-muted amino
acids, and chromatograms of the point-muted proteins completely losing activity are
shown. Asterisks suggest essential amino acids to show the HI activity, and
point-mutated amino acids with no effect on the HI activity are indicated by “WT”.
Black boxes under amino acid sequence of CaHI indicate amino acids conserved in all
proteins belonging to HI clade but not in HI-like clade. Gray boxes show amino acids
not conserved in HI clade, or conserved both HI and HI-like clades. 3-H and 2-H in
chromatograms indicate (Z)-3- and (E)-2-hexenal-DNPs, respectively. (B) Homology
modeling of CaHI to deduce catalytic amino acids. Catalytic amino acids locate in the
same pocket and near substrate (Z)-3-hexenal. PDB ID of template protein, identity
18

between CaHI and template protein, and QMEAN score are 2e9q, 23%, and 0.62,
respectively.
FIGURE 5. 1H-NMR spectra of authentic (upper) and D-labeled (lower)
(E)-2-hexenal-DNPs. The letters indicate the position of protons and their
corresponding signals. Arrows indicate integrated values of signals (that of H-1’ is set
to 1). Solvent signals are indicated by “x”.
FIGURE 6. Hypothetical catalytic mechanism of HI. (A) His-mediated isomerization
from (Z)-3-hexenal to (E)-2-hexenal catalyzed by HI. In this scheme, γ-nitrogen of His
is depicted as a representative example of the catalyst. (B) Inhibition of rCaHI activity
by suicidal substrate, 3-hexyn-1-al. Numbers in parentheses indicate pmol of
3-hexyn-1-al and enzyme used for analysis, respectively. Activity without 3-hexyn-1-al
was defined as 100% activity. Theoretically stoichiometrical relationship between
enzyme and suicidal substrate is indicated by a dotted line. (C) Suicidal substrate
inhibitory mechanism of HI. Also in this scheme, γ-nitrogen of His is depicted as a
representative example.
FIGURE 7. Induction of HIs and PINII by wounding treatment. After leaves of
tomato (A) and paprika (B) were wounded by tweezers, expressed genes were quantified
by qRT-PCR. Relative expression level of 0 time sample against internal standard gene
(the actin genes SlACT and CaACT for tomato and paprika samples, respectively) was
set to 1. Data are means ± SE (n = 3).
FIGURE 8. Overexpression of CaHI drastically changed (Z)-3-hexenal and
(E)-2-hexenal composition in transgenic tomatoes. (A) (Z)-3- and (E)-2-Hexenal
analysis in wild-type and CaHI-overexpressing tomato (oxCaHI) leaves. Volatiles were
collected by SPME, and then analyzed by GC-MS. SIM (m/z=98) chromatograms to
detect (Z)-3- and (E)-2-hexenals are shown. Inset, confirmation of enhanced expression
of CaHI in transgenic tomatoes by qRT-PCR (the actin gene SlACT was used as an
internal standard). Numbers indicate transgenic tomato lines showing high CaHI
expression. (B) SIM chromatogram (m/z=98) of volatiles from ripe tomato fruits.
19

Phospholipids and galactolipids in thylakoid membranesꢀ
Lipaseꢀ
Lipaseꢀ
13ꢀ
13ꢀ
COOH
COOH
Linolenic acidꢀ
Linoleic acidꢀ
13-Lipoxygenaseꢀ
13-Lipoxygenaseꢀ
Hydroperoxide lyaseꢀ
OOH
O
COOH
13-hydroperoxideꢀ
n-Hexanalꢀ
+NADPHꢀ
Hydroperoxide lyaseꢀ
Alkenal reductaseꢀ
(Z)-3:(E)-2-Hexenal
isomeraseꢀ
O
O
(Z)-3-Hexenalꢀ
(E)-2-Hexenalꢀ
Aldehyde reductase!
Aldo/keto reductase!
Alcohol dehydrogenaseꢀ
Aldehyde reductase!
Aldo/keto reductase!
Alcohol dehydrogenaseꢀ
+NAD(P)Hꢀ
+NAD(P)Hꢀ
OH
OH
(Z)-3-Hexenolꢀ
(E)-2-Hexenolꢀ
BAHD acyltransferaseꢀ +Acetyl-CoAꢀ
+Acetyl-CoAꢀ BAHD acyltransferaseꢀ
OAc(E)-2-Hexenyl acetateꢀ
OAc
(Z)-3-Hexenyl acetateꢀ
Fig. 1ꢀ

Aꢀ
0.07!
0.06ꢀ
0.05ꢀ
0.04ꢀ
0.03ꢀ
0.02ꢀ
0.01ꢀ
0ꢀ
aꢀ
bꢀcꢀdꢀeꢀfꢀgꢀhꢀiꢀ jꢀkꢀ
lꢀ
NaCl!
1 M!
80ꢀ
60ꢀ
0.6 Mꢀ
40ꢀ
20ꢀ
0ꢀ
0 Mꢀ
20ꢀ
60ꢀ
0ꢀ
10ꢀ
30ꢀ
40ꢀ
50ꢀ
Elution time (min)ꢀ
Bꢀ
Cꢀ
aꢀ bꢀ cꢀ dꢀ eꢀ
gꢀ hꢀ iꢀ jꢀ kꢀ lꢀ
fꢀ
Mꢀ
(kDa)ꢀ
(kDa)ꢀ
720ꢀ
150ꢀ
100ꢀ
75ꢀ
50ꢀ
37ꢀ
480ꢀ
25ꢀ
20ꢀ
144ꢀ
66ꢀ
Fig.2ꢀ

ZmHI-like
71
56
Germin and germin-like protein familyꢀ
GmHI-like1
OsHI-like2
OsHI-like1
98
29
CmHI-like
CsHI-like
AlHI-like
Germinꢀ
Miscellaneous
HI-likeꢀ
96
82
93
(40%)ꢀ
!ꢀ
AtGermin
A t G e r m i n
V v G e r m i n
AtHI-like1
(38%)ꢀ
!ꢀ
GmHI-like2
59
MtHI-like2
83
19
99
MtHI-like1
75ꢀ
HI and HI-likeꢀ
StHI-like1
90
Solanaceae!
HI-likeꢀ
34
SlHI-like1 (71%)ꢀ
98ꢀ
!ꢀ
99ꢀ
StHI-like2
StHI1
SlHI1
(81%)ꢀ
(79%)ꢀ
S t H I
72
90
32
Solanaceae!
HIꢀ
(79%)ꢀ
(80%)ꢀ
(100%)ꢀ
StHI2
100ꢀ
10
SlVicilin
36
SlHI2
60
93ꢀ
CaHI
CsHI1
CsHI4
CmHI
(35%)ꢀ
MtVicilin
76
98
96ꢀ
Vicilinꢀ
59
Cucurbitaceae!
HIꢀ
MsHIꢀ
CsHI3
96
82ꢀ
CsHI3
64ꢀ
91ꢀ
CsHI2
78ꢀ
100ꢀ
MsHI
MtHI1
(42%)ꢀ
5
81
26
95ꢀ
90ꢀ 83ꢀ
90ꢀ
Fabaceae !
HIꢀ
GmHI1
GmHI2
MtHI2
81
92
20
95ꢀ
OsHI
68ꢀ
OsHI1
OsHI3
(33%)ꢀ
56
96
Oryza!
HIꢀ
St11S
39
OsHI2
OsHI4
6
11S seed globulinꢀ
PtHI
Woody plant !
HIꢀ
VvHI
0.5ꢀ
AtHI-like2
0.2ꢀ
Fig. 3.ꢀ

Aꢀ
Bꢀ
Catalytic HKYꢀ
WTꢀ
H54Aꢀ
K60Aꢀ
3.8Åꢀ
H54ꢀ
Y128ꢀ
WTꢀ
3-Hexenalꢀ
K60ꢀ
*ꢀD *ꢀ
1:ꢀMDLILASKKA DKTIVEVEGV GGYYTWSRKF PVLSQKKLAA GLLVLQPRGF ALP
H
YA SS
K
IAYVIEGECIꢀ
Y128Aꢀ
WTꢀ
WTꢀ
*ꢀ
71:ꢀ
AGLISPEDSK EEVIKIQKGD SVPVPIGAT
S
WWYNGGDTRL SIIFLGE
S
GE YTPGEFC
Y
FF LTGAAGILNGꢀ
FSNELLAQTF HMTKTESEKL KKDQSSLNII IKISEGIKTP DPCNSGIHKL VFNLDGAKPS VEMKNGGVLT ꢀ
141:ꢀ
211:ꢀ
SVSVRDLPLL GDIGLSANRV VLEGGGMLGP LFTADSSVHL SYVTKGSGRV VIVGLFGKVV LDTKVDEGDL ꢀ
WTꢀ
WTꢀ
281:ꢀ
FFVPKFFPFV VEADEGGIEF FSV
K
TSSKQI YGALSGGPKS VFVAESPSIL EASLNMTPDF T
K
SFKSKIAK GAVIAPPꢀ
Fig. 4ꢀ

H-6ꢀ H-5ꢀ H-4ꢀ H-3ꢀ H-2ꢀH-1ꢀ H-1’ꢀH-Arꢀ
CH3-CH2-CH2-CH=CH-CH=N-NH-(C6H3)(NO2)2ꢀ
(D)ꢀ
CDCl3, 300 MHzꢀ
H-Arꢀ
H-5ꢀH-6ꢀ
xꢀ
2.32ꢀ
2.26ꢀ
Authentic!
(E)-2-Hexenal-DNPꢀ
H-2ꢀH-3ꢀ
H-1ꢀ
H-4ꢀ
1.00ꢀ
H-1’ꢀ
2.00ꢀ
xꢀ
xꢀ
H-5ꢀH-6ꢀ
H-Arꢀ
D-labeled!
(E)-2-Hexenal-DNPꢀ
H-2ꢀ H-3ꢀ
xꢀ
xꢀ
xꢀ
xꢀ
1.00ꢀ
1.23ꢀ
H-1ꢀ
H-1’ꢀ
H-4ꢀ
xꢀ
xꢀ
Fig. 5ꢀ

Aꢀ
Substrate-enzyme reactionꢀ
O
O
O-
H/D
H
H
N+
N
Enz
N
N
H
Enz
N
H
Enz
N
/D
in D2Oꢀ
H
D
N+
His54ꢀ
Enz
N
D
Bꢀ
Cꢀ
100ꢀ
80ꢀ
60ꢀ
40ꢀ
20ꢀ
0ꢀ
(1:100)ꢀ
(21:100)ꢀ
Suicidal substrate-enzyme reactionꢀ
(53:100)ꢀ
•
O
O
O
N
N
3-Hexyn-1-alꢀ
Enz
N
Enz
N
H
(107:100)ꢀ
His54ꢀ
0ꢀ 0.2ꢀ 0.4ꢀ 0.6ꢀ 0.8ꢀ 1.0ꢀ 1.2ꢀ
Ratio ([3-hexyn-1-al]/[enzyme])ꢀ
Fig. 6ꢀ

Aꢀ
Bꢀ
15ꢀ
10ꢀ
5ꢀ
8ꢀ
6ꢀ
4ꢀ
2ꢀ
SlHI1ꢀ
SlPINIIꢀ
1ꢀ
CaHIꢀ
0ꢀ
60ꢀ
0ꢀ
20ꢀ
CaPINIIꢀ
50ꢀ
40ꢀ
30ꢀ
20ꢀ
10ꢀ
0ꢀ
15ꢀ
10ꢀ
5ꢀ
0ꢀ
0ꢀ
2ꢀ
3ꢀ
4ꢀ
0ꢀ
2ꢀ
Time after wounding (h)ꢀ
4ꢀ
6ꢀ
8ꢀ
Time after wounding (h)ꢀ
Fig. 7ꢀ

Aꢀ
Bꢀ
8ꢀ
6ꢀ
4ꢀ
2ꢀ
0ꢀ
Wild typeꢀ
1 minꢀ
WTꢀ#28ꢀ#29ꢀ#37ꢀ#40ꢀ
oxCaHI #40ꢀ
oxCaHI #37ꢀ
oxCaHI #28ꢀ
ꢁꢂꢀ
oxCaHI #29ꢀ
1 minꢀ
oxCaHI #28ꢀ
Wild typeꢀ
oxCaHI #29ꢀ
Authentic (Z)-3-hexenalꢀ
Authentic (E)-2-hexenalꢀ
ꢁꢂꢀ
1 minꢀ
1 minꢀ
Retention time (min)ꢀ
Retention time (min)ꢀ
Fig. 8ꢀ

10
8
6
4
2
ND
ND
ND
ND
0
Fig. S1. Screening of plant materials. HI activity in crude extract prepared from each plant material was
determined by standard assay. Data are means ± SE (n=3). ND, not detected.

A
B
(Z)-3-Hexenal
(E)-2-Hexenal
(E)-2-Hexenal
(Z)-3-Hexenal
X
O
Before reaction
Before reaction
O
O
After reaction
After reaction
0
5
10
15
20
25
30
0
5
10
Retention time (min)
15
20
25
30
Retention time (min)
Fig. S2. Detection of HI activity by HPLC. Typical chromatograms of DNP-derivatized samples prepared
from reaction mixtures before and after reaction are shown. Arrows indicate peaks of which retention time
were identical to those of authentic compound-DNPs, respectively. Substrates are (Z)-3-hexenal (A), (E)-2-
hexenal (B), (E)-2-nonenal (C), and (Z,Z)-3,6-nonadienal (D). (E)-2-Hexenal was not isomerized (B).

C
D
(Z,Z)-3,6-nonadienal
(E,Z)-2,6-Nonadinal
(Z)-3-Nonenal
(E)-2-Nonenal
O
Before reaction
Before reaction
O
O
After reaction
After reaction
O
0
10
20
30
40
0
10
20
30
40
Retention time (min)
Retention time (min)
Fig. S2. (continued)