Enantioselectivity of the bioconversion of chiral citronellal during the inhibition of wheat seeds germination

Article abstract of DOI:10.1002/cbdv.201300131

Citronellal is one of the most prominent monoterpenes present in many essential oils. Low persistence of essential oils as bioherbicides has often been addressed because of the high volatility of these compounds. Bioconversion of citronellal by wheat seeds releases less aggressive and injurious compounds as demonstrated by their diminished germination. We demonstrated that optically pure citronellal enantiomers were reduced to optically pure citronellol enantiomers with retention of the configuration both in isolated wheat embryos and endosperms. Our findings reveal the potential of essential oils as allelopathic agents providing an insight into their mechanism of action and persistence. Copyright

Full text of DOI:10.1002/cbdv.201300131

CHEMISTRY & BIODIVERSITY – Vol. 11 (2014)
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Enantioselectivity of the Bioconversion of Chiral Citronellal during the
Inhibition of Wheat Seeds Germination
a
b
c
b
by Andrea Cavalieri ), Ravit Fischer ), Olga Larkov ), and Nativ Dudai* )
a) Department of Plant Science, University of Manitoba, 222 Agriculture Building, Winnipeg, R3T 2N2
Manitoba, Canada
b) Newe Yaꢀar Research Center, Agricultural Research Organization, 30095 Ramat Yishai, Israel
(
phone: þ972-4-9539556; e-mail: nativdud@gmail.com)
c) Department of Biology, Technion-Israel Institute of Technology, Haifa 32000, Israel
Citronellal is one of the most prominent monoterpenes present in many essential oils. Low
persistence of essential oils as bioherbicides has often been addressed because of the high volatility of
these compounds. Bioconversion of citronellal by wheat seeds releases less aggressive and injurious
compounds as demonstrated by their diminished germination. We demonstrated that optically pure
citronellal enantiomers were reduced to optically pure citronellol enantiomers with retention of the
configuration both in isolated wheat embryos and endosperms. Our findings reveal the potential of
essential oils as allelopathic agents providing an insight into their mechanism of action and persis-
tence.
Introduction. – The interest in the development of natural products as bioherbicides
has increased in the last decades [1–3]. As a result, researchers began focusing on plant
extracts, plant secondary metabolites, essential oils, and monoterpenes as agronom-
ically and economically important herbicides [4–6]. The inhibitory effects of essential
oils from a number of plants and pure monoterpenes on the germination of wheat seeds
have been demonstrated, as well as their possible application as bioherbicides by
spraying formulations on seeds sown in a soil media [4][7][8]. Additionally, we have
previously shown that wheat seeds exposed to a defined monoterpene, such as citral,
citronellal, pulegone, or carvacrol, are able to metabolize and partially detoxify it
[
8][9]. Unfortunately, it is not clear if the endogenous enzymes in the wheat seed are
substrate-specific or not, since the biological substrates are unknown. Many mono-
terpene substrates such as citronellal and their products (citronellol and citronellic
acid) are chiral, hence it seemed interesting to investigate if their biotransformations in
the seeds is enantioselective or not. In some herbs such as mint, basil, lavender, and
oregano that naturally contain chiral essential oils, enzymatic stereoselective formation
of monoterpenes has already been demonstrated [10–13]. In many cases, differential
effects of enantiomers have been noted in agrochemicals [14], pheromones, allelo-
chemicals [15], and taste or odor compounds [16–18]. Providing information on the
enantioselectivity of citronellal is a pivotal aspect. Citronellal is a specialized
metabolite of plants usually isolated as a non-racemic mixture by steam distillation
or solvent extraction from the oils of some Cymbopogon spp. or other essential oils
[
19][20]. Besides its use in the fragrance industry, citronellal is employed as an
ꢁ
2014 Verlag Helvetica Chimica Acta AG, Zꢂrich

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CHEMISTRY & BIODIVERSITY – Vol. 11 (2014)
intermediate in the synthesis of several terpenoids, and, when obtained from renewable
sources, this monoterpene is biodegradable [19]. Citronellal has various established
bioactivities such as serving as a mosquito repellent or as a germination inhibitor
[
9][20], but it is presently unknown if there is any enantioselectivity in their anti-
germination activity. We present here evidence that optically pure citronellal
enantiomers were reduced to optically pure citronellol enantiomers with retention of
the configuration both in isolated wheat embryos and endosperms.
Results and Discussion. – Essential Oils and Germination Inhibition. The response
of wheat seeds to each compound is depicted in Fig. 1. Fifty percent inhibition of
growth of radicles occurred at 38.4 and 40 nl/ml of (ꢀ)-(S)-citronellal and (þ)-(R)-
citronellal, respectively, compared with 56.8 and 64.8 nl/ml required for 50% inhibition
of germination of (ꢀ)-(S)-citronellol and (þ)-(R)-citronellol, respectively (Fig. 1). No
significant injury was detected for citronellic acid. Increasing the essential-oil
concentration resulted in a higher germination inhibition (Fig. 1), while the effect on
the radicle elongation was observed only for the highest concentration tested (data not
shown). An important feature highlighted by these results is how, starting from
citronellal, the inhibition ability of the compounds decreases considerably through the
process of oxidation or reduction. The second feature highlighted is the effect of
chirality sense of the compounds on the germination process. As a general tendency, the
(
ꢀ)-(R)-enatiomers had stronger inhibitions compared with those of (þ)-(S)-
enantiomers (Fig. 1). This trend is more evident at lower concentrations than at
higher concentrations.
Fig. 1. Effects of (þ)- and (ꢀ)-enantiomers on the Triticum aestivum seed germination

CHEMISTRY & BIODIVERSITY – Vol. 11 (2014)
421
Bioconversion of Compounds. Citronellal bioconversion during seed inhibition was
previously studied [9]. Results indicated that the main products of the transformation
are citronellol and citronellic acid (Scheme 1). The process takes place both in the
embryo and in the endosperm already within 24 h after the inbibition. To study the
enantioselectivity of this process, a chiral column to separate the enantiomers of
citronellal was used. The results evidence that both enantiomers of citronellal
underwent the bioconversion to their corresponding alcohols and, at a much lower
rate, to their corresponding acids in the embryo and in the endosperm (Fig. 2).
Enantiomerically pure (þ)-(R)-citronellal was reduced and oxidized to enantiomeri-
cally pure (þ)-(R)-citronellol and (þ)-(R)-citronellic acid, respectively. The same
result was observed with (ꢀ)-(S)-citronellal as a starting material. The lack of
racemization is well-expected, because both reduction and oxidation involve only the
C¼O group and did not influence the nearby stereogenic center. When racemic
citronellal was subjected to bioconversion, we observed a small but statistically
significant preference toward the specific enantiomer in the embryo and in the
endosperm. In our experiments, the racemic citronellal was reduced to (þ)-(R)-
citronellol (72% enantiomeric purity) in the embryos, and to (ꢀ)-(S)-citronellol (63%
enantiomeric purity) in the endosperms of wheat seeds. We observed only a low rate of
oxidation of citronellal enantiomers to citronellic acid in this phase.
Enantioselectivity of racemic citronellol has already been studied [21] using an
interface bioreactor. The findings indicated the enantioselective microbial oxidation of
racemic citronellol to optically active citronellal and citronellic acid, leaving the other
enantiomer of citronellol intact. Further studies [22] demonstrated how the microbial
oxidation of (þ)-(R)-citronellol to (þ)-(R)-citronellal and (þ)-(R)-citronellic acid
occurs in high yield without racemization in Rhodococcus equi JCM 6817 and
Geotrichum candidum JCM 01747, respectively.
Scheme 1. Main Products of the Bioconversion of Citronellal

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CHEMISTRY & BIODIVERSITY – Vol. 11 (2014)
Fig. 2. Enantiomeric ratios of citronellol obtained by bioconversion of racemic, and enatiomerically pure
þ)- or (ꢀ)-citronellal
(
The study of the ratio of the oxidation (citronellic acid) and the reduction
cilronellol) products evidenced that, both in the endosperm and in the embryo, most of
(
the bioconversion preferred the reduction route to citronellol (Fig. 3). Among the two
seed portions, the embryo performed overall better biotransformation, characterized
by a higher citronellol ratio (statistically significant p<0.05). Data showed that only up
to 12% (in the endosperm) or up to 7% (in the embryo) of citronellol was converted to
citronellic acid (Fig. 3). This could be explained by the respiration process taking place
mainly in the endosperm, compared to the embryo, and thus increasing the rates of
oxidation to citronellic acid. The experiment with two applied concentrations (2 and
4
ml) seemed to confirm this assumption. The biotransformation to citronellol was more
efficient at the lower concentration in the embryo (95%) compared with endosperm
89%; significant p<0.05), while, at the higher concentration, the citronellol ratio
(
displayed a lower but still significant (p<0.05) difference between the values both in
the embryo and endosperm (96 and 92%, resp.). The reason for the lower differences at
the higher concentration can be addressed by the effect that the higher concentration
has on the seeds viability. Inhibiting the germination and thus inhibiting the respiration,
the bioconversion process is shifted in favor of the citronellol ratio. The following
experiment indicated the influence of oxygen availability on the process. While in the
control air O was allowed inside the seed space by nonhermetic closing of the vial, the
2
atmosphere was replaced by N gas and closed hermetically in the other treatment. The
2
exclusion of O reduced the formation of the citronellic acid relative to the that of
2
citronellol. This result was significant (p<0.05) even if in the endosperm it was more
pronounced than in the embryo (Fig. 4).
In conclusion, the optically pure citronellal enantiomers were reduced to optically
pure citronellol enantiomers with retention of the configuration both in the embryos

CHEMISTRY & BIODIVERSITY – Vol. 11 (2014)
423
Fig. 3. Percentage of metabolism of citronellal in ten Triticum aestivum seeds (endosperms and emryos)
after application of 2 and 4 ml in the gaseous phase. The content of metabolites was measured 24 h after
sowing. The symbols in brackets represent the (þ)- or (ꢀ)-enantiomer.
and in the endosperms of wheat seeds. In the present study, we have examined the
biotransformation as a detoxification of citronellal. This process converts citronellal
into less aggressive and injurious compounds as demonstrated by their diminished
inhibition ability (Fig. 1) and lack of inhibition in radicle elongation (data not shown).
Our findings further support the potential of essential oils as allelopathic agents and
provide an insight into their mechanism of action and persistence.

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CHEMISTRY & BIODIVERSITY – Vol. 11 (2014)
Fig. 4. The effect of oxygen availability during bioconversion of citronellal enantiomers on the products
ratio. The content of metabolites was measured 24 h after sowing. O and N represent abundance of O2
and with N , respectively. The symbols in brackets refer to the (þ)- or (ꢀ)-enantiomer.
2
Experimental Part
Compounds. The essential oils tested, i.e., (þ)-(R)-citronellal, (ꢀ)-(S)-citronellal, (þ)-(R)-
citronellol, (ꢀ)-(S)-citronellol, (þ)-(R)-citronellic acid, and (ꢀ)-(S)-citronellic acid, were purchased
from Sigma-Aldrich. The compounds were subsequently identified by GC/MS analysis as described in
[
23].
Bioassay of Inhibition Induced by Essential Oils. Seeds of wheat (Triticum aestivum L. cv Dariel)
were germinated in Petri dishes (6-cm diameter) on three layers of filter paper (Whatman No. 3) wetted
with 2.5 ml of dist. H O. Petri dishes containing 20 seeds each were incubated in the dark at 278. To test
2
the inhibitory effects, a known amount of each compound was loaded (using a calibrated glass

CHEMISTRY & BIODIVERSITY – Vol. 11 (2014)
425
microcapillary) on a piece of filter paper attached (by double-sided adhesive tape) to the inner side of the
cover of the Petri dish. Up to 2 ml of compound and blank control were tested by this procedure. One ml of
essential oil per Petri dish was equivalent to 40 nl/ml. Each amount was tested in three replicates. After
incubation, the numbers of germinated seeds were counted, and the length of each radicle was measured.
The parameter to consider radical emergence as germinated is described in [4]; seeds with a protrusion of
non-differentiated cells without radical emergence were considered not germinated. Since compounds
inhibit both germination and growth, the effect of each compound on wheat seedlings was studied. The
amount of essential oil required to cause 50% inhibition of germination was determined, as described in
[
4], by interpolation from the curve obtained by plotting the means of the replicates vs. the amount of
essential oil applied.
Bioconversion of Compounds. Wheat seeds (Triticum aestivum L. cv Dariel) were germinated in 25-
ml glass vials, on three layers of filter paper (Whatman No. 1) wetted with 1.5 ml of dist. H O. Vials
2
containing 20 seeds each were incubated at 278 in the dark. Different paths of the bioconversion were
tested; in shortage of O (the vials were closed hermetically), in abundance of O (opening and closing
2
2
the vials every 3 h), and with N . To test the bioconversion of the each compound, a known amount of
2
every examined monoterpene was loaded (using a calibrated glass microcapillary) on a piece of filter
paper, which was attached to the inner side of the cover of the vial. Amounts of up to 4 ml of compound
were applied in this way. The compound per vial (1 ml) was equivalent to 40 nl/ml. Experiments were
t
conducted in five replicates. After 24 h, seeds were extracted with BuOMe, and the embryo and the
endosperm were separated.
Determination of Compounds in the Seed. The examined monoterpenes were (þ)-(R)-citronellal,
(
ꢀ)-(S)-citronellal, (þ)-(R)-citronellol, (ꢀ)-(S)-citronellol, (þ)-(R)-citronellic acid, and (ꢀ)-(S)-
citronellic acid. Seeds were washed by mild shaking for 5 s in dist. H O. Then, an extraction with
2
t
BuOMe containing 10 mg/ml isobutylbenzene as an internal standard was performed for 24 h with gentle
shaking at r.t. The samples were analyzed with a GC/MS 7890A apparatus equipped with a Hydrodex-b-
6
-TBDM (MACHERY-NAGEL, DE-Dꢂren), 25 mꢁ0.25-mm fused silica-gel cap. column; He was used
as the carrier gas; injection temp. was 2308, and the transfer line temp. was 2308. Column conditions were
08 for 10 min, followed by 1.58/min to 1308 and 108/min to 2008. The components were identified by co-
4
injection with authentic samples and by comparison of the EI-MS data obtained from computerized
libraries.
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Received April 19, 2013