ARTICLES
A&EB
THE BIOSYNTHETIC ORIGIN OF GERMINATION STIMULANTS FOR
OROBANCHE RAMOSA (L.) IN TOBACCO AND ARABIDOPSIS
I. Denev1, B. Deneva1,2, R. Batchvarova2
The University of Plovdiv, Department of Plant Physiology and Molecular Biology, Plovdiv, Bulgaria1
AgroBioInstitute, Sofia, Bulgaria2
Correspondence to: Ilia Denev
Email: [email protected]
ABSTRACT
The germination of root parasites like Orobanche and Striga depends on chemical products exuded from the roots of the host
plant, known as germination stimulants (GS). The predominant part of them has structural similarities with sesquiterpene
lactones. It is well known that sesquiterpenes are assembled by the cytosol-localized mevalonate pathway in which the key
precursor - isopentenyl diphosphate (IDP) is synthesised from mevalonic acid. Our earlier results however suggested that GS
originate from plastid- situated 2-C-Methyl-D-Erythritol-4-Phosphate (MEP pathway). Recently Matusova and co-authors al
(18) confirmed this and hypothesized that in maize GS originate from carotenoids.
In order to better understand the involvement of the two different isoprenoid biosynthetic pathways in GS formation we have
used separately and in combination specific chemical inhibitors for each of biosynthetic pathways. Our results indicate that
that indeed in Arabidopsis and tobacco the earliest precursors of GS originate from 1-deoxy-D-xylulose 5 phosphate pathway.
However application of carotenoid biosynthesis inhibitor – norflurazon did not influences the GS production. Therefore we
concluded that in tobacco and Arabodopsis GS are not derived from carotenoids but from an earlier intermediate of MEP
biosynthetic pathway, which is later exported from plastids to the cytosol, to be converted to the final product - GS.
Keywords: Orobanche, germination stimulants, isoprenoid
biosynthesis, Arabidopsis
Introduction
Parasitism appears at least eight times independently during the
evolution of higher plants and now about 1% of angiosperms
are not solitary but search out and form parasitic associations
with other plants (3, 20). Between them obligate root parasites
are considered the most evolutionary advanced (3, 20, 15). The
most successful parasitic strategy is the tight coordination of
early developmental stages of parasites with the life cycle of
host plant by using chemical signals (11, 19, 22). For example
the seeds of Orobanche and Striga species are capable to abort
their germination program even under favorite environmental
conditions if a certain concentration of specific chemicals,
exuded from the roots of host plants, is not present (2, 11, 25).
These seeds respond to very low concentrations (10-7¯10-15 M)
of those chemical compounds called germination stimulants
(GS) (24). Sub- and supra-optimal concentrations may inhibit
seed germination (14). A significant progress has been made
in identification of the chemical nature of GS in root exudates
from many hosts and some non-host plants of Striga (2, 21).
Two germination stimulants for Orobanche minor seeds:
alectrol and orobanchol, have been also isolated (26). The GS
for those two genera are strikingly similar (Fig. 1) (2, 21).
The molecular structure of all these compounds closely
resembles sesquiterpene lactones (they are called strigolactones)
(2) and therefore they were expected to be products of
isoprenoid metabolism in roots (8). Although the GS and their
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analogs were synthesized by organic synthesis (23, 24), their
biosynthetic origin remained unknown. The knowledge about
the biochemistry and genes responsible for GS production in
host plants may answer many questions about mechanisms of
co-evolution of parasites and their hosts and on the other hand
will lead to development of new practical measures for control
of crops-harming parasitic species (14).
Fig. 1. Chemical structure of so far identified germination stimulants for Striga
and Orobanche (2, 26)
It is well known that sesquiterpene lactones are produced
from farnesyl diphosphate (FDP) via germacrane A intermediate
(9, 10). The FDP is intermediate product of the mevalonate
pathway (Fig. 2) where the “building blocks” isopentenyl
diphosphate (IDP) units are synthesized from mevalonic acid
by HMG-CoA reductase (1). The enzyme is very sensitive
to chemical inhibitors like mevastatin (=mevinolin;=lovast
BIOTECHNOL. & BIOTECHNOL. EQ. 21/2007/1
atin). The presence mevinolin blocks the whole mevalonate
pathway (12)
In contrast the IDP units in alternative, plastid-situated
pathway, are synthesized from pyruvate and glyceraldehyde3-phosphate via 1-Deoxy-D-Xylulose-5-Phosphate and 2-CMethyl-D-Erythritol-4-Phosphate (MEP pathway) (16). The key
enzyme 1-deoxy-D-xylulose-5-phosphate reductoisomerase is
sensitive to fosmidomycin – an inhibitor which in 100 μmol.
l-1 block the MEP-pathway (27).
Then they were placed in new Petri dishes and 0,1 ml of diluted
root exudates were applied on double disks. The germination
tests were carried out with root exudates from control plants
and plants grown in a presence of inhibitors. Germination
percentages were assessed after incubation for 8 days at 26°C.
GR24 (0,4 mg/l) was used as a positive control. Root exudates
from non-host plants as well as sterile milli-Q water were
used as negative controls. All experiments were made in five
replicas.
Growing of host plants: Seeds of host plants were surface
sterilized in an aqueous solution containing 2% active chlorine
and 0,2% Tween 20. Seeds were carefully washed with sterile
milli-Q water and placed on 1,5% MS-agar (with 20 g/l sucrose).
For the experiments with inhibitors 100 μmol l-1 norflurazon
(Sandoz Ltd., Switzerland) or 10 μmol l-1 mevinolin (SIGMA –
M2147) or/and 100 μmol l-1 fosmidomycin (Molecular probes
– FR-31564) were added to the MS-agar.
Arabidopsis plants were grown for three weeks and tobacco
plants for five weeks (temperature 22o C, 16h light/ 8h dark
period).
Results and Discussion
Fig. 2. Biosynthetic pathways in plant cell with indicated steps of which
inhibitors and transgenes act. The figure was prepared by combining
information given in articles (1, 12, 16, 27).
In our previous investigations we found connection
between MEP pathway and formation of GS (6, 5, 7). Recently
(18) confirmed these results and hypothesized that in maize GS
originate from carotenoids degradation products.
Yet the maize is a monocotyledon plant with C4-type
photosynthesis and specific plastid biochemistry, while most
of the Orobanche hosts of are dicotyledon C3-type plants.
In order to better understand the involvement of the two
alternative biosynthetic pathways of isoprenoid in GS formation
we have designed an in vitro system to grow host plant in the
presence of the selective chemical inhibitors. Here we present
results about their effect on GS biosynthesis in tobacco and
Arabidopsis.
Materials and Methods
Plant material: Orobanche ramosa (L.) seeds were collected
from tobacco field in South-Bulgaria. Tobacco seeds are in
position of our lab. Arabidopsis thaliana seeds, ecotype Col
were obtained from Ohio Arabidopsis Research Center.
Germination tests: Seeds of O. ramosa (L.) were surface
sterilised in an aqueous solution containing 2% active chlorine
and 0,2% Tween 20. Seeds were carefully washed with sterile
nano-pure water and placed between 1 cm glass fibber filter
paper disks. These “sandwiches” were moistened with sterile
water and seeds were preconditioned at 26°C for 14 days
according to Mangnus et al. (17). After preconditioning the
sandwiches were taken out of conditioning Petri dishes and left
on sterile air steam until the surplus moisture was evaporated.
BIOTECHNOL. & BIOTECHNOL. EQ. 21/2007/1
In order to better understand the involvement of the two
alternative isoprenoid biosynthetic pathways in GS formation we
have used an in vitro system to grow host plant in the presence of
selective chemical inhibitors. Initially two inhibitors were used:
mevinolin, which blocks HMG-CoA reductase and respectively
the mevalonate pathway (12) and fosmidomycin – an inhibitor
of 1-deoxy-D-xylulose-5-phosphate reductoisomerase that
blocks the MEP-pathway (27).
First we have studied weather the inhibitors can directly
affect the germination of O. ramosa seeds. The germination
tests were carried out by treating disk with Orobanche seeds
with inhibitors (20 μmol.l-1 mevinolin, or/and 100 μmol.l1
of fosmidomycin) in 0,12 ml of sterile nano-pure water or
in combination with 0,5 mg. l-1 GR24 per disk. As a positive
control was used GR24 (0,5 mg.l-1) in sterile nano-pure
water and nano-pure water only as negative control. There
were no differences in seeds germination between variants
with inhibitors and those without (Fig. 3) and therefore we
concluded that the inhibitors do not influence directly the
Orobanche seeds germination.
Root exudates from experimental plants were collected
by transferring them from boxes with MS agar to multi-well
plate. Depending on the experiment each well was filled either
with 0,5 ml sterile water (for control plants) or with the same
amount of sterile water solution containing the inhibitors.
Plants were kept there for 24 h. Later their weight was
measured on analytical balance and the solutions from wells,
containing the root exudates, were diluted to equalize weight/
volume ratio. For instance if the weight of the plants kept in
a certain well was 200 mg, and the exudates volume was 0,5
ml the weight/volume ratio was calculated to be 400 mg/ml.
From this solution two dilutions were made – 10 mg/ml and 1
mg/ml that were applied on disks with Orobanche seeds to test
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amount of GS in exudates. Sterile nano-pure water was used as
negative control and GR24 (0,5 mg.l-1). The obtained results
are presented on figure 4.
tests (Fig. 5) did not confirmed observations of (18). On
a contrary – plants grown in the presence of norflurazone
produced slightly more GS than control plants.
Our results confirmed that the initial steps of GS formation
are much more sensitive to the inhibition of MEP pathway
rather than of Mev pathway. However is seems that the both
pathways are involved in the GS formation because the
maximum inhibition of GS formation was achieved when
plants were grown on MS20 agar with both inhibitors.
Fig. 3. Relative germination of preconditioned for 14 days Orobanche ramosa
(L) seeds incubated for further 8 days in sterile water or GR 24 (0,5 mg/l) with
and without inhibitors
Fig. 5. Relative germination of preconditioned for 14 days Orobanche ramosa
(L) seeds incubated for further 8 days with root exudates collected from
tobacco SRI (N. tobacum L.) and Arabidopsis thaliana (L) plants grown on
MS20 agar in the presence and absence (control) of norflurazone.
Fig. 4. Relative germination of preconditioned for 14 days Orobanche ramosa
(L) seeds incubated for further 8 days with root extracts collected from tobacco
SRI (N. tobacum L.) and Arabidopsis thaliana (L) plants grown on MS20 agar
in the presence and absence (control) of selective chemical inhibitors of
isoprenoid biosynthetic pathways.
The capability of root extracts from tobacco and
Arabidopsis plants grown in the presence of mevastatin to
induce germination of O. ramosa seeds was about 85 % of
the one of control plants. (Fig. 4). The plants grown in the
presence of fosmidomycin induce 2,5 times less germination
than the control plants while the root exudates from plants
grown in the presence of both inhibitors induced about 4
times less germination that the control plants. These results
are with concert with our earlier reports and with the data of
(18) and confirmed that the inhibition of plastid situated MEP
pathway decreases GS production mach more significantly that
inhibition of Mev pathway. Interestingly the maximum effect
on GS production was achieved when both inhibitors were
used together.
In order to check the reported by (18), effect of norflurazon
on GS formation, seeds of Arabidopsis and tobacco plants were
soaked in 100 μmol l-1 norflurazon and grown on MS20 agar
containing the same amount of the inhibitor. Root exudates
were collected as described above. Surprisingly germination
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Based on our results we can conclude that initial steps of
GS formation are not localized in cytoplasm and in particularly
in mevalonate biosynthetic pathway of isoprenoids. The GS
originate from the situated in plastids MEP pathway. However
it is difficult to accept that carotenoid degradation products
are origin of GS in tobacco and Arabidopsis. Judging by our
recent and earlier results (6, 5) it is more likely that carotenoid
biosynthetic branch competes for intermediates with the
branch from each GS originate. Further more, although the
original steps of GS production do not originate in mevalonate
pathway, we can hypothesize that at certain level the early
precursors of GS are exported from plastids to cytosol there
they are converted to the final product.
Fig. 6. Relative germination of preconditioned for 14 days Orobanche ramosa
(L) seeds incubated for further 8 days with root exudates collected from 14
days old green and etiolated Arabidopsis thaliana (L.)
BIOTECHNOL. & BIOTECHNOL. EQ. 21/2007/1
Conclusions
From the formal point of view it is surprising that compounds
which are emitted from roots originate from plastids, which
are much more abundant in leaves. However it is known that
roots also contains plastid. On the other hand the most typical
plastids – chloroplasts are biosynthetic origin of such important
isoprenoid hormones like abscisic acid and gibberellins, which
are distributed, thought the plant organism (4, 13). In fact the
life cycle of parasites like Striga and Orobanche, depends
(with exception of last stages of life cycle of Striga) entirely
on supply with photosynthesates, produced in host plants.
Therefore we can hypothesize that during the co-evolution of
these parasites were specialized to detect hosts by the products
of function of their plastids. As a prove of this hypothesis we
compared GS production in green and etiolated Arabidopsis
plants (Fig. 6) and found that etiolated plants produce 1,5 time
less GS then the green one at the same age. So in the context
of our hypothesis etiolated host 1,5 time “less attractive”
for Orobanche parasites than the green one with normally
functioning chloroplasts.
In general the differences between our results and these
obtained by others (18) can only suggest that the chemical
inhibitors are not precise enough tool to pinpoint the steps of
GS formation. It is very likely that inhibitors have side-effects
on general plant metabolism that masks the real origin of GS.
Only the tools of modern molecular biology can further unravel
the pathway of GS formation.
Acknowledgments
This research was supported by NATO- Science for Peace
program grant LST.CLG.980142; Bulgarian National Science
Foundation grants K1305/2003 and G4-00/2004
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