Download Saponins versus plant fungal pathogens

Journal of Cell and Molecular Biology 5: 13-17, 2006.
Haliç University, Printed in Turkey.
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Saponins versus plant fungal pathogens
Figen Mert-Türk
Çanakkale Onsekiz Mart University, Agricultural Faculty, Crop Protection Department, Çanakkale,
17100, Turkey
Received 10 May 2005; Accepted 26 September 2005
Abstract
The production of low-molecular-weight antimicrobial molecules within plants is one component of defense against
pathogens. Among them, preformed antimicrobial compounds (phytoanticipins) are the first biochemical barriers
against pathogens. Saponins (a group of phytoanticipins) are present constitutively in plants and play important roles
in plant defense. A variety of biological roles have been postulated for different saponins, involving allelopathic
activity, defense against insects and pathogens. The mechanism of antifungal action of saponins is not well
understood but it is believed that they complex with sterols in the cell membrane, leading to pore formation and
consequent loss of membrane integrity. Saponins that have been studied in detail in relation to their potential role in
plant defense against attack by phytopathogenic fungi are triterpenoid avenacins in oat roots and the steroidal
glycoalkaloids α-tomatine in the leaves of tomato. Here we present a review about the two saponins that have been
proved to have antimicrobial activity and have a role in plant defense.
Key words: Phytoanticipins, saponins, plant fungal pathogens, disease resistance, avenacins, α-tomatine
Bitki fungal hastal›klar›na karfl› saponinler
Özet
Küçük moleküler a¤›rl›¤a sahip antimikrobiyal moleküller bitkilerin patojenlere karfl› savunmas›nda önemli bir role
sahiptir. Bunlar aras›nda önceden varolan (fitoantisipinler) moleküller, patojenlere karfl› ilk bariyer olmalar›na
ra¤men, çok fazla ilgi görmemifllerdir. Saponinler (bir fitoantisipin grubu) bitki dokusunda do¤al olarak bulunurlar
ve bitki savunmas›nda önemli bir role sahiptir. Saponinler için çok farkl› biyolojik roller ortaya konulmufltur; bunlar
aras›nda allelopatik aktivite, böcek ve patojenler karfl› savunma say›labilir. Saponinlerin antifungal aktivitelerinin
mekanizmas› henüz tam olarak ayd›nlat›lmam›fl olmas›na karfl›n, hücre membran›nda bulunan sterollerle birleflerek,
hücre membran›n›n delinmesine ve böylece membran bütünlü¤ünün bozulmas›na sebep oldu¤u düflünülmektedir.
Bitkide fitopatojenik funguslara karfl›, savunmada potansiyel rollerinin detayl› çal›fl›ld›¤› saponinler aras›nda en
önemlileri yulaf köklerinde bulunan triterpenoid avenasinler ve domates yapraklar›nda bulunan α-tomatin’dir. Bu
makale antimikrobiyal aktiviteye ve bitki savunmas›nda bir role sahip bu iki saponin hakk›nda derleme bilgileri
vermektedir.
Anahtar Kelimeler: Saponinler, bitki fungal hastal›klar›, fitoantisipins, hastal›klara dayan›kl›l›k, avenasinler,
α-tomatin
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Figen Mert - Türk
Introduction
Saponins are secondary plant metabolites that occur in
a wide range of plant species (Hostettmann and
Marston, 1995). They are stored in plant cells as
inactive precursors but are readily converted into
biologically active antibiotics by plant enzymes in
response to pathogen attack. These compounds can
also be regarded as “preformed”, since the plant
enzymes that activate them are already present in
healthy plant tissues (Osbourn, 1996). The natural role
of saponins in plants is thought to be protection
against attack by pathogens and pets (Price et al. 1987;
Morrissey and Osbourn, 1999). These molecules also
have considerable commercial value and are processed
as drugs and medicines, foaming agents, sweeteners,
taste modifiers and cosmetics (Hostettmann and
Marston, 1995).
Saponins are glycosylated compounds that are
widely distributed in the plant kingdom and can be
divided into three major groups; a triterpenoid, a
steroid, or a steroidal glycoalkoloid. Triterponoid
saponins are found primarily in dicotyledonous plants
but also in some monocots, whereas steroid saponins
occur mainly in monocots, such as the Lilliaceae,
Droscoraceae and Agavaceae and in certain dicots,
such as foxglove (Hostettmann and Marston, 1995).
Oats (Avena spp.) are unusual because they contain
both triterpenoid and steroid saponins (Price et al
1987). Steroidal glycoalkaloids are found primarily in
members of the family Solanaceae, which includes
potato and tomato. The saponins produced by oats and
tomato have been studied in detail in relation to their
potential role in the defense of plants againts
phytopathogenic fungi (Osbourn, 1996).
1. Avenacins
The avenacins are a family of four structurally related
molecules, avenacins A-1, B-1, A-2, and B-2
(Crombie and Crombie, 1986a, Crombie et al. 1986).
Avenacin A-1 is the most abundant of the four in
extracts from young oat roots, comprising around 70%
of the total avenacin content (Crombie and Crombie,
1986b).
Avenacins, like many other saponins, have potent
antifungal activity (Turner, 1953; Crombie et al.
1986). The antifungal properties of saponins come
from the ability of these molecules to complex with
sterols in fungal membranes, so causing pore
formation and loss of membrane integrity (Morrisey
and Osbourn, 1999). Evidence of affinity for
membrane sterols comes from electron microscopy
studies of saponin treated membranes, which were
found to contain permenant lesions (Seeman, 1973).
These lesions are thought to be a micelle-like
aggregation of saponins and cholesterol in the plane of
the membrane, possibly with the saponin molecules
arranged in a ring with their hydrophobic moieties
combined with cholesterol around the outer perimeter
(Bengham and Horne, 1962; Seeman, 1973).
Electron microscobic analysis confirmed that
avenacin A-1 causes permeabilisation in a steroldependent manner and that it also affects membrane
fluidity (Armah et al., 1999). The presence of an
intact for these effects on artificial membrane and also
for effective antifungal activity (Armah et al., 1999).
Removal of a single D-glucose molecule from the
trisaccharide chain results in a substantial reduction in
biological activity. It is not clear how oats protect
themselves from the membrane-permeabilising effects
of the saponins that they produce. Avenacins, like
many other plant secondary metabolites, are thought
to be packed in the vacuoles of plant cells.
There has been considerable interest in the
importance of the triterpenoid avenacin saponins in
determining the resistance of oats to the root-infecting
fungus Gaeumannomyces graminis var. tritici, which
causes the disease known as “take-all disease”.
Although G. g. var. tritici causes severe yield losses in
wheat and barley, it is unable to infect oats, and unlike
the oat-attacking variety of G. graminis, G. g. var.
avenae, it is relatively sensitive to avenacins. Thus, the
resistance of oats to G. g. var. tritici has been attributed
to the presence of these saponins in oat roots (Turner,
1953).
The major avenacin, avenacin A-1, is localised in
the epidermal cell layer of oat root tips and also in the
lateral root initials, therefore it is ideally positioned to
represent a chemical barrier to invading soil-borne
microbes that damage plant tissue (Turner, 1961;
Osbourn, 1994) (Figure 1). Evidence for a role for
avenacin in disease resistance has come from
investigation of oat varieties that differ in saponin
content. There is very little natural variation in
avenacin content within Avena species (Osbourn et al.
1994; Mert-Türk et al., 2005). However at least one
diploid oat species (Avena longiglumis) has been
shown to lack avenacin A-1 and found that it is
significantly more susceptible to fungal diseases than
Saponins versus plant fungal pathogens
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Figure 1. The root tips of Avena species flouresce under UV light: (A) Cross-section from a young root; (B). Flouresced young
root tip of A. strigosa. Taken from Osbourn et al., 1994.
Figure 2. Germinated an oat (A) and wheat variants (B). Oat
root tips are flouresced under the UV, however not in wheat
variant (Mert-Türk et al., 2005)
its avenacin-producing relatives. Unfortunately A.
longiglumis does not cross with other oat species,
making genetic analysis of the interaction between
avenacin content and disease resistance difficult.
Avenacin A-1 flouresces under the UV light which is
unusual among saponins. An elegant experiment
model has been adopted to a diploid avenacin
producing oat species, A. strigosa, to investigate the
role of this compound in resistance. Artificial mutants
were generated from this species and root tips were
visualised under the UVlight. These saponin-deficient
(sad) mutants are compromised in their resistance to a
range of pathogens, providing strong evidence to
indicate that avenacins do indeed act as preformed
chemical defenses against pathogen attack
(Papadopoulou et al., 1999). The in vitro experiment
of oat root tip extract on cereal soil-bourn fungal
pathogens proved that it inhibits the mycelial growth
and germinated rate variably amoung the fungal
species tested (Mert-Türk, unpublished result).
The first step in avenacin biosynthesis is the
cyclisation of 2,3-oxidosqualence to β-amyrin
(Haralampidis et al., 2001). There is evidence that the
root tip is the main site of synthesis of β-amyrin. The
β-amyrin synthase activity is high in the root tips and
low or undetectable in older parts of the root
(Trajanowska et. al., 2001). Oat β-amyrin synthase
(AsbAS1) has been cloned and shown to correspond to
sad1, one of the genes defined by mutagenesis as
being required for avenacin biosynthesis
(Papadopoulou et al., 1999).
Avenacins are restricted to the genus Avena and the
closely related species Arrhenatherum elatius . Other
cultivated cereals appear to be generally deficient in
antifungal saponins of any kind (Osbourn, 2003; MertTürk et. al., 2005) (Figure 2). Orthologs of AsbAS1
are absent from modern cereals and may have been
lost during selection, rasing the possibility that this
gene could be exploited to enhance disease resistance
in crop plants (Haramlampidis et al., 2001).
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Figen Mert - Türk
2. α-Tomatine
The major saponin in tomato is the steroidal
glycoalkaloid, α-tomatine. α-Tomatine is, like the
avenacins, present in healthy plants in its biologically
active form. The levels of this saponin are particularly
high in the leaves, flowers and green fruits of tomato
(Roddick, 1974).
It is assumed that α-tomatine is present in tomato
leaves in the concentration around 1 mM which is
sufficient to inhibit the growth of many non-pathogens
of tomato. Therefore it would be expected that this
molecule could protect the tomato leaves from fungal
pathogens. However a number of tomato pathogens,
including Septoria lycopersici, Botrytis cinerea,
Fusarium oxysporum f.sp. lycopersici, Verticillium
albo-atrum and Alternaria solani, is able to produce
an enzyme that detoxify α-tomatine. The ability of
hydrolyzing sugar from α- tomatine is found to be
common in S. lycopersici, B. cinerea, F. oxysporum
f.sp. lycopersici. The in vitro experiments indicated
that the fungal pathogens of tomato are considerably
more tolerant to the compound than the non-pathogens
pointing out they had evolved together (reviewed by
Morrisey and Osbourn, 1999).
Despite the considerable variation in α-tomatine
levels in the genus Lycopersicon, relationship between
saponin content and disease resistance are not well
documented (Courtney and Lambeth, 1977; Rick et
al., 1994). It has been, however, suggested that αtomatine may play a secondary role in the varietyspecific resistance of tomato to incompatible races of
the biotroph Cladosporium fulvum and that release of
the saponin from leaf cells as a consequence of an
incompatible interaction may act to kill or contain the
pathogen (Dow and Callow, 1978).
Conclusion
The distribution of preformed antimicrobial
compounds within plants is often tissue spesific and
there is a tendency for these molecules to be
concentrated in the outer cell layers of plant organs,
suggesting that they may indeed act as deterrents to
pathogens and pests. They also have a variety of
commercial applicants including use as drugs and
medicines. The process of saponin biosynthesis is not
well understood despite the considerable interest in
this important group of natural products. This is due in
part to the complexity of the molecules and also to the
lack of pathway intermediates for biochemicals
studies. A more detailed understanding of these
secondary metabolite pathways and of the genes that
are involved will facilitate the development of the
plants with altered or novel saponin content, either by
classical plant breeding or by transformation-mediated
genetic modification.
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