13Journal of Cell and Molecular Biology 5: 13-17, 2006.Hali University, Printed in Turkey.

Saponins versus plant fungal pathogens

Figen Mert-Trkanakkale Onsekiz Mart University, Agricultural Faculty, Crop Protection Department, anakkale,

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 againstpathogens. Among them, preformed antimicrobial compounds (phytoanticipins) are the first biochemical barriersagainst pathogens. Saponins (a group of phytoanticipins) are present constitutively in plants and play important rolesin plant defense. A variety of biological roles have been postulated for different saponins, involving allelopathicactivity, defense against insects and pathogens. The mechanism of antifungal action of saponins is not wellunderstood but it is believed that they complex with sterols in the cell membrane, leading to pore formation andconsequent loss of membrane integrity. Saponins that have been studied in detail in relation to their potential role inplant defense against attack by phytopathogenic fungi are triterpenoid avenacins in oat roots and the steroidalglycoalkaloids -tomatine in the leaves of tomato. Here we present a review about the two saponins that have beenproved to have antimicrobial activity and have a role in plant defense.

Key words: Phytoanticipins, saponins, plant fungal pathogens, disease resistance, avenacins, -tomatine

Bitki fungal hastalklarna karfl saponinler

zet

Kk molekler arla sahip antimikrobiyal molekller bitkilerin patojenlere karfl savunmasnda nemli bir rolesahiptir. Bunlar arasnda nceden varolan (fitoantisipinler) molekller, patojenlere karfl ilk bariyer olmalarnaramen, ok fazla ilgi grmemifllerdir. Saponinler (bir fitoantisipin grubu) bitki dokusunda doal olarak bulunurlarve bitki savunmasnda nemli bir role sahiptir. Saponinler iin ok farkl biyolojik roller ortaya konulmufltur; bunlararasnda allelopatik aktivite, bcek ve patojenler karfl savunma saylabilir. Saponinlerin antifungal aktivitelerininmekanizmas henz tam olarak aydnlatlmamfl olmasna karfln, hcre membrannda bulunan sterollerle birleflerek,hcre membrannn delinmesine ve bylece membran btnlnn bozulmasna sebep olduu dflnlmektedir.Bitkide fitopatojenik funguslara karfl, savunmada potansiyel rollerinin detayl alflld saponinler arasnda ennemlileri yulaf kklerinde bulunan triterpenoid avenasinler ve domates yapraklarnda bulunan -tomatindir. Bumakale antimikrobiyal aktiviteye ve bitki savunmasnda bir role sahip bu iki saponin hakknda derleme bilgilerivermektedir.

Anahtar Kelimeler: Saponinler, bitki fungal hastalklar, fitoantisipins, hastalklara dayankllk, avenasinler,-tomatin

Introduction

Saponins are secondary plant metabolites that occur ina wide range of plant species (Hostettmann andMarston, 1995). They are stored in plant cells asinactive precursors but are readily converted intobiologically active antibiotics by plant enzymes inresponse to pathogen attack. These compounds canalso be regarded as preformed, since the plantenzymes that activate them are already present inhealthy plant tissues (Osbourn, 1996). The natural roleof saponins in plants is thought to be protectionagainst attack by pathogens and pets (Price et al. 1987;Morrissey and Osbourn, 1999). These molecules alsohave considerable commercial value and are processedas drugs and medicines, foaming agents, sweeteners,taste modifiers and cosmetics (Hostettmann andMarston, 1995).

Saponins are glycosylated compounds that arewidely distributed in the plant kingdom and can bedivided into three major groups; a triterpenoid, asteroid, or a steroidal glycoalkoloid. Tr i t e r p o n o i dsaponins are found primarily in dicotyledonous plantsbut also in some monocots, whereas steroid saponinsoccur 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 containboth triterpenoid and steroid saponins (Price et al1987). Steroidal glycoalkaloids are found primarily inmembers of the family Solanaceae, which includespotato and tomato. The saponins produced by oats andtomato have been studied in detail in relation to theirpotential role in the defense of plants againtsphytopathogenic fungi (Osbourn, 1996).

1. Avenacins

The avenacins are a family of four structurally relatedmolecules, 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 inextracts from young oat roots, comprising around 70%of the total avenacin content (Crombie and Crombie,1986b).

Avenacins, like many other saponins, have potentantifungal activity (Tu r n e r, 1953; Crombie et al.1986). The antifungal properties of saponins comefrom the ability of these molecules to complex withsterols in fungal membranes, so causing pore

formation and loss of membrane integrity (Morriseyand Osbourn, 1999). Evidence of affinity formembrane sterols comes from electron microscopystudies of saponin treated membranes, which werefound to contain permenant lesions (Seeman, 1973).These lesions are thought to be a micelle-likeaggregation of saponins and cholesterol in the plane ofthe membrane, possibly with the saponin moleculesarranged in a ring with their hydrophobic moietiescombined with cholesterol around the outer perimeter(Bengham and Horne, 1962; Seeman, 1973).

Electron microscobic analysis confirmed thatavenacin A-1 causes permeabilisation in a sterol-dependent manner and that it also affects membranefluidity (Armah et al., 1999). The presence of anintact for these effects on artificial membrane and alsofor effective antifungal activity (Armah et al., 1999).Removal of a single D-glucose molecule from thetrisaccharide chain results in a substantial reduction inbiological activity. It is not clear how oats protectthemselves from the membrane-permeabilising effectsof the saponins that they produce. Avenacins, likemany other plant secondary metabolites, are thoughtto be packed in the vacuoles of plant cells.

There has been considerable interest in theimportance of the triterpenoid avenacin saponins indetermining the resistance of oats to the root-infectingfungus Gaeumannomyces graminis var. tritici, whichcauses the disease known as take-all disease.Although G. g. var. tritici causes severe yield losses inwheat and barley, it is unable to infect oats, and unlikethe oat-attacking variety of G. graminis, G. g. var.avenae, it is relatively sensitive to avenacins. Thus, theresistance of oats to G. g. var. tritici has been attributedto the presence of these saponins in oat roots (Turner,1953).

The major avenacin, avenacin A-1, is localised inthe epidermal cell layer of oat root tips and also in thelateral root initials, therefore it is ideally positioned torepresent a chemical barrier to invading soil-bornemicrobes that damage plant tissue (Turner, 1961;Osbourn, 1994) (Figure 1). Evidence for a role foravenacin in disease resistance has come frominvestigation of oat varieties that differ in saponincontent. There is very little natural variation inavenacin content within Avena species (Osbourn et al.1994; Mert-Trk et al., 2005). However at least onediploid oat species (Avena longiglumis) has beenshown to lack avenacin A-1 and found that it issignificantly more susceptible to fungal diseases than

14 Figen Mert - Trk

its avenacin-producing relatives. Unfortunately A .longiglumis does not cross with other oat species,making genetic analysis of the interaction betweenavenacin content and disease resistance difficult. Avenacin A-1 flouresces under the UV light which isunusual among saponins. An elegant experimentmodel has been adopted to a diploid avenacinproducing oat species, A. strigosa, to investigate therole of this compound in resistance. Artificial mutantswere generated from this species and root tips werevisualised under the UVlight. These saponin-deficient(sad) mutants are compromised in their resistance to arange of pathogens, providing strong evidence toindicate that avenacins do indeed act as preformedchemical defenses against pathogen attack

(Papadopoulou et al., 1999). The in vitro experimentof oat root tip extract on cereal soil-bourn fungalpathogens proved that it inhibits the mycelial growthand germinated rate variably amoung the fungalspecies tested (Mert-Trk, unpublished result).

The first step in avenacin biosynthesis is thecyclisation of 2,3-oxidosqualence to - a m y r i n(Haralampidis et al., 2001). There is evidence that theroot tip is the main site of synthesis of -amyrin. The-amyrin synthase activity is high in the root tips andlow or undetectable in older parts of the root(Trajanowska et. al., 2001). Oat -amyrin synthase(AsbAS1) has been cloned and shown to correspond tosad1, one of the genes defined by mutagenesis asbeing required for avenacin biosynthesis(Papadopoulou et al., 1999).

Avenacins are restricted to the genus Avena and theclosely related species Arrhenatherum elatius . Othercultivated cereals appear to be generally deficient inantifungal saponins of any kind (Osbourn, 2003; Mert-Trk et. al., 2005) (Figure 2). Orthologs of AsbAS1are absent from modern cereals and may have beenlost during selection, rasing the possibility that thisgene could be exploited to enhance disease resistancein crop plants (Haramlampidis et al., 2001).

Saponins versus plant fungal pathogens 15

Figure 1. The root tips of Avena species flouresce under UV light: (A) Cross-section from a young root; (B). Flouresced youngroot tip of A. strigosa. Taken from Osbourn et al., 1994.

Figure 2. Germinated an oat (A) and wheat variants (B). Oatroot tips are flouresced under the UV, however not in wheatvariant (Mert-Trk et al., 2005)

2. -Tomatine

The major saponin in tomato is the steroidalglycoalkaloid, -tomatine. -Tomatine is, like theavenacins, present in healthy plants in its biologicallyactive form. The levels of this saponin are particularlyhigh in the leaves, flowers and green fruits of tomato(Roddick, 1974).

It is assumed that -tomatine is present in tomatoleaves in the concentration around 1 mM which issufficient to inhibit the growth of many non-pathogensof tomato. Therefore it would be expected that thismolecule could protect the tomato leaves from fungalpathogens. However a number of tomato pathogens,including Septoria lycopersici, Botrytis cinere a ,Fusarium oxysporum f.sp. lycopersici, Verticillium

albo-atrum and Alternaria solani, is able to producean enzyme that detoxify -tomatine. The ability ofhydrolyzing sugar from - tomatine is found to becommon in S. lycopersici, B. cinerea, F. oxysporumf.sp. lycopersici. The in vitro experiments indicatedthat the fungal pathogens of tomato are considerablymore tolerant to the compound than the non-pathogenspointing out they had evolved together (reviewed byMorrisey and Osbourn, 1999).

Despite the considerable variation in -tomatinelevels in the genus Lycopersicon, relationship betweensaponin content and disease resistance are not welldocumented (Courtney and Lambeth, 1977; Rick etal., 1994). It has been, however, suggested that -tomatine may play a secondary role in the variety-specific resistance of tomato to incompatible races ofthe biotroph Cladosporium fulvum and that release ofthe saponin from leaf cells as a consequence of anincompatible interaction may act to kill or contain thepathogen (Dow and Callow, 1978).

Conclusion

The distribution of preformed antimicrobialcompounds within plants is often tissue spesific andthere is a tendency for these molecules to beconcentrated in the outer cell layers of plant organs,suggesting that they may indeed act as deterrents topathogens and pests. They also have a variety ofcommercial applicants including use as drugs andmedicines. The process of saponin biosynthesis is notwell understood despite the considerable interest in

this important group of natural products. This is due inpart to the complexity of the molecules and also to thelack of pathway intermediates for biochemicalsstudies. A more detailed understanding of thesesecondary metabolite pathways and of the genes thatare involved will facilitate the development of theplants with altered or novel saponin content, either byclassical plant breeding or by transformation-mediatedgenetic modification.

References

Armah CN, Mackie AR, Roy C, Price K, Osbourn AE,Bawyer P and Ladha S. The membrane permeabilisingeffect of avenacin A-1 involves the reorganisation ofbilayer cholesterol. Biophys J. 76: 281-290, 1999.

Bengham AD and Horne RW. Action of saponin onbiological cell membranes. Nature 196: 952-953, 1962.

Courtney WH and Lambeth VN. Glycoalkaloid content ofmature fruit of Lycopersicon species. Hort Science. 13:9-25, 1977.

Crombie WM and Crombie L. Distribution of Avenacins A-1, A-2, B-1 and B-2 in oat roots: Their fungicidal activitytowards take-all disease. Phytochemistry. 25(9): 2069-2073, 1986.

Crombie WM and Crombie L. Pathogenicity of the take-allfungus to oats: its relationship to the concentration anddetoxification of the four avenacins. Phytochemistry. 25:2075-2083, 1986.

Crombie WM, Crombie L, Green JB and Lucas JA.Pathogenicity of the take-all fungus to oats; itsrelationship to the concentration and detoxification ofthe four avenacins. Phytochemistry. 25: 20-75-2083,1986.

Dow JM and Callow JA. A possible role for -tomatine inthe varietal-specific resistance of tomato toCladosporium fulvum. Phytopathology. 92: 211-216,1978.

Haralampidis K, Trojanowska M, Osbourn AE. Biosynthesisof thriterpenoid saponins in plants. Adv Biochem EngBiothecnology. 75:31-49, 2001.

Hostettmann KA and Marston A. Saponins. Chemistry andpharmacology of natural products. Cambridge UniverstyPress, Cambridge, United Kingdom, 1995.

Mert-Trk F, Egesel C and Gl MK. Avenacin A-1 contentof some local oat genotypes and in vitro effect ofavenacins on several soil-borne fungal pathogens ofcereals. Turk J Agric For . 29: 157-164, 2005.

Morrissey JP and AE Osbourn. Fungal resistance to plantantibiotics as a mechanism of pathogenesis. MicrobiolMol Biol Rev. 63:708-724, 1999.

Osbourn AE, Clarke BR, Lunness P, Scott PR and DanielsMJ. An oat species lacking avenacin is susceptible to

16 Figen Mert - Trk

infection by Gaeumannomyces graminis v a r. t r i t i c i .Physiol. Mol P Pathol . 45:457-467, 1994.

Osbourn AE. Preformed antimicrobial compounds and plantdefense againts fungal attack. Plant Cell. 8: 1821-1831,1996.

Osbourn AE. Saponins in cereals. Phytochemistry. 62:1-4,2003.

Papadopoulou K, Melton RE, Leggett M, Daniels MJ andOsbourn AE. Compromised disease resistance insaponin-deficient plants. P Natl Acad Sci USA. 96 (22):12923-12928, 1999.

Price KR, Johnson IT and Fenwick GR. The chemistry andbiological significance of saponins in food and feedingstufs. Crit Rev Food Sci Nutr. 26: 27-133, 1987.

Rick CM, Uhling JW and Jones AB. High -tomatinecontent in ripe fruit of Andean Lycopersicon esculantumvar. cerasiforme; devolopmental and genetic aspects.Proc Natl Acad Sci USA. 91, 12877-12881, 1994.

Roddick JG. The steroidal glycoalkaloid - t o m a t i n e .Phytochemistry. 13: 9-25, 1974.

Seeman P, Cheng Dand and Iles GH. Structure of membraneholes in osmatic and saponin hemolysis. J Cell Biol. 56:519-527, 1973.

Trajanowska MR, Osbourn AE, Daniels MJ and ThrelfallDR. Investigation of avenacin-deficient mutants o fAvena strigosa. Phytochemistry. 56:121-129, 2001.

Turner EMC. The nature of the resistance of oats to the take-all fungus. J Exp Bot. 11: 403-412, 1953.

Turner EMC. An enzymic basis for pathogen specificity inOphiobolus graminis. J Exp Bot. 4: 264-271, 1961.

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