secondary metabolites and plant defence
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Series Title Progress in Biological Control
Chapter Title Secondary Metabolites and Plant Defence
Chapter SubTitle
Copyright Year 2012
Copyright Holder Springer Science+Business Media B.V.
Family Name GoyalParticleGiven Name Shaily
Corresponding Author
SuffixDivision Laboratory of Bio-Molecular Technology, Department of BotanyOrganization M.L. Sukhadia UniversityAddress 313001, Udaipur, IndiaEmail
Family Name LambertParticleGiven Name C.
Author
SuffixDivision GESVAB – EA 3675, Institut des Sciences de la Vigne et du VinOrganization University of BordeauxAddress CS50008, 210, Chemin de Leysotte, F-33882, Villenave d’Ornon, FranceEmailFamily Name CluzetParticleGiven Name S.
Author
SuffixDivision GESVAB – EA 3675, Institut des Sciences de la Vigne et du VinOrganization University of BordeauxAddress CS50008, 210, Chemin de Leysotte, F-33882, Villenave d’Ornon, FranceEmailFamily Name MerillonParticleGiven Name J. M.
Author
SuffixDivision GESVAB – EA 3675, Institut des Sciences de la Vigne et du VinOrganization University of BordeauxAddress CS50008, 210, Chemin de Leysotte, F-33882, Villenave d’Ornon, FranceEmail [email protected]
Family Name RamawatParticle
Author
Given Name Kishan G.
SuffixDivision Laboratory of Bio-Molecular Technology, Department of BotanyOrganization M.L. Sukhadia UniversityAddress 313001, Udaipur, IndiaEmail [email protected]
Abstract Infected or elicited plants accumulate an array of plant defensive compounds. Now-a-days, it is wellaccepted that plant SECONDARY METABOLITES are involved in this plant defence system. The processof inducing resistance using elicitors is environmental friendly and is advantageous over the chemicalbased pesticides. It is like stimulation of the plant’s own “immune” potential rather than on suppression ofpathogens. The resistance developed in this way has prolonged effect. This strategy could be an alternativesolution to reduce the use of pesticides.
Keywords (separated by'-')
Plant defence - Elicitors - Isoflavones - Stilbenes
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J.M. Merillon and K.G. Ramawat (eds.), Plant Defence: Biological Control, Progress in Biological Control 12, DOI 10.1007/978-94-007-1933-0_5, © Springer Science+Business Media B.V. 2011
Abstract Infected or elicited plants accumulate an array of plant defensive compounds. Now-a-days, it is well accepted that plant SECONDARY METABOLITES are involved in this plant defence system. The process of inducing resistance using elicitors is environmental friendly and is advantageous over the chemical based pes-ticides. It is like stimulation of the plant’s own “immune” potential rather than on suppression of pathogens. The resistance developed in this way has prolonged effect. This strategy could be an alternative solution to reduce the use of pesticides.
Keywords Plant defence • Elicitors • Isoflavones • Stilbenes
5.1 Introduction
In nature, plants protect themselves against pathogen attack mainly by mechanical and chemical defences. Mechanical defences include structures such as spines, trichomes, thick cuticle, and hard, sticky, or smooth surfaces which prevent pathogens from picking for food or laying eggs. Chemical defences include a variety of substances that are toxic, repellent, or that render plant tissues indigestible to
S. Goyal (*) • K.G. RamawatLaboratory of Bio-Molecular Technology, Department of Botany, M.L. Sukhadia University, Udaipur 313001, Indiae-mail: [email protected]
C. Lambert • S. Cluzet • J.M. MerillonGESVAB – EA 3675, Institut des Sciences de la Vigne et du Vin, University of Bordeaux, CS50008, 210, Chemin de Leysotte, Villenave d’Ornon, F-33882, Francee-mail: [email protected]
[AU1]
Chapter 5Secondary Metabolites and Plant Defence
Shaily Goyal, C. Lambert, S. Cluzet, J.M. Merillon, and Kishan G. Ramawat
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animals [1]. Chemical defence due to secondary metabolites is prominently developed in plants, providing protection to the plant [2]. Mechanical and chemical defence can be either independent of each other [3] or they can work in combination such as glandular trichomes and secretory canals. They interact to entrap pathogen in sticky and toxic secretions [4]. Many plants produce resins, gum, lattices and mucilage, which are stored under pressure in networks of canals throughout the cortex of the stems and in the leaves, where they follow the vascular bundles. These secretions are rich in several secondary metabolites: for example, oleoresin present in Abies grandis is a complex mixture of monoterpenes, sesquiterpenes and diterpe-noid acids, used to deter insect pests and their symbiotic fungal pathogens [5]. Some Bursera species resins, rich in mostly monoterpenes and sesquiterpenes [6] are under considerable pressure, and so when a leaf is damaged, resin may be released in a spectacular syringe- like squirt. This squirt may travel up to 2 m and lasts a few seconds, so it represents a good example of mechanical and chemical defence inter-action [7]. Some species of Asclepias (milkweeds) latex contains cardenolides and cardiac glucosides which help the plant in defence response [8]. These toxic ste-roids have an interesting use in monarch butterflies. Adult monarch butterflies store the cardenolides they have built-up during their larval stage, feeding mostly on Asclepias. This stored cardenolide content in butterflies deters them from their vertebrate predators.
The chemical defence can be further classified as constitutive and inducible chemical defences. The term constitutive means that the defence is present in the plant whether the predator attacks or not. Many constitutive defence chemicals are produced by epidermal hairs that can trap and kill insect larvae. Inducible systems are those that are absent before a pathogen or predator attack, but are induced when the attack occurs. Defence related responses can occur in the plant organ originally attacked (local response) or in distant unaffected parts (systemic response). Examples of chemical defences can be found in article by Field and co-workers [9].
Plants can have a compatible response towards its pathogen, where their contact lead to a successful infection, or they can have a non-compatible response where a plant and pathogen contact lead to a non-successful infection. In incompatible inter-actions, infection by pathogens inducts a set of local responses in and around the infected host cell which can lead to cell death [10]. Thus, the pathogen may be “trapped” in dead cells and this prevents the infection from being spread. Local responses in the cells include oxidative burst, changes in cell wall composition that can inhibit penetration by the pathogen, and de novo synthesis of antimicrobial com-pounds such as phytoalexins and pathogenesis related (PR) proteins. Phytoalexins are mainly characteristics of the local response, PR proteins occur both locally and systemically [11].
The aim of this review is to reveal new avenues of research in the area of elicitor imparted secondary metabolites production in plants which provide them resistance against diseases.
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5.2 Secondary Metabolites and Defence
Plants (>300,000 species) and insects (likely >1,000,000 species) have co-evolved, still plants dominate the landscape [12]. This is due to the presence of secondary metabolites that make the plants both repellent and toxic to most pathogens, insects and other grazing animals. Secondary metabolites are the molecules that appear to be dispensable for normal growth, or are required only under particular conditions, whereas primary metabolites are involved in the physiological functions. These secondary products are the key components of active and potent defence mecha-nisms in plants [13, 14]. They are the active part of the chemical war between plants and their pathogens.
About 1–10% of the dry mass of some plants is made up of chemicals designed for defence against predators. Plants synthesize a huge array (around several tens of thousands) of different secondary metabolites [14]. Synthesizing a particular chem-ical so that it accumulates in the plant to a significant level has an associated cost. Various biosynthetic pathways are involved in secondary metabolites production and there is requirement of substantial amount of ATP. Besides their synthesis dur-ing the time of attack by pathogen, their storage in the vacuole requires energy as well. The energy for uphill transport and often for trapping the metabolite in the vacuole is provided by H+ – ATPase. In addition, some metabolites are transported into the vacuole with the help of ATP-binding cassette transporters (ABC-transporter) which depend on ATP [15]. It can be seen that if the cost of producing a defence compound is minimal and allows the plant that produces it to leave more offspring, then that plant has a greater evolutionary fitness than its non-defended colleagues. This can readily be demonstrated by partially defoliating plants or giving them a mild bacterial, fungal, or viral infection. Such plants grow much less vigorously and produce fewer seeds. However, if secondary metabolisms have not been very impor-tant in the biology of different organisms, evolution would not have selected and maintained the complex pathways leading to secondary metabolism [14].
Most of the secondary metabolites are derived from the isoprenoid, phenylpro-panoid, alkaloid or fatty acid/polyketide pathways [14, 16]. It is observed that related plant families generally make use of related chemical structures for defence, e.g. sesquiterpenes in the Solanaceae, stilbenes in the Vitaceae, isoflavones in the Leguminosae, sulfur-based glucosinolate–myrosinase in the Brassicaceae and limonoids among members of the families Meliaceae and Rutaceae. In plants, the best understood secondary metabolites are implicated in pathogen defence, sensing and signaling. This list is continuously growing by the extensive use of biochemical and genetic approaches to reveal the undiscovered metabolites and their complex signaling pathways that mediate plant disease resistance.
Pathogens, insects, and other parasites initially establish physical interactions with hosts via surface contact, and the plant surface initiate chemical signaling in response to it. The secondary metabolites content of the surface exempt of disease is also an important aspect to be studied in order to discover new defence molecules.
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For that, accurate methods of detection and quantification are required. Now-a days desorption electrospray ionization mass spectrometry (DESI-MS) have made possible fine scale evaluation of compounds on native surfaces of the plants. Lane and co-workers [17] reported presence of bromophycolides, antimicrobial compounds, on the surface of macroalga Callophycus serratus in sufficient quantity for inhibition of Lindra thalassiae, a marine fungal pathogen. Hamm and co-workers [18] have used laser desorption/ionisation time-of-flight mass spectrometry (LDI-ToFMS) to analyze phytoalexins at the surface of Vitis vinifera leaves. They have found that the amounts of resveratrol and pterostilbene are directly related to the degree of Plasmopara viticola contamination.
Several large groups, such as phenolics, alkaloids, terpenoids, iridoid glycosides, cardenolides, and cyanogenic glycosides have been implicated in plant defence systems. The literature on secondary metabolites is extensive. Here we summarized phenolics related to plant defence system.
5.3 Phenolics and Disease Resistance
Phenolics are represented by having at least one aromatic ring with one or more hydroxyl groups attached, and are widely present throughout the plant kingdom [19]. They are known to contribute to pigmentation of different organs along with their role against different biotic and abiotic stresses [20]. Phenolics occurring naturally in plant tissue can be classified into two groups, the flavonoids and the non-flavonoids.
Depending on the structural complexity of flavonoids (with an estimated 10,000 structurally different members), particularly on the oxidation state of the central ring C, flavonoids are themselves subclassified as flavonols, flavones, flavan-3-ols (catechins and their oligomers: proanthocyanidins), anthocyanidins, flavanones and isoflavones and those that are present in less quantity in diet are dihydroflavonols, flavan-3, 4-diols, chalcones, dihydrochalcones, and aurones [21] (Fig. 5.1). Majority of flavonoids exist naturally as glycosides. Both, the hydroxyl groups and sugars, increase water solubility of flavonoids [22]. Flavonoids in general are polyphenolic compounds comprising of 15 carbons, with 2 aromatic rings connected by a 3-carbon bridge (C
6–C
3–C
6). They consist mainly of 2-phenylchromans and also 3-phenyl-
chromans for isoflavonoids. The key enzyme for the formation of the flavonoid skeleton is chalcone synthase, which catalyses the stepwise condensation of three acetate units from malonyl-CoA with 4-coumaryl-CoA to the intermediate chalcone. Flavonoids play important role in defence against microorganisms and pests.
There are many instances which describe their potent role in disease resistant. A recent investigation by Koskimäki and co-workers [23] observed that accumulation of individual phenolic compounds could be specific for a particular infection. They demonstrated biosynthesis of different phenolic compounds in bilberry (Vaccinium myrtillus) after infection by a fungal endophyte (Paraphaeosphaeria sp.) and a pathogen (Botrytis cinerea). A study of barley mutants showed that proanthocyanidins
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B1
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3
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6
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8'9
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1'
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O
CA
O
O
Isoflavone
O
O
Flavone
OH
O
O
Flavonol
O
O Flavanones
OH
O+
Anthocyanidins
OH
O
Flavan-3-ol
Fig. 5.1 Basic flavonoid structure and its derivatives
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and even small amounts of dihydroquercetin are involved in the defence against Fusarium species [24]. The wild species of groundnut, Arachis kempff-mercadoi is resistant to tobacco armyworm Spodoptera liture due to its flavonols quercetin and its glycoside rutin [25]. Similarly, nematode resistance in banana is due to flavan-3,4-diols and condensed tannins [26]. Flavonoids also play a major role in postharvest resistance of fruits and vegetables [27]. High concentrations of flavonoids mainly in unripe fruits prevent them from pathogens; thus, ripe fruits are usually more sensible to fungal decay.
There are also several classes of non-flavonoids, dominated by phenylpropanoids containing only the C
6−C
3 phenylpropane skeleton and these compounds are directly
linked to lignin (polymer phenyl propanoid) biosynthesis in vascular plants. The most important examples are cinnamic acids and their derivatives such as chlorogenic acid, p-coumaric, ferulic and sinapic acids. Another class of non-flavonoid polyphenols which are less frequently found in diets (except for the grapes and peanuts) is the stilbenes with C6–C2–C6 skeletons [28, 29]. Hydroxycinnamic acids and flavonoid classes are widely present in higher plants whereas classes like isoflavones (e.g., Fabaceae) and stilbenes (e.g., 23 families only: Vitaceae, Cyperaceae, Dipterocarpaceae, Iridaceae, Fabaceae, Moraceae, Orchidaceae and Polygonaceae) are limited to particular families.
5.3.1 Isoflavones
Isoflavones are characterized by having the B-ring attached at C3 rather than the C
2
position [30] (Fig. 5.1). Till now about 1,600 isoflavones have been identified and the list is continuously growing [31]. The isoflavones like daidzein, genistein, and glycitein are synthesized via the phenylpropanoid pathway and stored in the vacuole as glucosyl- and malonyl glucose conjugates. The pathway to daidzein branches from the phenylpropanoid pathway, that is common to most plants, following the chalcone synthase reaction (Fig. 5.2) through a legume specific enzyme, chalcone reductase. Glycitein synthesis is likely to be derived from isoliquiritigenin. Genistein synthesis shares the naringenin intermediate with the flavonoid/anthocyanin branch of the phenylpropanoid pathway. In all cases, the unique aryl migration reaction to create the isoflavones is mediated by isoflavone synthase [32]. Oxidative rearrange-ment of naringenin (flavanone) with a 2.3-aryl shift yields the isoflavone. The initiating step in isoflavone formation may be an epoxidation catalysed by a cytochrome p-450-dependent mono-oxygenase. After structural rearrangement, aryl shift and addition of a hydroxyl ion to C-2, elimination of water by a dehydratase gives the isoflavone structure. Details of isoflavones structure can be found in the article of Veitch, 2007 [31].
These compounds were initially recognized for their roles in plant disease resistance and as signal molecules to promote Rhizobium nodulation [16]. They also serve as precursors for the production of major phytoalexins during plant –microbe interactions [33] and inhibit pathogen attack [34–37]. Isoflavones have demonstrated
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efficient antimicrobial and antifungal activities. The isoflavones like daidzein inhibits the growth of Fusarium culmorum, while glycitein and formononetin can reduce mycelial development in Aspergillus ochraceus [38]. Biochanin A and genistein exhibit antifungal activity against Rhizoctonia solani and Sclerotium rolfsii [39]. Antimicrobial and antifunagl properties of various isoflavones have been well demonstrated. Isoflavones from stem bark of Flemingia paniculata [40] and from F. stropbilifera [41] showed significant antibacterial activity. Extract of Tamarix gallica containing quercetin [42], Prunus Americana containing isoflavones [43] and Glycirrhiza glabra containing glabridin [44] showed promising antimicrobial activity. On elicitation by Aspergillus sojae, Soybean produced antifungal glyceol-lins [45] effective against Fusarium oxysporum, Phytophthora capsici, Sclerotina sclerotiorum and Botrytis cinerea, while lactofen induced isoflavones were correlated with defence responses [46].
Phenylalanine
Cinnamate
p - Coumarate
p - Coumarate CoA
Liquiritigenin
Naringenin
Dihydroflavonol
AnthocyaninsCondensed tannins
Genistein
Glyceollins
Glycitein
Daidzein
Cinnamic acid 4 - hydroxylase
Chalcone isomerase
Isoflavone hydroxylaseIsoflavone reductase
Isoflavone synthase
Anthocyaninsynthase
4 reductase
-
IsoliquiritigeninFlavone
Flavonol
Phenylalanineammonia lyase
-
4-Coumarate:CoA ligase
Chalcone synthase
Chalcone reductase
Flavone synthase
Flavan - 3,4 - diol
Flavan-3,4 - diol
+ 3 malonylCoAChalcone isomerase
Isoflavone synthase
Fig. 5.2 Biosynthetic route of isoflavones production
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5.3.2 Stilbenes
Stilbenes are a group of phenylpropanoid-derived compounds characterized by a 1,2-diphenylethylene backbone (C
6–C
2–C
6). Stilbenes exist in the stereo isomeric
forms (E and Z forms) depending on the position of where the functional groups are attached in relation to one another on either side of the double bond. Stilbenes constitute an important group of natural products that are of particular interest owing to their wide range of biological activities [47]. Combretastatins, piceatannol, pinosylvin, rhapontigenin, pterostilbene and resveratrol are some of the naturally occurring stilbenes. Of these, combretastatins and resveratrol have been extensively studied. Most plant stilbenes are derivatives of the basic unit trans-resveratrol (3,5,4-0-trihydroxy-stilbene). From this relatively simple structure, over a thousand stilbenoid compounds have been characterized, resulting from different chemical substitutions patterns like methylation, glycosylation or isoprenylation, in addition to oxidative condensations of monomers into dimers (for example viniferins) and subsequent condensations of these [48]. All higher plants seem to be able to synthe-size malonyl-CoA and CoA-esters of cinnammic acid derivatives, but only few plant species are able to produce stilbenes, as the stilbene synthase (STS), the fundamen-tal enzyme of stilbene synthesis, is present in a limited number of plant species, for example Vitis spp., Arachis hypogea, Pinus spp., Rheum spp. and Fallopia spp. STS genes exist as a family of related genes in these plants [49]. Stilbene synthase cata-lyzes, in a single reaction, the biosynthesis of the stilbene backbone from three malonyl-CoA and one CoA-ester of a cinnamic acid derivative (Fig. 5.3). Grapevine genome contains more than 20 STS genes [50] and nearly all of them are expressed in grape following infection with Plasmopara viticola [51].
Some plant species, such as Fallopia japonica (formely Polygonum cuspidatum), pine (Pinus spp.) and grapevine (Vitis spp.) constitutively accumulate large amounts of stilbenes [49]. However, most studies concerning stilbene biosynthesis have been conducted on peanut, grapevine and pine. Induction of stilbenes synthesis is well known in response to a wide range of abiotic and biotic stresses. As example in grapevine, upon infection with different fungal pathogens, including powdery mildew (Erysiphe necator) [52], downy mildew (P. viticola) [53, 54], or gray mold (B. cinerea) [55, 56], coordinated activation of STS and upstream enzymes in this pathway occurred.
Stilbenes can accumulate in plant tissues to concentrations necessary to inhibit fungal growth [57, 58]. Stilbenes like pinosylvin and pinosylvin 3-O-methyl ether, which occur naturally in conifers, have strong antifungal activity in in vitro assays. These compounds are active in vitro against Coriolus versicolor and Gloeophyllum trabeum, two wood-destroying fungi [59]. Other stilbene like resveratrol inhibits conidial germination of Botrytis cinerea (the gray mold agent on grapes) [53] and also reduces the germination of sporangia of Plasmopara viticola (the downy mil-dew agent) whereas its glucoside piceid reduces fungal spore germination [60] at concentrations compatible with the activity range of other phytoalexins. It is inter-esting to note that trans-resveratrol, piceids, viniferins and pterostilbene concentrations
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reach up to 50–400 mg/g DW in infected grapevine leaves [49, 61]. Pterostilbene, the dimethylated form of resveratrol, had a five-fold higher activity than resveratrol in inhibiting fungal growth in vitro, indicating that methylation of hydroxyphenyl groups could lead to increased biocidal activity of phenolics [62].
To demonstrate the role of these phytoalexins in plant disease resistance, the stilbene synthase VST1 gene fused to an alfalfa pathogen-inducible promoter was introduced in 41B grapevine rootstock. Resulting transgenic plants produced more resveratrol under biotic and abiotic stress conditions and showed reduced symptoms after infection with B. cinerea [63]. Two stilbene synthase genes VST1 and VST2 from grapevine (Vitis vinifera L.) and the pinosylvin synthase gene PSS from pine (Pinus sylvestris L.) were stably transferred into bread wheat. Upon inoculation with the biotrophic pathogen Puccinia recondita f.sp. tritici several VST transgenic wheat lines showed a significant reduction of disease symptoms compared to wild-type
Phenylalanine
Phenylalanine ammonia lyase(PAL)
Cinnamic acid
Cinnamate 4-hydroxylase(C4H)
p-coumaric acid
4-coumarate CoA ligase(4CL)
p-coumaroyl-CoA+ 3 malonyl-CoA
Chalcone synthaseStilbene synthase
Tetraketide intermediate
Stilbene synthaseChalcone synthase
Chalcone Resveratrol
Fig. 5.3 Steps of stilbene biosynthesis
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plants. The reduction of disease symptoms was even more obvious after inoculation with the facultative biotrophic pathogen Septoria nodorum Berk [64]. Similarly, the transfer of stilbene synthase genes to tobacco, tomato, barley and rice leads to the accumulation of resveratrol and the resistance of the resulting transgenic plants to fungal pathogens [65–67]. In some cases, such as in kiwifruits and poplars , the heterologous expression of stilbene synthase did not result in an improved pathogen resistance, but resveratrol glucosides (less active on fungi than the aglycon form) were accumulated [68, 69].
Resveratrol is one of the most extensively studied natural products. Plethora of studies have demonstrated that resveratrol has preventive effect against a wide variety of diseases including cancer, cardiovascular diseases, as well as AIDS [70, 71].
5.3.3 Mechanism of Secondary Metabolites Action
Flavonoids and phenylpropanoids are widely distributed in plants and exhibit different mode of action against the pathogens. It is interesting to know that hundreds of clinical antifungal drugs in use, target only 6 different processes. Mostly they act as analogues of cellular signal compounds or substrates. They affect various physio-logical process and the parts of the pathogens like biomembranes, enzyme inhibi-tion, estrogenic properties and DNA alkylation [72]. These molecules usually have several phenolic hydroxyl groups in common, which can dissociate in negatively charged phenolate ions. Phenolic hydroxyl groups form hydrogen and ionic bonds with proteins and peptides. The higher the number of hydroxyl groups, the stronger the astringent and denaturing effect [73].
Proteins can only work properly if they have the correct three-dimensional structure, called conformation. Conformational changes alter their properties and can prevent effective crosstalk between proteins, and between proteins and DNA or RNA. Most secondary metabolites interact with proteins in one or another way by binding, complexing, denaturing, thereby changing protein conformations. Most secondary metabolites form covalent bond with protein, often by binding to free amino-, SH- or OH- groups, e.g., phenylpropanoids binds to amino groups, SH reagents and epoxides couple to free SH groups. The covalent modification can lead to a conformational change and thus loss of activity; or protein turnover is altered because proteases can no longer break down the alkylated protein. Polyphenols (phenylpropanoids, flavonoids, catechins, tannins, lignans, quinines, anthraquinones) interact with proteins by forming hydrogen bonds and the much stronger ionic bonds with electronegative atoms of the peptide bonds and or the positively charged side chains of basic amino acids (lysine, histidine, arginine). A single of these non-covalent bonds is quite weak. But because several of them are formed concomi-tantly when a polyphenols encounters a protein, a change in protein conformation or a loss in protein flexibility is likely to occur that commonly leads to protein inac-tivation. Since most polyphenols are quite polar and therefore, hardly absorbed after oral intake, they are usually not regarded as serious toxins [74].
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The effect of two stilbene compounds, pinosylvin and resveratrol, on the growth of several fungi was evaluated in plate tests. Wood decay tests were carried out with birch and aspen samples impregnated with the two stilbenes. In plate experiments, resveratrol had an enhancing effect on growth at concentrations where pinosylvin was already enough to prevent the growth of most fungi studied [75]. Looking at the efficient mode of action of plant SECONDARY METABOLITES against insects and pathogens pesticides, they are gaining increased attention and interest among those concerned with environment friendly, safe, and integrated crop management approaches [76].
5.4 Elicitation
Infected or elicited plants accumulate an array of plant defencive compounds. Now-a-days, it is well accepted that plant SECONDARY METABOLITES are involved in this plant defence system [77]. In 1982, Wolters and Eilert [78] reported for the first time that in rue callus cultures the acridone alkaloid content increased when it was co-cultivated with fungi. Through the years, fungal cell wall components, microbial preparations, various heavy metals, UV irradiation or ultrasound treatment are able to enhance SECONDARY METABOLITES accumulation in plants. Plants treated with non-specific elicitors develop a general defence mechanism. This induced defence is a phenotypic trait. The process of inducing resistance using elicitors is environmental friendly and is advantageous over the chemical based pesticides. It is based on induction of the native “immune” potential of the host plant rather than on suppression of phytopathogens. The resistance developed in this way has prolonged effect. This strategy could be an alternative solution to reduce the use of pesticides.
Elicitors are physical or chemical factors which when comes in contact in small concentrations to a living plant cell system triggers morphological and physiological responses such as phytoalexins accumulation. Elicitation is the process in which elicitor induct a sequence of reaction in the living cell, particularly related to biosynthesis of metabolites such as phytoalexins.
On the basis of their nature, elicitors can be divided into two types namely biotic and abiotic. Abiotic elicitors are the substances of non –biological origin, like organic salts and physical factors acting as elicitors (Table 5.1). Biotic elicitors are substances with biological origin, like polysaccharides, derived from plant cell walls (pectin or cellulose), plant gums and microorganisms (chitins or glucans) and glycoproteins or intracellular proteins whose functions are coupled to receptors and act by activating or inactivating a number of enzymes or ion channels [79]. Biotic elicitors can be further divided on the basis of their source into exogenous and endogenous groups. Exogenous elicitors are considered as the primary signals in plant pathogen interactions. They originate in the pathogen itself, mostly have a limited mobility within plant tissues, and evoke a response in cells in the immediate vicinity to the pathogen. Endogenous elicitors are of plant origin and arise as a
296
297
298
299
300
301
302
303
304
305
306
307
308
309
310
311
312
313
314
315
316
317
318
319
320
321
322
323
324
325
326
327
328
329
330
331
332
333
334
335
336
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result of the interaction with the pathogen. Most appear to be apoplastic and their function may be to modulate the extent of the response in the surrounding tissue. This modulation can be exerted independently of the presence of exogenous elici-tors or in a synergistic manner [80].
Table 5.1 List of commonly used biotic and abiotic elicitors
Abiotic elicitors2-amino-2-deoxy-d-galactopyranose
(galactosamine)Freezing and thawing cycles
2-amino-2-deoxy-d-glucose (glucosamine) Fungicides (Maneb, Butylamine, Benomyl)6,1¢,6¢-triamino-6,1¢,6¢-trideoxysucrose
(saccharosamine)Gentamycin
b-cyclodextrin Herbicides (Acifluorofen)Activated carbon Methyl jasmonateArachidonic acid Nitric oxideColchicine Oxidative stress, amino acid starvationCopper Sulphate Salicylic acidCopper/Cadmium/Aluminium Chloride Sodium ferric ethylenediamine di-(o-hydroxyphe-
nylacetate) FeEDDHACu+2, Cd+2, Ag+ Trifluoroethyl salicylate (TFESA)Curdlan UV lightDenatured proteins (RNase) Vanadium/Vanadyl sulphateDiethyl amino ethyl dichlorophenyl ether XAD-4Electromagnetic treatment Xanthan
Biotic elicitorsArachidonic acid LaminarinAlginate oligomers MonilicolinAlginate oligomers Pectic acidAlteromonas macleodii PectinAspergillus niger/sojae Phytopthora megasperma/cryptogea/sojaeBlue green algae crude Plants gumsBotrytis cinerea PolyaminesCellulase Poly-L-lysineChitosan Pythium aphanidermatumColletotrichum trifolii Rhizoctonia bataticolaCuscuta extract Rhizoctonia solaniDiaporthe phaseolorum Rhizopus arrhizusEicosapentanoic acid Sacharomyces cerevisiaeEnterobacter sakazaki Spodoptera frugiperdaExogenous cork pieces Streptomyces melanosporofaciensFlagellin SyringolideFusarium conglutanis SysteminGlucans Trichoderma virideGlucomannose ulvaGlycoproteins Verticillum dahliaeHarpins Volicitin (N-linolenoyl –L-glutamine)Hemicellulase Yeast elicitorHepta-b –glucosides
337
338
339
340
t1.1
t1.2
t1.3
t1.4
t1.5
t1.6
t1.7
t1.8
t1.9
t1.10
t1.11
t1.12
t1.13
t1.14
t1.15
t1.16
t1.17
t1.18
t1.19
t1.20
t1.21
t1.22
t1.23
t1.24
t1.25
t1.26
t1.27
t1.28
t1.29
t1.30
t1.31
t1.32
t1.33
t1.34
t1.35
t1.36
t1.37
t1.38
t1.39
t1.40
t1.41
t1.42
t1.43
Uncor
recte
d Pro
of
5 Secondary Metabolites and Plant Defence
In order to initiate defence, elicitors must be recognized by plant receptors localized to the plasma membrane or the cytoplasm. Elicitors subsequently or indi-rectly activate the corresponding effectors such as G-proteins, lipases and kinases, which then transduce the elicitor signal to downstream defence responses. The defence reaction involves synthesis of pathogenesis related proteins or defence secondary metabolites [81].
The influx of Ca2+ is a critica1 event in elicitor induced signal transduction that leads to accumulation of plant secondary metabolites. For example, treatment of grapevine cells with various elicitors rapidly triggers Ca2+ influx, alkalinization of extracellular medium, oxidative burst, activation of MAP kinases and protein phosphorylation/dephosphorylation events. These early events are followed by the induction of defence gene expression (including PAL and STS), resulting in the production of resveratrol, piceid and є-viniferins [15, 82, 83].
5.5 In Vitro and In Vivo Studies
5.5.1 Abiotic Elicitors
5.5.1.1 Jasmonic Acid
Jasmonic acid (JA), an oxylipin-like hormone with its more active derivative methyljasmonate (MeJA) is derived from oxidized linolenic acid. Intensive investi-gations by many laboratories about the signal cascade of elicitation process resulted in the identification of JA and its derivatives as important elicitors [84]. It plays a key role in the elicitation of defence signaling pathways involved in resistance to pathogens, especially necrotrophs. JA is also used in a large number of cell and organ culture systems to increase the secondary metabolite yields [85].
Fungal diseases are a major problem in grapevine cultivation around the world. In order to limit these infections some alternative eco-friendly strategies like elicita-tion of plants with elicitors has been adapted. Several studies report the stimulation of stilbene production by exogenous application of MeJA in grapevine cell cultures (Table 5.2). Some in vivo studies demonstrated that MeJA-treated leaves showed increased transcript levels, coding pathogenesis related proteins and coding enzymes involved in phytoalexin biosynthesis (phenylalanine ammonia lyase and stilbene synthase) (Table 5.3). This was correlated with the accumulation of stilbenes (antimicrobial compounds). The eliciting activity of MeJA was confirmed by enhanced tolerance of grapevine foliar cuttings and vineyard against powdery mildew (75% and 73%, respectively) [109]. On the basis of these original results, MeJA could therefore, act as an efficient elicitor in an alternative strategy of grapevine protection.
Similarily, MeJA treated in vitro cultures of plants like Pueraria montana, P. candollei, P. tuberosa, Medicago truncatula accumulated significantly increased
341
342
343
344
345
346
347
348
349
350
351
352
353
354
355
356
357
358
359
360
361
362
363
364
365
366
367
368
369
370
371
372
373
374
375
376
377
378
Uncor
recte
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of
S. Goyal et al.
Tabl
e 5.
2 E
ffec
t of
elic
itors
on
the
stilb
enes
con
tent
of
in-v
itro
gro
wn
plan
ts
S. N
o.Pl
ant s
peci
esC
ultu
re ty
pes
Elic
itors
use
dPr
oduc
tsR
efer
ence
s
1C
ayra
tia
trif
olia
Roo
t cul
ture
sY
east
ext
ract
, sal
icyl
ic a
cid,
met
hyl j
asm
onat
e,
ethr
elPi
ceid
, res
vera
trol
, vi
nife
rin,
am
pelo
psin
[86]
2C
ayra
tia
trif
olia
Cel
l cul
ture
Salic
ylic
aci
d, m
ethy
l jas
mon
ate,
eth
rel a
nd y
east
ex
trac
t, sa
licyl
ic a
cid
and
angi
ospe
rm p
aras
ite
Cus
cuta
Pice
id, r
esve
ratr
ol,
vini
feri
n, a
mpe
lops
in[8
7, 8
8]
3Vi
tis
rupe
stri
s an
d Vi
tis
vini
fera
cvs
In v
itro
pla
nts
UV
irra
diat
ion,
alu
min
um c
hlor
ide,
and
Bot
ryti
s ci
nere
aR
esve
ratr
ol[8
9]
4Vi
tis
spp
Non
-em
bryo
geni
c ca
llus
UV
-C ir
radi
atio
nR
esve
ratr
ols
and
pice
ids
[90]
5Vi
tis
vini
fera
Cel
l cul
ture
sM
ethy
l jas
mon
ate
tran
s-R
esve
ratr
ol a
nd
pice
ids
[85,
91]
6Vi
tis
vini
fera
Cel
l cul
ture
Cyc
lode
xtri
nsR
esve
ratr
ol[9
2] 7
Viti
s vi
nife
raC
ell c
ultu
reC
hito
san
Res
vera
trol
[93,
94]
8Vi
tis
vini
fera
Cel
l cul
ture
Met
hylja
smon
ate,
cyc
lode
xtri
nsR
esve
ratr
ol[8
3, 9
5] 9
Viti
s vi
nife
raC
ell c
ultu
reD
imet
hyl b
-cyc
lode
xtri
nR
esve
ratr
ol[9
6, 9
7]10
Viti
s vi
nife
raC
ell c
ultu
reSa
licyl
ic a
cid,
Na-
orth
ovan
adat
e, ja
smon
ates
, ch
itosa
n an
d th
e m
onom
ers
d-gl
ucos
amin
e
and
N-a
cety
l-d-
gluc
osam
ine,
am
pici
llin
and
rifa
mpi
cin
Res
vera
trol
[98]
11Vi
tis
vini
fera
Cal
lus
cultu
res
UV
irra
diat
ion
tran
s-R
esve
ratr
ol[9
9]12
Vitis
vin
ifera
cv.
Bar
bera
Cel
l cul
ture
Jasm
onic
aci
d, m
ethy
ljasm
onat
e an
d N
a-or
thov
anad
ate
tran
s- a
nd c
is-r
esve
ratr
ol;
tran
s-re
sver
atro
l and
pi
ceid
s
[100
, 101
]
13Vi
tis v
inife
ra c
vs M
iche
le
Palie
ri a
nd R
ed G
lobe
Cel
l cul
ture
sM
ethy
l jas
mon
ate
tran
s-Pi
ceid
and
e-v
inif
erin
[102
]
t2.1
t2.2
t2.3
t2.4
t2.5
t2.6
t2.7
t2.8
t2.9
t2.1
0
t2.1
1
t2.1
2
t2.1
3
t2.1
4
t2.1
5
t2.1
6
t2.1
7
t2.1
8
t2.1
9
t2.2
0
t2.2
1
t2.2
2
t2.2
3
t2.2
4
t2.2
5
t2.2
6
Uncor
recte
d Pro
of
5 Secondary Metabolites and Plant Defence
amount of isoflavones in comparison to control cultures. The isoflavone like daidzein is the precursor to the major phytoalexins including medicarpin which are produced in Medicago and Glycine, respectively [32]. There are reports for the increased production of these metabolites by using MeJa and other biotic elicitors (Table 5.4). There are studies on Medicago truncatula which demonstrated that on infection with fungal pathogen Macrophomina phaseolina, genes involved in flavonoid and isoflavonoid biosynthesis were strongly up-regulated in the shoot. In addition, some genes in jasmonates (JAs) or ethylene (ET) pathways were not strongly induced in infected root tissue. Treating plants with methyl jasmonate (MJ) induced partial resistance in M. truncatula plants [130].
5.5.1.2 Ethephon
Ethephon is an ethylene-releasing compound. It has been known for a long time as an inducer of phenylpropanoid biosynthesis which may be related to a general wound and/or stress response. An ethephon related increase of PAL activity had
Table 5.3 Effect of elicitors on the stilbenes content of in-vivo grown plants
S. No. Plant speciesPlant parts elicited Elicitors used Products References
1 Vitis vinifera Whole plants Laminarin Resveratrol and e-viniferin
[103]
2 Vitis vinifera sylvestris, Vitis vinifera sativa
Postharvest grapes
Ultraviolet C Total stilbenes [104]
3 Vitis vinifera Plants Plasmopara viticola infection, ultraviolet light, and AlCl
3
Pterostilbene [105, 106]
4 Vitis vinifera L. cv. Barbera
Berries Aspergilli japonicus, A. ochraceus, A. fumigatus and isolates of A. carbonarius
trans-Resveratrol [107, 108]
5 Vitis vinifera Plants; berries Methyl jasmonate Stilbenes [109, 110]6 Vitis vinifera Grapevine
detached leaves and grapevine foliar cuttings
Ethephon Resveratrol, piceid, viniferins pterostilbene
[111]
7 Vitis vinifera cv. Barbera
Berries Aspergillus carbonarius
trans-Resveratrol and piceatannol
[112]
8 Vitis vinifera Flowers, berries UV radiation Resveratrol [113]
379
380
381
382
383
384
385
386
387
388
389
390
391
392
t3.1
t3.2
t3.3
t3.4
t3.5
t3.6
t3.7
t3.8
t3.9
t3.10
t3.11
t3.12
t3.13
t3.14
t3.15
t3.16
t3.17
t3.18
t3.19
t3.20
t3.21
t3.22
t3.23
t3.24
t3.25
t3.26
t3.27
t3.28
t3.29
t3.30
Uncor
recte
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of
S. Goyal et al.
Tabl
e 5.
4 E
ffec
t of
elic
itors
on
the
isofl
avon
es c
onte
nt o
f in
-vit
ro g
row
n pl
ants
S. N
o.Pl
ant s
peci
esC
ultu
re ty
pes
Elic
itors
Prod
ucts
Ref
eren
ces
1A
lbiz
zia
kalk
ora
Roo
t cul
ture
sSt
rain
s of
Rhi
zobi
um s
pD
aidz
ein
and
geni
stei
n[1
14]
2C
icer
ari
etin
umC
allu
s/tis
sues
Hyp
nea
mus
cifo
rmis
(re
d al
gae)
Form
onon
etin
, maa
ckia
in, n
arin
gin
and
nari
ngin
mel
onat
e[1
15]
3G
lyci
ne m
axH
airy
roo
tsF
usar
ium
sol
ani
Gen
istin
, dai
dzin
, gly
citin
and
thei
r m
alon
yl c
onju
gate
s an
d ag
lyco
nes,
cou
mes
trol
and
gl
yceo
llin
[116
]
4G
lycy
rrhi
za e
chin
ata
Cel
l cul
ture
sY
east
ext
ract
Form
onon
etin
and
dai
dzei
n[1
17]
5L
upin
us a
lbus
Seed
lings
Puri
fied
yeas
t cel
l wal
lPr
enyl
ated
isofl
avon
e ag
lyco
nes
[118
] 6
Man
ihot
esc
ulen
taC
ell c
ultu
res
Yea
st e
xtra
ctPh
enyl
prop
anoi
ds a
nd s
copo
letin
[119
] 7
Med
icag
o tr
unca
tula
Cel
l cul
ture
sY
east
ext
ract
and
met
hyl j
asm
onat
eFo
rmon
onet
in a
nd b
ioch
anin
-A
med
icar
pin
and
daid
zin
[120
]
8P
hase
olus
vul
gari
sSe
edlin
gsC
uCl 2,
chito
san,
gen
tam
ycin
, sac
char
o-sa
min
e, g
alac
tosa
min
e an
d gl
ucos
amin
ePh
aseo
llin,
cou
mes
trol
, gen
iste
in
and
daid
zein
[121
]
9P
sora
lea
cory
lifo
lia
Hai
ry r
oots
Yea
st e
xtra
ct, c
hito
san,
sal
icyl
ic a
cid
Dai
dzei
n an
d ge
nist
ein
[122
]10
Pso
rale
a co
ryli
foli
aC
ell c
ultu
res
Yea
st e
xtra
ct, c
hito
san,
sal
icyl
ic a
cid
Dai
dzei
n an
d ge
nist
ein
[123
]11
Pue
rari
a ca
ndol
lei v
ar.
cand
olle
i and
P. c
ando
llei
va
r. m
irifi
ca
Cel
l cul
ture
Cop
per
sulf
ate,
met
hyl j
asm
onat
e (M
eJA
),
and
yeas
t ext
ract
, chi
tosa
n, la
min
arin
Isofl
avon
es[1
24]
12P
uera
ria
cand
olle
iH
airy
roo
tsM
ethy
l jas
mon
ate,
chi
tosa
n, s
alic
ylic
aci
d,
Agr
obac
teri
um, a
nd y
east
ext
ract
Isofl
avon
es[1
25]
13P
uera
ria
mon
tana
Hyd
ropo
nica
lly
grow
n se
edlin
gsC
ork
piec
es, X
AD
-4, a
nd m
ethy
l jas
mon
ate
Dai
dzei
n, g
enis
tein
, dai
dzin
, ge
nist
in, a
nd p
uera
rin
[126
]
14P
uera
ria
tube
rosa
Cel
l cul
ture
sY
east
ext
ract
, sal
icyl
ic a
cid,
met
hyl
jasm
onat
e, e
thre
lPu
erai
n, g
enis
tin, d
aidz
ein
and
geni
stin
[127
, 128
]
15Tr
ifol
ium
pra
tens
eSe
edlin
gsC
hito
hexo
se C
oppe
r C
hlor
ide
Form
onon
etin
e-7-
O-g
luco
syl-
6″-
mal
onat
e an
d M
aack
iain
-3-
O-g
luco
syl-
6″-m
alon
ate.
[129
]
t4.1
t4.2
t4.3
t4.4
t4.5
t4.6
t4.7
t4.8
t4.9
t4.1
0
t4.1
1
t4.1
2
t4.1
3
t4.1
4
t4.1
5
t4.1
6
t4.1
7
t4.1
8
t4.1
9
t4.2
0
t4.2
1
t4.2
2
t4.2
3
t4.2
4
t4.2
5
t4.2
6
t4.2
7
t4.2
8
t4.2
9
t4.3
0
Uncor
recte
d Pro
of
5 Secondary Metabolites and Plant Defence
been described long back [131–133]. There are some in vivo and in vitro studies which demonstrate increased stilbene and isoflavone accumulation in the plants by ethephon/ethrel treatment (Tables 5.2–5.4). In a study, Belhadj and co-workers [111] treated grapevine foliar cuttings (Cabernet Sauvignon) with ethylene- releasing ethephon. This resulted in an increase in the number of pathogenesis-related protein gene copies (CHIT4c, PIN, PGIP, and GLU) and in an enhancement of phytoalexin biosynthesis by inducing the PAL and STS genes that correlated with the accumula-tion of stilbenes (antimicrobial compounds). Moreover, ethephon treatment triggered the protection of grapevine detached leaves and grapevine foliar cuttings against Erysiphe necator, the causal agent of powdery mildew (64% and 70%, respectively). These studies emphasize the major role of ethylene in grapevine defence.
Production of isoflavones like puerarin, daidzein, genistin and genistein were also increased in ethephon treated cell cultures of Pueraria tuberosa [128]. Effects of ethephon on isoflavones production in different plants still need attention.
5.5.1.3 UV Light
Resveratrol production and expression of the genes related to resveratrol biosynthesis were investigated in the skins of three Vitis vinifera cultivars. Resveratrol concentra-tion in the skins of all the grapes increased significantly when exposed to ultraviolet (UV-C, 254 nm) irradiation [134]. In another study, it was demonstrated that in V. rupestris UV irradiation induced a high, constant level of STS mRNA production which was correlated to resveratrol accumulation [89]. Another group of workers evaluated grape flowers and green berries of 72 grape genotypes for their ability to produce resveratrol in response to UV radiation. This was used to establish a selection criterion for screening genotypes for resistance to gray mold and powdery mildew. There was a strong negative correlation between UV-induced resveratrol production and susceptibility to Botrytis infection [135]. Callus culture of V. vinifera were exposed to 254 nm UV light. About 15 min of UV irradiation period was found to be effective for induction of (62 mg/g callus fresh weight) trans-resveratrol produc-tion [99]. Thus, UV light can be used as an efficient elicitation source for stilbene production (Tables 5.2 and 5.3).
5.5.1.4 Cyclodextrins
Cyclodextrins (CD) are cyclic oligosaccharides of 6,7 or 8 a –d- glucopyranoside residues linked by a 1→4 glucosidic bonds, which are called a-, b-, and g- CD. They have an hydrophilic external surface and hydrophobic central cavity that can trap a polar compounds [136]. The production of cyclodextrins is relatively simple and involves treatment of ordinary starch with a set of easily available enzymes [137]. Dimethyl -b-cyclodextrin (DIMEB), an oligosaccharide consisting of 2,6-methylated cyclic a (1→4)- linked glucopyranose moieties, has shown to be capable of inducing stilbene biosynthesis in liquid Vitis vinifera cell cultures, also
393
394
395
396
397
398
399
400
401
402
403
404
405
406
407
408
409
410
411
412
413
414
415
416
417
418
419
420
421
422
423
424
425
426
427
428
429
430
431
Uncor
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d Pro
of
S. Goyal et al.
in the absence of pathogenic organisms [96] (Table 5.2). This molecule seems to mimic a defence elicitor which enhances the physical barriers of the cell, stops cell division and induces phytoalexin synthesis [97].
A grapevine liquid cell culture system was used to examine the properties of CDs as inducers of defence responses. This work shows that the chemically pure heptakis (2,6-di-O-methyl)-b CD caused a dramatic extracellular accumulation of the phytoalexin resveratrol and changes in peroxidase activity and isoenzymatic pattern. Other modified CDs tested on several grapevine cell lines resulted in different eliciting capacities of CDs and different sensibilities of the cell lines. The spent medium of elicited cultures containing polyphenolic compounds released by plant cells was shown to disturb Botrytis cinerea growth in a plate assay [92].
5.5.1.5 Phosphites
Phosphite is a neutralized solution of the phosphonate anion [138]. Phosphite contains one less oxygen (O) than phosphate, making its chemistry and behavior quite different. Phosphite is less chemically stable and more soluble than phosphate, when applied to plants, it is quickly absorbed by leaves, roots and branches, thus high concentrations can be toxic for plants. It is able to move in both, xylem and phloem. Unlike phosphates, phosphites stimulate the pathogen defence mechanisms in plants and have antifungal activity by inducing production of phytoalexins. It is effective against fungi like Plasmopara viticola, Pythium sp. and Phytophthora nicotianae. The relatively limited fungicidal effect – combined with its ability to stimulate plants to make a broad spectrum of biologically active metabolites – makes phosphite relatively benign to the environment and safe to use [139]. Now-a- days there are many reports on phosphite induced cellular responses to pathogen challenge and suppressed pathogen ingress in both in vitro and in vivo cultures [140, 141]. Different mechanism of phosphite actions were postulated by Grant and co-workers [142]. With the indirect actions, phosphite is hypothesized to cause the pathogen to produce elicitors or inhibit its production of suppressors, allowing plant defence responses to halt invasion by the pathogen [143].
5.5.1.6 Pulsed Electric Field
Pulsed electric field (PEF) an external stimulus or stress, is proposed as a promising new abiotic elicitor for stimulating secondary metabolite biosynthesis in plant cell cultures. The effects of PEF on growth and secondary metabolite production by plant cell culture were investigated by using suspension cultures of Taxus chinensis as a model system. A significant increase in intracellular accumulation of taxuyun-nanine C (Tc), a bioactive secondary metabolite, was observed by exposing the cells in the early exponential growth phase to a 30-min PEF [144]. The effects of PEF and ethephon on growth and secondary metabolites accumulation were also investi-gated in suspension culture of Vitis vinifera L. cv. Gamay Fréaux as a model system.
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After the treatments production levels of extracellular phenolic acids, 3-O-glucosyl-resveratrol was increased in the cultures [145]. In another recent study, isoflavones production in the Glycine cell culture increased with pulsed electric field (PEF) application at 1.6 kV, and aglycone forms were influenced to a greater extent [146]. These results show that PEF induced a defence response of plant cells and may have altered the cell/membrane’s dielectric properties.
5.5.2 Biotic Elicitors
5.5.2.1 Chitosan
Chitosan is a polysaccharide called poly [b-(1→ 4)-2-amino-2-deoxy -d-glucopyranose]. It is a plant defence booster derived from deactylation of chitin which is extracted from the exoskeleton of crustaceans such as shrimps and crabs, as well as from the cell walls of some fungi [147, 148]. The primary unit in the chitin polymer is poly [b-(1→4)-2-acetamido-2-deoxy -d-glucopyranose]. The units are combined by 1,4 glycosidic linkages, forming a long chain linear polymer. Removal of most of the acetyl groups of chitin by treatment with strong alkalis yields chitosan [149]. Agricultural applications of chitosan are for stimulation of plant defence. The chitosan molecule triggers a defence response within the plant, leading to the formation of physical and chemical barriers against invading pathogens [150].
Chitosan conferred a high protection of grapevine leaves against grey mould caused by Botrytis cinerea. Treatment of grapevine leaves by chitosan led to marked induction of lipoxygenase (LOX), phenylalanine ammonia-lyase (PAL) and chitinase activities, three markers of plant defence responses. Strong reduction of B. cinerea infection were achieved with 75–150 mg/l chitosan [151]. In some studies it was observed that grapevines with higher assays of chitinase or ß-1,3-glucanase had greater resistance to powdery mildew, and when combined had even greater field resistance against powdery mildew (Uncinula necator) [152].
In an investigation, chitosan increased the amounts of genistein and 2- hydroxygenistein monoprenyls in roots of white lupin and in the exudates [153]. Further studies indicated that chitosan triggers either the de-novo synthesis of phe-nolic compounds as the first defensive line designed to inhibit growth of the fungus and b-1,3-glucans act as a second mechanical barrier for blocking potential invasion by fungal cells and protecting the tissue against phytotoxic substances [154, 155]. Chitosan coating of litchi fruits increased their content of flavonoids and resistance to browning and postharvest decay [156]. Chitosan has also been used in cell cul-tures of Vitis vinifera and Psoralea corylifolia, Pueraria candollei to enhance the stilbene and isoflavone production, respectively (Tables 5.2 and 5.4). Besides its use in in vitro studies, chitosan is also used in in vivo studies to increase the isoflavones production (Table 5.5). Chitosan is a nontoxic biodegradable material, acting as an elicitor. Thus, has the potential to become a new class of plant protecting agent, assisting towards the goal of sustainable agriculture.
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Tabl
e 5.
5 E
ffec
t of
elic
itors
on
the
isofl
avon
es c
onte
nt o
f in
-viv
o gr
own
plan
ts
S. N
o.Pl
ant s
peci
esPl
ant p
art e
licite
dE
licito
rPr
oduc
tsR
efer
ence
s
1C
icer
ari
etin
umSe
edlin
gsR
educ
ed g
luta
thio
neB
ioch
anin
A, f
orm
onon
etin
and
m
edic
arpi
n an
d m
aack
iain
, ho
mof
erre
irin
and
cic
erin
Arm
ero
et a
l. [1
57].
2G
lyci
ne m
axSe
eds
Lip
o-ch
itool
igos
acch
arid
es,
chito
san,
Str
epto
myc
es
mel
anos
poro
faci
ens
stra
in
EF-
76 a
nd y
east
ext
ract
Dai
dzei
n, g
enis
tein
, gly
cite
inA
l-Ta
wah
a et
al.
[158
].
3G
lyci
ne m
axSe
eds
and
seed
lings
Asp
ergi
llus
soja
e ce
ll w
all e
xtra
ctG
lyce
ollin
sB
oué
et a
l. [1
59].
4G
lyci
ne m
axC
otyl
edon
sD
iapo
rthe
pha
seol
orum
f. s
p.
mer
idio
nali
sD
aidz
ein,
gen
iste
in a
nd
glyc
eolli
ns, a
pige
nin
Mod
olo
et a
l. [1
60].
5G
lyci
ne m
axSe
eds
b-G
luca
n fr
om P
hyto
phth
ora
soja
eD
aidz
ein,
cou
mes
trol
, gen
iste
in,
lute
olin
and
api
geni
nK
retz
schm
ar e
t al.
[161
]
6G
lyci
ne m
axC
otyl
edon
tiss
ueb-
Glu
can
from
Phy
toph
thor
a so
jae
Gly
ceol
linA
bbas
i and
Gra
ham
[16
2] 7
Lot
us ja
poni
cus
Seed
lings
Red
uced
glu
tath
ione
Ves
titol
Shim
ada
et a
l. [1
63]
8L
upin
us a
ngus
tifo
lius
Seed
lings
Ple
ioch
aeta
set
osa
Gen
iste
in, 2
prim
e-hy
drox
ygen
iste
inB
edna
rek
et a
l. [1
64]
9M
edic
ago
sati
vaPl
antle
tsC
olle
totr
ichu
m tr
ifol
iiM
edic
arpi
nSa
lles
et a
l. [1
65]
10M
edic
ago
trun
catu
laPl
antle
tsP
hom
a m
edic
agin
isFo
rmon
onet
in 7
-O-g
luco
side
and
m
alon
ylat
ed f
orm
onon
etin
7-
O-g
luco
sid
Jasi
ński
et a
l. [1
66]
11Tr
ifol
ium
pra
tens
ePl
antle
tsY
east
ext
ract
and
chi
tosa
nG
enis
tein
, dai
dzei
n, f
orm
onon
etin
an
d bi
ocha
nin
ASi
vesi
nd a
nd S
egui
n [1
67]
t5.1
t5.2
t5.3
t5.4
t5.5
t5.6
t5.7
t5.8
t5.9
t5.1
0
t5.1
1
t5.1
2
t5.1
3
t5.1
4
t5.1
5
t5.1
6
t5.1
7
t5.1
8
t5.1
9
t5.2
0
t5.2
1
t5.2
2
t5.2
3
t5.2
4
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recte
d Pro
of
5 Secondary Metabolites and Plant Defence
5.5.2.2 Laminarin
The molecule laminarin (also known as laminaran) is a linear glucan found in brown algae. Laminarin is clearly a high-energy carbohydrate. It is used as a food reserve in the same way that chrysolaminarin is used by phytoplankton. It is made up of b(1→3)-glucan with b(1→6)-linkages and a b(1→3):b(1→6) ratio of 3:1 [168]. In a study, b-1,3-glucan laminarin derived from the brown algae Laminaria digitata was shown to be an efficient elicitor of defence responses in grapevine cells and plants. It also effectively reduced B. cinerea and P. viticola development on infected grapevine plants. Defence reactions elicited by laminarin in grapevine cells include calcium influx, alkalinization of the extracellular medium, an oxidative burst, acti-vation of two mitogen-activated protein kinases, expression of 10 defence-related genes with different kinetics and intensities, increases in chitinase and b-1,3-gluca-nase activities, and the production of two phytoalexins (resveratrol and e-viniferin). When applied to grapevine plants, laminarin reduced infection of B. cinerea and P. viticola by approximately 55% and 75%, respectively [103]. In another study, laminarin increased the isoflavones accumulation in cell cultures of Pueraria candollei (Table 5.4).
5.5.2.3 Yeast Extract
Yeast extract is the common name for various forms of processed yeast products made by extracting the yeast cell contents (removing the cell walls). Yeast has been proved to be an efficient elicitor for the increased accumulation of isoflavone and stilbene in different plants (Tables 5.2, 5.4, and 5.5). Yeast extract-treated suspension cultures of a new cell line, AK-1, of Glycyrrhiza echinata were induced to produce an isoflavonoid phytoalexin (medicarpin). From these cells, putative full-length cDNAs encoding cytochrome P450s, (2S)-flavanone 2-hydroxylase and isoflavone 2¢-hydroxylase, were cloned [169]. A cDNA encoding UDP-glucose: formononetin 7-O-glucosyltransferase, designated UGT73F1, was cloned from yeast extract-treated Glycyrrhiza echinata L. cell-suspension cultures. Recombinant UGT73F1 was expressed as a histidine-tag fusion protein in Escherichia coli. The purified recombinant enzyme was selective for isoflavone, formononetin and daidzein as substrates [117]. Besides this, there are various reports where yeast extract was the most efficient elicitor in comparison to other elicitors [125].
5.6 Conclusions
The application of biotic and abiotic elicitors in developing plant resistance is still in the early stages of use. Currently, our knowledge is mainly based on the experi-mental trials. There are many reports which prove there efficacy as potent natural pesticides. It is well established that these elicitors impart disease resistance by
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elevating or developing some secondary metabolites in the plants. Effect of several elicitors on the production of stilbenes in different Vitis species and other stilbenes containing plants has been summarized in Tables 5.2 and 5.3. Similarly, the recent studies on the effect of different biotic and abiotic elicitors on the isoflavones production have been summarized in Tables 5.4 and 5.5.
Numbers of studies, especially concerning grapevine inoculated with various pathogens, have established a positive correlation between stilbene levels and pathogen resistance. More evidence supporting the role of stilbene (resveratrol) in resistance to pathogen infection was supplied by the transfer of stilbene synthase genes in plants that do not produce stilbenes, such as tobacco and alfalfa [65, 67].
Isoflavones function in both, the symbiotic relationship with rhizobial bacteria and the plant defence response. The importance of isoflavones can be judged by different reports where in order to increase the disease resistance in plant, produc-tion of certain type of isoflavones was enhanced by genetic manipulations [170]. The non-legume plants like Arabidopsis, Nicotiana tabacum and Zea mays (for human consumption) were metabolic engineered for isoflavones production. Due to complexities in regulation of inter-related biochemical pathways, metabolic engineering to affect the isoflavones biosynthetic capacity of a target plant tissue, presents a challenge [171]. However, by using elicitors this process can be simplified and can become more practically feasible in fields.
Use of elicitors has an added advantage over the chemicals used to prevent plants from diseases. There are increasing evidences, that elicitors could be used in the future as alternatives to traditional pesticides for managing pathogens and pests in agriculture and nursery production of forest trees. However, inappropriate use of elicitor treatments can change the chemical composition of the treated plant material. Therefore, with agricultural crops, suitable concentration of elicitors should be use which may not affect human health.
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Author QueriesChapter No.: 5 0001210227
Queries Details Required Author’s Response
AU1 Kindly confirm the corresponding author and provide e-mail id.
AU2 Kindly provide page numbers for the reference “Ramanujan (2008)”.
AU3 Please provide volume no. and page range for Cheng et al. (2011).
AU4 Please update the reference “Tamm et al. (2011)”.