**1. Introduction**

362 Lipid Metabolism

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Plant disease resistance can be defined as the ability of the plant to prevent or restrict pathogen growth and multiplication. All plants, whether they are resistant or susceptible, respond to pathogen attack by the induction of a coordinated resistance strategy. Acceleration and/or amplification of the plant responses by the application of resistance inducers could provide a biologically, environmentally and commercially viable alternative to existing pathogen control methods [1].

Among pathogenic fungi, the obligate parasite *Blumeria graminis* f. sp. *tritici* (*Bgt*) is responsible for wheat (*Triticum aestivum*) powdery mildew, one of the most damaging foliar diseases of this crop, especially in Northern Europe. Worldwide yield losses due to wheat powdery mildew would be about 30% without chemical treatments, so that an extensive use of conventional fungicides is undertaken. Moreover, populations of *Bgt* resistant to the main chemical fungicides (ergosterol biosynthesis inhibitors, EBIs and 2-aminopyridines) are rising, and these resistant strains emerged all over most European territories [2]. New disease management strategies based on the use of molecules that induce plant resistance *via* the elicitation of defence responses are therefore developed in order to reduce the use of conventional fungicides. These strategies match the growing concern about the consequences of the use of fungicides on both health and environment [3,4].

Induced partial resistance against *B. graminis* f.sp. *tritici* has been obtained in wheat with different elicitors and resistance inducers. Infection level was reduced to 57% and 58% relative to controls when nonacetylated and acetylated oligogalacturonides, respectively, were sprayed on wheat 48h before inoculation with *Bgt* [5]. Trehalose, a non-reducing disaccharide found in a wide variety of organisms, confers a 60% protection level against

<sup>© 2013</sup> Reignault et al., licensee InTech. This is an open access chapter distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

powdery mildew [6]. It has also been shown that a double spraying of wheat plantlets with salicylic acid (SA) confers a 65% protection level against powdery mildew [7]. Prophylactic efficacies of Iodus 40® and heptanoyl salicylic acid (HSA) against wheat powdery mildew have been tested [8]. Iodus 40®, a commercial product, is used to decrease wheat powdery mildew damage in the field. Its active ingredient is laminarin, a storage β-1,3-D-glucan (polysaccharide), extracted from the brown alga *Laminaria digitata*. It induces protection in grapevine against *Botrytis cinerea* and *Plasmopara viticola* [9] as well as in wheat against powdery mildew [8]. HSA is synthesized by esterification of 2-OH benzoic acid by heptanoic acid [7]. Plantlets treated twice exhibited 60% and 100% protection levels, respectively [8]. A long up-to-run-off spraying of wheat leaves with Milsana®, an ethanolic extract from leaves of the giant knotweed *Reynoutria sachaliensis*, 48h before inoculation led to a 97% protection level against powdery mildew [10]. No direct effect against the fungus has been noticed for any of these elicitors [8] except for Milsana® which exhibited a direct fungistatic effect on *B. graminis* conidia germination [10]. It is now necessary to understand the mode of action and the cascade of cellular and molecular events triggered by these wheat resistance inducers.

Lipids as Markers of Induced Resistance in Wheat: A Biochemical and Molecular Approach 365

**Figure 1.** Formation and attenuation of phosphatidic acid (PA) [16]

**Figure 2.** Lipid messengers derived from hydrolysis of plasma membrane [22]

In the last fifteen years, SA itself has been described as playing a key role in the activation of defence systems against pathogens in plants. Despite several reports [11-14], works focusing on SA as a resistance inducer are far from being as extensive as those concerning BTH, a functional analogue of SA, and, as far as we know, a single one involved wheat [8].

Plant lipids and lipid metabolic pathways have been shown to be of crucial importance during a plant-pathogen interaction. Many changes in membrane lipids are known to occur in plants at the site of infection. Moreover, lipids and lipid metabolites, released from membranes, function and act as signal molecules in the activation of plant defence responses [15].

Over the past few years, it has become increasingly clear that phosphatidic acid (PA) is involved in stress signaling because it is rapidly and transiently formed in response to various environmental stimuli [16]. PA could be generated by 2 distinct pathways as shown in figure 1: a first one involves phospholipase D (PLD) acting hydrolytically on membrane phospholipids, particularly phosphatidylcholine (PC) and phosphatidylethanolamine (PE); a second one involves phospholipase C (PLC) acting sequentially with diacylglycerol kinase (DGK) *via* diacylglycerol (DAG) phosphorylation [17].

Phospholipid-signaling pathways are complex, interrelated, and involve numerous enzymes and substrates [18]. As an ubiquitous enzyme family, phospholipases play various roles in stress responses [19]. Beside PLC and PLD, a main class of phospholipases A (PLA) hydrolyze phospholipids (such as PC) into the corresponding free fatty acid and lysophospholipid (such as lysoPC). Such a fatty acid can be a precursor for oxylipin biosynthesis, and lysoPC may be involved in multiple cellular processes [20]. One important finding on functions of lysoPC is that it can activate H+-ATPase in the tonoplast and cause cytoplasmic acidification, which is shown to activate defense responses and phytoalexin production [21]. The lipid messengers derived from hydrolysis of the plasma membrane are illustrated in figure 2.

**Figure 1.** Formation and attenuation of phosphatidic acid (PA) [16]

364 Lipid Metabolism

wheat resistance inducers.

illustrated in figure 2.

powdery mildew [6]. It has also been shown that a double spraying of wheat plantlets with salicylic acid (SA) confers a 65% protection level against powdery mildew [7]. Prophylactic efficacies of Iodus 40® and heptanoyl salicylic acid (HSA) against wheat powdery mildew have been tested [8]. Iodus 40®, a commercial product, is used to decrease wheat powdery mildew damage in the field. Its active ingredient is laminarin, a storage β-1,3-D-glucan (polysaccharide), extracted from the brown alga *Laminaria digitata*. It induces protection in grapevine against *Botrytis cinerea* and *Plasmopara viticola* [9] as well as in wheat against powdery mildew [8]. HSA is synthesized by esterification of 2-OH benzoic acid by heptanoic acid [7]. Plantlets treated twice exhibited 60% and 100% protection levels, respectively [8]. A long up-to-run-off spraying of wheat leaves with Milsana®, an ethanolic extract from leaves of the giant knotweed *Reynoutria sachaliensis*, 48h before inoculation led to a 97% protection level against powdery mildew [10]. No direct effect against the fungus has been noticed for any of these elicitors [8] except for Milsana® which exhibited a direct fungistatic effect on *B. graminis* conidia germination [10]. It is now necessary to understand the mode of action and the cascade of cellular and molecular events triggered by these

In the last fifteen years, SA itself has been described as playing a key role in the activation of defence systems against pathogens in plants. Despite several reports [11-14], works focusing on SA as a resistance inducer are far from being as extensive as those concerning BTH, a

Plant lipids and lipid metabolic pathways have been shown to be of crucial importance during a plant-pathogen interaction. Many changes in membrane lipids are known to occur in plants at the site of infection. Moreover, lipids and lipid metabolites, released from membranes,

Over the past few years, it has become increasingly clear that phosphatidic acid (PA) is involved in stress signaling because it is rapidly and transiently formed in response to various environmental stimuli [16]. PA could be generated by 2 distinct pathways as shown in figure 1: a first one involves phospholipase D (PLD) acting hydrolytically on membrane phospholipids, particularly phosphatidylcholine (PC) and phosphatidylethanolamine (PE); a second one involves phospholipase C (PLC) acting sequentially with diacylglycerol kinase

Phospholipid-signaling pathways are complex, interrelated, and involve numerous enzymes and substrates [18]. As an ubiquitous enzyme family, phospholipases play various roles in stress responses [19]. Beside PLC and PLD, a main class of phospholipases A (PLA) hydrolyze phospholipids (such as PC) into the corresponding free fatty acid and lysophospholipid (such as lysoPC). Such a fatty acid can be a precursor for oxylipin biosynthesis, and lysoPC may be involved in multiple cellular processes [20]. One important finding on functions of lysoPC is that it can activate H+-ATPase in the tonoplast and cause cytoplasmic acidification, which is shown to activate defense responses and phytoalexin production [21]. The lipid messengers derived from hydrolysis of the plasma membrane are

functional analogue of SA, and, as far as we know, a single one involved wheat [8].

function and act as signal molecules in the activation of plant defence responses [15].

(DGK) *via* diacylglycerol (DAG) phosphorylation [17].

**Figure 2.** Lipid messengers derived from hydrolysis of plasma membrane [22]

Adaptation of higher plants to biotic and abiotic stress is often accompanied by the occurrence of lipid peroxidation and metabolites which derived therefrom are called oxylipins. Lipid peroxydation may be the result of a coordinated action of enzymes or the result of auto-oxidation (Figure 3). Oxylipins are potent signaling molecules in the defense response in plants [23]. The synthesis of oxylipins is first catalyzed by lipoxygenases (LOXs), which add molecular oxygen to polyunsaturated fatty acids (PUFAs) to yield the corresponding fatty acid hydroperoxides that are substrates for other enzymes (figure 4) [24]. Based on their regiospecificity, the dioxygenation occurs at C-9 or C-13 and LOXs have been thus classified as 9- and 13-LOX, which yield 9- or 13-hydroperoxides, respectively [25]. In the case of linolenic acid C18:3 and 13-LOX, the resulting product is 13-HPOT (hydroperoxy octadectrienoic acid) [15]. These LOX-derived hydroperoxides can be converted through different reactions of the LOX pathway, particularly by an allene oxide synthase (AOS) leading to jasmonic acid (JA). Most of the LOX-derived compounds are considered as acting in plant defense reactions: indeed, C6 volatiles induce defense-related genes expression [26], divinyl ethers are antifungal [27], and JA is an important signaling compound that is involved in plant response to biotic stress [28,29]. Jasmonates are primarily derived from the C18:3 FA, which is released from membrane lipids via the activity of phospholipase A1.

Lipids as Markers of Induced Resistance in Wheat: A Biochemical and Molecular Approach 367

Their expression is induced by microbial elicitors and upon exposure to pathogen. The high expression level of these PLA genes precedes the accumulation of JA in pathogen-inoculated or elicitor-treated tissues. Activation of PLA has also been reported in response to TMV

**Figure 4.** Enzymatic and non-enzymatic mechanisms leading to the synthesis of oxylipins in plants [15]

FAs not only serve as the major source of reserve energy but also consist of complex lipids, which are essential components of cellular membrane lipids. Increasing evidence also shows the involvement of FAs and their derivatives in signaling and altering normal and diseaserelated physiologies in microbes, insects, animals, and plants. In plants, FAs modulate a variety of responses to biotic and abiotic stresses. For instance, PUFAs levels in chloroplastic membranes affect membrane lipid fluidity and determine the plant's ability to acclimatize to temperature stress [34]. Linolenic acid (18:3) is involved in protein modifications in heatstressed plants [35]. FAs also regulate salt, drought, and heavy metal tolerance as well as wound-induced responses and defense against insect and herbivore feeding in plants [36]. FA metabolic pathways play significant roles in defense against pathogens. Classically, only passive roles were assigned to FAs in plant defense such as providing biosynthetic precursors for cuticular components (studies of FA metabolic mutants also reveal an active signaling role for the cuticle in plant defense) or JA, well known for its role in wound responses and plant defense against insect pathogens. However, recent works demonstrate more direct roles for FAs and their breakdown products in inducing various modes of plant defenses. Both 16- and 18-carbon FAs participate in defense to modulate basal, effector-

Furthermore, lipid transfer proteins (LTPs), located in the cell wall, participate in the *in vitro* transfer of phospholipids between membranes and can bind acyl chains. Based on these

triggered, and systemic immunity in plants [37].

infection in tobacco [32] and elicitor treatment of cultured parsley cells [33].

**Figure 3.** Schematic illustration of biosynthetic pathway of JA and other related oxylipins [22]

The phospholipase A (PLA) superfamily which catalyzes the hydrolysis of membrane phospholipids, acts up-stream the LOX to generate the corresponding PUFAs and lysophospholipids [30]. PLA may be involved in the release of free fatty acids for the biosynthesis of JA during the activation of plant defence responses. Indeed, three tobacco genes that encode putative members of the patatin family of PLAs, were identified [31]. Their expression is induced by microbial elicitors and upon exposure to pathogen. The high expression level of these PLA genes precedes the accumulation of JA in pathogen-inoculated or elicitor-treated tissues. Activation of PLA has also been reported in response to TMV infection in tobacco [32] and elicitor treatment of cultured parsley cells [33].

366 Lipid Metabolism

activity of phospholipase A1.

Adaptation of higher plants to biotic and abiotic stress is often accompanied by the occurrence of lipid peroxidation and metabolites which derived therefrom are called oxylipins. Lipid peroxydation may be the result of a coordinated action of enzymes or the result of auto-oxidation (Figure 3). Oxylipins are potent signaling molecules in the defense response in plants [23]. The synthesis of oxylipins is first catalyzed by lipoxygenases (LOXs), which add molecular oxygen to polyunsaturated fatty acids (PUFAs) to yield the corresponding fatty acid hydroperoxides that are substrates for other enzymes (figure 4) [24]. Based on their regiospecificity, the dioxygenation occurs at C-9 or C-13 and LOXs have been thus classified as 9- and 13-LOX, which yield 9- or 13-hydroperoxides, respectively [25]. In the case of linolenic acid C18:3 and 13-LOX, the resulting product is 13-HPOT (hydroperoxy octadectrienoic acid) [15]. These LOX-derived hydroperoxides can be converted through different reactions of the LOX pathway, particularly by an allene oxide synthase (AOS) leading to jasmonic acid (JA). Most of the LOX-derived compounds are considered as acting in plant defense reactions: indeed, C6 volatiles induce defense-related genes expression [26], divinyl ethers are antifungal [27], and JA is an important signaling compound that is involved in plant response to biotic stress [28,29]. Jasmonates are primarily derived from the C18:3 FA, which is released from membrane lipids via the

**Figure 3.** Schematic illustration of biosynthetic pathway of JA and other related oxylipins [22]

The phospholipase A (PLA) superfamily which catalyzes the hydrolysis of membrane phospholipids, acts up-stream the LOX to generate the corresponding PUFAs and lysophospholipids [30]. PLA may be involved in the release of free fatty acids for the biosynthesis of JA during the activation of plant defence responses. Indeed, three tobacco genes that encode putative members of the patatin family of PLAs, were identified [31].

**Figure 4.** Enzymatic and non-enzymatic mechanisms leading to the synthesis of oxylipins in plants [15]

FAs not only serve as the major source of reserve energy but also consist of complex lipids, which are essential components of cellular membrane lipids. Increasing evidence also shows the involvement of FAs and their derivatives in signaling and altering normal and diseaserelated physiologies in microbes, insects, animals, and plants. In plants, FAs modulate a variety of responses to biotic and abiotic stresses. For instance, PUFAs levels in chloroplastic membranes affect membrane lipid fluidity and determine the plant's ability to acclimatize to temperature stress [34]. Linolenic acid (18:3) is involved in protein modifications in heatstressed plants [35]. FAs also regulate salt, drought, and heavy metal tolerance as well as wound-induced responses and defense against insect and herbivore feeding in plants [36]. FA metabolic pathways play significant roles in defense against pathogens. Classically, only passive roles were assigned to FAs in plant defense such as providing biosynthetic precursors for cuticular components (studies of FA metabolic mutants also reveal an active signaling role for the cuticle in plant defense) or JA, well known for its role in wound responses and plant defense against insect pathogens. However, recent works demonstrate more direct roles for FAs and their breakdown products in inducing various modes of plant defenses. Both 16- and 18-carbon FAs participate in defense to modulate basal, effectortriggered, and systemic immunity in plants [37].

Furthermore, lipid transfer proteins (LTPs), located in the cell wall, participate in the *in vitro* transfer of phospholipids between membranes and can bind acyl chains. Based on these properties, LTPs are thought to be involved in membrane biogenesis and regulation of intracellular FA pools [38]. Many roles were suggested for LTPs: involvement in cutin formation, embryogenesis, symbiosis and adaptation of plants to various environmental conditions [39]. Among them, defensive role of LTPs has been proposed. Indeed, LTPs have been naturally classified as members of pathogenesis-related (PR) proteins belonging to the group PR-14 [40]. Some members of this family have the ability to inhibit the growth of fungal pathogens in barley and maize [41], in sunflower against *Fusarium solani* [42], in transgenic rice against *Magnaporthe grisea*, *Rhizoctonia solani* and *Xanthonomas oryzae* [43]. In transgenic wheat expressing *Ace-AMP,* the corresponding encoded LTP showed enhanced antifungal activity against *Bgt* [44]. *Ltp3F1,* a novel gene encoding an antifungal protein against *Alternaria sp*., *Curcularia lunata*, *Bipolaris oryzae* and *Sarocladium oryzae* was characterized from wheat [45].

Lipids as Markers of Induced Resistance in Wheat: A Biochemical and Molecular Approach 369

Wheat (*Triticum aestivum*) cultivar Orvantis was used throughout the experiments. It was provided by Benoit C.C. (Orgerus, France). This cultivar is fully susceptible to the MPEBgt1 powdery mildew isolate. First leaf of ten-day-old wheat plantlets was infiltrated with salicylic acid (1g/L) solution using a hypodermic syringe without needle. Infiltrated area was delineated with a marker pen. Control plantlets were infiltrated with distillated

Ten-day-old wheat seedlings were treated with solutions of Iodus 40® (1g/L), HSA (1g/L), Milsana® (0.3% v/v) and trehalose (15g/L) as described by Renard-Merlier *et al*. [46]. Treatments consisted in "up-to-run-off" sprayings. Two days after inducer treatments, seedlings to be inoculated were sprayed with conidia of *Bgt* suspended in Fluorinert FC43 at

**2.2. RNA extraction and quantification of gene expression by real-time PCR** 

repeated twice with similar results and representative results are presented.

The results are the mean of three biological repetitions.

**2.4. Fatty acid extraction and analysis** 

SA and water-infiltrated wheat leaves were sampled at 3, 6, 9, 12, 15, 18, 21, 24, 48, 72 and 96 hours after infiltration (hai) and stored at -80oC until use. Total RNA was extracted from 100 mg plant tissue using RNeasy Plant Mini Kit (Quiagen, The Netherlands) with some modifications of the protocol. cDNA synthesis was carried out using High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, USA) according to the manufacturer's protocol. Real Time qPCR was performed using ABI Prism 7300 detection system (Applied Biosystems, USA). The *tub* and *ef1α* genes, encoding respectively for tubulin and elongation factor ef1alpha, were used as reference genes. The relative expression of the target genes was evaluated in SA-infiltrated wheat leaves compared with water-infiltrated leaves and normalized to the *tub* and *ef1a* expression level. The analyses were performed using the relative expression software tool REST® as described in [47]. The experiments were

LOX was assayed as described in [10] according to [48] and [49] with slight modifications.

Total cellular FAs extraction and purification were performed by the authors in [46] using

Free FAs, PLFA and PL extraction was carried out according to the method described in [51]. Data shown are the results of the first experiment, which need to be confirmed by a

adapted protocols from [50]. The results are means of three independent repetitions.

**2. Material and methods** 

**2.1. Treatments application** 

a concentration of 5.106 spores.mL-1.

water.

**2.3. LOX assay** 

biological repetition.

In this review, we will discuss further and extend the study conducted by Renard-Merlier *et al*. [46], where a global investigation of total FA content in relation to treatment with four inducers of resistance and to powdery mildew infection was undertaken. Previous studies established that lipid metabolism is altered by Milsana®, Iodus 40®, HSA, SA and trehalose [8,10]; therefore, our work aimed to characterize their impact at the total FA level. During a time course experiment, content (quantitative analysis) and percentage (qualitative analysis) of FAs were compared in treated plants and in controls, as well as in non-inoculated (ni) plants and *Bgt*-challenged plants (i). Previous results will be considered and discussed relatively to new findings.

Moreover, the effect of one resistance inducer, namely SA, on lipid metabolism is evaluated by molecular and biochemical approaches.

Phospholipids being the major membrane components, we investigated PC, PE, DAG and PA content variation in wheat leaves infiltrated with salicylic acid (SA). SA can modulate the content variation of these compounds, reservoirs from which biologically active lipids and precursors of oxidized lipids are released.

At the transcriptional level, a PLC-encoding gene expression was investigated in an attempt to assign any participation of this pathway in the phospholipids equilibrium described above.

We also investigated free FAs and PLFAs content variations in SA-infiltrated wheat leaves; this pool of lipids is quite interesting since it ensures several functions, from being an energy source to acting as cellular messengers; the latter being highly related to resistance induction in plants. The lipoxygenase response to SA-infiltration, at the molecular and enzymatic level, was also evaluated; this enzyme activity is important for oxylipins biosynthesis in plants, because of its position upstream the cascade of enzymatic lipid peroxydation.

An LTP-encoding gene expression was also monitored, taking into account the possible antifungal activity of LTPs as well as their ability to bind and transport membrane lipids, thus participating in lipid-mediated signaling mechanisms.
