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Keywords:

  • Blumeria graminis;
  • oxidative burst;
  • priming;
  • sulphated polysaccharide;
  • Ulva fasciata;
  • ulvan

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

The capacity of ulvan, a sulphated heteropolysaccharide, to prime the chitin- and chitosan-elicited oxidative burst in wheat and rice cells was tested. Gas-liquid chromatography showed that ulvan was composed of rhamnose, xylose, glucuronic acid, glucose and galactose. It contained very low amounts of proteins and ca. 19% sulphate groups. The polymer did not elicit the production of hydrogen peroxide in suspension-cultured wheat or rice cells. Furthermore, in both cell cultures, the simultaneous addition of ulvan and chitin hexamer or chitosan polymer did not significantly alter the intensity of the oxidative burst caused by the elicitors alone. However, pretreatment of wheat cells with ulvan increased the chitin-elicited oxidative burst about five- to sixfold, and that elicited by chitosan about twofold. In rice cells, the production of H2O2 elicited by chitin or chitosan was strongly primed by pretreatment with the same concentration of ulvan, increasing the burst triggered by the elicitors alone by 150 and 80 times, respectively. Pretreatment of whole plants with ulvan significantly reduced the symptom severity of Blumeria graminis infection, by 45% in wheat and by 80% in barley. Thus, the priming activity of ulvan on the oxidative burst correlates with a decrease of disease symptoms in infected plants. This is apparently the first report on priming activity of a natural algal polysaccharide.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Plants are continuously in contact with pathogenic microorganisms attacking them. However, they are typically able to defend themselves after pathogen recognition via the perception of signal molecules, named elicitors or pathogen/microbe-associated molecular patterns (PAMPs or MAMPs) (Trouvelot et al., 2008). It has been discussed that these molecules can mimic pathogen invasion (Ménard et al., 2004). Plants possess a broad range of possible reactions to elicitor recognition, including rapid responses such as a transient oxidative burst, alkalinization of the extracellular medium, or influx of calcium. Slower defence mechanisms include production of antimicrobial compounds such as phytoalexins, reinforcement of the plant cell wall, and induction of pathogenesis-related (PR) proteins (Ménard et al., 2004). Often, these resistance reactions are accompanied by programmed host cell death, in which case the totality of the resistance responses are named ‘hypersensitive response’ (HR) (Klarzynski et al., 2000; Mercier et al., 2001).

Elicitors can originate either from the pathogen (PAMP/MAMP) or from the host plant itself (pathogen-induced molecular pattern or PIMP); accordingly, they are named exogenous and endogenous elicitors, respectively. Endogenous elicitors typically are oligogalacturonans derived from pectins, or sometimes cellodextrins, xyloglucans or polyglucans derived from cellulose or hemicellulose (Aziz et al., 2007). Exogenous elicitors from fungal cell walls are e.g. chitin fragments (Baier et al., 1999; Ortmann & Moerschbacher, 2006), oligomers and polymers of chitosan (Santos et al., 2008) or oligoglucans (Shinya et al., 2006). In these and many other cases, the elicitor-active compounds were shown to be carbohydrates. A number of reports have shown that glycans obtained from red and brown marine macroalgae can also act as signal molecules to protect land plants against fungal, bacterial or viral infections, either by acting as elicitors of defence responses or by triggering a state of so-called induced resistance (Klarzynski et al., 2000, 2003; Aziz et al., 2003; Ménard et al., 2005; Laporte et al., 2007; Trouvelot et al., 2008).

Induced plant disease resistance is often associated with an enhanced ability to rapidly and effectively mobilize cellular defence responses to subsequent pathogen attack, a state called ‘primed’ (Conrath et al., 2002). A priming effect has been described for some specific bacterial components, mainly lipopolysaccharides (LPS) of Gram-negative bacteria (Newman et al., 2007). In a number of reports, these compounds are important signal molecules for perception of pathogens and have been shown to not directly mobilize plant defence responses, but to enhance the speed and/or strength of various responses upon biotic or abiotic stress (Newman et al., 2007). Similarly, exopolysaccharides (EPS) isolated from the plant-growth-promoting rhizobacterium Pantoea agglomerans primed wheat and rice cell cultures for subsequent elicitation by chitin oligomers or chitosan polymers (Ortmann et al., 2006). Direct application of EPS to the suspension-cultured cells did not induce the oxidative burst, but pretreatment of the cells with EPS for some hours primed the chitin hexamer-induced burst (Ortmann & Moerschbacher, 2006; Ortmann et al., 2006).

New approaches for the management of plant disease are being explored and one important option is the induction of resistance in the host using natural signal molecules, i.e. priming compounds. Whilst polysaccharides derived from brown and red algae are known to possess signal character in higher plants, little information is available today on the elicitor or resistance-inducing activity of polysaccharides obtained from green algae. This paper describes the ability of the naturally sulphated polysaccharide ulvan from the green alga Ulva fasciata to prime wheat and rice cells for subsequent elicitor recognition, and to enhance resistance in wheat and barley plants to powdery mildew attack.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Elicitors

Chitin hexamer and chitosan polymers were used as general elicitors of plant pathogen defence at a final concentration of 0·1 or 1 μg mL−1 and 0·1, 1 or 10 μg mL−1, respectively. Chitin hexamer was purchased from Accurate Chemical & Scientific Corporation. Chitosan with an average degree of acetylation of 8% and an average degree of polymerization of 400 was prepared previously as described by Vander et al. (1998).

Ulvan extraction

The green seaweed U. fasciata was collected from the coast of South Brazil (Florianópolis, Santa Catarina State) in September 2004. Briefly, after carefully washing with tap water to remove contamination and epiphytes, the algae were air-dried, ground into a fine powder and stored at −20°C before use. For ulvan extraction, dry algae (25 g) were autoclaved in 1 L distilled water at 110°C for 1 h. The hot aqueous solution was separated from the algal residues by successive filtrations and concentrated to about 200–300 mL. Soluble compounds were precipitated by the addition of three volumes of ethanol for a period of 48 h at −20°C (Paradossi et al., 1999; Cluzet et al., 2004). The precipitate was recovered by filtration and air-dried at 50°C. The dried fraction was weighed, dissolved in water, dialysed against distilled water for 20 h using molecular weight cutoff 12–14 kDa dialysis membrane (Serva Biochemica) and then freeze-dried.

Plant material

Cell-suspension cultures

Wheat (Triticum aestivum cv. Prelude-Sr5) cell-suspension cultures, established by Gotthardt & Grambow (1992) were maintained in 50 mL MS medium (Murashige & Skoog, 1962) supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D), casein hydrolysate and sucrose as described by Ortmann & Moerschbacher (2006). Rice (Oryza sativa) cell-suspension cultures were grown in 50 mL MS medium supplemented with 2,4-D and sucrose as described by Ortmann et al. (2006). The cell cultures were kept at 26°C in the dark under agitation.

Plants

Wheat (cv. Kanzler) and barley (Hordeum vulgare cv. Villa) plants were grown on peat soil with clay (Einheitserde Type T, Balster Einheitserdewerk GmbH) in small pots (180 mL). In each pot 10–15 seeds were sown. Plants were kept in a greenhouse chamber at 80% relative humidity, with a photoperiod of 16 h light (sodium vapour lamps, Osram 400W) at 19°C and 8 h dark at 18°C until use.

Carbohydrate analysis

Total carbohydrate content was determined by anthrone-sulphuric acid assay (Morris, 1948) and rhamnose was used as a standard. To determine the monosaccharide composition, a polysaccharide fraction was hydrolysed with 1·5 m methanolic-HCl (16 h at 80°C). The generated methyl glycosides were converted into their trimethylsilylated derivates and analysed by gas-liquid chromatography (GLC) (Chaplin, 1986). The gas chromatograph HP-6890 (Hewlett Packard Co.) was equipped with a flame ionization detector (FID) and fused silica capillary column. The carrier gas was nitrogen with a flow rate of 1 mL min−1. The temperature of the detector was kept at 260°C and the injector temperature was 230°C. Myo-inositol was used as an internal standard, and chromatographic peaks were identified and quantified by comparison with reference sugars. Iduronic acid was not identified because of the lack of an appropriate standard.

Protein and sulphate determination

Protein contents of ulvan were estimated by colorimetry according to Bradford (1976). Bovine serum albumin was used as a standard. Sulphate content was determined from 2 mg ulvan treated with 1 n HCl at 105°C for 5 h using the barium-chloride–gelatin method of Dodgson & Price (1962). Turbidity was measured at 500 nm.

Oxidative burst measurement in cell cultures

The cells were used 3–4 days after subcultivation in a fresh medium, gently separated from the medium through a sintered glass filter. Aliquots of 300 mg cells were suspended in 5 mL pre-incubation medium [3% sucrose (w/v) and 10 mm MES in 5% (v/v) culture medium, pH 5·8] in a six-well plate and incubated under culture conditions for 5 h as described before by Ortmann et al. (2006). Oxidative burst was determined using a method based on the H2O2-dependent chemiluminescence of luminol according to Warm & Laties (1982) using a luminometer (Lumat LB 9501 Berthold). At different time intervals following the treatments, the hydrogen peroxide concentration was quantified as follows: aliquots of 200 μL wheat or rice cells were mixed with 700 μL 50 mm phosphate buffer (pH 7·9). The reaction was started by automatically adding 100 μL luminol (Sigma; 1·21 mm in phosphate buffer) and 100 μL 14 mm potassium hexacyanoferrate (Fluka) followed by light detection (10 s integration time, 430 nm). The micromolar (μm) H2O2 concentration was determined using a standard calibration curve.

Effect of priming activity on powdery mildew symptoms

To determine whether priming had an effect of disease symptom development, plants were treated with ulvan prior to infection with the pathogen Blumeria graminis. Ulvan extract was dispersed in water with the help of an Ultra Turrax disperser (20 000 r.p.m.). Whole barley and wheat plants (6 days old, grown as indicated above) were sprayed (airbrush at 1·2 bar, nozzle diameter 2·5 mm) twice (5 and 1 days before inoculation) with 0·1 or 1 mg mL−1 ulvan solution or water until runoff. As a positive control, plants were also sprayed with 0·5 mg mL−1 a.i. of the fungicide Folicur® 250EW (Bayer CropScience AG). One day after the second treatment, wheat and barley plants were inoculated with conidia of B. graminis f. sp. tritici or hordei, respectively. Conidia were taken from plants already infected by B. graminis for 10 days. Disease evaluation was performed 8 days after inoculation. Symptoms on ulvan- or Folicur®-treated leaves were visually evaluated in terms of percentage infection and compared among treatments and with the water-treated control leaves. In wheat plants, the first leaves were evaluated, whilst in barley plants, only the second leaves were evaluated. The experiment was performed independently three times for wheat and twice for barley. Percentage efficacy was determined by applying Abbott’s formula (Abbott, 1925).

Statistical analysis

Differences between treatments were analysed statistically using Student’s t-test or the non-parametric Mann–Whitney test, as indicated in the legends to figures.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Chemical composition of ulvan from U. fasciata

Ulvan was obtained from the green seaweed U. fasciata by hot-water extraction and recovered by precipitation with the addition of ethanol. About one-fifth of the algal dry weight was recovered as ulvan extract (Table 1). Chemical analysis of this material revealed that it was at least two-thirds carbohydrate. Analysis of the monosaccharide composition by GLC, using trimethylsilylated methyl-glycosides, showed that the ulvan fraction was mainly composed of rhamnose, xylose and glucuronic acid, with small amounts of glucose and galactose, as described previously (Lahaye & Robic, 2007; Robic et al., 2009). Further chemical analyses revealed c. 20% sulphate and very low quantities of protein contamination (<1%). As iduronic acid was not quantified for lack of an appropriate standard, a large part of the missing 13·6% may in fact be accounted for by this monosaccharide.

Table 1.   Chemical composition of the water-soluble polysaccharide (ulvan) extracted from Ulva fasciata
Yield (%)aSulphate (%)b,cProteins (%)b,dCarbohydrates (%)b,eSugar composition (mol%)f
RhaXylGlcAGlcGal
  1. aPercentage of algal dry weight.

  2. bPercentage of ulvan dry weight.

  3. cSulphate content determined according to Dodgson & Price (1962).

  4. dProtein content determined as described by Bradford (1976).

  5. eCarbohydrate content determined as the sum of monosaccharides determined using GLC.

  6. fMonosaccharide composition determined by GLC of the trimethylsilylated methyl-glycosides. Rha, rhamnose; Xyl, xylose; GlcA, glucuronic acid; Glc, glucose; Gal, galactose.

  7. Data shown are from one representative out of three independent experiments.

21·118·90·9>66·646·533·414·73·91·6

Priming by ulvan pretreatment of wheat cells for the elicitation of an oxidative burst by chitin hexamer and chitosan polymer

When wheat cells were treated with a final concentration of 0·2–200 μg ulvan mL−1, no induction of an oxidative burst reaction was detected (Fig. 1a). However, a small burst was observed after treatment with chitin hexamer (final concentration of 0·1 or 1 μg mL−1) (Fig. 1a) or chitosan polymer (final concentration of 0·1, 1 and 10 μg mL−1) (Fig. 3a). H2O2 production reached its peak value in the first 6–12 min of reaction, started to decrease immediately after this and returned to its initial level after about 30–40 min for the chitin hexamer (Fig. 1) and after 30 min for the chitosan-induced burst (Fig. 3).

image

Figure 1.  (a) Oxidative burst reaction of wheat cell-suspension cultures induced by 0·1 or 1 μg mL−1 chitin hexamer elicitor; (b) effect on H2O2 production of simultaneous addition of chitin hexamer (1 μg mL−1) and ulvan (2, 20 or 200 μg mL−1); and (c) priming effect on the chitin hexamer (0·1 μg mL−1) oxidative burst after pretreatment for 3 h with ulvan at 20, 100 or 200 μg mL−1. Hydrogen peroxide concentration was monitored by luminol chemiluminescence. Data given are from one representative of three independent experiments with similar results.

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image

Figure 3.  (a) Elicitation of the oxidative burst of wheat cell-suspension cultures induced by 0·1, 1 or 10 μg mL−1 chitosan with or without the simultaneous addition of 200 μg mL−1 ulvan; (b) priming effect on the chitosan (0·1, 1 or 10 μg mL−1) oxidative burst after pretreatment for 3 h with ulvan at 200 μg mL−1. Hydrogen peroxide concentration was monitored by luminol chemiluminescence. Data given are from one representative of three independent experiments with similar results.

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When ulvan (2–200 μg mL−1) was added simultaneously with 1 μg mL−1 chitin hexamer to wheat cells, the chitin-induced burst was slightly increased by the 20- and 200-μg mL−1 treatments, but not by the 2-μg mL−1 ulvan treatment (Fig. 1b). When the cells were treated with 200 μg ulvan mL−1 and chitosan (0·1, 1 or 10 μg mL−1) at the same time, the oxidative burst kinetics did not significantly change compared with chitosan treatment alone at the respective concentrations (Fig. 3a).

Finally, it was tested whether ulvan was able to prime the elicitor-induced oxidative burst. For this reason, the wheat cells were pretreated with 20, 100 or 200 μg ulvan mL−1 for a period of 3 h. After the pre-incubation time, the cells were treated with chitin hexamer (0·1 μg mL−1) and the concentration of H2O2 was determined. The chitin oxidative burst was notably higher than with the elicitor alone or with simultaneous addition of ulvan and the elicitor. Hydrogen peroxide production started immediately upon addition of the elicitor and reached a maximum after 6–7 min (Fig. 1c).

Ulvan (200 μg mL−1) clearly primed the chitin hexamer oxidative burst five-sixfold in wheat cells, H2O2 production increasing from 2·5 to 13·8 μm (Fig. 2). Induction of hydrogen peroxide production was also observed when wheat cells were pre-incubated for 3 h with ulvan (200 μg mL−1) and then chitosan was added. The addition of the elicitor at 10 μg mL−1 resulted in the production of 13·5 μm H2O2 (Fig. 3b), whilst the same chitosan concentration without ulvan pretreatment produced only 6·4 μm H2O2 (Fig. 3b).

image

Figure 2.  Priming effect on the development of the oxidative burst of chitin hexamer (0·1 μg mL−1) in wheat cell-suspension cultures after 3 h of pretreatment with ulvan (200 μg mL−1). Hydrogen peroxide concentration was monitored by luminol chemiluminescence. Values are mean ± SD of three independent experiments (n = 3). Differences between chitin, ulvan and ulvan+chitin treatments are statistically significant (t-test *< 0·05, **< 0·01).

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Priming by ulvan pretreatment of rice cells for the elicitation of an oxidative burst by chitin hexamer and chitosan polymer

Similar results as described above for wheat cells were also seen when using suspension-cultured rice cells. No oxidative burst was induced by treatment with ulvan (200 μg mL−1) alone (Fig. 4a), but when using chitin hexamer (1 μg mL−1) or chitosan polymer (1 μg mL−1) alone, a slight elicitation was measured (Fig. 4a,b). Furthermore, the simultaneous addition of ulvan (200 μg mL−1) and either chitin hexamer or chitosan polymer (treatments indicated with asterisks on Fig. 4) to the cells did not significantly increase H2O2 production compared with that induced by the respective elicitor alone (Fig. 4a,b). By contrast, pre-incubation with the same ulvan concentration as before primed the rice cells, inducing a strong oxidative burst reaction upon chitin or chitosan treatment. The response observed was higher after the addition of chitin hexamer than after chitosan treatment. These responses were ulvan-dose-dependent. The addition of 200 or 100 μg ulvan mL−1 resulted in a significant burst, whereas the production of H2O2 was only weakly induced by pretreatment with 50 or 20 μg ulvan mL−1. The reaction started after the addition of the elicitors and reached its peak within 14 min. After 35 min of reaction, the H2O2 concentration had come down to initial levels (Fig. 4a,b).

image

Figure 4.  (a) Dose-dependent priming of the chitin hexamer (1 μg mL−1) oxidative burst in rice cell-suspension cultures after 3 h of preincubation with 20, 50, 100 or 200 μg mL−1 ulvan. Treaments of ulvan (200 μg mL−1) or chitin hexamer (1 μg mL−1), or simultaneous addition of both (*), were included for comparison. (b) Dose-dependent priming effect of the chitosan (1 μg mL−1)-induced oxidative burst reaction in rice cell-suspension cultures after 3 h of preincubation with 20, 50, 100 or 200 μg mL−1 ulvan. For comparison, H2O2 generation was measured after addition of ulvan (200 μg mL−1) or chitosan (1 μg mL−1) alone, or after simultaneous addition of both (*). Hydrogen peroxide concentration was monitored by luminol chemiluminescence. Data given are from one representative of three independent experiments with similar results.

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Resistance to powdery mildew enhanced in barley and wheat plants by ulvan pretreatment

The relevance of ulvan as a priming agent of defence responses in planta was examined using wheat and barley plants. When infiltrated into wheat leaves (0·1–20 mg mL−1, data not shown) or sprayed onto wheat and barley plants, the sulphated polysaccharide did not induce any phytotoxic effects or necrosis. Thus, after a visual inspection of leaf tissue, no damage or cell death was observed.

The question of whether the ulvan-induced priming of an oxidative burst observed in suspension-cultured cells was indicative of ulvan-enhanced disease resistance in intact plants was investigated using B. graminis infections. Whole wheat and barley plants were sprayed with water, the fungicide Folicur® (0·5 mg mL−1) or ulvan solution (0·1 or 1 mg mL−1), allowed to recover for 5 days, and then sprayed again with the same solution as before. One day after the second treatment, plants were inoculated with B. graminis conidia. Eight days after inoculation, the symptoms on primary wheat leaves or on secondary barley leaves were compared visually (percentage infection) with the water control and Folicur®-treated leaves (Fig. 5). The control plants showed strong infection, with symptoms covering the leaves almost entirely (Fig. 6c). By contrast, plants treated with ulvan, regardless of the concentration tested (0·1 and 1 mg mL−1), exhibited symptoms of infection reduced by up to 46% in wheat (Fig. 6a,b) and by up to 82% in barley plants, compared with controls. Thus, the strong ulvan-induced priming effect on the oxidative burst in suspension-cultured cells correlated with decreased symptom development in inoculated plants.

image

Figure 5.  Ulvan stimulation of resistance to Blumeria graminis infection, measured as percentage efficacy (Abbott, 1925), in wheat (Triticum aestivum cv. Kanzler) and barley (Hordeum vulgare) plants. Whole plants were pretreated (sprayed) twice with 0·1 or 1 mg mL−1 ulvan solution or water. For comparison, other plants were sprayed with 0·5 mg mL−1 fungicide Folicur®. Plants were inoculated with B. graminis conidia 1 day after the second spray and disease was evaluated 8 days post-inoculation. For wheat, values are mean ± SD of three independent experiments (n = 30). Bars followed by the same letter are not significantly different using Mann–Whitney test (< 0·01). For barley, values are mean ± SD of two independent experiments (n = 14). Bars followed by the same letter are not significantly different using Mann-Whitney test ( 0·02).

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image

Figure 6.  Ulvan stimulation of resistance in wheat plants (Triticum aestivum cv. Kanzler) against Blumeria graminis f. sp. tritici infection. Whole plants were pretreated (sprayed) twice with (a) 0·1 or (b) 1 mg mL−1 ulvan solution or (c) water. For comparison, other plants were sprayed with (d) 0·5 mg mL−1 fungicide Folicur®. Plants were inoculated with B. graminis conidia 1 day after the second spray and representative photographs taken 8 days post-inoculation.

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Oligo- and polysaccharides derived from brown and red marine macroalgae, an inexpensive possible source of sulphated complex carbohydrates (Mercier et al., 2001; Ménard et al., 2005), have been used as elicitors of active plant defence responses, such as the oxidative burst (Aziz et al., 2003; Klarzynski et al., 2003). The addition of β-1,3-glucans with a degree of polymerization of 10, purified from the marine brown alga Laminaria digitata, induced the production of H2O2 with a maximum level 20 min after elicitation in grapevine cell suspensions (Aziz et al., 2007). Klarzynski et al. (2003) reported on the elicitor activity of sulphated oligofucans isolated from the brown alga Pelvetia canaliculata in tobacco cell cultures. The fucan preparations induced an alkalinization and the production of H2O2 in the extracellular medium, an accumulation of salicylic acid (SA), and they induced local and systemic resistance to Tobacco mosaic virus (TMV). According to Ménard et al. (2005), λ-carrageenan, a highly sulphated galactan found in cell walls of red algae (e.g. Gigartina sp.), trigged the production of SA, jasmonic acid and ethylene, and induced defence genes encoding chitinase and sesquiterpene cyclase. Almost 100% protection against Colletotrichum trifolii was obtained in the legume Medicago truncatula in response to one or two ulvan treatments, and the PR10-1 gene was induced from 7 up to 10 days after treatment (Cluzet et al., 2004).

In contrast to this elicitor activity of ulvan in the dicotyledonous M. truncatula, treatment of monocotyledonous wheat and rice cells with ulvan did not elicit an oxidative burst, but instead acted as a priming inducer. Even at high concentration (200 μg mL−1), ulvan did not induce H2O2 generation, and a simultaneous application of the same ulvan concentration along with the elicitor chitin hexamer or chitosan did not strongly influence the elicitor-induced oxidative burst. However, pre-incubation for 3 h with ulvan primed wheat and, even more strongly, rice cells for the elicitation of an oxidative burst by the subsequent treatment with chitin hexamer or chitosan. This is apparently the first example of priming plant cells by a natural algal polysaccharide. A similar phenomenon, i.e. elicitor activity in dicotyledonous plants but priming activity in monocotyledonous plants, was previously reported for a bacterial exopolysaccharide (Ortmann et al., 2006), hinting at related modes of action for these two very different polysaccharides.

Priming in plants is the induction of a physiological situation of enhanced ability to mobilize pathogen or elicitor-induced defence responses. As a consequence, primed cells respond more rapidly or more efficiently to abiotic or biotic stress than naive cells (Goellner & Conrath, 2008). Priming is typically induced by PAMPs, such as fragments of cell wall of Phytophthora sojae, or bacterial lipo- or exopolysaccharides, modulating plant defence responses (Ortmann & Moerschbacher, 2006; Newman et al., 2007; Goellner & Conrath, 2008). A primed state, also called potentiation, can also be obtained with elicitors used at concentrations insufficient to induce measurable responses alone (Val et al., 2008). Interestingly, priming can also be observed in mammalian cells, and Castro et al. (2004) reported that Ulva rigida water-soluble extract can prime the respiratory burst activity of turbot phagocytes. In that study, the cells pretreated with the seaweed extract and then incubated with phorbol myristate acetate (PMA) showed higher activity than control cells stimulated by PMA alone, suggesting a priming effect. The cell responses increased with the concentration of extract and with the time of incubation, reaching a maximum 3 h after the treatment. Whether or not the same active domain in ulvan is responsible for priming in plant and animal cells remains to be investigated.

Consistent with its strong priming activity in suspension-cultured monocotyledonous cells, ulvan also protects wheat and barley plants against infection by the pathogenic fungus B. graminis. In preliminary experiments, a small effect (20% efficacy) was also observed when rice plants were sprayed with ulvan solution (1 mg mL−1) and infected later with the rice blast pathogen (Pyricularia oryzae) (data not shown).

Ulvan did not have in vitro antibacterial activity against Pseudomonas syringae (data not shown) or Gram-positive bacteria (Paulert et al., 2007); nor did it have any antifungal activity against Colletotrichum lindemuthianum, Colletotrichum trifolii or Candida albicans (Cluzet et al., 2004; Paulert et al., 2007). Moreover, ulvan did not show a direct elicitor effect in wheat and rice cell-suspension cultures (this study), nor did it induce the pathogenesis-related proteins chitinase and thaumatin-like protein when injected into wheat leaves (data not shown). Thus, it can be assumed that the plant protection is the result of the ability of the ulvan polymer to enhance defence reactions in monocotyledonous plants, priming them to respond more effectively to the infection.

The mode of action of ulvan as a priming inducer is unknown at present. One revealing observation is the fact that ulvan primes the cells for better recognition of both chitin and chitosan elicitors. Chitin oligomers are recognized as elicitors by many plants such as alfafa, tobacco, wheat and rice (Baier et al., 1999; Ortmann et al., 2006), probably by interaction with a high-affinity binding site for chitin fragments on the membrane surface (Baureithel et al., 1994). On the other hand, chitosan polymers have been proposed to act by physicochemical interaction with the membrane as a result of their strong positive charge (Kauss et al., 1989). Some priming inducers, such as P2 derived from Phytophthora parasitica, the proteinaceous elicitor β-megaspermin purified from Phytophthora megasperma, or λ-carrageenan, appear to act via the elicitation of local necroses (Klarzynski et al., 2000; Mercier et al., 2001; Cluzet et al., 2004).

Since ulvan did not induce HR when infiltrated into wheat (data not shown) or into M. truncatula leaves (Cluzet et al., 2004), it would appear that HR is not a prerequisite for the development of enhanced resistance. Similarly, the infiltration of laminarin caused no tissue damage or cell death in tobacco leaves, but enhanced resistance against the soft rot bacterial pathogen Erwinia carotovora (Klarzynski et al., 2000). To better understand structure–activity relationships and the mode of action of ulvan as a priming inducer in monocotyledonous plants, oligosaccharides obtained by partial hydrolysis of ulvan and their biological activities are currently being analysed.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

RP is grateful to CNPq, Brazil for financial support. We would like to thank Professor Dr Marciel Stadnik (UFSC, Brazil) for kindly providing the ulvan sample and U. Beike for skillful technical assistance.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  • Abbott WS, 1925. A method for computing the effectiveness of an insecticide. Journal of Economic Entomology 18, 2657.
  • Aziz A, Poinssot B, Daire X et al. , 2003. Laminarin elicits defense responses in grapevine and induces protection against Botrytis cinerea and Plasmopara viticola. Molecular Plant-Microbe Interactions 16, 111828.
  • Aziz A, Gauthier A, Bézier A et al. , 2007. Elicitor and resistance-inducing activities of β-1,4 cellodextrins in grapevine, comparison with β-1,3 glucans and α-1,4 oligogalacturonides. Journal of Experimental Botany 58, 146372.
  • Baier R, Schiene K, Kohring B, Flaschel E, Niehaus K, 1999. Alfafa and tobacco cells react differently to chitin oligosaccharides and Sinorhizobium meliloti nodulation factors. Planta 210, 15764.
  • Baureithel K, Felix G, Boller T, 1994. Specific, high affinity binding of chitin fragments to tomato cells and membranes – competitive inhibition of binding by derivatives of chitooligosaccharides and a nod factor of Rhizobium. The Journal of Biological Chemistry 269, 179318.
  • Bradford MM, 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry 72, 24854.
  • Castro R, Zarra I, Lamas J, 2004. Water-soluble seaweed extracts modulate the respiratory burst activity of turbot phagocytes. Aquaculture 229, 6778.
  • Chaplin MF, 1986. Gas-liquid chromatography. In: ChaplinMF, KennedyJF, eds. Carbohydrate Analysis, a Practical Approach. Oxford, UK: IRL Press, 2333.
  • Cluzet S, Torregrosa C, Jacquet C et al. , 2004. Gene expression profiling and protection of Medicago truncatula against a fungal infection in response to an elicitor from green algae Ulva spp. Plant, Cell and Environment 27, 91728.
  • Conrath U, Pieterse CMJ, Mauch-Mani B, 2002. Priming in plant–pathogen interactions. Trends in Plant Science 7, 2106.
  • Dodgson KS, Price RG, 1962. A note on the determination of the ester sulphate content of sulphated polysaccharides. Biochemical Journal 84, 10610.
  • Goellner K, Conrath U, 2008. Priming: it’s all the world to induced disease resistance. European Journal of Plant Pathology 121, 23342.
  • Gotthardt U, Grambow HJ, 1992. Near isogenic wheat suspension cultures: establishment, elicitor induced peroxidase activity and potential use in the study of host/pathogen interaction. Journal of Plant Physiology 139, 65965.
  • Kauss H, Jeblick W, Domard A, 1989. The degrees of polymerization and N-acetylation of chitosan determine its ability to elicit callose formation in suspension cells and protoplasts of Catharanthus roseus. Planta 178, 38592.
  • Klarzynski O, Plesse B, Joubert J-C et al. , 2000. Linear β-1,3 glucans are elicitors of defense responses in tobacco. Plant Physiology 124, 102737.
  • Klarzynski O, Descamps V, Plesse B, Yvin J-C, Kloareg B, Fritig B, 2003. Sulfated fucan oligosaccharides elicit defense responses in tobacco and local and systemic resistance against tobacco mosaic virus. Molecular Plant-Microbe Interactions 16, 11522.
  • Lahaye M, Robic A, 2007. Structure and functional properties of ulvan, a polysaccharide from green seaweeds. Biomacromolecules 8, 176574.
  • Laporte D, Vera J, Chandía NP, Zúniga EA, Matsuhiro B, Moenne A, 2007. Structurally unrelated algal oligosaccharides differentially stimulate growth and defense against tobacco mosaic virus in tobacco plants. Journal of Applied Phycology 19, 7988.
  • Ménard R, Alban S, Ruffray P et al. , 2004. β-1,3 glucan sulfate, but not β-1,3 glucan, induces the salicylic acid signaling pathway in tobacco and Arabidopsis. The Plant Cell 16, 302032.
  • Ménard R, Ruffray P, Fritig B, Yvin J-C, Kauffmann S, 2005. Defense and resistance-inducing activities in tobacco of the sulfated β-1,3 glucan PS3 and its synergistic activities with the unsulfated molecule. Plant Cell Physiology 46, 196472.
  • Mercier L, Lafitte C, Borderies G, Briand X, Esquerré-Tugayé M-T, Fournier J, 2001. The algal polysaccharide carrageenans can act as an elicitor of plant defence. New Phytologist 149, 4351.
  • Morris DL, 1948. Quantitative determination of carbohydrates with Dreywood′s anthrone reagent. Science 107, 2545.
  • Murashige T, Skoog F, 1962. A revised medium for rapid growth and bioassays with tobacco tissue culture. Physiologica Plantarum 15, 47397.
  • Newman M-A, Dow JM, Molinaro A, Parrilli M, 2007. Priming, induction and modulation of plant defence responses by bacterial lipopolysaccharides. Journal of Endotoxin Research 13, 6984.
  • Ortmann I, Moerschbacher BM, 2006. Spent growth medium of Pantoea agglomerans primes wheat suspension cells for augmented accumulation of hydrogen peroxide enhanced peroxidase activity upon elicitation. Planta 224, 96370.
  • Ortmann I, Conrath U, Moerschbacher BM, 2006. Exopolysaccharides of Pantoea agglomerans have different priming and eliciting activities in suspension-cultured cells of monocots and dicots. FEBS Letters 580, 44914.
  • Paradossi G, Cavalieri F, Pizzoferrato L, Liquori AM, 1999. A physico-chemical study on the polysaccharide ulvan from hot water extraction of the macroalga Ulva. International Journal of Biological Macromolecules 25, 30915.
  • Paulert R, Smania Jr A, Stadnik MJ, Pizzolatti MG, 2007. Antimicrobial properties of extracts from the green seaweed Ulva fasciata Delile against pathogenic bacteria and fungi. Algological Studies 123, 12330.
  • Robic A, Bertrand D, Sassi J-F, Lerat Y, Lahaye M, 2009. Determination of the chemical composition of ulvan, a cell wall polysaccharide from Ulva spp. (Ulvales, Chlorophyta) by FT-IR and chemometrics. Journal of Applied Phycology 21, 4516.
  • Santos ALW, El Gueddari NE, Trombotto S, Moerschbacher BM, 2008. Partially acetylated chitosan oligo- and polymers induce an oxidative burst in suspension cultured cells of the gymnosperm Araucaria angustifolia. Biomacromolecules 9, 34115.
  • Shinya T, Ménard R, Kozone I et al. , 2006. Novel β-1,3-, 1-6-oligoglucan elicitor from Alternaria alternata 102 for defense responses in tobacco. FEBS Journal 273, 242131.
  • Trouvelot S, Varnier A-L, Allègre M et al., 2008. A β-1,3 glucan sulfate induces resistance in grapevine against Plasmopara viticola through priming of defense responses, including HR-like cell death. Molecular Plant-Microbe Interactions 21, 23243.
  • Val F, Desender S, Bernard K, Potin P, Hamelin G, Andrivon D, 2008. A culture filtrate of Phytophthora infestans primes defense reaction in potato cell suspensions. Biochemistry and Cell Biology 98, 6538.
  • Vander P, Vårum K, Domard A, El Gueddari NE, Moerschbacher BM, 1998. Comparison of the ability of partially N-acetylated chitosans and chitooligosaccharides to elicit resistance reactions in wheat leaves. Plant Physiology 118, 13539.
  • Warm E, Laties GG, 1982. Quantification of hydrogen peroxide in plant extracts by the chemiluminescence reaction with luminol. Phytochemistry 21, 82731.