Partially Acetylated Chitosan Oligo- and Polymers Induce an Oxidative

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Biomacromolecules 2008, 9, 3411–3415

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Partially Acetylated Chitosan Oligo- and Polymers Induce an Oxidative Burst in Suspension Cultured Cells of the Gymnosperm Araucaria angustifolia Andre´ Luis Wendt dos Santos,† Nour Eddine El Gueddari,† Ste´phane Trombotto,‡ and Bruno Maria Moerschbacher*,† Institute of Plant Biochemistry and Biotechnology, University of Muenster, D-48143 Muenster, Germany, and Laboratoire des Mate´riaux Polyme`res et des Biomate´riaux, UMR CNRS 5223 Inge´nierie des Mate´riaux Polyme`res, Universite´ Claude Bernard Lyon 1, F-69622 Villeurbanne Cedex, France Received September 12, 2008; Revised Manuscript Received October 22, 2008

Suspension-cultured cells were used to analyze the activation of defense responses in the conifer A. angustifolia, using as an elicitor purified chitosan polymers of different degrees of acetylation (DA 1-69%), chitin oligomers of different degrees of polymerization (DP 3-6), and chitosan oligomer of different DA (0-91%). Suspension cultured cells elicited with chitosan polymers reacted with a rapid and transient generation of H2O2, with chitosans of high DA (60 and 69%) being the most active ones. Chitosan oligomers of high DA (78 and 91%) induced substantial levels of H2O2, but fully acetylated chitin oligomers did not. When cultivated for 24-72 h in the presence of 1-10 µg mL-1 chitosan (DA 69%), cell cultures did not show alterations in the levels of enzymes related to defense responses, suggesting that, in A. angustifolia, the induction of an oxidative burst is not directly coupled to the induction of other defense reactions.

Introduction Plants are constantly confronted with the presence of potentially pathogenic microorganisms and changing and possibly adverse environmental conditions. To cope with such situations, plants have developed a sophisticated defense response system to protect themselves against biotic and abiotic stresses.1 Among the many mechanisms developed by plants, the rapid and transient production of large amounts of reactive oxygen species, termed “oxidative burst”, appears to be one of the earliest reactions of plant cells to pathogen attack and abiotic stresses.2,3 Reactive oxygen species produced by plant cells include the superoxide anion radical ( · O2-), hydrogen peroxide (H2O2), and the hydroxyl radical (•OH).4 H2O2, the most stable form of the reactive oxygen species, has been associated with a broad range of defense reactions including (a) as a local signal for hypersensitive cell death,5 (b) as short-range messenger for induction of defense gene expression,6 (c) as a cosubstrate for oxidative cross-linking and immobilization of cell wall structural proteins, phenolic acids, and monolignols,7 and (d) as an antimicrobial agent.8 The involvement of an oxidative burst in disease resistance has been shown in a variety of dicotyledonous plants9 and, more recently, in some monocotyledonous plants.10,11 However, little evidence for an oxidative burst in gymnosperms has been presented so far.12 Quantitative analyses of the oxidative burst can best be achieved in suspension-cultured plant cells treated with a defined elicitor, for example, chitin oligomers. Even though plant cell cultures represent systems of reduced complexity compared to intact plants, they proved very attractive models in analyzing isolated compounds for their ability to interact with the plant defense system on a molecular level.13 * To whom correspondence should be addressed. Fax: +49 251 83-28371. E-mail: [email protected]. † University of Muenster. ‡ Universite´ Claude Bernard Lyon 1.

Chitin, a linear polysaccharide composed of (1f4)-linked 2-acetamido-2-deoxy-β-D-glucopyranose (GlcNAc, A-unit), is de-N-acetylated to chitosan, a polycationic polysaccharide composed of (1f4)-linked units of both GlcNAc and 2-amino2-deoxy-β-D-glucopyranose (GlcN, D-unit), in the cell walls of some fungi.14,15 Chitin and chitosan have been shown to possess elicitor activity inducing an oxidative burst and other plant defense systems in a wide range of angiosperms.16,17 Despite the evolutionary distance between gymnosperms and angiosperms (about 300 million years), some of the coordinated defense responses observed in chitin or chitosan-elicited angiosperm species are also present in gymnosperms, suggesting that the signaling pathway mediating chitin or chitosan-induced transcription are highly conserved in the plant kingdom.18-20 Although the activation of some defense response systems in woody plants has been characterized,21 mechanisms of disease resistance in conifers are poorly understood and restricted to members of the family Pinaceae. Similarly, relatively little is known on potential elicitors of induced disease resistance mechanisms in gymnosperms.22 In general, typical surface exposed components of microbial cell walls, such as chitin, can be expected to trigger resistance in host plants. The aim of the present work was to test cell suspension cultures of Araucaria angustifolia (Bert) O. Ktze (Brazilian pine tree), a subtropical conifer species of the Araucariaceae family, concerning the induction of an oxidative burst and other defense-related responses using as elicitors purified chitosan polymers and oligomers with known degrees of polymerization and acetylation and chitin oligomers.

Experimental Section Initiation of an Embryogenic Culture. Embryogenic cultures were initiated from immature zygotic embryos of A. angustifolia cultivated on BM basal salts23 containing 5 µM 2,4-dichlorophenoxy acetic acid, 2 µM 6-benzylaminopurine, and 2 µM kinetin and maintained in the dark at 25 ( 2 °C, as already reported.24

10.1021/bm801025g CCC: $40.75  2008 American Chemical Society Published on Web 11/18/2008

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Establishment of a Cell Suspension Culture. Cell suspension cultures were established according to the previously described protocol,25 with minor modifications. Embryogenic cultures (500 mg) were dispersed in Petri dishes (60 × 15 mm) containing 5 mL of liquid MSG26 culture medium supplemented with 1.46 g L-1 L-glutamine (MSG-0). Tissue clumps were broken up using forceps and scalpel. The resulting cell suspension was transferred to 100 mL flasks containing 20 mL of MSG-0 medium and cultivated for 9 days in the dark at 25 ( 2 °C at 120 rpm in orbital shakers. At the end of this period, cell suspension cultures were fractionated by sieving through metal meshes with pore sizes of 160 µm. The cell fraction smaller than 160 µm was washed in liquid MSG-0 medium, and aliquots of 4 mL were transferred to 200 mL flasks containing 50 mL of MSG-0 medium, then cultivated as above. Cell suspensions were subcultured in intervals of 12 days for four months prior to elicitation experiments. Cultivation of Cells for Oxidative Burst Assays. For quantification of extracellular hydrogen peroxide, cell suspensions were cultivated as described earlier.11 A total of 8 days after subculturing, suspension cells were collected using a sintered glass filter and washed twice with liquid MSG-0 medium. Then, aliquots of 300 mg were transferred to 5 mL of assay medium (5% (v/v) MSG-0 medium in 10 mM MES (pH 5.8) supplemented with 3% (w/v) sucrose) and cultivated on a rotatory shaker for 5 h prior to elicitor addition. Assay for Hydrogen Peroxide (H2O2) Quantification. H2O2 generation was quantified in the assay medium of cells by luminoldependent chemiluminescence as already reported.27 A total of 200 µL of assay medium containing A. angustifolia cells was mixed with 600 µL of potassium phosphate buffer (50 mM, pH 7.9) and transferred into a luminometer (Lumat LB 9501/16, Germany). A total of 100 µL of luminol (1.21 mM in potassium phosphate buffer, 50 mM, pH 7.9) and 100 µL of potassium hexacyanoferrate(III) (14 mM) were injected automatically before light detection (10 s integration time, 430 nm). Chemiluminescence was determined in relative light units, and conversion to µM H2O2 was carried out as described earlier.11 Quantification of Extracellular Chitinase, β-1-3-glucanase, and peroxidase activity, and intracellular phenylalanine ammonialyase activity. Extracellular Chitinase and β-1-3-glucanase activities were determined using as a substrate the polymers carboxymethylchitin-remazol brilliant violet (CM-chitin-RBV, Lo¨we Biochemica), and carboxymethyl-Curdlan-remazol brilliant blue R (CM-Curdlan-RBB, Lo¨we Biochemica), respectively.28 Peroxidase and intracellular phenylalanine ammonia-lyase activities were determined as described previously.29 Concentration of Extracellular Proteins, Isoelectric Focusing, and Detection of Extracellular Chitinase Activity. After 5 h of cultivation and prior to elicitor addition, assay medium containing A. angustifolia cells was collected for analysis of extracellular Chitinase activity. Cells were separated from the assay medium by filtering through analytical filter paper (70 mm) and then through a 0.46 µm menbrane filter (Schleicher & Schuell, Germany). Aliquots of 2 mL from filtered assay medium were desalted on prepacked Sephadex G-25 columns (PD-10, Amersham Bioscience) equilibrated with water. Eluates were lyophilized and resuspended in water. Protein concentration was determined by the dye-binding method of Bradford using bovine serum albumin as a standard.30 For isoelectric focusing, extracellular proteins were separated over the pH range 3-10 in a polyacrylamide gel containing Ampholine (Amersham Bioscience). Proteins showing chitinolytic activity were detected using an overlay gel containing 0.01% (w/v) glycol chitin.31 After incubation in 100 mM sodium acetate buffer (pH 5.0) for 90 min at 37 °C, overlay gels were incubated for 5 min in solution containing 0.01% (w/v) calcofluor M2R (Sigma) in 500 mM Tris-HCl (pH 8.9). After 5 min, the calcofluor solution was removed and the overlay gel was rinsed overnight in distilled water. Lytic zones were visualized and recorded under UV light.

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Figure 1. Growth curve of suspension cultured cells of A. angustifolia maintained in MSG medium free of growth regulators (MSG-0), showing the increase in dry weight over 12 days of culture. The bars indicate the standard deviation from mean values derived from three independent experiments.

Inhibition of Extracellular Chitinase Activity with Allosamidin. Allosamidin was generously provided by Dr. S. Sakuda, Department of Applied Biological Chemistry, University of Tokio. Allosamidin was dissolved in 0.1 N acetic acid, filter sterilized, and added to the assay medium in a concentration of 10-6 M.32 After 5 h, the assay medium was collected and the inhibition of Chitinase activity was tested using an overlay gel containing glycol chitin. Elicitor Preparation. A degree of acetylation (DA) series of chitosans with constant DPn (number average degree of polymerization) of about 190 but varying in their DA from 1 to 69% was prepared previously by partial de-N-acetylation of highly acetylated chitosan polymers.33 The acetyl groups were distributed randomly along the linear polymer chains, and the polymers were all fully water-soluble. This DA series was generously provided by Prof. Dr. Kjell M. Vårum, Department of Biotechnology, Norwegian University of Science and Technology, Trondheim, Norway. Chitin oligomers with DP (degree of polymerization) from 3 to 6 were generously provided by Dr. K. Hicks, U.S. Departament of Agriculture, Eastern Regional Research Center, Wyndmoor, U.S.A. Chitosan oligomers mixtures (3 < DP < 8) with different DA from 0 to 91% were generated and characterized as described previously, by partial re-N-acetylation of a given mixture of GlcN-oligomers so that all samples had identical DP distributions.34

Results Growth of Suspension Cultured Cells of A. angustifolia. Despite the absence of growth regulators in the medium, suspension cultured A. angustifolia cells could be established and maintained for a period of over one year. After three days of culture, cells entered the logarithmic growth phase and they did not reach the stationary phase even after 12 days (Figure 1). During 12 days of cultivation, suspension cultured cells of A. angustifolia produced a more than 6-fold increment in dry weight. Dynamic of Hydrogen Peroxide (H2O2) Production in Suspension Cultured A. angustifolia Cells. A quantitative assay based on the H2O2-dependent chemoluminescence of luminol was used to monitor the time-dependent generation of extracellular H2O2 after application of elicitor in suspension cultured cells of A. angustifolia. The chitosan-induced production of H2O2 was detectable within 6 min after elicitation, with maximum levels normally occurring between 12 and 24 min postelicitation for elicitor concentrations ranging from 0.01 to 1 µg mL-1, and at 36 min for the lowest concentration tested (0.001 µg mL-1; Figure 2). After this period, the generation of H2O2 was reduced progressively. When cultivated for up to 72 h in MSG-O liquid medium containing 1-10 µg mL-1 chitosan with DA of 69%, suspension cultured cells of A. angustifolia

Defense Responses in A. angustifolia

Figure 2. Time-response curves of H2O2 generation by suspension cultured cells of A. angustifolia elicited with chitosan polymers with a degree of acetylation 15%, at 0.001, 0.01, 0.1, and 1 µg mL-1. Data given are mean values derived from three independent experiments.

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Figure 4. Time-response curves of H2O2 generation by suspension cultured cells of A. angustifolia elicited with chitin oligosaccharides of degree of polymerization 3, 4, 5, and 6 in a concentration of 0.1 µg mL-1. Data given are mean values derived from three independent experiments.

Figure 5. Isoelectric focusing of extracellular proteins in suspension cultured cells of A. angustifolia followed by activity staining using an overlay gel containing glycol chitin. (A) Extracellular chitinolytic proteins present in the assay medium after five hours of incubation; (B) extracellular chitinolytic proteins present in the assay medium supplemented with allosamidin 10-6 M after five hours of incubation. An equal amount (3 µg) of extracellular protein was loaded in each lane. Figure 3. Influence of (A) the degree of acetylation (1 and 35%) and concentration (0.001-1 µg mL-1) and (B) degree of acetylation (1-69%) at 0.01 µg mL-1 on the elicitor activity of chitosan polymers for generation of H2O2 in suspension cultured cells of A. angustifolia. Data given are mean values after 24 min of cell elicitation derived from three independent experiments.

did not show increases in the activities of extracellular Chitinase, glucanase, or peroxidase, nor of intracellular phenylalanine ammonia-lyase activities (data not shown). The production of H2O2 (µM) was dependent on the type of elicitor used (chitosan polymers, chitosan oligomers, chitin oligomers), on the elicitor concentration applied (0.001-1 µg mL-1), and on the degree of acetylation of the elicitor (Figure 3A). Dose response curves of chitosans with different degrees of acetylation, ranging from 1 to 69%, showed that all chitosans tested elicited an oxidative burst in suspension cultured cells of A. angustifolia. With increasing DA, elicitor activity of the chitosan increased, with the chitosan of DA 69% being the most potent inducer of an oxidative burst (Figure 3B). Chitosan polymers with higher DA are water-insoluble and, therefore, could not be tested for their elicitor activities. However, the

application of glycol-chitin (a soluble derivative of chitin) in the same concentration range resulted in H2O2 levels lower than those observed for chitosans with DA 60% and 69% (data not shown). As chitosan polymers with DA above 70% are insoluble in water, we tested the elicitor activities of fully acetylated chitin oligomers. Purified chitin oligomers with DP ranging from 3 to 6 did not induce the production of substantial amounts of H2O2 (Figure 4). The production of H2O2 was only weakly induced at both concentrations tested (0.01-0.1 µg mL-1), and neither the speed of H2O2 generation nor the maximum H2O2 level reached correlated with the DP of the chitin oligomers. Analysis of extracellular proteins produced by A. angustifolia cells during the incubation in assay medium revealed the presence of three proteins showing chitinolytic activity (Figure 5). These proteins could be involved in the degradation of the chitin oligomers used in our experiments, and this fact would explain the low levels of H2O2 generation observed. To test this hypothesis, suspension cells of A. angustifolia were cultivated in the presence of allosamidin, a specific inhibitor of

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Figure 6. (A) Time-response curves of H2O2 generation by suspension cultured cells of A. angustifolia elicited with DP 3-8 mixtures of chitosan oligomers of degree of acetylation ranging from 0 to 91% in a concentration of 0.1 µg mL-1. (B) Influence of the degree of acetylation (0-91%) on the generation of H2O2 in suspension cultured cells of A. angustifolia 24 min after elicitation with DP 3-8 mixtures of chitosan oligomers and with the GlcNAc pentamer (DA 100%) at 0.1 µg mL-1. Data given are mean values derived from three independent experiments.

family 18 chitinases.35 However, under the conditions tested, allosamidin did not inhibit the activity of these chitinolytic proteins (Figure 5). We finally analyzed the elicitor activities of mixtures of partially acetylated chitosan oligomers. All mixtures tested had identical DP distributions ranging from DP 3 to DP 8, but they differed in their DA, which ranged from 0 to 91%. When these oligomer mixtures were used as elicitors (0.1 µg mL-1), the generation of H2O2 increased progressively with increasing DA up to 78% (Figure 6A,B). As observed for glycol-chitin, the chitosan oligomers with DA 91% exhibited slightly lower elicitor activities than the DA 78% oligomers.

Discussion In this report, the time-dependent generation of extracellular H2O2 following the application of well characterized, partially acetylated chitosan polymers, chitosan oligomers, and fully acetylated chitin oligomers to suspension cultured cells of A. angustifolia was determined. The application of chitosan polymers and chitosan oligomers induced an oxidative burst, and the elicitor activity increased with increasing DA. However, very highly acetylated chitosan oligomers had slightly decreased elicitor activities, and fully acetylated chitin oligomers barely elicited any oxidative burst. The rapid and transient oxidative burst induced by chitosans in suspension cultured cells of the gymnosperm A. angustifolia resembled the oxidative burst observed in suspension cultured cells of dicot and monocot angiosperm species.11-37 The levels of H2O2 reached were low when compared to other reports.11-36 However, when highly purified and homogeneous elicitors are

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used, the intensity of the oxidative burst induced can reach in some cases only 10% or less of that induced by crude elicitor preparations, suggesting the existence of additional signal perception mechanisms in the plant cell involved in the full induction of that response.38-40 In our experiments, chitosan polymers with DA 69% and chitosan oligomers with DA 78% elicited the highest generation of H2O2. To our knowledge, such a thorough comparison between chitosan polymers and oligomers of different DA has not been previously reported for the induction of an oxidative burst in suspension cultured cells, nor for any other of the many biological activities of chitosan. In chitosan-elicited leaves of Triticum aestiVum, the activity of enzymes related to plant defense, namely phenylalanine ammonia-lyase and peroxidase, increased with increasing DA of the chitosan.16 Conversion of chitin into chitosan by de-N-acetylation has been proposed as a fungal strategy to protect cell walls of pathogenic hyphae from hydrolysis by host chitinases, avoiding the generation of an autocatalytic defense response system in the invaded host tissue.14 The elicitor activity of chitosan in T. aestiVum is supposed to reside in the acetylated regions of the molecule since fully deacetylated chitosan is inactive.41 Also, fully acetylated chitin oligomers exhibited activity in T. aestiVum, while fully deacetylated chitosan oligomers did not.16 It is important to note that both the chitosan polymers and the chitosan oligomers we used were generated using chemical means of de-N-acetylation and re-N-acetylation, respectively. In both cases, the resulting chitosans are characterized by random patterns of acetylation (PA).33,34 Based on theoretical considerations, we have argued that the PA of chitosans is likely to have a major effect on the biological activities of both chitosan polymers and chitosan oligomers.42 However, experimental verification of this hypothesis awaits the generation and characterization of chitosans with nonrandom PA, for example, using chitosan modifying enzymes or chemical synthesis. The perception of chitin oligosaccharides released from fungal pathogens by plant cells can induce a broad range of plant defense reactions16-19 including the production of reactive oxygen species11-43 in many plant species. In Oryza satiVa, chitin oligosaccharides with DP between 6 and 8 induce plant defense reactions,19 and in Glycine max, the generation of reactive oxygen species was directly dependent on the degree of polymerization of the chitin oligomers.42 However, in the experiments with the gymnosperm cells reported here, almost no H2O2 generation was observed when chitin oligomers with DP ranging from 3 to 6 were used as elicitors. The presence of extracellular proteins showing chitinolytic activity prior to elicitor addition can be responsible for the degradation of the chitin oligomers, resulting in the low production of H2O2. Unfortunately, allosamidin did not inhibit the activity of these proteins, most likely because they belong to family 19 of chitinases, and thus, we could not confirm this hypothesis. The generation of reactive oxygen species is usually associated with the hypersensitive response following perception of pathogen avirulence signals.4 However, when cultivated for up to 72 h in the presence of 1-10 µg mL-1 chitosan of DA 69%, the most potent elicitor of an oxidative burst, suspension cultured cells of A. angustifolia did not show increases in the activities of extracellular plant PR proteins or phenylalanine ammonialyase. In contrast, the application of chitosan to suspension cultured cells of the gymnosperms Pinus taeda and Pinus elliottii induced the activation of defense responses, including lignification, increased activities of plant defense proteins (chitinases and glucanases), production of ethylene, and induction of gene

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products associated with inducible defense responses in angiosperms.18-2022-44 It is interesting to note that in suspension cultured cells of Oryza satiVa, the oxidative burst was not necessary for expression of Chitinase and phenylalanine ammonia-lyase genes,45 indicating that the rapid induction of oxidative burst and the induction of other defense reactions can be independent of each other, as also apparent in A. angustifolia.

Conclusion The rapid and transient production of huge amounts of reactive oxygen species, the so-called oxidative burst, is one the earliest reactions of plant cells against pathogen attack. In this report, the generation of hydrogen peroxide, one of the components of the oxidative burst, was quantified in suspension cultured cells of A. angustifolia elicited with partially acetylated chitosan oligomers and polymers of different degrees of acetylation, and chitin oligomers of different degrees of polymerization. We observed that H2O2 production was strongly dependent on the degree of acetylation of the chitosan oligoand polymers, with highly acetylated chitosans being most active. Interestingly, and in contrast to many other plant species, fully acetylated chitin oligomers were inactive as an elicitor. It remains to be seen whether failure to recognize chitin oligomers is a feature common to other gymnosperm species as well or unique to A. angustifolia. Acknowledgment. We are thankful to Dr. Shohei Sakuda for kindly providing the Chitinase inhibitor allosamidin, to Prof. Dr. Kjell M. Vårum for kindly providing the chitosan DA series, and to Dr. Kevin Hicks for kindly providing the chitin oligomer DP series. We also thank Uschi Beike and Imke Ortman for their technical assistance during A. angustifolia cell suspension culture establishment and hydrogen peroxide quantification, respectively. This work was supported by a German Academic Exchange Service (DAAD) fellowship to A.L.W.d.S. and by CAPES (Brazil).

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