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Elicitation of peroxidase activity and lignin biosynthesis in pepper suspension cells by Phytophthora capsici
J. Plant Physiol. 158. 151 – 158 (2001) Urban & Fischer Verlag http://www.urbanfischer.de/journals/jpp
Elicitation of peroxidase activity and lignin biosynthesis in pepper suspension cells by Phytophthora capsici Catalina Egea1, Ahmed Sid Ahmed2, Milagros Candela3, Maria Emilia Candela2 * 1 2 3
Department of Agrarian Production, E.T.S.I.A., Polytechnic University of Cartagena, Paseo Alfonso XIII no52, 30201 Cartagena, Spain Department of Plant Biology, Faculty of Biology, University of Murcia, Campus de Espinardo, 30100 Espinardo (Murcia), Spain Department of Genetics, Faculty of Biology, University Complutense of Madrid, 28040 Madrid, Spain
Summary Cell suspension cultures of three varieties of Capsicum annuum L., each with a different degree of sensitivity to the fungus Phytophthora capsici, responded to elicitation by both lyophilized mycelium and fungus filtrate with a hypersensitive reaction. They showed the synthesis or accumulation of PR-proteins with peroxidase (EC 1.11.1.7) activity and the accumulation of lignin-like polymer (as measured by derivatization with thioglycolic acid). The cultivation medium was optimised for both plant and fungus growth in order to avoid any stress during their combination. The resistant pepper variety, Smith-5, showed a more intense response to the elicitor preparations than the sensitive varieties, Americano and Yolo Wonder. This was particularly evident when the cell suspensions were elicited with the filtrate. After elicitation, the cell walls thickened through the accumulation of lignin, as can be observed by staining microscope preparations with methylene blue. Elicitation also reduced the level of total peroxidase activity in the susceptible varieties, while such activity increased in resistant varieties, and was accompanied by de novo expression of acidic peroxidase isoenzymes in the extracellular and cell wall fractions. Of note was the PR protein of pI 5.7 showing peroxidase activity, which was induced by both elicitor types in the elicited cell suspensions of the resistant variety alone, making it a marker of resistance. The increases in the activity of these peroxidases in the resistant variety are in concordance with the accumulation of lignin observed 24 h after inoculation by both elicitors from the fungus. The possible role of these isoenzymes in lignin biosynthesis, used to reinforce the cell walls against fungal penetration of the cells, is discussed. These results are in accordance with those previously observed in plant stem sections. Key words: Capsicum annuum – lignification – peroxidase – Phytophthora capsici – PR-proteins – resistance
Introduction The responses of plants to fungal infection are generally characterised by metabolic and structural changes asso* E-mail corresponding author: [email protected]
ciated with the occurrence of defence reactions. The fungus Phytophthora capsici L. causes blight in peppers (Capsicum annuum L.). A destructive phase of this disease, occurring at the crown of the stem, results in wilting and the development of a hypersensitive reaction (HR) as a defense response. Previous studies of the interaction between P. capsici and three 0176-1617/01/158/02-151 $ 15.00/0
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peppers cultivars, showing different degrees of susceptibility to the fungus, enable us to establish a method of studying and measuring factors involved in the resistance of plants to fungal attack. These studies demonstrated that the defensive reaction of C. annuum is involved in the pattern of phenolic acids showed by Candela et al. (1995), pathogenesis related proteins (PR) shown by Alcazar et al. (1995), as well as in the production of phytoalexin capsidiol shown by Egea et al. (1996 a). Genetic factors in both the host and target cells and the microorganism determine the specificity of these local responses and their rate of expression, which are often very effective in limiting the spread of the infection. During the investigation of the possible correlation between higher levels of resistance and a more rapid and intensive response, cell lines of three pepper cultivars were established in order to solve the problem of the high number of plants required for in vivo studies of stem cells. An additional advantage, shown by Dixon (1985), is that cell suspension cultures may be rapidly and uniformly exposed to exogenously added elicitors. Among the enzymes more directly involved in lignin biosynthesis are peroxidases, which have been studied by Gaspar et al. (1985) in different tissues and organs in relation to different physiological processes such as root formation, flower initiation, abscission, as well as response to wounding and disease. The present paper compares the defensive response in suspension-cultured cells of three pepper cultivars of varying sensitivity to Phytophthora capsici, a fungus responsible for cell necrosis in the so-called hypersensitive response (HR). The aim was to analyze the induction of peroxidase activity and cell wall thickening caused by lignin accumulation, and relate these activities with higher levels of resistance and a more intensive HR.
Materials and Methods Plant material and tissue-culture conditions Seeds from three Capsicum annuum varieties Smith-5 (S-5, resistant), Americano (intermediate), and Yolo Wonder (YW, susceptible) were surface sterilised by immersion in a 10 % calcium hypochlorite solution for 20 min, followed by four 5 min washes in sterile distilled water. Seeds were sown on the surface of MS, Murashige and Skoog (1962) medium, pH 5.8, supplemented with 3 % sucrose and 0.8 % (w/v) Difco agar, and germinated under 16 h photoperiod at 25 ˚C. Cotyledon explants from 5-week-old seedlings were excised in approximately 1-cm segments. Proximal and distal parts of cotyledons, with the upper surface in contact with the medium, were cultivated in 50-mL flasks containing MS medium supplemented with 5 % sucrose, 8 % PDA (potato-dextrose-agar), and 2,4-dichlorophenoxy acetic acid (2,4-D) (1mg L –1) (MS-PDA). Starting from friable callus (ca. 1 g), pepper cells were cultured in suspension in the above-described medium without agar (MS-PD), with orbital stirring at 125 rpm, for three weeks before being treated with elicitors. The cell suspension cultures were maintained in the
above conditions and subcultured every three weeks by transferring inocula to fresh media. Plant cell culture growth was measured as packed cell volume (PCV) and expressed as a percentage of the total volume. For the experiments, 100-mL flasks containing 20 mL of MSPD medium (with a PCV of 60 %) were elicited with hyphae or filtrate from the fungus. Each datum point represents the average of three flasks and each experiment was repeated at least twice.
Elicitor preparation and elicitation procedure The fungus Phytophthora capsici isolate 17 (Germoplasm Bank of CRIA, Consejería de Agricultura, Comunidad Autónoma de la Región de Murcia, Spain) was grown on solid (MS-PDA) medium. From a fiveday-old culture, 1 cm2 mycelium was inoculated on 100 mL liquid (MSPD) medium. The culture was kept at 25 ˚C for 30 days. Two kinds of elicitor were used: (a) lyophilized fungus mycelium (120 µg mL –1 of fresh medium) (M) and, (b) cell-free filtrate (F) filtered through a 0.2 µm Whatman GF/C filter disk. Checks were conducted to insure that the lyophilized mycelium did not continue to grow in a medium free of plant cells. Elicitation was performed by adding 10 mL of elicitor preparation (M or F) to 20 mL of aliquots from 3-week-old cell suspension cultures. Controls were carried out using the same medium without fungus, showed by Garcia-Pérez et al. (1998).
Microscope preparations Samples were taken from the elicited and non-elicited (control) cultures for microscopic examination. For this, one drop of cells from the healthy culture medium and from fungus-elicited medium was placed on a slide. Necrotic and healthy cells were taken from the latter and stained for optic microscope examination with 0.5 % aqueous solution of methylene blue for 1min, as described by Collins et al. (1989).
Analysis of PR-proteins The proteins were analyzed 0 and 24 h after elicitation with lyophilized mycelium or filtrate from the fungus in the cells and medium. The differential extraction of proteins revealed two fractions: an extracellular (from medium) and a cell wall fraction (from cells). Extracellular fraction: 2.5 volumes of 100 % ethanol were added to the medium (without cells) at 4 ˚C overnight to precipitate the proteins. This was followed by centrifugation at 10 000 × g for 30 min at 4 ˚C. The precipitates were considered as the extracellular fraction. Cell wall fraction: cells (2 g) were ground with liquid nitrogen using a mortar and pestle, and the fine powder extracted as described by Egea et al. (1996 b). The precipitate was resuspended five times in 7 mL of 0.05 mol/L acetate buffer, pH 5.0 in the presence of Tritón X–100 (1 % w/v), before being centrifuged at 1000 × g for 10 min at 4 ˚C. The resulting precipitates were washed three times in the same buffer without Triton X–100, and then centrifuged in the same way. The precipitate of this last centrifugation was considered as the purified cell wall fraction. To separate the enzyme bound to the cell walls, the fractions were incubated for 30 min at 4 ˚C in 0.05 mol/L acetate buffer, pH 5.0, containing 1mol/L KCl, and then centrifuged at 1000 × g for 10 min at 4 ˚C. The resulting supernatant was considered as the protein fraction ionically bound to the cell walls. The supernatants were dialysed over-
Elicitation of peroxidase activity and lignin biosynthesis in pepper suspension cells night at 4 ˚C with two changes of 0.05 mol/L acetate buffer, pH 5.0. The samples were stored at – 20 ˚C until use. Samples of two fractions were concentrated by centrifugation in Centripep 10 AMICON (Amicon, USA) before analysis. Total protein was measured according to the method of Lowry et al. (1951). The proteins from two fractions were analysed by isoelectric focusing (IEF) using PhastSystem separation equipment (Pharmacia, Uppsala, Sweden). IEF was carried out as described by Candela et al. (1994). A total of 5 µg protein was applied per line. To determine the pI, we made a calibrated straight line in which the marker pI was represented with respect to its own Rf. The standards from Pharmacia (Uppsala, Sweden) were in a gel parallel to the separation samples.
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lapse, accompanied by a substantial reduction in the packed cell volume. Differences were observed between the three varieties, especially between the resistant variety, S-5, and both sensitive varieties, A and YW. Differences according to the type of elicitation used (mycelium or filtrate) were also noted. In the case of S-5 inoculated with filtrate, HR was instantaneous, whereas in the sensitive varieties, in which slight cell necrosis was observed, the reaction occurred later and to a lesser degree (Fig. 1).
Microscope preparations Peroxidase assay Total peroxidase activity was assayed by spectrophotometry at 593 nm, as described by Alcazar et al. (1995), using 4-methoxy-αnaphtol (4-MN) as hydrogen donor. Peroxidase activity was expressed as µmol of substrate transformed × mg –1 of total protein x min –1. The results were showed as increase with respect to control (suspension cell not inoculated). Peroxidase isoenzymes were detected in gels by dipping the gels for 20 min in Petri dishes containing 20 mL of 0.1 mol/L Tris-acetate buffer, pH 5.0, 1 mmol/L 4-MN, and 0.33 mmol/L H2O2, and held in darkness at 25 ˚C. Peroxidase isoenzymes were developed as blue bands. The IEF gels were scanned for transmittance at 613 nm using a PhastImage Densitometer from Pharmacia (Uppsala, Sweden).
Determination of lignin-like polymers Lignin-like polymers were measured at 0 and 24 hours after elicitation with mycelium or filtrate; 4 g of cells were homogenized (Polytron) in 160 mL of absolute MeOH at top speed for 1 min. The homogenate was filtered through Whatman no 4 filter paper and the residue dried at 60 ˚C for 24 h. The dried alcohol-insoluble residue, which contained both true lignin and phenolic acids esterified to the cell walls, was used for lignin determination. For this, HCl (5 mL, 2 N) and 0.5 mL thioglycolic acid (TGA) were added to 100 mg of residue, and the mixture was placed in boiling water for 4 h. The mixture was then centrifuged at 10 000 × g for 10 min, and the precipitate was washed with 5 mL distilled water, and again centrifuged. The precipitate was suspended in 5 mL 0.5 N NaOH, shaken at room temperature for 2 h, and then centrifuged at 10 000 × g for 10 min. Concentrated HCl (1 mL) was added to the supernatant and the lignin-thioglycolic acid allowed to precipitate at 4 ˚C for 4 h. After centrifugation at 10 000 × g for 10 min, the precipitate was dissolved in 0.5 N NaOH, again centrifuged, and the absorbance of TGA derivatives in the supernatant was measured at 280 nm as described by Bruce and West (1989).
Results Cell response to elicitor The addition of filtrate or mycelium from P. capsici to pepper suspension cells produced a marked hypersensitive response (HR), which was characterised by rapid cell browning and col-
The healthy cells of the non-elicited cultures, or those from the non-necrotic area of elicited culture suspensions, had a similar appearance, (same size and shape in the 3 vars.) showing the cell membrane and the typical thin primary walls of cells in suspension (Fig. 2 A and 2 C). After inoculation, a defence response was exhibited in all varieties as a cell wall formation on the cell membrane. The principal difference was the extent of the necrotic reaction, higher in the Smith-5 cells (resistant) than in the Yolo Wonder cells (susceptible) (Fig. 2B and 2D). Although Smith-5 cell (Fig. 2D) appears like a differential xylem element, this could not be the case because the cells originated from a suspension cell culture where the cells were not part of a tissue. The changes in the cell surface appear only after the inoculation with the fungus elicitor. However, the cells from necrotic areas had brown walls that had rapidly thickened due to the accumulation of (possibly) lignin-like polymers. To identify these accumulations, we carried out a lignin analysis and an analysis of the enzymes involved in lignin biosynthesis (such as peroxidases).
PR-proteins with peroxidase activity Proteins with peroxidase activity were detected in the extracellular and cell wall fractions of the three varieties of Capsicum annuum after elicitation with mycelium and filtrate from P. capsici (Figs. 3 – 6 and Tables 1 and 2).
Extracellular fraction After elicitation with both types of elicitor, total peroxidase activity increased (with respect to the values observed in nonelicited tissue) in S-5 (S-5C = 52.2, S-5IM = 166.1, S-5IF = 101.1 µmol (mg of total protein) –1) and A (AC = 56.2, AIM = 105.4, AIF = 91.2 µmol (mg of total protein) –1), while in YW it decreased (YWC = 99.2, YWIM = 93.2, YWIF = 86.9 µmol (mg of total protein) –1) (Fig. 3). This increase was very pronounced in var. S-5, where inoculation with mycelium and filtrate increased the activity 3.2 and 1.9 times, respectively, over pre-inoculation values. In var. Americano, the activity is 1.9 and 1.2 times. In YW, it decreased slightly with respect to control values.
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Catalina Egea et al. the PR-protein of pI 5.7, showing peroxidase activity which was detected in the cell suspensions of the resistant variety elicited with both elicitors (Fig. 4 and Table 1).
Cell wall fraction
Figure 1. Bottom view of flask containing liquid culture of Capsicum annuum, var. Susceptible Yolo Wonder (left) and var. Resistant Smith5 (right), 1 hour after inoculation with filtrate of Phytphthora capsici. H: Healthy cells, N: necrotic cells
Compared with the values observed in the non-elicited tissue (Fig. 5), total peroxidase activity in S-5 increased strongly when elicited with mycelium and filtrate (S-5C = 4.5, S-5IM = 12.0, S-5IF = 12.4 µmol (mg of total protein) –1). Such activity fell in both the other varieties (AC = 11.1, AIM = 7.7, AIF = 4.3 µmol (mg of total protein) –1) (YWC = 12.5, YWIM = 7.3, YWIF = 5.3 µmol (mg of total protein) –1), the fall in Americano and YW being more pronounced with filtrate. After IEF separation of peroxidases from cell wall fraction (Fig. 6), one basic (pI 9.0) and one acidic (pI 3.5) isoperoxidase were detected in all three varieties. Quantification of these isoenzymes (Table 2) showed that the pI 9.0 isoperoxidase increased its activity in var. S-5 after elicitation, while the same activity fell in the other two varieties after inoculation with filtrate, especially in A. Isoenzyme pI 3.5 activity was only induced with infection in S-5, while in Americano and YW, although it was present in healthy tissue, it fell upon elicitation. Isoenzymes pI 6.1 and 5.6 were detected in Americano and S-5 but not in YW. Again of note is the behaviour of isoenzyme pI 5.6 in var. S-5, which showed higher values with both elicitors than in var. Americano (Table 2).
Lignins production Lignin formation was ascertained by measuring the absorbance at 280 nm of the derived TGA (Fig. 7). The basal levels were higher in S-5 than in the other two varieties studied.
Figure 2. Optic micrographs of Capsicum annuum cells stained with methylene blue. A and C): The healthy cells of the non-elicited cultures of Yolo Wonder (susceptible) and Smith-5 (resistant), respectively. B and D): Cells of Yolo Wonder (susceptible) and Smith-5 (resistant), respectively, after inoculation with filtrate of P. capsici. Note the thickening of the cell wall in Smith-5 (D) resulting from the hypersensitive reaction with the fungus.
After IEF separation and staining for peroxidases activity (Fig. 4), four isoperoxidases were detected in the three varieties, one basic of pI 9.0, and three acidic of pI 4.1, 3.9, and 3.5. Although these isoenzymes were constitutive in all three varieties, densitometric quantification (Table 1) showed that their activity, particularly that of pI 9.0 and 4.1, rose considerably in S-5 whether elicited with mycelium or filtrate, while in YW such activity fell with both types of elicitation. Of note is
Table 1. Densitometric quantification of peroxidase isoenzymes from the extracellular fraction of suspension cell culture of Capsicum annuum vars. Smith-5 (S-5), Americano (A), and Yolo Wonder (YW), at 0 (C: control) and 24 h after inoculation with mycelium (IM) and filtrate (IF) from Phytophthora capsici, separated by IEF and stained with 4-MN. pI: isoelectric point. Values are in µmol (mg of total protein) –1. Each assay was repeated at least twice. Sample
pI 9.0
5.7
4.1
3.9
3.5
YW C YW IM YW IF
40.80 34.99 35.09
– – –
15.92 14.82 7.86
1.59 1.48 1.65
40.89 41.90 42.28
AC A IM A IF
13.88 42.24 31.29
– – –
1.77 18.29 14.91
0.82 4.68 2.37
39.75 40.20 42.58
S-5 C S-5 IM S-5 IF
15.40 53.18 42.30
– 5.32 4.95
5.58 49.32 17.79
– – 6.72
31.22 58.24 29.35
Elicitation of peroxidase activity and lignin biosynthesis in pepper suspension cells
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Figure 3. Changes in total peroxidase activity in the extracellular fraction of suspension cell culture of Capsicum annuum vars. Yolo Wonder (YW), Americano (A), and Smith-5 (S-5), inoculated with mycelium (IM) and filtrate (IF) of Phytophthora capsici. The changes are expressed as increase (positive numbers) or decrease (negative numbers) with respect to controls.
Discussion
Figure 4. Peroxidase isoenzymes from the extracellular fraction of a suspension cell culture of Capsicum annuum cvs. Smith-5 (S-5), Americano (A) and Yolo Wonder (YW), at 0 (C: control) and 24 h after inoculation with mycelium (IM) and filtrate (IF) from Phytophthora capsici, separated by IEF and stained with 4-MN. pI: isoelectric point.
There was an increase in absorbance in all three varieties after elicitation with mycelium or filtrate. In YW the increase was slight and did not differ with elicitor type, while in S-5 the increase was more pronounced after elicitation with filtrate. In this case, the basal levels practically doubled, and the final levels were approximately three times those observed in YW after elicitation with the same elicitor.
A complex series of cellular processes enables plants to defend themselves against fungal diseases. These processes apparently form an integrated set of resistance mechanisms, which are activated by the microorganism. When the host plant recognizes the microbial signal, target cells respond by inducing several local responses, including localized cell death, known as HR, deposition of callose and lignins, and the synthesis of phytoalexin and pathogenesis-related proteins showed by Lamb (1994) and Kuc (1995). Bowles (1990) showed that the induction of this defence response in plant tissues is presumed to be mediated by an initial recognition process between plant and pathogen, one involving the detection of signal molecules or elicitors by the plant cell. Cell suspension cultures of three varieties of Capsicum annuum, with different degrees of sensitivity to the fungus Phytophthora capsici, responded to elicitation by both lyophilised mycelium and fungus filtrate. The cells of all three pepper varieties produced a hypersensitive reaction when elicited by the fungus, which differed in the speed and the extent of necrosis (much faster in the resistant var. S-5) (Figs.1 and 2). Candela et al. (1995) showed that these differences were reflected in the length of necrotic tissue measured in the stems of the same varieties of plants. This confirmed the suitability of cell suspensions as a system for studying the defensive response in plants since they provide an unlimited source of repeatable material exempt from the fluctuations in the availability of “in vivo” cells. Analysis of the proteins with peroxidase activity confirmed that the cell suspensions elicited with both mycelium and filtrate synthesised or accumulated peroxidases, both in the extracellular medium (Figs. 3 – 4 and Table 1) and cell wall fraction (Figs. 5 – 6 and Table 2). This lends weight to the
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Figure 5. Changes in total peroxidase activity in the cell wall fraction of a suspension cell culture of Capsicum annuum vars. Yolo Wonder (YW), Americano (A) and Smith-5 (S-5), inoculated with mycelium (IM) and filtrate (IF) of Phytophthora capsici. The changes are expressed as increase (positive numbers) or decrease (negative numbers) with respect to controls.
Table 2. Densitometric quantification of peroxidase isoenzymes from the cell wall fraction of a suspension cell culture of Capsicum annuum vars. Smith-5 (S-5), Americano (A), and Yolo Wonder (YW), at 0 (C: control) and 24 h after inoculation with mycelium (IM) and filtrate (IF) from P. capsici, separated by IEF and stained with 4-MN. pI: isoelectric point. Values are in µmol (mg of total protein) –1. Each assay was repeated at least twice. Sample
Figure 6. Peroxidase isoenzymes from the cell wall fraction of a suspension cell culture of Capsicum annuum cvs. Smith-5 (S-5), Americano (A), and Yolo Wonder (YW), at 0 (C: control) and 24 h after inoculation with mycelium (IM) and filtrate (IF) from Phytophthora capsici, separated by IEF and stained with 4-MN. pI: isoelectric point.
theory that peroxidase plays an important function in secondary cell wall biosynthesis by polymerizing hydroxy and methoxycinnamic alcohols into lignin, and forming rigid cross-links between cellulose, pectic, hydroxyproline-rich glycoproteins, and lignin, as showed by Grisebach (1981). These events were observed in cultures of all three varieties studied, although with different degrees of intensity, and they agree with
pI 9.0
6.1
5.6
3.5
YW C YW IM YW IF
6.58 7.32 5.28
– – –
– – –
5.88 – –
AC A IM A IF
5.98 4.12 3.89
0.21 0.20 0.09
0.19 0.38 0.27
4.72 2.95 –
S-5 C S-5 IM S-5 IF
4.03 6.35 6.42
0.27 0.39 0.49
0.24 0.23 0.49
– 5.02 4.99
the observations of Mohan and Kolattukudy (1990), who detected a variation of peroxidase activity in both compatible and incompatible plant pathogen interactions. Thus, peroxidase may play a role in the defence response of plants to pathogen attack showed by Ye et al. (1990) and Graham and Graham (1991). In the interaction between Capsicum annuum and P. capsici, a variation in extracellar peroxidase activity was observed in the three varieties studied after inoculation with both elicitors. The greatest change in peroxidase activity (Fig. 3) was found in S-5 in which the presence of the isoenzyme of pI 5.7 was of note. The fact that the isoenzyme was only detected in this variety after inoculation (but not in Ame-
Elicitation of peroxidase activity and lignin biosynthesis in pepper suspension cells
Figure 7. Induction of phenol polymer/lignin deposition in a suspension cell culture of Capsicum annuum vars. Smith-5 (S-5), Americano (A), and Yolo Wonder (YW), at 0 (C: control) and 24 h after inoculation with mycelium (M) and filtrate (F) from Phytophthora capsici.
ricano or YW) (Fig. 4 and Table 1) suggests that it might serve as a marker of resistance to P. capsici. As regards the cell wall fraction, the variations in activity with respect to the control were more pronounced in S-5 than in A and YW in which there is a decreasing with both types of inoculation (Fig. 5). One reason for this may be the disappearance of the activity from the cell wall in the most susceptible variety (YW). This could be due to the inactivation of these isoenzymes during lignin formation because it has been reported by Ferrer and Ros Barcelo (1994) that peroxidase turnover and inactivation is favoured during the oxidation of phenols. Peroxidases have been found in all vascular (lignifying) plants studied so far. Secretory (vacuolar and cell wall located) plant peroxidases are glycoproteins, and are acid or basic depending on their pI. While basic peroxidases are simultaneously located in the cell wall and vacuoles, acidic peroxidases are normally restricted to the cell wall. Acidic peroxidases have a greater affinity for cinnamyl alcohol than basic peroxidases, and their reactivity with it is stronger as showed by Ros Barcelo et al. (1997). It seems that peroxidases may play a certain role in the polymerization of cinnamyl alcohols because inmunocytochemical studies by Kim et al. (1988) have revealed that peroxidase is located in lignifying cell walls. Similarly, our results suggest that the isoperoxidases detected in the cell wall of the resistant variety (Table 2) are important for lignin production since the increase in activity with respect to the control was more pronounced than in the sensitive variety (Fig. 5). However, although quantitative correlations between peroxidase activity and lignification have frequently been reported in the literature, e.g. lignifying poplar tissues (Baier et al. 1993), lignifying needles from Norway spruce (Polle et al. 1994) or lignifying Zinnia tracheary elements (Sato et al. 1995), in no case was a specific peroxidase, basic or acidic, exclusively involved in the correlation
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between lignification and peroxidase activity. From all these studies emerged the idea that more than one individual peroxidase isoenzyme may participate in lignification. This observation may perhaps explain why in the C. annuum – P. capsici interaction different peroxidase isoenzymes are induced in both the cell wall and extracellular fraction. The accumulation of peroxidase in suspension culture cells from var. S-5 might be a sign of a defense reaction prior to fungal invasion, and suggests that its catalytic capacity acts as a barrier against infection by increasing lignification reactions. Indeed, the reinforcement of cell walls as measured by lignin production is three times greater in suspension cells from the resistant variety than in the sensitive variety (Fig. 7). We consider the synthesis of lignin in elicited cultures to be a plant disease resistance mechanism that is induced by the elicitor. In conclusion, when cell suspensions of Capsicum annuum were elicited with two different P. capsici elicitors, the peroxidase activity of the resistant variety increased. This may be related to lignin biosynthesis, which might prevent fungal penetration in the cell, thus slowing down the infection process. This did not occur in the sensitive varieties, in which the advance of the fungus was unstoppable. Acknowledgements. This work was partly supported by the Project PB98-0377 from CICYT Spain.
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Egea C, Alcázar MD, Candela ME (1996 b) β-1,3-glucanase and chitinase activities as pathogenesis-related proteins in the defense reaction of Capsicum annuum cultivars infected with cucumber mosaic virus. Biol Plantarum 38: 437– 443 Ferrer MA, Ros Barcelo A (1994) Inactivation of cell wall acidic peroxidase isoenzymes during the oxidation of coniferyl alcohol in Lupinus. Phytochem 36: 1161–1163 García-Pérez MD, Egea C, Candela ME (1998) Defense response of pepper (Capsicum annuum) suspension cells to Phytophthora capsici. Physiol Plant 103: 527– 533 Gaspar T, Penel C, Castillo FJ, Greppin H (1985) A two-step control of basic and acidic peroxidases and its significance for growth and development. Physiol Plant 64: 418 – 423 Graham MY, Graham TL (1991) Rapid accumulation of anionic peroxidases and phenolic polymers in soybean cotyledon tissue following treatment with Phytophthora megasperma f. sp. glycinea wall glucan. Plant Physiol 97: 1445–1455 Grisebach H (1981) Lignin. In: Conn EE (ed) The Biochemistry of Plants, Academic Press, New York. Vol 7, pp 451– 478 Kim SH, Terry ME, Hoops P, Dauwalder M, Roux SJ (1988) Production and characterization of monoclonal antibodies to wall-localizaed peroxidases from corn seedlings. Plant Physiol 88: 1446–1453 Kuc JA (1995) Phytoalexin, stress, metabolism, and disease resistance in plants. Annu Rev Phytopathol 33: 275 – 297
Lamb CJ (1994) Plant disease resistance genes in signal perception and transduction. Cell 76: 419 – 422 Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein measurement with the Folin phenol reagent. J Biol Chem 193: 265 – 275 Mohan R, Kolattukudy PE (1990) Differential expression of a suberization-associated anionic peroxidase gene in near-isogenic resistant and sensitive tomato lines by elicitors of Verticillium albo-atrum. Plant Physiol 92: 276 – 280 Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassay with tobacco tissue cultures. Physiol Plant 15: 473 – 497 Polle A, Otter T, Seifert F (1994) Apoplastic peroxidases and lignification in needles of Norway spruce (Picea abies L.). Plant Physiol 106: 53 – 60 Ros Barceló A (1997) Lignification in Plant Cell Walls. International Review of Cytology 176: 87–132 Sato Y, Sugiyama M, Komamine A, Fukuda H (1995) Separation and characterization of the isoenzymes of the wall-bound peroxidase from cultured Zinnia cells during tracheary elements differentiation. Planta 196: 141–147 Ye XS, Pan SQ, Kuc J (1990) Activity, isoenzyme pattern and cellular localization of peroxidase as related to systemic resistance of tobacco to blue mold (Peronospora tabacina). Phytopathol 80: 1295–1299
Elicitation of peroxidase activity and lignin biosynthesis in pepper suspension cells by Phytophthora capsici Catalina Egea1, Ahmed Sid Ahmed2, Milagros Candela3, Maria Emilia Candela2 * 1 2 3
Department of Agrarian Production, E.T.S.I.A., Polytechnic University of Cartagena, Paseo Alfonso XIII no52, 30201 Cartagena, Spain Department of Plant Biology, Faculty of Biology, University of Murcia, Campus de Espinardo, 30100 Espinardo (Murcia), Spain Department of Genetics, Faculty of Biology, University Complutense of Madrid, 28040 Madrid, Spain
Summary Cell suspension cultures of three varieties of Capsicum annuum L., each with a different degree of sensitivity to the fungus Phytophthora capsici, responded to elicitation by both lyophilized mycelium and fungus filtrate with a hypersensitive reaction. They showed the synthesis or accumulation of PR-proteins with peroxidase (EC 1.11.1.7) activity and the accumulation of lignin-like polymer (as measured by derivatization with thioglycolic acid). The cultivation medium was optimised for both plant and fungus growth in order to avoid any stress during their combination. The resistant pepper variety, Smith-5, showed a more intense response to the elicitor preparations than the sensitive varieties, Americano and Yolo Wonder. This was particularly evident when the cell suspensions were elicited with the filtrate. After elicitation, the cell walls thickened through the accumulation of lignin, as can be observed by staining microscope preparations with methylene blue. Elicitation also reduced the level of total peroxidase activity in the susceptible varieties, while such activity increased in resistant varieties, and was accompanied by de novo expression of acidic peroxidase isoenzymes in the extracellular and cell wall fractions. Of note was the PR protein of pI 5.7 showing peroxidase activity, which was induced by both elicitor types in the elicited cell suspensions of the resistant variety alone, making it a marker of resistance. The increases in the activity of these peroxidases in the resistant variety are in concordance with the accumulation of lignin observed 24 h after inoculation by both elicitors from the fungus. The possible role of these isoenzymes in lignin biosynthesis, used to reinforce the cell walls against fungal penetration of the cells, is discussed. These results are in accordance with those previously observed in plant stem sections. Key words: Capsicum annuum – lignification – peroxidase – Phytophthora capsici – PR-proteins – resistance
Introduction The responses of plants to fungal infection are generally characterised by metabolic and structural changes asso* E-mail corresponding author: [email protected]
ciated with the occurrence of defence reactions. The fungus Phytophthora capsici L. causes blight in peppers (Capsicum annuum L.). A destructive phase of this disease, occurring at the crown of the stem, results in wilting and the development of a hypersensitive reaction (HR) as a defense response. Previous studies of the interaction between P. capsici and three 0176-1617/01/158/02-151 $ 15.00/0
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peppers cultivars, showing different degrees of susceptibility to the fungus, enable us to establish a method of studying and measuring factors involved in the resistance of plants to fungal attack. These studies demonstrated that the defensive reaction of C. annuum is involved in the pattern of phenolic acids showed by Candela et al. (1995), pathogenesis related proteins (PR) shown by Alcazar et al. (1995), as well as in the production of phytoalexin capsidiol shown by Egea et al. (1996 a). Genetic factors in both the host and target cells and the microorganism determine the specificity of these local responses and their rate of expression, which are often very effective in limiting the spread of the infection. During the investigation of the possible correlation between higher levels of resistance and a more rapid and intensive response, cell lines of three pepper cultivars were established in order to solve the problem of the high number of plants required for in vivo studies of stem cells. An additional advantage, shown by Dixon (1985), is that cell suspension cultures may be rapidly and uniformly exposed to exogenously added elicitors. Among the enzymes more directly involved in lignin biosynthesis are peroxidases, which have been studied by Gaspar et al. (1985) in different tissues and organs in relation to different physiological processes such as root formation, flower initiation, abscission, as well as response to wounding and disease. The present paper compares the defensive response in suspension-cultured cells of three pepper cultivars of varying sensitivity to Phytophthora capsici, a fungus responsible for cell necrosis in the so-called hypersensitive response (HR). The aim was to analyze the induction of peroxidase activity and cell wall thickening caused by lignin accumulation, and relate these activities with higher levels of resistance and a more intensive HR.
Materials and Methods Plant material and tissue-culture conditions Seeds from three Capsicum annuum varieties Smith-5 (S-5, resistant), Americano (intermediate), and Yolo Wonder (YW, susceptible) were surface sterilised by immersion in a 10 % calcium hypochlorite solution for 20 min, followed by four 5 min washes in sterile distilled water. Seeds were sown on the surface of MS, Murashige and Skoog (1962) medium, pH 5.8, supplemented with 3 % sucrose and 0.8 % (w/v) Difco agar, and germinated under 16 h photoperiod at 25 ˚C. Cotyledon explants from 5-week-old seedlings were excised in approximately 1-cm segments. Proximal and distal parts of cotyledons, with the upper surface in contact with the medium, were cultivated in 50-mL flasks containing MS medium supplemented with 5 % sucrose, 8 % PDA (potato-dextrose-agar), and 2,4-dichlorophenoxy acetic acid (2,4-D) (1mg L –1) (MS-PDA). Starting from friable callus (ca. 1 g), pepper cells were cultured in suspension in the above-described medium without agar (MS-PD), with orbital stirring at 125 rpm, for three weeks before being treated with elicitors. The cell suspension cultures were maintained in the
above conditions and subcultured every three weeks by transferring inocula to fresh media. Plant cell culture growth was measured as packed cell volume (PCV) and expressed as a percentage of the total volume. For the experiments, 100-mL flasks containing 20 mL of MSPD medium (with a PCV of 60 %) were elicited with hyphae or filtrate from the fungus. Each datum point represents the average of three flasks and each experiment was repeated at least twice.
Elicitor preparation and elicitation procedure The fungus Phytophthora capsici isolate 17 (Germoplasm Bank of CRIA, Consejería de Agricultura, Comunidad Autónoma de la Región de Murcia, Spain) was grown on solid (MS-PDA) medium. From a fiveday-old culture, 1 cm2 mycelium was inoculated on 100 mL liquid (MSPD) medium. The culture was kept at 25 ˚C for 30 days. Two kinds of elicitor were used: (a) lyophilized fungus mycelium (120 µg mL –1 of fresh medium) (M) and, (b) cell-free filtrate (F) filtered through a 0.2 µm Whatman GF/C filter disk. Checks were conducted to insure that the lyophilized mycelium did not continue to grow in a medium free of plant cells. Elicitation was performed by adding 10 mL of elicitor preparation (M or F) to 20 mL of aliquots from 3-week-old cell suspension cultures. Controls were carried out using the same medium without fungus, showed by Garcia-Pérez et al. (1998).
Microscope preparations Samples were taken from the elicited and non-elicited (control) cultures for microscopic examination. For this, one drop of cells from the healthy culture medium and from fungus-elicited medium was placed on a slide. Necrotic and healthy cells were taken from the latter and stained for optic microscope examination with 0.5 % aqueous solution of methylene blue for 1min, as described by Collins et al. (1989).
Analysis of PR-proteins The proteins were analyzed 0 and 24 h after elicitation with lyophilized mycelium or filtrate from the fungus in the cells and medium. The differential extraction of proteins revealed two fractions: an extracellular (from medium) and a cell wall fraction (from cells). Extracellular fraction: 2.5 volumes of 100 % ethanol were added to the medium (without cells) at 4 ˚C overnight to precipitate the proteins. This was followed by centrifugation at 10 000 × g for 30 min at 4 ˚C. The precipitates were considered as the extracellular fraction. Cell wall fraction: cells (2 g) were ground with liquid nitrogen using a mortar and pestle, and the fine powder extracted as described by Egea et al. (1996 b). The precipitate was resuspended five times in 7 mL of 0.05 mol/L acetate buffer, pH 5.0 in the presence of Tritón X–100 (1 % w/v), before being centrifuged at 1000 × g for 10 min at 4 ˚C. The resulting precipitates were washed three times in the same buffer without Triton X–100, and then centrifuged in the same way. The precipitate of this last centrifugation was considered as the purified cell wall fraction. To separate the enzyme bound to the cell walls, the fractions were incubated for 30 min at 4 ˚C in 0.05 mol/L acetate buffer, pH 5.0, containing 1mol/L KCl, and then centrifuged at 1000 × g for 10 min at 4 ˚C. The resulting supernatant was considered as the protein fraction ionically bound to the cell walls. The supernatants were dialysed over-
Elicitation of peroxidase activity and lignin biosynthesis in pepper suspension cells night at 4 ˚C with two changes of 0.05 mol/L acetate buffer, pH 5.0. The samples were stored at – 20 ˚C until use. Samples of two fractions were concentrated by centrifugation in Centripep 10 AMICON (Amicon, USA) before analysis. Total protein was measured according to the method of Lowry et al. (1951). The proteins from two fractions were analysed by isoelectric focusing (IEF) using PhastSystem separation equipment (Pharmacia, Uppsala, Sweden). IEF was carried out as described by Candela et al. (1994). A total of 5 µg protein was applied per line. To determine the pI, we made a calibrated straight line in which the marker pI was represented with respect to its own Rf. The standards from Pharmacia (Uppsala, Sweden) were in a gel parallel to the separation samples.
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lapse, accompanied by a substantial reduction in the packed cell volume. Differences were observed between the three varieties, especially between the resistant variety, S-5, and both sensitive varieties, A and YW. Differences according to the type of elicitation used (mycelium or filtrate) were also noted. In the case of S-5 inoculated with filtrate, HR was instantaneous, whereas in the sensitive varieties, in which slight cell necrosis was observed, the reaction occurred later and to a lesser degree (Fig. 1).
Microscope preparations Peroxidase assay Total peroxidase activity was assayed by spectrophotometry at 593 nm, as described by Alcazar et al. (1995), using 4-methoxy-αnaphtol (4-MN) as hydrogen donor. Peroxidase activity was expressed as µmol of substrate transformed × mg –1 of total protein x min –1. The results were showed as increase with respect to control (suspension cell not inoculated). Peroxidase isoenzymes were detected in gels by dipping the gels for 20 min in Petri dishes containing 20 mL of 0.1 mol/L Tris-acetate buffer, pH 5.0, 1 mmol/L 4-MN, and 0.33 mmol/L H2O2, and held in darkness at 25 ˚C. Peroxidase isoenzymes were developed as blue bands. The IEF gels were scanned for transmittance at 613 nm using a PhastImage Densitometer from Pharmacia (Uppsala, Sweden).
Determination of lignin-like polymers Lignin-like polymers were measured at 0 and 24 hours after elicitation with mycelium or filtrate; 4 g of cells were homogenized (Polytron) in 160 mL of absolute MeOH at top speed for 1 min. The homogenate was filtered through Whatman no 4 filter paper and the residue dried at 60 ˚C for 24 h. The dried alcohol-insoluble residue, which contained both true lignin and phenolic acids esterified to the cell walls, was used for lignin determination. For this, HCl (5 mL, 2 N) and 0.5 mL thioglycolic acid (TGA) were added to 100 mg of residue, and the mixture was placed in boiling water for 4 h. The mixture was then centrifuged at 10 000 × g for 10 min, and the precipitate was washed with 5 mL distilled water, and again centrifuged. The precipitate was suspended in 5 mL 0.5 N NaOH, shaken at room temperature for 2 h, and then centrifuged at 10 000 × g for 10 min. Concentrated HCl (1 mL) was added to the supernatant and the lignin-thioglycolic acid allowed to precipitate at 4 ˚C for 4 h. After centrifugation at 10 000 × g for 10 min, the precipitate was dissolved in 0.5 N NaOH, again centrifuged, and the absorbance of TGA derivatives in the supernatant was measured at 280 nm as described by Bruce and West (1989).
Results Cell response to elicitor The addition of filtrate or mycelium from P. capsici to pepper suspension cells produced a marked hypersensitive response (HR), which was characterised by rapid cell browning and col-
The healthy cells of the non-elicited cultures, or those from the non-necrotic area of elicited culture suspensions, had a similar appearance, (same size and shape in the 3 vars.) showing the cell membrane and the typical thin primary walls of cells in suspension (Fig. 2 A and 2 C). After inoculation, a defence response was exhibited in all varieties as a cell wall formation on the cell membrane. The principal difference was the extent of the necrotic reaction, higher in the Smith-5 cells (resistant) than in the Yolo Wonder cells (susceptible) (Fig. 2B and 2D). Although Smith-5 cell (Fig. 2D) appears like a differential xylem element, this could not be the case because the cells originated from a suspension cell culture where the cells were not part of a tissue. The changes in the cell surface appear only after the inoculation with the fungus elicitor. However, the cells from necrotic areas had brown walls that had rapidly thickened due to the accumulation of (possibly) lignin-like polymers. To identify these accumulations, we carried out a lignin analysis and an analysis of the enzymes involved in lignin biosynthesis (such as peroxidases).
PR-proteins with peroxidase activity Proteins with peroxidase activity were detected in the extracellular and cell wall fractions of the three varieties of Capsicum annuum after elicitation with mycelium and filtrate from P. capsici (Figs. 3 – 6 and Tables 1 and 2).
Extracellular fraction After elicitation with both types of elicitor, total peroxidase activity increased (with respect to the values observed in nonelicited tissue) in S-5 (S-5C = 52.2, S-5IM = 166.1, S-5IF = 101.1 µmol (mg of total protein) –1) and A (AC = 56.2, AIM = 105.4, AIF = 91.2 µmol (mg of total protein) –1), while in YW it decreased (YWC = 99.2, YWIM = 93.2, YWIF = 86.9 µmol (mg of total protein) –1) (Fig. 3). This increase was very pronounced in var. S-5, where inoculation with mycelium and filtrate increased the activity 3.2 and 1.9 times, respectively, over pre-inoculation values. In var. Americano, the activity is 1.9 and 1.2 times. In YW, it decreased slightly with respect to control values.
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Cell wall fraction
Figure 1. Bottom view of flask containing liquid culture of Capsicum annuum, var. Susceptible Yolo Wonder (left) and var. Resistant Smith5 (right), 1 hour after inoculation with filtrate of Phytphthora capsici. H: Healthy cells, N: necrotic cells
Compared with the values observed in the non-elicited tissue (Fig. 5), total peroxidase activity in S-5 increased strongly when elicited with mycelium and filtrate (S-5C = 4.5, S-5IM = 12.0, S-5IF = 12.4 µmol (mg of total protein) –1). Such activity fell in both the other varieties (AC = 11.1, AIM = 7.7, AIF = 4.3 µmol (mg of total protein) –1) (YWC = 12.5, YWIM = 7.3, YWIF = 5.3 µmol (mg of total protein) –1), the fall in Americano and YW being more pronounced with filtrate. After IEF separation of peroxidases from cell wall fraction (Fig. 6), one basic (pI 9.0) and one acidic (pI 3.5) isoperoxidase were detected in all three varieties. Quantification of these isoenzymes (Table 2) showed that the pI 9.0 isoperoxidase increased its activity in var. S-5 after elicitation, while the same activity fell in the other two varieties after inoculation with filtrate, especially in A. Isoenzyme pI 3.5 activity was only induced with infection in S-5, while in Americano and YW, although it was present in healthy tissue, it fell upon elicitation. Isoenzymes pI 6.1 and 5.6 were detected in Americano and S-5 but not in YW. Again of note is the behaviour of isoenzyme pI 5.6 in var. S-5, which showed higher values with both elicitors than in var. Americano (Table 2).
Lignins production Lignin formation was ascertained by measuring the absorbance at 280 nm of the derived TGA (Fig. 7). The basal levels were higher in S-5 than in the other two varieties studied.
Figure 2. Optic micrographs of Capsicum annuum cells stained with methylene blue. A and C): The healthy cells of the non-elicited cultures of Yolo Wonder (susceptible) and Smith-5 (resistant), respectively. B and D): Cells of Yolo Wonder (susceptible) and Smith-5 (resistant), respectively, after inoculation with filtrate of P. capsici. Note the thickening of the cell wall in Smith-5 (D) resulting from the hypersensitive reaction with the fungus.
After IEF separation and staining for peroxidases activity (Fig. 4), four isoperoxidases were detected in the three varieties, one basic of pI 9.0, and three acidic of pI 4.1, 3.9, and 3.5. Although these isoenzymes were constitutive in all three varieties, densitometric quantification (Table 1) showed that their activity, particularly that of pI 9.0 and 4.1, rose considerably in S-5 whether elicited with mycelium or filtrate, while in YW such activity fell with both types of elicitation. Of note is
Table 1. Densitometric quantification of peroxidase isoenzymes from the extracellular fraction of suspension cell culture of Capsicum annuum vars. Smith-5 (S-5), Americano (A), and Yolo Wonder (YW), at 0 (C: control) and 24 h after inoculation with mycelium (IM) and filtrate (IF) from Phytophthora capsici, separated by IEF and stained with 4-MN. pI: isoelectric point. Values are in µmol (mg of total protein) –1. Each assay was repeated at least twice. Sample
pI 9.0
5.7
4.1
3.9
3.5
YW C YW IM YW IF
40.80 34.99 35.09
– – –
15.92 14.82 7.86
1.59 1.48 1.65
40.89 41.90 42.28
AC A IM A IF
13.88 42.24 31.29
– – –
1.77 18.29 14.91
0.82 4.68 2.37
39.75 40.20 42.58
S-5 C S-5 IM S-5 IF
15.40 53.18 42.30
– 5.32 4.95
5.58 49.32 17.79
– – 6.72
31.22 58.24 29.35
Elicitation of peroxidase activity and lignin biosynthesis in pepper suspension cells
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Figure 3. Changes in total peroxidase activity in the extracellular fraction of suspension cell culture of Capsicum annuum vars. Yolo Wonder (YW), Americano (A), and Smith-5 (S-5), inoculated with mycelium (IM) and filtrate (IF) of Phytophthora capsici. The changes are expressed as increase (positive numbers) or decrease (negative numbers) with respect to controls.
Discussion
Figure 4. Peroxidase isoenzymes from the extracellular fraction of a suspension cell culture of Capsicum annuum cvs. Smith-5 (S-5), Americano (A) and Yolo Wonder (YW), at 0 (C: control) and 24 h after inoculation with mycelium (IM) and filtrate (IF) from Phytophthora capsici, separated by IEF and stained with 4-MN. pI: isoelectric point.
There was an increase in absorbance in all three varieties after elicitation with mycelium or filtrate. In YW the increase was slight and did not differ with elicitor type, while in S-5 the increase was more pronounced after elicitation with filtrate. In this case, the basal levels practically doubled, and the final levels were approximately three times those observed in YW after elicitation with the same elicitor.
A complex series of cellular processes enables plants to defend themselves against fungal diseases. These processes apparently form an integrated set of resistance mechanisms, which are activated by the microorganism. When the host plant recognizes the microbial signal, target cells respond by inducing several local responses, including localized cell death, known as HR, deposition of callose and lignins, and the synthesis of phytoalexin and pathogenesis-related proteins showed by Lamb (1994) and Kuc (1995). Bowles (1990) showed that the induction of this defence response in plant tissues is presumed to be mediated by an initial recognition process between plant and pathogen, one involving the detection of signal molecules or elicitors by the plant cell. Cell suspension cultures of three varieties of Capsicum annuum, with different degrees of sensitivity to the fungus Phytophthora capsici, responded to elicitation by both lyophilised mycelium and fungus filtrate. The cells of all three pepper varieties produced a hypersensitive reaction when elicited by the fungus, which differed in the speed and the extent of necrosis (much faster in the resistant var. S-5) (Figs.1 and 2). Candela et al. (1995) showed that these differences were reflected in the length of necrotic tissue measured in the stems of the same varieties of plants. This confirmed the suitability of cell suspensions as a system for studying the defensive response in plants since they provide an unlimited source of repeatable material exempt from the fluctuations in the availability of “in vivo” cells. Analysis of the proteins with peroxidase activity confirmed that the cell suspensions elicited with both mycelium and filtrate synthesised or accumulated peroxidases, both in the extracellular medium (Figs. 3 – 4 and Table 1) and cell wall fraction (Figs. 5 – 6 and Table 2). This lends weight to the
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Figure 5. Changes in total peroxidase activity in the cell wall fraction of a suspension cell culture of Capsicum annuum vars. Yolo Wonder (YW), Americano (A) and Smith-5 (S-5), inoculated with mycelium (IM) and filtrate (IF) of Phytophthora capsici. The changes are expressed as increase (positive numbers) or decrease (negative numbers) with respect to controls.
Table 2. Densitometric quantification of peroxidase isoenzymes from the cell wall fraction of a suspension cell culture of Capsicum annuum vars. Smith-5 (S-5), Americano (A), and Yolo Wonder (YW), at 0 (C: control) and 24 h after inoculation with mycelium (IM) and filtrate (IF) from P. capsici, separated by IEF and stained with 4-MN. pI: isoelectric point. Values are in µmol (mg of total protein) –1. Each assay was repeated at least twice. Sample
Figure 6. Peroxidase isoenzymes from the cell wall fraction of a suspension cell culture of Capsicum annuum cvs. Smith-5 (S-5), Americano (A), and Yolo Wonder (YW), at 0 (C: control) and 24 h after inoculation with mycelium (IM) and filtrate (IF) from Phytophthora capsici, separated by IEF and stained with 4-MN. pI: isoelectric point.
theory that peroxidase plays an important function in secondary cell wall biosynthesis by polymerizing hydroxy and methoxycinnamic alcohols into lignin, and forming rigid cross-links between cellulose, pectic, hydroxyproline-rich glycoproteins, and lignin, as showed by Grisebach (1981). These events were observed in cultures of all three varieties studied, although with different degrees of intensity, and they agree with
pI 9.0
6.1
5.6
3.5
YW C YW IM YW IF
6.58 7.32 5.28
– – –
– – –
5.88 – –
AC A IM A IF
5.98 4.12 3.89
0.21 0.20 0.09
0.19 0.38 0.27
4.72 2.95 –
S-5 C S-5 IM S-5 IF
4.03 6.35 6.42
0.27 0.39 0.49
0.24 0.23 0.49
– 5.02 4.99
the observations of Mohan and Kolattukudy (1990), who detected a variation of peroxidase activity in both compatible and incompatible plant pathogen interactions. Thus, peroxidase may play a role in the defence response of plants to pathogen attack showed by Ye et al. (1990) and Graham and Graham (1991). In the interaction between Capsicum annuum and P. capsici, a variation in extracellar peroxidase activity was observed in the three varieties studied after inoculation with both elicitors. The greatest change in peroxidase activity (Fig. 3) was found in S-5 in which the presence of the isoenzyme of pI 5.7 was of note. The fact that the isoenzyme was only detected in this variety after inoculation (but not in Ame-
Elicitation of peroxidase activity and lignin biosynthesis in pepper suspension cells
Figure 7. Induction of phenol polymer/lignin deposition in a suspension cell culture of Capsicum annuum vars. Smith-5 (S-5), Americano (A), and Yolo Wonder (YW), at 0 (C: control) and 24 h after inoculation with mycelium (M) and filtrate (F) from Phytophthora capsici.
ricano or YW) (Fig. 4 and Table 1) suggests that it might serve as a marker of resistance to P. capsici. As regards the cell wall fraction, the variations in activity with respect to the control were more pronounced in S-5 than in A and YW in which there is a decreasing with both types of inoculation (Fig. 5). One reason for this may be the disappearance of the activity from the cell wall in the most susceptible variety (YW). This could be due to the inactivation of these isoenzymes during lignin formation because it has been reported by Ferrer and Ros Barcelo (1994) that peroxidase turnover and inactivation is favoured during the oxidation of phenols. Peroxidases have been found in all vascular (lignifying) plants studied so far. Secretory (vacuolar and cell wall located) plant peroxidases are glycoproteins, and are acid or basic depending on their pI. While basic peroxidases are simultaneously located in the cell wall and vacuoles, acidic peroxidases are normally restricted to the cell wall. Acidic peroxidases have a greater affinity for cinnamyl alcohol than basic peroxidases, and their reactivity with it is stronger as showed by Ros Barcelo et al. (1997). It seems that peroxidases may play a certain role in the polymerization of cinnamyl alcohols because inmunocytochemical studies by Kim et al. (1988) have revealed that peroxidase is located in lignifying cell walls. Similarly, our results suggest that the isoperoxidases detected in the cell wall of the resistant variety (Table 2) are important for lignin production since the increase in activity with respect to the control was more pronounced than in the sensitive variety (Fig. 5). However, although quantitative correlations between peroxidase activity and lignification have frequently been reported in the literature, e.g. lignifying poplar tissues (Baier et al. 1993), lignifying needles from Norway spruce (Polle et al. 1994) or lignifying Zinnia tracheary elements (Sato et al. 1995), in no case was a specific peroxidase, basic or acidic, exclusively involved in the correlation
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between lignification and peroxidase activity. From all these studies emerged the idea that more than one individual peroxidase isoenzyme may participate in lignification. This observation may perhaps explain why in the C. annuum – P. capsici interaction different peroxidase isoenzymes are induced in both the cell wall and extracellular fraction. The accumulation of peroxidase in suspension culture cells from var. S-5 might be a sign of a defense reaction prior to fungal invasion, and suggests that its catalytic capacity acts as a barrier against infection by increasing lignification reactions. Indeed, the reinforcement of cell walls as measured by lignin production is three times greater in suspension cells from the resistant variety than in the sensitive variety (Fig. 7). We consider the synthesis of lignin in elicited cultures to be a plant disease resistance mechanism that is induced by the elicitor. In conclusion, when cell suspensions of Capsicum annuum were elicited with two different P. capsici elicitors, the peroxidase activity of the resistant variety increased. This may be related to lignin biosynthesis, which might prevent fungal penetration in the cell, thus slowing down the infection process. This did not occur in the sensitive varieties, in which the advance of the fungus was unstoppable. Acknowledgements. This work was partly supported by the Project PB98-0377 from CICYT Spain.
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