levels of 4-hydroxystilbene-oxidizing isoperoxidases related to constitutive disease resistance in...

Phmt Cell, Tissue and Organ Culture 29: 63-70, 1992. © 1992 Kluwer Academic Publishers'. Printed in the Netherlands.

Levels of 4-hydroxystilbene-oxidizing isoperoxidases related to constitutive disease resistance in in vitro.cultured grapevine

Antonio A. Calder6n, Jos6 M. Zapata, Maria A. Pedrefio, Romualdo Mufioz & Alfonso Ros Barcel6* Department of Plant Biology, University of Murcia, Campus of Espinardo E-30071 Murcia, Spain (* requests for offprints)

Received 25 April 1991; accepted in revised form 28 October 1991

Key words: axillary bud culture, cell culture, peroxidase isoenzymes, viniferin synthesis, Vitis vinifera, (Vitis vinifera x Vitis rupestris) x Vitis riparia

Abstract

A zymographic screening of peroxidases (EC 1.11.1.7) capable of oxidizing 4-hydroxystilbene was carried out by means of the peroxidase-catalyzed oxidative coupling of 4-hydroxystilbene and 4- aminoantipyrine. This screening reveals that only a few isoperoxidases are active in oxidizing 4- hydroxystilbene to viniferin-type compounds in in vitro cultures of grapevine. Unlike total peroxidase activity measured with 4-methoxy-a-naphthol, the levels of total peroxidase activity measured using 4-hydroxystilbene are related to disease resistance against downy mildew in axillary bud cultures of Vitis vinifera and (Vitis vinifera x Vitis rupestris) x Vitis riparia. From this observation, and using the above zymographic assay, it was found that a 4-hydroxystilbene-oxidizing isoperoxidase was overexpressed in both leaves and stems of the hybrid in relation to the increase in disease resistance of this species. These results suggest that constitutive 4-hydroxystilbene-oxidizing isoperoxidases may participate through their role in viniferin synthesis in the constitutive resistance mechanism that grapevines show against downy mildew.

Abbreviations: 4-AAP-4-aminoantipyrine, HRP-horseradish peroxidase, 4-HS-hydroxystilbene, HSPrx - 4-hydroxystilbene-oxidizing peroxidase, 4-MN - 4-methoxy-c~-naphthol

Introduction

4-Hydroxystilbenes constitute a group of natural- ly occurring plant phenolics involved in prevent- ing diseases caused by fungal pathogens such as Botrytis cinerea (grey mould) and Plasmopara viticola (downy mildew) in grapevines (Langcake 1981). These compounds are considered to be the precursors of viniferins, a class of stress metabolites with strong antifungal activity (Langcake 1981). The synthesis of viniferins re- quires a set of enzymes. Resveratrol synthase cata- lyzes the synthesis of the stilbene backbone from phenylpropanoid precursors and malonyl-CoA

(Kindl 1986; Schr6der & Schr6der 1990). The subsequent oxidative coupling of resveratrol (la, scheme I) yielding ~-viniferin (IIa, scheme I) involves peroxidases (Langcake & Pryce 1977).

Resveratrol synthase is inducible under stress conditions (e.g. treatment with abiotic elicitors or pathogen attack) (Fritzemeier & Kindl 1981), while peroxidase appears to be a constitutive enzyme, with levels of expression more dependent on the developmental stage of the cells rather than on exogenous factors (Cuenca et al. 1989; Garcia-Florenciano et al. 1990).

Grapevine cell cultures are useful in studies on the oxidation of 4-hydroxystilbenes (Calder6n et

64

OH

HO ~, ~ la

SCHEME I

al. 1990a; Hoos & Blaich 1988). We have em- ployed these cultures as a standardized system to investigate the activity of peroxidases in catalyz- ing the oxidative coupling of two 4-hydroxystilbene (Ib, scheme I) backbones to viniferin- type compounds (IIb, scheme I) (Calder6n et al. 1990a). However, grape peroxidase exists in mul- tiple forms (isoenzymes) (Garcfa-Florenciano et al. 1990), and not all peroxidase isoenzymes might have the same activity in oxidizing 4- hydroxystilbenes. This specificity is a major problem in defining the functions of peroxidases in grapevines.

The purpose of the present study was to test the application of a zymographic assay of isoperoxidases active in oxidizing 4-hydroxystilbene (Calder6n et al. 1990b) to the screening of peroxidase isoenzymes from in vitro cultures of Vitis vinifera. Optimal reaction conditions were first checked. Evidence is presented which suggest that, similar to viniferins (Langcake 1981; Pool et al. 1981), 4-hydroxystilbene-oxidizing isoperoxidases from axillary bud cultures of (Vitis vinifera x Vitis rupestris) x Vitis riparia are overexpressed, compared with the Vitis vinifera parent species, in both leaves and stems of the hybrid in relation to the increase in disease resistance of this cultivar to downy mildew.

Materials and methods

Chemicals

4-Hydroxystilbene (4-HS) was obtained from Janssen Chimica (Beerse, Belgium). Its purity was checked by elemental analysis and mass spectroscopy. Dioxane and H20 2 were pur- chased from Merck (Darmstadt, Germany).

Horseradish peroxidase (grade I) was obtained from Boehringer Mannheim (Barcelona, Spain), 4-methoxy-a-naphthol (4-MN) from Aldrich- Chemie (Steinheim, Germany) and 4-aminoantipyrine (4-AAP) from Sigma (St. Louis, MO, USA). All other chemicals used in this work were obtained from various commercial sup- pliers, and were of the highest purity available.

Callus cultures and peroxidase fractions

Grapevine (Vitis vinifera cv. Muscat) callus cultures were kindly supplied by Drs. J.C. Pech and A. Latch6 (ENSA, Toulouse, France) and were cultured on a Murashige and Skoog basal salt medium (Murashige & Skoog 1962), supple- mented with 250mg 1-1 casein hydrolysate, 30g 1 -I sucrose, 2mg 1 ~ kinetin, 0.1mg 1 naphthalene acetic acid and the vitamins described by Morel (Morel 1970). Calli were grown in 250 ml flasks containing 100 ml of medium for 20 days at 25°C, and with a 14/10-h photoperiod o f 6 W m z

Soluble peroxidase fractions from grapevine callus were obtained as described elsewhere (Garcfa-Florenciano et al. 1991).

Axillary bud cultures and peroxidase fractions

Shoot cuttings of the following varieties were taken from greenhouse-grown vines and established as axillary bud cultures in the laboratory:

A) Vitis vintfera cv. Monastrell (clon 35), and B) 1203 C (Monastrell x Martin) x Gloria (clon 99), a hybrid resulting from the crossing of (Vitis vinifera x Vitis rupestris) x Vitis riparia.

Cultivar A was chosen for its intermediate susceptibility to the downy mildew Plasmopara viticola, while cultivar B was selected for a test of the expression of the resistance character. Cultivar B represented a hybrid between resistant -Vitis riparia cv. Gloria (Langcake 1981)-, and susceptible -Vitis vinifera cv. Monastrell genotypes.

Axillary bud cultures of both cultivars were established by the method described by Lee & Wetzstein (1988) with modifications. Nodal segments (ca. 10mm) were dissected from greenhouse-grown vines. Explants were surface steril-

ized with vigorous stirring for 10min in 7% (w/v) calcium hypochlorite containing 0.1% (w/ v) Tween 20, and then rinsed three times in sterile distilled water. Nodal segments were trimmed at both ends, and cultured in 25 x 250mm culture tubes containing 20ml of Murashige and Skoog medium (Murashige & Skoog 1962) modified for macroelements, which were at half strength. Plants were grown in vitro under a 14-h photoperiod of 15 W m - 2 of light intensity at 25°C.

Fully expanded leaves or entire stems of 1- month-old vitroplants were used in all the ex- periments. The tissue was homogenized in 0.2 M Na-phosphate buffer, pH 6.5, containing 6 mM ascorbic acid and 50 mg ml -~ polyvinylpolypyr- rolidone. The homogenate was centrifuged at 20,000 g for 20min and the supernatant was dialyzed overnight against 50 mM Tris-HCl buffer, pH 7.5, this constituting the peroxidase frac- tion used in all the corresponding studies.

Spectrophotometric measurements

These were performed using a Kontron Uvikon- 940 spectrophotometer. The assay of peroxidase activity using 4-MN as a substrate was carried out as previously described (Ferrer et al. 1990). The activity of peroxidase in oxidizing 4-HS to viniferin-type compounds was assayed as re- ported by Calder6n et al. (1990a).

lsoelectrofocusing and zymographic assays

Isoelectrofocusing (IEF) of horseradish and grapevine peroxidases was performed at 4°C using a 2103PS LKB power supply. Polyac- rylamide gels for disc-IEF were prepared and polymerized according to Calder6n et al. (1990b). Usually, 100 pA of solution containing the LKB Ampholines (pH 3.5-10) carrier, 60% sucrose and enzyme, were layered on each gel, and focusing of protein was allowed as already described (Calder6n et al. 1990b). To stain the peroxidase isoenzymes capable of oxidizing 4- HS, a staining solution consisting of 24 mM 4- AAP, 15% (v/v) dioxane, 0.25 mM 4-HS (prepared from a 10mM stock solution in HPLC- grade methanol), 5 mM H202, and 85 mM Na- phosphate-citrate-borate buffer, pH 7.5 or 3.0,

65

was used. Staining was carried out at 25°C for 20 min, and gels were destained in water. The color was stable for 3h. Controls were performed in the absence of 4-HS.

Staining of peroxidase isoenzymes with 4-MN was carried out as previously described (Ferrer et al. 1990). The stained gels were scanned at 620 nm using a Joyce-Loebl MK II scanner den- sitometer equipped with a linear transport. The relative activity of isoperoxidases was calculated from the area under each isoperoxidase peak, and this was measured with a planimeter. Stained gels were photographed on a CAMAG Reprostar ® illumination system using a Pixcolor AR film for color prints.

Results and discussion

Optimization of the screening reaction

The peroxidase-catalyzed oxidative coupling of 4-HS and 4-AAP at pH 7.5 yields a chromogenic product with an absorption maximum at 370- 380nm (Calder6n et al. 1990b). However, the use of this dye reaction for testing grapevine isoperoxidases capable of oxidizing 4-HS at optimal physiological conditions raises several meth- odological problems. On the one hand, grapevine isoperoxidases are located in vacuoles (Garcfa-Florenciano et al. 1991) with a vacuolar pH of around 3.0 (Moskowitz & Hrazdina 1981). On the other hand, 4-AAP can be oxidized at acidic pH by peroxidases to yield a dye product known as antipyrine red (Faust & Mikulewicz 1967), which may mask the peroxidase-catalyzed oxidative coupling of 4-HS and 4-AAP.

In an attempt to optimize the coupling reaction between 4-HS and 4-AAP, and to test its validity at the pH of the grapevine vacuolar sap, a study was performed on the visible spectrum characteristics of the reaction product. Results for pH 3.0 and pH 5.0 show that at both pH- values the dye product has an absorption maximum at 520 nm. Using the increases in absorbance at 520 nm within the first min as a measure of the oxidation rate of 4-HS by peroxidases, the rate of the peroxidase-catalyzed oxidative coupling of the two substrates was studied at different pH's. The results (Fig. 1), illustrate that at

66

u

C3

0 " - ~ E

o l -

1.50

1.00

0.50

0.00 2.0 3.0 4.0 5.0 6.0 7.0

pH

Fig. 1, Effect of the pH of assay media on the rate of formation of antipyrine red (A), and on the rate of formation of the dye product resulting from the oxidative coupling of 4-HS and 4-AAP (B), in reaction media composed of 24 mM 4-AAP, 5mM H20 2, 1 nkat horseradish peroxidase, in 85 mN Na-phosphate-citrate-borate buffer of variable pH, and 15% (v/v) dioxane. In (B) the concentration of 4-HS was 0.25 mM. Oxidation rates were measured as increases in absorbance at 520 nm between 15 and 30 s after the reaction was begun by the addition of enzyme. Values are means -+SE of at least three independent determinations.

acidic pH it was possible to detect and to measure the formation of antipyrine red (curve A, Fig. 1), but that antipyrine red levels were insuf- ficient to mask the peroxidase-catalyzed oxidative coupling of 4-HS and 4-AAP (curve B, Fig. 1).

In addition, it was found that in contrast to pH 7.5, the assay of this enzymatic activity at pH 3.0 leads to an improvement in the detection limit of the assay from 0.20nkat to 0.02nkat as calculated according to Ingle (1974). These results suggest that the zymographic assay of isoperoxidases active in oxidizing 4-HS is also very sensitive at the acidic pH of the grapevine vacuolar sap.

In order to check this apparent improvement in sensitivity of the dye reaction with regard to the original method (Calder6n et al. 1990b), the screening of horseradish peroxidase isoenzymes capable of oxidizing 4-HS was carried out at pH 3.0 (Fig. 2A). By applying amounts of horseradish peroxidase (0.25 nkat) that are near the vi- sual detection limit at pH 7.5 (Fig. 2A, lane 3), all the horseradish peroxidase isoenzymes were clearly visible when the coupling reaction was carried out at pH 3.0 (arrowheads, Fig. 2A, lane 2). For both pH-values, the isoenzyme pattern was similar to that obtained with the highly specific and sensitive substrate 4-MN (Fig. 2A, lane 1).

Application of the reaction to isoperoxidases from grapevine cell cultures

When the staining reaction was tested in order to screen grapevine isoperoxidases isolated from Muscat cell cultures, an unsuspected substrate specificity was observed. Only three isoperoxidases (arrowheads, Fig. 2B, lane 2a) could oxidize 4-HS to a great extent. Other grapevine isoperoxidases (Fig. 2B, lane 1; isoperoxidases not marked) were incapable of oxidizing 4-HS, al- though some of these isoperoxidases were found in a proportion (judging by the stain with 4-MN) similar to that found for isoperoxidases active in oxidizing 4-HS.

A control for the 4-HS peroxidatic reaction was carried out at pH 3.0, since 4-AAP in itself can be oxidized by peroxidases to antipyrine red at acidic pH (Fig. 1). However, this control did not reveal isoperoxidase bands of sufficient color intensity (Fig. 2B, lane 2b) to mask the 4-HS- dependent peroxidatic reaction shown by Muscat grapevine isoperoxidases (Fig. 2B, lane 2a). In contrast to pH 3.0, Muscat grapevine isoperoxidases stained at pH 7.5 showed only faint yellow- orange bands at the limit of detection (Fig. 2B, lane 3).

Screening of grapevine isoperoxidases in axillary bud cultures that differ in disease resistance to downy mildew

In order to test the application of the assay to the screening of grapevine isoperoxidases involved in disease resistance through their partici- pation in viniferin synthesis, two grapevine cultivars were chosen for their different susceptibilities to the downy mildew Plasmopara viticola: a Monastrell cultivar (V. vinifera), of intermediate susceptibility and a highly resistant hybrid (cv. 1203 C) resulting from the crossing of Monastrell vines with V. riparia. These cultivars were established as axillary bud cultures in the laboratory (Fig. 3).

Unlike observations for total peroxidase activity measured with 4-methoxy-a-naphthol, the levels of basal peroxidase activity measured using 4-hydroxystilbene were related to disease resistance against downy mildew in axillary bud cultures of these grapevines (Table 1).

In order to test whether these differences were

67

Fig. 2B.

Fig. 2. Staining of horseradish (A) and Muscat grapevine (B) peroxidase isoenzymes with 4-MN (1), 4-HS and 4-AAP at pH 3.0 (2 and 2a), 4-AAP at pH 3.0 (2b), and 4-HS and 4-AAP at pH 7.5 (3). Vertical arrow indicates the direction of protein migration during isoelectrofocusing.

Fig,. 3. Excised nodes from both 1203 C (A) and Monastrell (B) cultivars grown in a modified Murashige & Skoog medium (see Materials and methods) after 1 month of culture.

68

Fig. 4. (A) Staining with 4-MN (1 and 3), 4-HS and 4-AAP at pH 3.0 (2a), and control at pH 3.0 in the absence of 4-HS (2b), of peroxidase isoenzymes from leaves (1, 2a and 2b), and stems (3) of the cultivar 1203 C. (B) Staining with 4-MN of peroxidase isoenzymes from leaves (la and lb) and stems (2a and 2b) of both 1203 C (la and 2a) and Monastrell (lb and 2b) cultivars. (C) Staining with 4-HS and 4-AAP at pH 3.0 (la) and control at pH 3.0 in the absence of 4-HS (lb) of peroxidase isoenzymes from Monastrell cultivar leaves. Vertical arrow indicates the direction of the protein migration during isoelectrofocusing. HSPrx = 4- hydroxystilbene-oxidizing peroxidase.

Table 1. Basal levels of total peroxidase activity (nkat g FW -~) measured using 4-methoxy-a-naphthol (4-MN) and 4-hydroxystilbene (4-HS), and levels of the activity (nkat g FW ~) of HSPrx in leaves and stems of susceptible (cv. Monastrell) and resistant (cv. 1203 C) grapevines.

Peroxidase HSPrx activity activity

4-MN 4-HS

leaves cv. Monastrell 342 ± 16 86 ± 4 29 ± 1 cv. 1203 C 461 ± 21 136 ± 6 58 + 3

stems cv. MonastrelI 39i ± f3 65 ± 2 25 -+ 2 cv. 1203 C 375 +- 14 117 ± 4 57 ± 3

due to a substrate specificity of the constitutive peroxidase isoenzymes, a screening of grapevine isoperoxidases capable of oxidizing 4 HS was carried out in both leaves and stems of 1 month- old vitroplants.

The results revealed that, as in grapevine cell cultures, soluble isoperoxidases extracted from both leaves and stems of the highly resistant cultivar 1203 C show a strong substrate specificity in the oxidation of 4-HS (Fig. 4A). Of all the wide array of constitutive isoenzymes present in leaves (Fig. 4A, lane 1), only a strong basic isoperoxidase was capable of oxidizing 4-HS to any great extent (Fig. 4A, arrowhead, lane 2a). An identical substrate specificity was also shown by isoperoxidases located in stems of the cv. 1203 C, which showed a similar isoenzyme pattern to that found in leaves (Fig. 4A, lane 3).

On the basis of the pI of this isoenzyme, this strong basic isoperoxidase was also identified in both leaves and stems of the Monastrell progenitor (Fig. 4B, arrowhead, lanes lb and 2b), this isoperoxidase also constituting in this cultivar the only isoenzyme capable of oxidizing 4-HS to any great extent (Fig. 4C), For this reason, this strong basic isoperoxidase present in leaves and stems of both cultivars was now named HSPrx.

Evidence of a relationship between the level of HSPrx and resistance of the two cultivars to downy mildew was obtained from quantification of densitometric recording of the isoenzyme patterns shown in Fig. 4B. Thus, it was found that the level of HSPrx in both leaves and stems was

69

significantly higher in cv. 1203 C than in the Monastrell progenitor (Table 1).

These results are in accordance with different susceptibilities of the two cultivars to downy mildew and seem to be related with the expression of the resistance introduced into the V. vinifera progenitor by the crossing with both V. riparia and V. rupestris genotypes. This is under- lined by the strong qualitative similarity between the isoenzyme patterns of both the hybrid (clon 99) and the susceptible parent species (Fig. 4B), an unusual fact even between different cultivars of the same Vitis species (manuscript in prepa- ration),

Finally, an attempt was made to correlate the levels of HSPrx in both the cv. 1203 C and the Monastrell progenitor with the ability of soluble protein extracts to oxidize 4-HS to viniferin-type compounds. The results showed a good correla- tion (r =0.957; statistically significant at p < 0.05) between the basal level of HSPrx (calculated from the isoenzyme pattern using 4-MN as substrate) and the ability of soluble protein extracts (measured spectrophotometrically) to ox idize 4-HS to viniferin-type compounds (Table 1)_

These results suggest that constitutive 4- hydroxystilbene-oxidizing isoperoxidases may participate through their role in viniferin synthe sis in the constitutive resistance mechanism that grapevines show against downy mildew.

Acknowledgements

This work was in part supported by a grant from the Comisi6n Interministerial de Ciencia y Tec nologia, Spain, project number A G R 296/89. A.A. Calder6n has a fellowship (BPP) from the Ministerio de Educaci6n y Ciencia. Thanks are given to Drs. J.C. Pech and A. Latch6 (ENSA, Toulouse, France) for providing the Muscat grape cell cultures, and to the Centro Regional de lnvestigaciones Agrarias of Murcia for providing the grapevine plants used in these studies.

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70

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levels of 4-hydroxystilbene-oxidizing isoperoxidases related to constitutive disease resistance in...

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