Changes in thylakoid protein patterns and antioxidant levels in two wheat cultivars with different sensitivity to sulfur dioxide

Environmental and Experimental Botany

ELSEVIER

Environmental and Experimental Botany 37 (1997) 125-135

Changes in thylakoid protein patterns and antioxidant levels in two wheat cultivars with different sensitivity to sulfur dioxide A. Ranieri a*, A. Castagna a, G. Lorenzini b, G. F.

Soldatini a

"Istituto di Chimica A#raria, Universit~ di Pisa, Via San Michele degli Scalzi 2, 1-56124 Pisa, Italy bDipartimento di CDSL, Sezione di Patologia Vegetale, Universit&di Pisa, Pisa, Italy

Received 14 April 1995; revised 6 March 1996; accepted 15 July 1996

Abstract Plants of two wheat cultivars (Triticum aestivum L., cultivars 'Mec' and 'Chiarano'), each with a different sensitivity to sulfur dioxide (SO2), were exposed continuously to 2, 23, 64 and 96 nl 1-~ SO2 for 4 months. To investigate whether the higher resistance to SO2 of Chiarano was due to increased activities of detoxification systems, some antioxidant enzymatic and non-enzymatic systems were analyzed. Moreover, to test whether the different responses of the two cultivars to SO2 were related to different changes at thylakoid level, we analyzed the thylakoid polypeptide patterns by two-dimensional electrophoresis. Two-dimensional gels of thylakoid membranes revealed a more pronounced generalized decrease of spot optical density in the more SO2-sensitive Mec; following SO2 fumigation, changes were observed in the area with molecular masses 26-29 kDa and isoelectric point (pI) 5-6 and molecular masses 24-27 kDa and pI 5.7-5.8. A significant decrease in optical density of a spot with a molecular mass of 32 kDa and pI 5.4, reported to be DI protein of the photosystem II reaction centre, was observed in the leaves of both cultivars fumigated with 96 nl 1- ~SO2. Catalase activity was unaffected by SO2 exposure. Following SO2 fumigation, superoxide dismutase activity decreased in Chiarano; but remained unchanged in Mec. Both Cultivars were found to increase guaiacol and ascorbic peroxidase activities as a response to SO2. The redox status of ascorbic acid was similar in both cultivars and was unaffected by SO2, However, the content of ascorbic acid was higher in the more tolerant Chiarano than in Mec at all SO2 concentrations. The data reported here confirmed the different sensitivities to SO2 of the two cultivars, as demonstrated by thylakoid protein analysis, and suggest that this difference depends on a differential ability to maintain elevated levels of ascorbic acid rather than on increasing detoxifying enzyme activities. © 1997 Elsevier Science B.V. Keywords: Antioxidants; SO2; Thylakoid proteins; Triticum aestivum; Wheat

1. IntroduCtion Sulfur dioxide (SO2)is a pollutant widely diffused in the world today. Its effects on vegetation and

*Corresponding author.

agriculture, and its role in the f o r m a t i o n o f acid rain, continue to be controversial. After penetration t h r o u g h the stomata, SO2 dissolves in the aqueous m e d i u m surrounding the plant cells, generating the toxic molecular species sulfite and bisulfite. Sulfite and bisulfite react directly with disulfide bridges o f structural and enzymatic proteins [1], causing

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A. Ranieri et al. / Environmental and Experimental Botany 37 (1997) 125-135

a decrease in their functions. The detoxification reaction of sulfite to sulfate, which takes place by reactions initiated by light and is mediated by the photosynthetic electron transport chain [2], leads to the formation of superoxide anions (O2"-), hydroxyl radicals (OH.) and hydrogen peroxide (H20~) [2]. These highly oxidant molecular species, together with the toxic sulfite, can damage the lipids and proteins of cell membranes [3]. Chloroplasts are one of the main targets for the action of SO2, which affects their functionality, inducing a loss of net CO2 assimilation and a decline in photosynthetic rate. This could be due either to an action on 'light' [4] or 'dark' photosynthetic reactions [5]. It has been reported that the toxic action of SO2 can act on the photosynthetic electron transport system, principally through molecular alteration of photosystem II (PSII) [4]; particularly affected is the D1 protein [6], which is an integral part of this photosystem and usually has a high turnover rate [7]. Previous research [8, 9] demonstrated that two wheat cultivars (Triticum aestivum L. cultivars 'Mec' and 'Chiarano') showed different sensitivities to SO2 applied for 4 months in a range of concentrations from 0 to 120 nl 1-1 [8, 9]. Mec showed greater sensitivity to the pollutant since it exhibited significant reductions in net CO2 assimilation, stomatal conductance and several growth parameters, while Chiarano was either unaffected or only slightly affected [9, 10]. Furthermore, in Chiarano, the content of the glutathione 'pool', one of the main antioxidant metabolites, was about twice as high as that of Mec [11]. Since the photosynthetic machinery is particularly sensitive to SO2 [12, 13], in the present study we have analyzed the thylakoid protein pattern by two-dimensional gel electrophoresis in these two differently SO2-sensitive wheat cultivars to test whether different changes occur at the thylakoid level. It is known that plants react to oxidative stressors by increasing some antioxidant systems [14-17]. We hypothesized that the enhancement of the activities of the detoxification system is responsible for the low sensitivity to SO2 of Chiarano, and conversely that the lack of this response explains the sensitivity to SO2 of Mec.

2. Materials and methods

2.1. Study design Seeds of two wheat cultivars ('Mec' and 'Chiarano') were planted in black polyethylene containers filled with an organic compost. Seven-dayold seedlings were moved to fumigation chambers and fumigated with the following concentrations of SO2 (average_SD): 2+0.3, 23__3.5, 64-t-4.8 and 96__+9.3 nl 1-1. SO2 was mixed with charcoal-filtered air entering the chambers by means of electron thermal mass flow controllers (Hastings) driven by a miroprocessor-controlled module (MKS 147B). The concentration of SO2 in the chambers was continuously sampled and monitored by an automatic analyzer based on a fluorescence method (Model 8850; Monitor Labs, San Diego, CA, USA) [18]. After 4 months of continuous fumigation, when the plants had reached the post-anthesis stage, the flag leaves of all the plants were collected together for each treatment and subsequently divided into three fractions. The fumigation experiment was performed once.

2.2. Chloroplast and thylakoid isolation Chloroplasts were extracted in a grinding medium consisting of 350 mM sucrose, 25 mM HepesKOH pH 7.6 and 2 mM ethylenediaminetetraacetic acid (EDTA), and held at - 20°C until an ice-slurry formed. Fresh leaves were homogenized in the isolation buffer (1:5, w/v) using a Polytron homogenizer (2 x 3 s bursts at 75% full speed). The homogenate was filtered through eight layers of muslin and centrifuged twice at 4000 x 9 for 1 min at 0°C. Chloroplast intactness was assessed using phase contrast microscopy (n = 3). To obtain thylakoid membranes, the pellet containing the chloroplasts was resuspended in 10 mM Tris-HCl (pH 7.0), incubated on ice for 5 min and centrifuged for 5 min at 5000 x # at 4°C. The pellet was collected and washed twice in 10 mM Tricine-NaOH (pH 7.0), 300 mM sucrose and 5 mM MgCI2 to remove stromal proteins (n = 3) [19].

A. Ranieri et al. / Environmental and Experimental Botany 37 (1997) 125-135

2.3. Two-dimensional electrophores& of thylakoid membranes Acetone-depigmented thylakoids were separated by two-dimensional electrophoresis (2-D PAGE) according to the method of O'Farrell [20], modified according to Hochstrasser et al. [21], to improve the resolution of the protein spots as follows: the dried thylakoid pellet was solubilized (1:1, w/v) in an isoelectrofocusing buffer (0.1 g dithiothreitol, /DTT), 0.4 g 3-[(3-cholamidopropyl)-dimethylammonio]-l-propane-sulfonate, 5.4 g urea, 300 #1 ampholine pH 5-8, 200 #1 ampholine pH 3.5-10, 6.5 ml distilled H20); after centrifugation the supernatant was loaded onto isoelectrofocusing (IEF) rod gels (1.5 mm i.d.). For comparison among the treatments, an equal amount of protein was loaded onto each IEF gel. The best resolution of polypeptide spots was obtained by loading 25 #g of protein. Isoelectrofocusing was performed at a controlled constant temperature (18°C) with a constant voltage of 200 V for 2 h, followed by 500 V for 5 h and 1000 V overnight, using 10 mM H3PO4 as the anode solution and 100 mM NaOH as the cathode solution. The IEF gels were then equilibrated for a few seconds in 125 mM Tris-HC1 (pH 6.8) containing 4% (w/v) sodium dodecyl sulfate (SDS). The second dimension was a SDS-PAGE, performed according to Laemmli [22] except that a 1020% acrylamide gradient was used. Silver-stained gels were scanned with a Molecular Dynamics computing densitometer 300B (Sunnyvale, CA, USA) using the software program MELANIE 1 for quantifying protein spots. Determination of the molecular mass and isoelectric point (pI) of the polypeptides was made using commercial standards (n=3).

2.4. Enzyme extraction Leaves (2 g) were homogenized with quartz sand, liquid nitrogen, 10% (w/w) polyvinylpyrrolidone and 5 ml of a grinding medium prepared as described below. The extraction buffer for superoxide dismutase (SOD), guaiacol peroxidase (POD) and catalase (CAT) contained 220 mM Tris-HC1 (pH 7.4), 250 mM sucrose, 50 mM KCI, 1 mM MgC12, 1% fl-mercaptoethanol and 0.01% (w/v)

127

phenylmethylsulfonyl fluoride (PMSF) [23]. The extraction of ascorbate peroxidase (AP) was performed according to the method of Foyer et al. [24], except that 1 mM ascorbate was added to the grinding medium to avoid enzyme inactivation; the supernatant was dialyzed against H20 for 2 h at 4°C. The grinding medium for AP contained 100 mM Tricine-NaOH (pH 8.0), 50 mM KCI, 10 mM MgCI2, 1 mM EDTA, 1 mM DTT, 1 mM ascorbic acid, 0.1% (w/v) Triton X-100 and 0.01% (w/v) PMSF. Each extract was centrifuged (24 000 × 9, 20 min, 2°C); the supernatants were passed through Sephadex G-25 columns (PD-10 columns; Pharmacia, Germany) and used for the assays. Enzyme extraction was performed three times for each enzyme tested.

2.5. Enzyme assays Superoxide dismutase (SOD) The assay was performed according to the method of Constantine et al. [25] based on the fact that the enzyme extract possesses the ability to inhibit the reduction of nitroblue tetrazolium (NBT). One unit of enzyme activity is defined as the amount of enzyme inhibiting 50% of NBT photoreduction.

Gua&col peroxidase (POD) The reaction medium contained 20 mM sodium acetate (pH 5.0), 30 mM H202, 2 mM guaiacol and an appropriate amount of enzyme extract. The rate of guaiacol oxidation was recorded at 470 nm [26]. One unit of enzymatic activity was defined as the amount of the enzyme which produced, under the assay conditions, a change in absorbance of 0.01 in 1 min. The activity was calculated using the extinction coefficient of 2.55 mmol-~ cm-~ for guaiacol.

Catalase (CA 73 The assay mixture contained 65 mM phosphate buffer, pH 7.2 (KH2POa-Na2HPO4), 12.5 mM H202 and a sufficient amount of the supernatant to allow a decrease in absorbance, due to the decomposition of H2Oz, from 0.450 to 0.400 at 240 nm in less than 60 s [27].

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Ascorbate peroxidase (AP) The activity was assayed following the decline in absorbance at 290 nm due to the oxidation of ascorbic acid in the first 30 s from the start of the reaction. An appropriate aliquot of supernatant was added to 100 mM Hepes-KOH (pH 7.8), 1 mM EDTA, 0.5 mM ascorbate and 0.1 mM H202 [28]. The activity was calculated using the extinction coefficient of 2.8 mmol-~ cm-~ for ascorbate. All enzymatic activities, with the exception of SOD and CAT values, were expressed as follows: 1 U (unit) is equivalent to 1 #mol of substrate transformed min-1 cm-l. SOD and CAT activities were expressed according to the definitions reported by Fridovich [29] and LUck [27], respectively. For POD and AP, SI units were used. All enzymatic activities were expressed as 'specific activity' (i.e. on a protein basis). 2.6. Protein content The protein concentration of each extract (enzyme and thylakoid samples) was determined according to Bradford [30] using the Bio-Rad Protein Assay Kit II, using bovine serum albumin as a standard.

2.7. Ascorbate (ASA) and dehydroascorbate (DHA) determination Leaves were homogenized in liquid nitrogen with 5 % metaphosphoric acid (1:2, w/v) and centrifuged at 14000 x # (20 min, 2°C). The supernatant was collected and used for the assay. ASA quantification is based on the reduction of Fe 3+ to Fe 2÷ by ASA and the consequent reaction of Fe 2÷ with ~,~'-bipyridyl to yield a pink complex that absorbs at 525 nm. Total ascorbate ( A S A + D H A ) was determined through the reduction of DHA to ASA by DTT [31].

2.8. Statistical analysis Enzyme activities, metabolite contents and 2-D analysis of thylakoid proteins were determined in triplicate for each sample. To determine whether a significant relationship exists between the SO2 treatments and the various measurements, analysis

of variance by a general linear model was performed using orthogonal components modified for unequal-spaced steps to test the trend. All main effects and interactions were tested for level of significance (P=0.05) using F statistics. Regression equations for enzymes and metabolites were determined, and the control values were compared by ttest [32].

3. Results

3.1. Two-dimensional analysis of thylakoid polypeptides Since 2-D electrophoretograms of the thylakoids from plants subjected to the lower and medium concentrations of SO2 (23 and 64 nl 1-l, respectively) did not show any significant changes compared with the control samples, two-dimensional analyses are reported here only for plants treated with the highest SO2 level (Figures 1 and 2). From densitometric analyses, silver-stained gels revealed a significant decline of about three and a half times in optical density of spots with a molecular mass of 32 kDa and pI of 5.4 following SO2 fumigation of Mec and Chiarano plants (Figures 1 and 2). However, the generalized decrease of the total spot density was about 51% in Mec and 10% in Chiarano in comparison to the respective controls (Figures 1 and 2). Large changes were observed in treated plants of Mec in the area with molecular masses 26-29 kDa and pI 5-6, and molecular masses 24-27 kDa and pI 5.7-5.8; other proteins either disappeared or were considerably reduced in the SO2-treated tissue.

3.2. Enzyme assays The interaction between cultivar and S02 treatment was significant for all the enzymes tested (Tables 1 and 2), so the response to SO2 differed between the two cultivars, with the exception of CAT activity. Increasing SO2 concentration significantly decreased SOD activity in Chiarano but had no effect on Mec (Figure 3). Peroxidase activities (POD and AP) were significantly stimulated by SO/treatment in both cultivars, the increase being

A. Ranieri et al. / Environmental and Experimental Botany 37 (1997) 125-135

a

129

b

$7_

32_

20~

kDa

7.2

pH

4.8

7.2

pH

4.8

Fig. 1. Two-dimensional silver-stained gels of thylakoid polypeptides from Mec plants treated with (a) filtered air or (b) 96 nl 1- mSO2. Arrows indicate spots with significant differences in optical density between the treatments.

a

b

$7_

32_

2OI

kDa

7.2

pH

4.8

7.2

pH

4.8

Fig. 2. Two-dimensional silver-stained gels of thylakoid polypeptides from Chiarano plants treated with (a) filtered air or (b) 96 nl 1SO2. Arrows indicate spots with significant differences in optical density between the treatments.

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Table 1 Results oforthogonal analysis of superoxide dismutase (SOD) and catalase (CAT) for SO2 treatment, cultivar treatment and interactions Source of variation

DF

SOD MS

F

CAT P

MS

F

P

SOs exposure Linear Quadratic Cubic

3 1 1 1

0.163 0.199 0.080 0.210

3.80 4.66 1.86 4.89

* * NS *

0.799 1.260 1.086 0.051

1.98 3.12 2.69 0.13

NS NS NS NS

Cultivar

1

6.505

151.62

**

224.726

557.08

**

SOs exposure x cultivar Linear x eultivar Quadratic x cultivar Cubic x cultivar

3 1 1 1

0.337 0.954 0.016 0.042

7.86 22.23 0.37 0.97

** ** NS NS

1.443 0.038 3.681 0.610

3.58 0.09 9.12 1.51

* NS ** NS

16

0.043

Error

0.403

DF, degrees of freedom; MS, mean square; F, Fisher test; NS, non-significant; *, significant at P = 0.05; **, significant at P = 0.01.

Table 2 Results of orthogonal analysis ofperoxidase (POD) and ascorbic peroxidase (AP) for SO2 treatment, cultivar treatment and interactions Source of variation

DF

POD

AP

MS

F

P

MS

F

P

SO2 exposure Linear Quadratic Cubic

3 1 1 1

344.553 699.998 108.385 225.275

149.62 303.96 47.06 97.82

* ** ** **

0.195 0.473 0.053 0.058

81.79 198.68 22.14 24.55

* ** ** **

Cultivar

I

195.625

84.95

**

0.002

0.68

NS

SO2 exposure x cultivar Linear x cultivar Quadratic x cultivar Cubic x cultivar

3 1 1 1

21.949 17.743 46.854 1.252

9.53 7.70 20.35 0.54

** * ** NS

0.092 0.026 0.144 0.107

38.72 10.82 60.59 44.76

** ** ** **

16

2.303

Error

0.002

DF, degrees of freedom; MS, mean square; F, Fisher test; NS, non-significant; *, significant at P=0.05; **, significant at P=0.01.

m o r e p r o n o u n c e d in M e c ( F i g u r e 3). N o i n t e r a c t i o n w a s f o u n d b e t w e e n SO2 t r e a t m e n t a n d c u l t i v a r s f o r C A T a c t i v i t y ; t h i s w a s f o u n d t o b e h i g h e r in M e c t h a n in C h i a r a n o ( F i g u r e 3), b u t a p p e a r e d n o t t o b e a f f e c t e d b y t h e SO2 t r e a t m e n t ( T a b l e 3).

3.3. A s c o r b a t e and dehydroascorbate levels The interaction between SO 2 and cultivar treatments was significant only for the reduced form of a s c o r b i c a c i d ( T a b l e 4). I n C h i a r a n o , t h e A S A levels

A. Ranieri et al. / Environmental and Experimental Botany 37 (1997) 125-135

131 18

SOD

CAT 15

12

9

7

x7

~7

6

I

5

0 I

[

I.

I

I

POD

I

I

O

AP v

40.

1.0

V

32

24

0.8

0.6

./ 16

I

v

I

I

[

I

I

1

I

I

2

23

64

96

2

23

64

96

nl 1

-i

0,4

SO

Fig. 3. Antioxidant enzymes of (-) Mec and (V) Chiarano plants following SO2 treatment. Regression equations: SOD, y=2.889+0.008x, y=2.354-0.003x; CAT, y = 12.985-0.007x, y=6.774-0.005x; POD, y=23.518+0.172x, y = 19.940+0.125; AP, y = 0.497 + 0.005x, y = 0.565 + 0.003. The first and second equations for each enzyme refer to Mec and Chiarano, respectively.

appeared not to be significantly affected by the treatment (Figure 4). In Mec, the ASA content decreased with increasing SO2 levels (Figure 4). No interaction between cultivars and SO2 treatment was found for D H A levels, the two linear regressions being parallel to each other (Figure 4). In both cultivars the DHA levels decreased with increasing SO2 levels. No changes were observed in redox status, expressed by the ratio ASA/(ASA + DHA) [33]. The two cultivars appeared to be very different in their constitutive levels of ASA and D H A (Table 5). In the controls, ASA levels (5.98 in Chiarano

and 4.29 in Mec) and D H A levels (1.18 in Chiarano and 0.54 in Mec) were significantly different at P = 0.01 when compared by Student's t-test.

4. Discussion

The two cultivars (Mec and Chiarano) had previously shown different responses to SO2, indicating that Chiarano was possibly more resistant to SO2 than Mec [8, 9, 10]. One possible explanation for the different behaviour of these two cultivars was a postulated differential response of the photo-

A. Ranieri et al. / Environmental and Experimental Botany 37 (1997) 125-135

132

Table 3 Absolute values (U mg ' protein) of enzyme activities in the cultivars Mec and Chiarano following SO2 fumigation Treatment (nl 1-~)

SOD

CAT

POD

AP

Mec 2 23 64 96

2.903 a 3.032a 3.500a 3.600a

13.51 a 12.50a 11.67a 12.93 a

22.167c 31.677b 29.863 b 42.103 a

0.548 b 0.677b 0.521 b 1.126a

Chiarano 2 23 64 96

2.361 a 2.159b 2.420a 1.930c

6.73a 6.50a 6.89 a 6.01 a

21.687c 24.170b 20.510c 36.603 a

0.550b 0.635 b 0.811 a 0.810a

S02 fumigation, a significant decrease was found in the optical density of the spots focusing in the area with molecular masses of 26-29 kDa and pI 5-6. Remy et al. [34] reported that in pea plants the polypeptides of the light-harvesting complex (also named antenna) of PSII focus in this region. Guillemaut et al. [35] found a similar decrease in the PSII antenna proteins of decaying spruce needles as compared with apparently healthy needles. In both cultivars, SO2 was found to decrease the optical density of spots with molecular mass 32 kDa and pI 5.4. This peptide had previously been [36] identified as D1 protein of the PSII reaction centre by immunological analysis on 2-D gels of thylakoid samples of sunflower and pumpkin plants. The fact that in the present experiment this protein was affected in both sensitive and less sensitive plants is in agreement with other reports indicating that the D1 protein is particularly sensitive to adverse environmental conditions which may affect its content, inducing either an accumulation or a degradation [6, 37, 38]. Since a decrease in stomatal conductance has been reported for the more sensitive Mec [10], mechanisms other than 'avoidance' (i.e. reduction in SO2 absorption) could be evoked to explain the different responses of the two cultivars to SO2. Apparently, the lower stomatal conductance did

SOD, superoxide dismutase; CAT, catalase; POD, peroxidase; AP, ascorbic peroxidase. In each block, values followed by the same letter are not significantly different at P = 0.05.

synthetic machinery, and particularly of the photosynthetic membranes, to SO2. From the results obtained in this work, the thylakoid 2-D protein pattern of leaves fumigated with 96 nl 1-1 of SO2 for 4 months revealed that the two cultivars reacted differently to SO2. Overall changes in the thylakoid protein pattern were much greater in the more sensitive Mec. In this cultivar, following

Table 4 Results of orthogonal analysis of ascorbate (ASA) and dehydroascorbate (DHA) for SO2 treatment, cultivar treatment and interactions Source of variation

DF

ASA

DHA

MS

F

P

MS

F

P

SO2exposure Linear Quadratic Cubic

3 1 1 1

1.845 0.476 4.714 0.344

240.98 62.16 615.84 44.94

** ** ** **

0.072 0.165 0.012 0.037

13.59 30.91 2.33 6.94

** ** NS *

Cultivar

1

28.886

3773.92

**

1.848

345,45

**

SO2 exposure × cultivar Linear × cultivar Quadratic x cultivar Cubic x cultivar

3 1 1 1

0.267 0.752 0.045 0.004

34.87 98.31 5.85 0.47

** ** * NS

0.020 0.009 0.004 0.049

3.82 1.63 0.67 9.17

* NS NS **

16

0.008

Error

0.005

DF, degrees of freedom; MS, m e a n square; F, Fisher test; NS, non-significant; *, significant at P = 0.05; **, significant at P = 0.01.

133

A. Ranieri et al. / Environmental and Experimental Botany 37 (1997) 125-135 8

1.6

DHA

ASA

1.2"~

7 ~0

II

Q 0

0.8

O ffQ I

V

~

V

.I

I

I

I

3

23

64

96

0.4

V 2

I

I

I

I

2

23

64

96

- 1

0.0

nl 1 SOa Fig. 4. Ascorbic(ASA)and dehydroascorbic(DHA) acid levelsof(V) Mec and (o) Chiarano plants followingSO2treatment. Regression equations: ASA, y = 1773- 0.002x, y = 5.480+ 0.002x; DHA, y = 0.511 - 0.002x, y = 1.106- 0.002x. The first and second equations for each metabolite refer to Mec and Chiarano, respectively.

Table 5 Absolute values (#tool g- ~fresh weight) of ascorbic acid (ASA) and dehydroascorbic acid (DHA), and the redox status (ASA/(ASA+ DHA)) of cultivars Mec and Chiarano following SO2 fumigation Treatment (nl 1-')

ASA

DHA

ASA/(ASA+ DHA)

4.29 a 3.00 b 2.73 c 3.34 b

0.54 a 0.45 a 0.35 a 0.38 a

0.89 a 0.87 a 0.87 a 0.90 a

5.98a 5.01 b 5.23 b 5.92 a

1.18a 0.92 b 1.02 ab 0.82 b

0.84a 0.84 a 0.84 a 0.88 a

Mec 2 23 64 96 Chiarano 2 23 64 96

In each block, values followed by the same letter are not significantly different at P = 0.05.

not prevent the more sensitive Mec from being injured. An alteration at the level of thylakoid proteins was particularly evident in Mec following the treatment with SOz, and this may be a more relevant mechanism to the cultivar's sensitivity to SO2. To test the hypothesis that, under prolonged SO2 fumigation, the effects of oxygen-derived molecular species are of major concern in determining molec-

ular alteration, we attempted to correlate our data concerning the modification of thylakoid protein patterns with the ability shown by the two wheat cultivars to scavenge the oxygen molecular species. A lack of resistance to free radical attack has already been found to accompany both qualitative and quantitative changes in the 2-D chloroplast protein patterns of yellow needles of decaying spruce and fir compared with those of healthy green ones [35]. Sen Gupta et al. [17] suggested that the increase and maintenance of the active antioxidant metabolism may protect the photosynthetic apparatus of poplar trees during exposure to the oxidative stress induced by ozone (03) exposure. SOD has been considered a key enzyme in the defense system of plants against oxidative stress [29], but contradictory data have also been reported, showing increases [17, 39] or declines [40] in SOD activity under SO2 and 03 pollution. In our experiments, under SO2 fumigation, SOD activity decreased in the less sensitive Chiarano, while no significant change was detected in Mec. SOD activity was higher in the more sensitive Mec than in Chiarano, irrespective of SO2 treatment. From these data, changes in SOD activity do not explain the different sensitivities of the two cultivars. The same appears to be true for catalase activity, since it was not affected by SO2 treatment in either Chiarano or Mec. Our data showed that both cultivars

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A. Ranieri et al. / Environmental and Experimental Botany 37 (1997) 125-135

reacted to SO2 with increasing POD and AP activities, with the SO2-sensitive Mec showing a more pronounced response to the highest SO2 level. These data are in accord to those reported by Benes et al. [41], who found a higher increase in AP and POD activities, following 03 exposure, in needles of an O3-sensitive clone of ponderosa pine in comparison to a tolerant one. It appeared that the ability to increase peroxidase (POD and AP) activities was not the basis of the major resistance to SO2 of Chiarano. The redox status was not affected by SO2 and showed similar values in both cultivars. However, we observed different behaviour in the two cultivars as regards the content of ascorbic acid: the constitutive level of ascorbic acid was higher (+ 39%) in Chiarano than in Mec, and it remained higher at all SO2 concentrations in the SO2-resistant cultivar. The lower pool of ascorbate detected in Mec is indicative of a reduced capacity to counteract oxidative stress conditions since ascorbic acid acts not only as the substrate for the enzymatic mechanism that removes H202, but is also capable of reducing activated oxygen non-enzymatically, alone or in a cooperative interaction with tocopherols [42]. Data obtained by this research showed different behaviours in the two wheat cultivars under longterm fumigation with SO2: the major alterations in the thylakoid protein pattern were observed in the more sensitive Mec. On the basis of the data obtained, the hypothesis formulated that the basis for the lower sensitivity to SO2 of Chiarano was related to an enhanced activation of the detoxification enzymes can be rejected; the more sensitive Mec showed the more pronounced responses from the detoxification enzymes. Instead, the lower sensitivity of Chiarano appears to be related to a higher constitutive level of ascorbic acid, which remained elevated at all SO2 concentrations.

Acknowledgements This research was supported by the National Research Council of Italy, Special Project RAISA, Sub-project N.2, and by exMURST, 60%.

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