2
.
8
. Protein content determination The protein content of IWF and RCM was
determined by the protein – dye binding method of Bradford [25], using bovine serum albumin as
standard. The spectrofotometrical reading was measured at 595 nm.
2
.
9
. POD isoenzyme determination Isoperoxidases were resolved by isoelectric fo-
cusing IEF, in a horizontal slab apparatus Bio- Rad, on acrylamide gel using a pH gradient of
3.5 – 10. Samples were dialysed overnight in 0.05 Na-phosphate buffer pH 7.0, lyophilised and
then dissolved in 1 glycine. The resuspended samples, after filtration with 0.45 mm pore size
filters Whatman, were loaded on 5 polyacry- lamide gel, containing 4.8 acrylamide, 0.2
N,N-methylene-bis-acrylamide, 5 glycerol, 5 ampholine pH 3.5 – 10. To the solution 10 ml
were added 3 ml of TEMED and 70 ml of 10 APS. To estimate the pI of separated isoenzymes,
standard proteins of known pI Sigma were run on the same gels.
The IEF run was carried out at constant voltage of 100 V for 15 min, followed by 15 min at 200 V
and 45 min at 450 V. The POD isoenzymes were visualised by incubating gel in 0.5 benzidine and
0.03 H
2
O
2
in 4.5 acetic acid modified from Ref. [26] and after the reaction was stopped by
immersing the gel in 7 acetic acid for 30 s, gels were immediately photographed. This benzidine
stain was chosen since this substrate is slightly less selective towards peroxidase isoforms than other
hydrogen donors and rapidly precipitates on gels.
2
.
10
. Ascorbate extraction and determination For determination of ascorbic ASA and dehy-
droascorbic DHA acid, the residual cell material was homogenised in a mortar with liquid nitrogen,
quartz sand and a solution of 5 metaphosphoric acid 1:2, wv and centrifuged at 14 000 × g for
20 min.
The total amount of ASA + DHA in intercellu- lar washing fluid was quantified immediately after
its extraction to minimise the ascorbate oxidase AAO-dependent oxidation of ASA during mea-
surements. The absence of such oxidation of ASA was confirmed by adding 10 mM sodium azide an
inhibitor of AAO to the buffer used to obtain IWF [27]. The quantitative determination was car-
ried out according to Okamura [28] and Law et al. [29]. An aliquot of supernatant or IWF was added
to 10 TCA wv and after addition of 5 M NaOH, the mixture was centrifuged at 12 000 × g
for 2 min. To quantify ASA, 150 mM phosphate buffer pH 7.4 was added to the supernatant. For
the total amount of ASA + DHA, 10 mM DTT was supplied, after incubation with 10 mM DTT
for 15 min at room temperature, 0.5 of N-ethyl- maleimide solution was added. Then, each sample
was supplied with 10 TCA wv, 44 H
3
PO
4
solution vv, 4 a-adipyridyl wv in 70 methanol and 3 FeCl
3
wv. After vigorous stirring, the samples were kept at 37°C for 60 min
and then the absorbance was read at 525 nm against a standard curve of pure ASA Sigma in
the 0 – 40 nmol range.
2
.
11
. Statistic A minimum of 12 plants per treatment were
used in all experiments. Values shown in the tables are the means of eight determinations 9 S.D.
Comparison between means was evaluated by t- test and the P = 0.05 level of error.
3. Results
At the end of ozone exposure, sunflower plants did not show any visible symptoms of injury on
leaf surfaces, confirming previous data on the likeness of chlorophyll content of O
3
-treated and untreated leaves [30].
Accidental contamination of IWF with intracel- lular proteins during the infiltration procedure was
tested by analysing the activity of the cytosolic and chloroplastic enzyme markers G6PDH and
GAPDH respectively. Regardless of the treatment, the relative activity of these marker enzymes in the
IWF was always B 0.1 of the total activity in the RCM data not shown.
3
.
1
. Redox state At apoplastic level the total ascorbate content
raised + 112 as a consequence of O
3
fumiga- tion Table 1. Within the ascorbate pool, both the
reduced and the oxidised form underwent a signifi-
Table 1 Reduced ASA, oxidized DHA, total ASA+DHA ascorbate content and redox state of ascorbate ASAASA+DHA of
extracellular IWF and intracellular RCM extracts of control and O
3
-treateed sunflower plants
a
RCM IWF
Control O
3
-treated Control
O
3
-treated ASAASA+DHA
0.7190.02b 123.8911.1a
123.8911.1a 123.8911.1a
ASA 143.2910.8b
88.494.2a 9.4 9 0.4a
11.490.2b 119.398.8b
2.4 9 0.2a 35.392.8a
8.5 9 0.2b DHA
262.5912.4b ASA+DHA
11.890.6a 123.8911.1a
19.990.5b 0.5490.03a
0.7990.01b 0.5790.02a
ASAASA+DHA 0.7190.02b
a
Values are expressed as nmol g
− 1
fw and mmol g
− 1
fw for extracellular and intracellular samples, respectively. Within each fraction IWF or RCM values followed by different letters are statistically different at P = 0.05 n = 5.
ing O
3
exposure, while the syringaldazine – POD activity underwent a significant increase by 23.4
Table 2. At simplastic level, the guaiacol – POD activity
showed no significant variation in the ozonated plants as compared with the untreated ones Table
2. The activity of soluble, ionically- and cova-
lently-bound PODs was assessed using syringal- dazine as electron donor, owing to the preferential
involvement of these fractions in the lignification process. All three fractions tested showed a signifi-
cant similar increase in Syr – POD activity follow- ing
O
3
fumigation, with
the soluble
POD increasing by 94, the ionically-bound POD by
112 and the covalently-bound POD by 82, respectively Table 3.
cant increase, DHA showing a more pronounced stimulation than ASA + 238 and + 62 in com-
parison to their respective controls. As a conse- quence of these different increments the redox
state changed from 0.71 in the control to 0.54 in the ozonated plants − 24. Similar reduction in
the redox state of ascorbate was also exhibited at symplastic level − 29 where the oxidised and
the reduced form underwent an increase, respec- tively, by 255 and 21 in the fumigated sunflower
plants Table 1.
3
.
2
. POD acti6ity With regard to the extracellular unspecific POD,
the guaiacol – POD activity did not change follow-
Table 2 Guaiacol G–POD and syringaldazine Syr–POD peroxidase
activity in the intercellular washing fluid IWF and intracel- lular soluble fraction RCM of sunflower leaves treated with
O
3
150 ppb, 4 d, 4 h per day
a
Enzyme activity IWF
RCM G–POD
Samples Syr–POD
G–POD 1.47 9 0.2a
4.04 9 0.20a Control
90.4 9 1.5a 3.98 9 0.17a
O
3
-treated 111.5 9 4.3b
1.27 9 0.2a
a
Enzyme activity is expressed as DA
470
guaiacol–POD min
− 1
mg
− 1
proteins and DA
530
syringaldazine–POD min
− 1
mg
− 1
proteins. Within each fraction IWF or RCM values followed by different letters are statistically different at
P = 0.05 n = 5. Table 3
Effect of fumigation with O
3
150 ppb, 4d, 4h per day on soluble S, ionically IB and covalently CB cell wall bound
peroxidases of sunflower leaves
a
Samples Enzyme activity
I.B. Soluble
C.B. 34.5 9 1.7a
7.4 9 0.6a 145.1 9 6.7a
Control 67.0 9 1.1b
15.7 9 0.7b O
3
-treated 264.1 9 11.5b
a
Enzyme activity, determined using syringaldazine as elec- tron donor, is expressed as DA
530
min
− 1
mg
− 1
proteins, with the exception of CB activity, which is expressed as DA min
− 1
mg
− 1
dry weight of residual cell wall material. Within each fraction S, IB or CB values followed by different letters are
statistically different at P = 0.05 n = 5.
Fig. 1. Benzidine staining of peroxidase isoforms of extracel- lular IWF and intracellular RCM leaf fluids of control C
and O
3
-treated O
3
sunflower plants. A total of 50 mg of proteins for IWF and 30 mg of proteins for RCM fractions
were resolved by a pH 3.5 – 10 isoelectrofocusing gel. Isolec- trophoretic analysis was performed in triplicate.
untreated ones, particularly evident for the most acidic and the most basic isoforms. Beside, an
additional anionic band seems to be present as a consequence of O
3
exposure. In the ionically- bound fraction, only a few bands with POD activ-
ity are visible. Also, in this fraction, the increase in the staining intensity was accompanied by qualita-
tive differences between the two treatments be- cause of the presence of an anionic protein band in
the O
3
-treated sample that, on the contrary, was not visible in the control. As regards the cova-
lently-bound POD profiles, only slight quantitative differences in the staining intensity were evident
between treated and untreated plants.
4. Discussion and conclusions
