enzymatic, able to prevent or to mitigate O
3
injury have been reported [10]. Among antioxidant
metabolites, ascorbic acid, in addition to playing a key role in detoxification of H
2
O
2
as a specific substrate for ascorbate peroxidase APX, is capa-
ble of direct scavenging of O
3
molecules and O
3
- derived ROS [7,8,11 – 13]. Although the pool of
ascorbate present in the apoplast is only a small proportion of the total leaf content, it is suggested
that its increase following ozone exposure may afford protection against the dangerous effect of
this pollutant [12,14].
In the process of detoxification of H
2
O
2
is par- ticularly important the large family of peroxidases
POD, which includes either the specific ascorbate peroxidase enzyme APX and the so called unspe-
cific peroxidases POD. Previous data, obtained in our laboratory on sunflower plants subjected to
O
3
exposure, have shown a differential stimulation of APX isoforms, indicating an enhancement in
the activity at both the apoplastic and symplastic level, while stromal and thylakoid-bound chloro-
plastic APX activity remained unchanged [15]. The unspecific POD, besides their antioxidant role, is
involved in many metabolic functions. In the cell wall, PODs are present in soluble, ionically- and
covalently-bound forms and, in addition to a detoxificant role as scavengers of H
2
O
2
, they have been reported to be involved in a number of
physiological processes which regulate cell growth by catalysing the formation of cross-links between
extensin and feruloyated polysaccarides and the polymerisation of lignin precursors [16 – 20]. The
cell wall stiffening has been attributed mainly to peroxidases whose activity can be detected by
using, in the enzymatic assay mixture, syringal- dazine as a specific substrate [16].
In the present work, the apoplastic ascorbate pool content of sunflower plants exposed to O
3
and the cell redox state, measured as reduced versus total ascorbate ratio, was determined. Be-
side, the behaviour of unspecific PODs following O
3
exposure was tested, measuring their activity by using different electron donors such as guaiacol
and syringaldazine, both in the extracellular IWF and in the intracellular RCM leaf fluids, as well
as in the soluble S, ionically IB and covalently CB cell wall-bound fractions. In addition, the
POD isoforms of each fraction, along with the apoplastic and symplastic ones, were separated by
isoelectrofocusing analysis.
2. Materials and methods
2
.
1
. Plant material Sterilised sunflower seeds Heliantus annuus L.,
cv Hor were germinated, in the dark, in Petri dishes for 3 days and the seedlings were grown in
perlite for a week. After this period, plants were transferred and grown for 4 weeks in a greenhouse
at 17 – 25°C night – day, R.H. between 60 and 80, with a 14 h photoperiod and a photosyn-
thetic photon flux density of 530 mmol m
− 2
s
− 1
PAR: 400 – 700 nm. Only uniform plants with eight fully expanded
leaves were selected : 35 days after sowing. All biochemical analyses were carried out on fully
expanded middle-aged leaves from control and treated plants.
2
.
2
. Fumigation treatment O
3
fumigation was performed in air conditioned chambers 0.48 m
3
. Temperature was maintained at 20 9 1°C and R.H. at 85 9 5. A photon flux
density at plant height of 530 mmol m
− 2
s
− 1
was provided by incandescent lamps. O
3
was generated by electric discharge passing pure oxygen through
a Fisher ozone generator 500 Fisher Labor und Verfahrenstechnik,
Meckenheim, Germany.
Ozone concentration in the fumigation chambers was continuously monitored with a Monitor Labs
Analyzer mod. 8810 Monitor Labs, San Diego, CA operating on the principle of UV absorption
and interfaced with a personal computer. Control plants were grown in charcoal-filtered air cham-
bers under the same conditions. Plants were pre- adapted to the chamber conditions for 48 h and
half of them were exposed to 150 ppb O
3
for 4 days 4 h per day, while the remainders, un-
treated, were used as a control.
2
.
3
. Preparation of the apoplastic fluid Freshly-harvested intact leaves 10 g were
rinsed with distilled water and vacuum infiltrated − 65 kPa, three cycles of 30 s each in 50 ml of 66
mM K-phosphate buffer pH 7.0 and 100 mM KCl. After infiltration, the leaves were wiped and
centrifuged 1500 × g for 10 min at 4°C. The apoplastic fluid IWF was recovered and tested
for peroxidase activity and protein concentration
[21]. The residual cell material RCM was im- mersed in liquid nitrogen and stored at − 80°C
until use.
2
.
4
. Extraction of enzymes from residual cell material
For POD determination, the RCM was crushed in liquid nitrogen and homogenised at 4°C in 0.22
M Tris – HCl pH 7.4, PMSF 50 mgml, 10 mM b-mercaptoethanol and 0.1 insoluble PVP. After
centrifugation at 24 000 × g for 10 min at 4°C, the supernatant was recovered and dialysed overnight
against 2 mM Tris – HCl pH 7.4 and tested for enzyme activity [22].
To test the activity of the cytoplasmatic and chloroplastic enzyme markers, glucose-6-P dehy-
drogenase G6PDH, EC 1.1.1.49 and glyceralde- hyde-3-P dehydrogenase GAPDH, EC 1.2.1.12,
the RCM was homogenised with liquid nitrogen in a cold buffer solution 1:4, wv consisting of 50
mM HEPES pH 7.4 containing 0.25 M sucrose, 70 mM KCl, 10 mM MgCl
2
, 1 mM EDTA, 1 wv BSA, 10 mM b-mercaptoethanol bME and
phenylmethylsulfonyl fluoride PMSF 50 mg ml
− 1
. NH
4
SO
2
70 of saturation at 4°C was added to the surnatant obtained after centrifuga-
tion of the homogenate at 39 000 × g for 15 min at 4°C, to precipitate the proteins. After centrifu-
gation 17 000 × g for 15 min at 4°C, the pellet obtained was resuspended in 10 mM HEPES
buffer at pH 7.4, containing 10 mM MgCl
2
, 50 mM KCl, 0.5 mM EDTA and 1 mM bME and
dialysed overnight. The extract obtained was utilised for the analysis of the enzymatic activity
[15].
2
.
5
. Marker enzyme acti6ity The incubation medium for the G6PDH activity
determination consisted
of 86.3
mM tri-
etanolamine-HCl buffer pH 7.6, 6.7 mM MgCl
2
, 12 mM glucose-6-P, 0.37 mM NADP
+
and a suitable aliquot of IWF or RCM. The absorbance
was read at 340 nm. The activity was expressed as mmol of NADP
+
reduced per minute and referred to the protein content of the extract. The activity
assay for GAPDH was carried out at 25°C, recording the decreasing of the absorbance at 340
nm. The reaction mixture contained 50 mM Tris – HCl buffer pH 7.8, 10 mM MgCl
2
, 5 mM EDTA, 3 mM ATP, 100 mM NADPH, 5 mM
3-P-glycerate, 0.45 U ml
− 1
P-glycerate KINASE and an aliquot of RCM or IWF [15].
2
.
6
. Separation of soluble, ionically and co6alently-bound peroxidases
The separation of soluble, ionically- and cova- lently-bound POD was carried out according to
Grison and Pilet [23]. Freshly harvested leaves were homogenised at 4°C with 66 mM Na-phos-
phate pH 6.1 and centrifuged at 800 × g for 5 min. The
supernatant was
again centrifuged
at 10 000 × g for 5 min and the recovered second
supernatant was considered the soluble fraction. The first pellet was washed twice with phosphate
buffer, twice with water and after shaking continu- ously for 1 h at 4°C in Triton X-100, it was again
rinsed five times with water. The pellet obtained was treated with 1 M CaCl
2
for 1 h and cen- trifuged at 800 × g for 10 min at 4°C. The result-
ing supernatant was the ionically-bound fraction. The pellet was washed several times with distilled
water and incubated at room temperature for 16 h with 0.3 cellulase, 0.3 macerase and 0.3 cellu-
lolysin in 50 mM Na-acetate buffer pH 5.5 to obtain the covalently-bound fraction, after cen-
trifugation at 800 × g for 10 min. The residual cell wall material was dried at 80°C and weighed.
2
.
7
. POD assay POD activities were measured spectrophotomet-
rically using two different substrates, guaiacol and syringaldazine.
The incubation mixture for the determination of guaiacol – POD activity consisted of 20 mM Na-
acetate buffer pH 5.0, 2 mM guaiacol, 30 mM H
2
O
2
and a suitable portion of plant tissues ex- tract. Increase in absorbance was recorded at 470
nm [22]. Syringaldazine – POD activity was assayed by
the increase in absorbance at 530 nm in a reaction medium containing 200 mM Na-K phosphate
buffer pH 6.0, 2.5 mM H
2
O
2
and 2 mM syringal- dazine [24]. For each enzymatic fraction, the POD
activity was measured with reference to the protein content with the exception of covalently-bound
POD activity, which was expressed on the basis of the dry weight of residual cell wall materials, as
reported by Pandolfini et al. [24].
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
