tion and plasma membrane ruptures in the outer root cap and meristematic cells [13].
The physiological mechanisms of Mn toxicity and tolerance are still not well known. Many
reports suggest that Mn excess may cause the induction of oxidative stress [4]. In this case, as a
consequence of the uptake of toxic concentrations of the metal, several defense enzymes such as
SOD, ascorbate peroxidase ASPX, glutathione reductase GR, guaiacol peroxidase GPX, are
induced in the chloroplasts and cytosol as protec- tion against oxidative stress [14 – 17].
Peroxidases POD are oxido-reductases which occur practically in all plants. They are present as
several isoenzymes which are found in soluble, ionically bound and covalently bound forms [18].
Their form and distribution is, probably, related to different physiological functions [19,20] An in-
crease of POD activity has been considered as a metabolic response under various stress conditions
including heavy metal stress [21]. In the cell wall- located bound POD activity plays a key role in
controlling the production and deposition of lignin in vascular tissue [22,23]. In non-lignified primary
cell walls, POD may be involved in the cross-link- ing of cinnamic acids such as ferulic acid, the
production of which is implicated in the enhance- ment of cell adhesion in parenchyma tissues [24 –
26]
POD may also prevent oxidative damage to plasmamembranes, acting as peroxide radical
scavengers [14]. A previous research established an in vitro pro-
cedure for the selection of Nicotiana tabacum var. BEL W3 callus lines and regenerated plants toler-
ant to high doses of Mn 2 and 5 mM. The performance of the Mn-tolerant plants with regard
to several morphological, anatomical and cytologi- cal characteristics, in comparison to the Mn-sus-
ceptible regenerated plants, was described [12]. In particular, Mn was also shown to affect the num-
ber of xylem elements and the degree of lignifica- tion, which differed from plants grown in the
presence of 2 and 5 mM Mn compared to the control. Moreover, in these plants, damaged
chloroplasts and a reduction in their number were also observed, especially for those treated with 5
mM Mn.
With an aim of better explaining these previous results and to characterize Mn-tolerant plants
from the physiological point of view, the present study was planned to determine in selected plants
growing in the presence of Mn at 2 and 5 mM, 1 the activity of extracellular POD in the stems
using two electron donors such as guaiacol and syringaldazine, 2 the activity of SOD, ASPX and
GR in the leaves.
2. Materials and methods
2
.
1
. Plant material and culture conditions The selection procedure of N. tabacum var. BEL
W3 plants tolerant to 2 and 5 mM Mn has been previously
reported [12].
Briefly, Nicotiana
tabacum callus cultures were initiated from sterile germinated seeds placed in Murashige and Skoog’s
MS medium [27], containing the standard Mn concentration 0.1 mM and containing 1 mg l
− 1
Naphtaleneacetic acid NAA, 1 mg l
− 1
kinetin, 30 g l
− 1
sucrose, 0.8 Difco Bacto Agar callusing and shooting medium. Plants were selected for
tolerance to Mn by regeneration from callus cul- tures which, starting from 0.1 mM Mn control,
were transferred monthly to MS medium where the level of Mn was progressively increased to 20
mM. At the end of each month the surviving calli with shoot-forming capacity were recorded and
used for the successive subculture. At Mn concen- trations higher than 5 mM the surviving calli were
able to grow for only 3 – 4 months subcultured monthly. On the contrary, in the presence of 2
and 5 mM Mn the selected tolerant calli contin- ued to grow and regenerate shoots for at least
8 – 10 months. During this period, the shoots formed from these Mn-tolerant callus lines were
removed and used for rooting. To induce root formation, 4 – 5 cm high shoots were removed
from the control and selected calli and transferred into Magenta vessels SIGMA, St. Louis, MO
containing 50 ml of MS medium supplemented with 1 mg l
− 1
Indole-3-butyric acid IBA control plantlets or 1 mg l
− 1
IBA plus 2 and 5 mM Mn Mn-tolerant plantlets, respectively. Moreover, a
part of control plantlets were induced to root and grow also in the presence of 2 and 5 mM Mn
Mn-sensitive plants to evaluate their perfor- mance in comparison to the Mn-tolerant plants.
After 3 weeks, the well rooted plantlets five plantlets for each treatment type, chosen at ran-
dom were cut at the base of the stem and divided
into leaves and stems for enzyme extraction and for the determination of the Mn and Fe content
MnFe ratio in the leaves. The cultures were maintained in a growth
room at 25 9 1°C under a 16 h light photoperiod 35 mmol m
− 2
s
− 1
provided by Sylvania Day- light fluorescent tubes F36W154-ST.
2
.
2
. Extraction of extracellular enzymes and enzymic assays
2
.
2
.
1
. Stem All stems of N. tabacum, previously weighed,
were vacuum infiltrated with 66 mM K-phos- phate buffer pH 7.0 5 ml g
− 1
FW containing 100 mM KCl and then centrifuged at 1500 g for
10 min at 4°C, as described by Castillo and Greppin [23]. A fraction designated as ‘extracellu-
lar fluid’ was obtained containing extracellular free and ionically bound wall POD. The residual
plant material was then homogenized with cold mortar and pestle, using cold absolute acetone 2
ml g
− 1
FW plus quartz sand, until no fibrous residue could be seen. The homogenate was then
filtered in vacuo and the resulting acetone pow- ders were dissolved in a 30 mM Tris buffer pH
7.6 2 mg g
− 1
FW containing 5 mM mercap- toethanol, 0.5 vv Tween 20, 0.1 wv insol-
uble PVP and 1 mM EDTA and centrifuged at 800 g for 10 min. The pellet was washed twice in
30 ml Tris buffer pH 7.6 and then suspended in the same buffer containing 2 vv Triton X-100
and centrifuged at 800 g for 10 min. The pellet, after washing in distilled water and Tris buffer,
was treated with 1 mM NaCl for 2 h at room temperature and centrifuged at 800 g for 10 min.
The supernatant gave the fraction containing the ‘ionically bound wall POD fraction’. The pellet
was rinsed several times in distilled water, treated with 0.5 wv cellulase and 2.5 pectinase in
0.1 M Na-acetate buffer, pH 5.0 for 15 h 30 min at room temperature and centrifuged at 800 g for
10 min. The supernatant represented the fraction containing the ‘covalently bound wall POD’. The
activity of POD was assayed in the extracellular fluid and in the ionically and covalently bound
wall fractions, using guaiacol and syringaldazine as substrates.
GPX EC. 1.11.1.7 activity was assayed in 60 mM K-phosphate buffer pH 6.1 containing 28
mM guaiacol and 5 mM H
2
O
2
[28]. The increase in absorbance was recorded at 470 nm. Syringal-
dazine POD activity was assayed in 0.1 M NaK phosphate buffer pH 6.0 containing 4.2 × 10
− 2
mM syringaldazine dissolved in methanol – diox- ane 1:1 vv and 1.6 mM H
2
O
2
[28]. The increase in absorbance was recorded at 530 nm. Guaiacol
and syringaldazine POD activities were referred measured with reference to the protein content
of each sample and expressed as DA
470
min
− 1
mg
− 1
protein and DA
530
min
− 1
mg
− 1
protein, respectively. All enzyme activities were tested us-
ing a Perkin – Elmer UV 55IS spectrophotometer. Protein concentration was determined by Bio-
Rad Protein Assay Biorad with bovine plasma globulin as a standard
2
.
2
.
2
. Leaf Crude extracts of leaves were obtained from
homogenization with cold acetone 2 ml g
− 1
FW as described for the stems. The resulting acetone
powders obtained after drying the sample were dissolved in 30 mM Tris buffer pH 7.6 3 ml g
− 1
FW containing 5 mM mercaptoethanol, 0.5 v v Tween 20, 0.1 wv insoluble PVP and 1
mM EDTA, and then kept at 4°C for 90 min. The extracts were filtered with Miracloth Cal-
biochem and centrifuged at 15 000 g at − 4°C for 20 min. The supernatant was collected for the
enzyme assays. GPX EC. 1.11.1.7 activity was assayed at 470 nm with 28 mM guaiacol, 5 mM
H
2
O
2
and 50 – 100 ml of the extract in 60 mM K-phosphate buffer pH 6.1 [28]. ASPX EC.
1.11.1.11 activity was measured at 290 nm by the method of Kato and Shimizu [29]. The assay
mixture contained 0.5 mM Na-ascorbate, 1 mM H
2
O
2
, 0.1 mM EDTA and 100 ml of the extract in 50 mM K-phosphate buffer pH 7.0 GR EC.
1.6.4.2 activity was determined at 340 nm using the method of Foster and Hess [30] in 100 mM
K-phosphate buffer pH 7.0 containing 150 mM NADPH, 500 mM GSSG, 1.0 mM EDTA and
200 ml of the extract. SOD EC. 1.15.1.1 activity was assayed at 550 nm by the method of Mc
Cord and Fridovich [31]. The assay mixture con- tained 50 mM K-phosphate buffer pH 7.8, 0.1
mM EDTA, 50 mM xanthine, 10 mM ferricy- tochrome c, enough xanthine oxidase to cause
D
A
550
min between 0.03 and 0.04, and 100 ml of the extract. One unit of enzyme activity was
defined as described by Giannopolitis and Ries [32]. Protein concentration was determined as de-
scribed above.
2
.
3
. Mn and Fe analyses Mn and Fe concentrations mg g FW were
determined in leaves of Mn-tolerant and Mn-sensi- tive plants by atomic absorption spectrophotome-
try Perkin – Elmer 370 after the samples had been rinsed in distilled water, dried at 80°C for 24 h and
wet-ashed in a nitric and perchloric acid mixture 5:2 vv on an electric thermostatic plate at 300 –
350°C.
2
.
4
. Statistical analyses All measurements were made in triplicate. All
values reported have been expressed as means of triplicates 9 SE. The significance of differences be-
tween control and treated mean values have been evaluated by Student’s t-test and reported in the
tables as P 5 0.05 and P 5 0.01.
3. Results
