the mixture were placed in a water

the mixture were
placed in a water bath for 20 min at 70°C and then 30 min in cold water.
Finally, the absorbance of solutions was read at 485 nm by the
spectrophotometer. Soluble sugars content were calculated using glucose for the
standard curve and were expressed as mg g-1 of dry leaf weight.

Enzymes
extraction and electrophoresis

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     The
activities of SOD and CAT were determined in native PAGE (Polyacrylamide gel
electrophoresis). Enzyme extraction from the leaves was performed according to
Valizadeh et al. 2011. Considering this fact that enzymes are very vulnerable
to high temperatures and would be deformed, all extraction stages and loading
the gel were performed in 0-4°C. The crude extract of fresh and healthy leaves
was prepared with separate mortars and pestles in a Tris-HCl extraction buffer
with a pH of 7.5 (Tris 50 mM, sucrose 5%, ascorbic acid 50 mM, sodium
metabisulfite 20 mM, PEG 2% and 2-mercaptoethanol 0.1%), using 0.5 mg ?l-1
sample (1w:2v) which centrifuged at 4 ºC and 10000 rpm for 10 min Valizadeh et
al. 2011. Enzyme extracts were immediately absorbed onto 3×5 mm wicks cut from
Whatman 3 mm filter paper and loaded onto 7.5% horizontal slab polyacrylamide
gels (0.6 × 15 × 12 cm), prepared by Poulik buffer Soltis and Soltis 1990,
using TBE (Tris-Borate-EDTA) electrode buffer (pH=8.8). Electrophoresis was
carried out at 4°C for 3 hours (constant current of 30 mA, and voltage of about
180 V). Subsequently, two slices of slab gel were separated. The staining for
determination of SOD and CAT was carried out according to Soltis and Soltis
1990. For detection of SOD isoforms, the gels were incubated in 50 ml
Tris-HCl buffer (pH=8) containing 2 mg riboflavin, 1 mg EDTA and 10 mg nitro
blue tetrazolium (NBT) for 30 min in the dark condition and then developed for
30-45 min under moderate light intensity. For CAT staining, another layer of
slab gel was soaked in a solution containing 60 ml double-distilled water
(dd H2O) and 30 µl H2O2 for 20 min in
darkness. Then this solution rinsed and stained in a freshly prepared solution
containing 60 ml double-distilled water (dd H2O), 600 mg
potassium ferricyanide and 600 mg ferric chloride for 15-20 min. After
completing the staining, isozyme bands which appeared on the gel were
photographed and then scored. After scanning fixed gels, enzymes activity were
quantified by MCID 0.7 Analysis evaluation. Area and density of each isozyme
bands were measured by the software, and then multiplication of area to
intensity of the bands (D × A) were considered as isozyme densitometric
activity.

Statistical
analysis

The
data were tested for homogeneity and normality of residuals using the
Kolmogorov-Smirnov and Bartlett tests, respectively. Analysis of variance of
the data was carried out by MSTAT-C and SPSS-20, considering year as a random
factor. The mean values of the traits were compared by applying Duncan multiple
range tests at p ? 0.05, and the figures were drawn by Excel software.