Glutathione determination: The amount of
reduced
glutathione
was
determined
spectrophotometrically at 412 nm using Glutathione
Assay Kit (Sigma-Aldrich). Leaf samples of 0.1g
were taken from 3 variants of plants - control,
drought-exposed and rehydrated plants, and ground
in liquid nitrogen. After adding 5% sulfosalicylic
acid, the mixture was stirred, kept at 2-8°C for 10
min and centrifuged at 10, 000g. The obtained
supernatant was used for the reaction. The content
of the reaction mixture was as follows: 95 mM
potassium phosphate buffer (pH 7.0), 0.95 mM
EDTA, 0.038 mg/ml (48µM) NADPH, 0.031
mg/ml DTNB, 0.115 units/ml glutathione reductase
and 0.24% 5-sulfosalicylic acid. To construct the
calibration curve, solutions (0.5 - 20 µg/ml) of GSH
of various concentrations were prepared in 0.4 M
Tris-HCl buffer (pH 8.5).
Ascorbic acid determination: In an acidic
environment, ascorbic acid reduces potassium
hexacyanoferrite
(Fe
+3
)
to
potassium
hexacyanoferrate (Fe
+2
). Samples were taken from
3 variants of plants and frozen in liquid nitrogen.
Then 1 ml of citrate buffer (pH 3.69) was added to
samples of 0.5g, stirred and centrifuged for 5 min at
12,500g. The reaction mixture was prepared as
follows: 25 µl K
3
[Fe(CN)
6
] (1%) and 25 µl 2% NaF
were added to 500 µl or 0.5 ml of the plant extract.
After keeping for 5 min, 1.9 ml of dH
2
O, 50 µl
FeCl
3
were added, kept for 5-7 min and optical
density was measured spectrophotometrically at
680 nm (Matei et al., 2012).
Extraction of the enzyme. 0.5g of the leaf
sample was ground in liquid N
2
. 100 mM Na-
phosphate (pH 7.8) buffer containing 1 mM EDTA,
2 mM FMSF, 1% PVP and 0.1% Triton X-100 was
used as a homogenization medium. Then the
sample was centrifuged at 4
0
C, for 20 min, at
15,000 g and the obtained supernatant was used for
the APX and GR assays.
