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Plant Gene and Trait, 2013, Vol.4, No.20, 109
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above conditions for 48 h. Control plants were grown
in Hyponex solution only. After treatment data were
taken from the leaf samples and immediately used.
3.3 Extraction and analysis of ascorbate (AsA) and
reduced glutathione (GSH) and reduced glutathione
(GSSG)
Treated mustard leaves (0.5 g fresh weight) were
homogenized in 1.5 mL ice-cold acidic extraction
buffer (6% metaphosphoric acid containing 1 mM
EDTA) using a mortar and pestle. Homogenates were
centrifuged at 11,500× g for 15 min at 4 °C and the
supernatant was collected for analysis of ascorbate
and glutathione.
The AsA content was determined following the
method of (Hossain et al., 2010). The supernatant was
neutralized with 0.5 M K-phosphate buffer (pH 7.0).
The AsA was assayed spectrophotometrically at 265
nm in 100 mM K-phosphate buffer (pH 5.6) with 0.5
unit of ascorbate oxidase (AO). A specific standard
curve with AsA was used for quantification. The
glutathione pool was assayed according to previously
described methods (Hossain et al., 2010) utilizing 0.4
ml of aliquots of supernatant neutralized with 0.6 ml
of 0.5 M K-phosphate buffer (pH 7.5). Based on
enzymatic recycling, glutathione is oxidized by
5,5′-dithio-bis (2-nitrobenzoic acid)(DTNB) and
reduced by NADPH in the presence of GR, and
glutathione content was evaluated by the rate of
absorption changes at 412 nm of 2-nitro-5-thiobenzoic
acid (NTB) generated from the reduction of DTNB.
The amount of GSSG was determined after removal
of GSH by 2-vinylpyridine derivatization. Standard
curves were generated with reduced and oxidized
glutathione.
3.4 Enzyme extraction and activity assays
For extraction of enzymes, leaf samples (0.5 g)
were homogenized in pre-chilled mortar and pestle
by using a extraction buffer of 1 ml of 50 mM
ice-cold K-phosphate buffer (pH 7.0) containing
100 mM KCl, 1 mM ascorbate, 5 mM
β
-mercap-
toethanol and 10% (w/v) glycerol. The homogenates
were centrifuged at 11,500× g for 10 min and the
resulted supernatants were used for determination of
enzyme activity and protein content. All procedures
were performed at 0~4°C.
Ascorbate peroxidase (APX) activity was measured
according to Hossain et al (2010) by monitoring the
rate of oxidation of AsA at 290 nm for 1 min.
Monodehydroascorbate reductase (MDHAR) activity
was measured by using 0.5 units of AO and the
oxidation rate of NADPH was followed at 340 nm
(Hossain et al., 2013a). Dehydroascorbate reductase
(DHAR) activity was measured by following the
formation of AsA from DHA at 265 nm using GSH
(Hossain et al., 2013a). Glutathione reductase (GR)
activity was assayed in the presence of 1 mM GSSG
by measuring the rate of NADPH oxidation, which is
accompanied by a rapid decrease in absorbance at 340
nm (Hossain et al., 2010). Catalase (CAT) activity was
measured according to the method of Hossain et al.
(2009) by monitoring the decrease of absorbance at
240 nm for 1 min caused by the decomposition of
H
2
O
2
. Glutathione S-transferase (GST) activity was
measured as described by Hossain et al (2010) using 1
mM 1-chloro-2,4- dinitrobenzene (CDNB) as a
substrate and the increase in absorbance was measured
at 340 nm for 1 min. Glutathione peroxidase (GPX)
activity was measured as described by Hossain et al.
(2010) using H
2
O
2
as a substrate. The oxidation of
NADPH was recorded at 340 nm for 1 min.
Glyoxalase I (Gly I) assay was carried out according
to Hossain et al. (2009) using MG as a substrate.
Glyoxalase II (Gly II) activity was determined
according to the method of Hossain et al. (2010) by
monitoring the formation of GSH at 412 nm for 1 min.
3.5 Measurement of lipid peroxidation
The level of lipid peroxidation was measured in leaf
tissue by estimating MDA, using thiobarbituric acid
(TBA) as the reactive material following the method
of (Hossain and Fujita, 2010). The concentration of
MDA was calculated by using an extinction co-
efficient of 155 mM
−1
cm
−1
and expressed as nmol of
MDA g
−1
fresh weight.
3.6 Measurement of H
2
O
2
To determine H
2
O
2
concentration, 0.5 g of leaf tissue
was homogenized in 3ml of 50 mM K-phosphate
buffer pH (6.5) at 4°C and centrifuged at 11,500× g
for 15 min. A 3-ml sample of supernatant was mixed
with 1 ml of 0.1% titanium chloride (TiCl
4
) in 20%
(v/v) H
2
SO
4
(v/v). The mixture was then centrifuged
at 11,500x g for 15 min. The absorbance was
measured at 410 nm to determine the H
2
O
2
content
(Є=0.28 μM
−1
cm
−1
) and expressed as μmol g
−1
fresh