Plant Materials and Growth Condition: Seeds of mungbean (Vigna radiata L. cv. BARI Mung-3) plants were placed in a petridish containing six layer of moist filter paper and kept in germinator in dark place for three days. Then, germinated seedlings were transferred into growth chamber under controlled conditions (light, 350 μmol·photon·m−2·s−1; temperature, 25 ± 2 °C; relative humidity, 65%–70%); 10,000-fold diluted Hyponex solution (Hyponex, Osaka, Japan) was applied as nutrient. Two sets of four-day-old seedlings were pretreated with Spd (0.25 mM) for 24 h. These pre-treated seedlings were then exposed to 6 °C temperature at the fifth day. Other two sets of seedlings were exposed to 6 °C temperature (without Spd pretreatment). Control seedlings were grown with Hyponex solution. Another two sets of seedlings were grown with Spd without any stress. All treatments were considered for 2 and 3 days and after that data on different parameters were taken following the standard methodology.
Measurement of Growth Parameter: Plant height was taken from each set of seedlings and expressed as cm. Ten randomly selected fresh seedlings from each treatment were dried at 80 °C for 48 h, then weighed and considered as dry weight (DW), which was expressed in gram (g).
Measurement of Relative Water Content: Relative water content (RWC) of leaf was measured according to Barrs and Weatherly. Fresh weight (FW), turgid weight (TW) and dry weight (DW) of leaves were taken, RWC was calculated by following formula:
Measurement of Chlorophyll Content: Leaves supernatant were extracted with 80% v/v acetone (centrifuging at 5000× g), absorbances were taken with UV-visible spectrophotometer at 663 and 645 nm for chl a and chl b content, respectively; chl contents were calculated according to Arnon.
Measurement of Proline Content: Proline (Pro) was appraised according to Bates et al. Leaves were homogenized in 3% sulfosalicylic acid, centrifuged at 11,500× g. Filtrate was mixed with acid ninhydrin with glacial acetic acid and phosphoric acid. After incubating the mixture at 100 °C for 1 h and cooling, toluene was added, after several minutes chromophore containing toluene was read spectrophotometrically at 520 nm.
Histochemical Detection of Hydrogen Peroxide and Superoxide: The H2O2 and O2•− were localized histochemically by staining leaves with 1% 3,3-diaminobenzidine (DAB) and 0.1% nitroblue tetrazolium (NBT) solution, respectively. Leaves were immersed in those solutions until brown spots appeared due to the reaction of DAB with H2O2 or dark blue spots appeared due to the reaction of NBT with O2•−. After that, leaves were blanched in boiling ethanol to observe the spots.
Measurement of Lipid Peroxidation: The level of lipid peroxidation was measured by estimating MDA, using thiobarbituric acid (TBA) according to Heath and Packer with modifications.
Measurement of Hydrogen Peroxide Content: Hydrogen peroxide was assayed according to Yu et al., extracting leaves in potassium-phosphate buffer (pH 6.5) (centrifuging at 11,500× g), then reacting with a mixture of TiCl4 in 20% H2SO4 (v/v) and was measured spectrophotometrically at 410 nm.
Measurement of Methylglyoxal Level: Leaves were homogenized in 5% perchloric acid and centrifuged at 4 °C for 10 min at 11,000× g. The supernatant was decolorized by adding charcoal. The supernatant was neutralized by saturated solution of potassium carbonate at room temperature. The neutralized supernatant was used for MG estimation by adding sodium dihydrogen phosphate and N-acetyl-l-cysteine to a final volume of 1 mL. Formation of the product N-α-acetyl-S-(1-hydroxy-2-oxo-prop-1-yl)cysteine was recorded after 10 min at a wavelength of 288 nm according to Wild et al. The MG content within the plant sample was calculated by using standard curve of known concentration of MG and expressed as µmol·g−1 FW.
Extraction and Measurement of Ascorbate and Glutathione: Leaves (0.5 g) were homogenized in 5% meta-phosphoric acid containing 1 mM EDTA (centrifuged at 11,500× g) for 15 min at 4 °C and the supernatant was collected for analysis of ascorbate and glutathione. Ascorbate content was determined following the method of Huang et al., with some modifications as described by Hasanuzzaman et al.. The glutathione pool was assayed according to previously described methods with modifications as described by Paradiso et al., and Hasanuzzaman et al., Standard curves with known concentrations of GSH and GSSG were used. The content of GSH was calculated by subtracting GSSG from total GSH.
Protein Determination: The protein concentration of each sample was determined following the method of Bradford using BSA (bovine serum blood) as a protein standard.
Enzyme Extraction and Assays: Leaves were homogenized in K-P buffer (pH 7.0) containing KCl, ascorbate, β-mercaptoethanol and glycerol. Homogenates were centrifuged at 11,500× g, supernatants were assayed.
CAT (enzyme commission number, EC: 1.11.1.6) activity Decrease of absorbance (by decomposition of H2O2) at 240 nm was recorded for 1 min. Reaction was initiated with enzyme extract; activity was calculated from extinction coefficient 39.4 M−1·cm−1.
APX (EC: 1.11.1.11) activity assay: Reaction buffer solution contained 50 mM K-P buffer (pH 7.0), 0.5 mM AsA, 0.1 mM H2O2, 0.1 mM EDTA, and enzyme extract (final volume 700 μL). Reaction was started by adding H2O2. Activity was measured at 290 nm for 1 min using an extinction coefficient 2.8 mM−1·cm−1.
MDHAR (EC: 1.6.5.4) activity: Reaction mixture contained 50 mM Tris-HCl buffer (pH 7.5), 0.2 mM NADPH, 2.5 mM AsA, 0.5 unit of AO and enzyme solution (final volume 700 μL). Reaction was started by adding AO. Absorbance was taken at 340 nm; activity was calculated from change in for 1 min using an extinction coefficient of 6.2 mM−1·cm−1.
DHAR (EC: 1.8.5.1) activity: Reaction buffer contained 50 mM K-P buffer (pH 7.0), 2.5 mM GSH, and 0.1 mM DHA. Activity was calculated from change in absorbance at 265 nm for 1 min using extinction coefficient of 14 mM−1·cm−1.
GR (EC: 1.6.4.2) activity: Reaction mixture contained 0.1 M K-P buffer (pH 7.0), 1 mM EDTA, 1 mM GSSG, 0.2 mM NADPH; enzyme solution (final volume 1 mL). Reaction was initiated with GSSG; decrease in absorbance at 340 nm was recorded for 1 min (calculated using extinction coefficient 6.2 mM−1·cm−1).
GPX (EC: 1.11.1.9) activity was measured as described by Elia et al. with slight modification as described by Hasanuzzaman et al. The reaction mixture consisted of 100 mM K-P buffer (pH 7.0), 1 mM EDTA, 1 mM NaN3, 0.12 mM NADPH, 2 mM GSH, 1 unit GR, 0.6 mM H2O2 (as a substrate) and 20 μL of sample solution. The oxidation of NADPH was recorded at 340 nm for 1 min and the activity was calculated using the extinction coefficient of 6.62 mM−1·cm−1.
Glyoxalase I (EC: 4.4.1.5): Assay mixture contained 100 mM K-P buffer (pH 7.0), 15 mM magnesium sulphate, 1.7 mM GSH and 3.5 mM MG (final volume 700 μL). Reaction was started by adding MG; increase in absorbance was recorded at 240 nm for 1 min. Activity was calculated using extinction coefficient 3.37 mM−1·cm−1.
Glyoxalase II (EC: 3.1.2.6): Formation of GSH at 412 nm was monitored in 1 min. Reaction mixture contained 100 mM Tris–HCl buffer (pH 7.2), 0.2 mM DTNB, 1 mM S-d-lactoylglutathione (SLG) (final volume of 1 mL). Reaction was started by SLG; activity was calculated using extinction coefficient 13.6 mM−1·cm−1.
Measurement of Free Polyamine Content: Endogenous free PAs were estimated according to Kotzabasis. In brief, leaf tissue (0.1 g) was homogenized in 1 mL of 5% (v/v) cold perchloric acid (PCA). The homogenates were kept at 2 °C for 2 h and centrifuged at 15,000× g for 20 min. The supernatant was collected and stored at 2 °C. Aliquots (200 μL) of supernatant was mixed 1:1 (v/v) with 12 N HCL and hydrolyzed for 16 h at 110 °C in flame sealed ampoules. The hydrolyzed products were centrifuged at 3000× g to remove carbonized material and then evaporated at 70 °C. The dried pellet was redissolved in 200 μL of 5% perchloric acid. The nonhydrolyzed PCA supernatant containing free PAs was subjected to benzoylation in alkaline medium. The benzoyl-PAs were extracted with diethyl ether and then evaporated to dryness in a water bath. The benzoyl-PAs were redissolved in methanol and free PAs were analyzed by HPLC with a UV-vis spectrophotometric detector at 254 nm, and then determined using a standard curve of PAs (Put, Spd and Spm) and expressed as µmol·g−1·FW.
Statistical Analysis: All data obtained were subjected to analysis of variance (ANOVA) and the mean differences were compared by a Duncan’s multiple range test (DMRT) using XLSTAT v.2010 software. Differences at p < 0.05 were considered significant.