*2.2. Biochemical and Physiological Characters*

The physiological and biochemical characters were recorded at 80 days from transplanting.

#### 2.2.1. Relative Water Content (RWC%)

According to Sanchez et al. [56], twenty leaf discs were used to determine RWC, the fresh weight (FW) for the discs was determined, the same discs were saved in petri dishes containing distilled water for 1 h to determine the turgid weight (TW), after that the discs were dried for 24 h at 80 ◦C to determine the dry weight (DW). Relative water content (RWC%) was calculated as follows: RWC = (FW − DW)/(TW − DW) × 100.

#### 2.2.2. Determination of Chlorophyll *a* and *b* Concentrations

The extraction was done using *N*-*N* Dimethyl formamide, whereby 5 cm<sup>3</sup> *N*-*N* Dimethyl formamide was added to 1 g fresh leaves and kept in the refrigerator overnight, and the absorbance was measured at 647 and 664 nm according to Moran [57].

#### 2.2.3. Electrolyte Leakage Assay (EL%)

Ten discs (1 cm2) of sweet pepper leaves were placed into flasks containing deionized water (25 cm3). Flasks were shaken for 20 h, initial electrical conductivity was recorded for each vial and then flasks were immersed in a water bath at 80 ◦C for 1 h. The vials were shaken for 20 h at 21 ◦C. Final conductivity was measured for each flask. Electrolyte leakage % was calculated according to Szalai et al. [58] with the following formula: initial conductivity/final conductivity × 100.

#### 2.2.4. Chlorophyll Fluorescence Parameter

Chlorophyll *a* fluorescence parameter (*Fv*/*Fm* ratio) was measured at 80 days from the sowing using a chlorophyll fluorometer (PEA, Hansatech Instrument Ltd., version 1.21, Norfolk, UK).

According to Schreiber [59], middle-aged sweet pepper leaves were placed in the dark for 30 min to stimulate the reaction of photosystem II. The minimum chlorophyll fluorescence (*Fo*) was measured using a measuring beam of <0.1 μmol m−2·s<sup>−</sup>1. The maximum fluorescence (*Fm*) was determined after a 1 s saturating pulse (>3500 μmol·m−2·s<sup>−</sup>1). Variable fluorescence (*Fv*) was determined by the difference between the maximum fluorescence and the minimum fluorescence (*Fm* − *Fo*). The maximum efficiency of PSII was determined as the ratio of (*Fv*) to (*Fm*) as follows: *Fv*/*Fm* = (*Fm* − *Fo*)/*Fm*.

#### 2.2.5. Proline Determination

According to Bates et al. [60], proline was assayed in sweet pepper plants, 0.5 g fresh leaf in 3% sulphosalicylic acid and centrifuged for 20 min at 3000× *g*. Then, 2 cm<sup>3</sup> of glacial acetic acid and 2 cm3 ninhydrin reagent was boiled with 2 cm<sup>3</sup> supernatant for 1 h, the reaction was completed in an ice bath, and proline was separated using toluene. Proline was determined as μg·g−<sup>1</sup> FW using a spectrophotometer at 520 nm.

#### 2.2.6. Determination of Lipid Peroxidation

According to Davenport et al. [61], lipid peroxidation was measured as malondialdehyde (MDA) using 100 mg fresh leaves in 1% trichloro acetic acid and centrifuged at 10,000× *g* for 5 min. 0.5% thiobarbituric acid was then added, and mixture was boiled at 95 ◦C for half an hour. The samples were placed on an ice bath and centrifuged for 5 min at 5000× *g*, the measurements were done using spectrophotometer at 532 and 600 nm. MDA (nmol·g−<sup>1</sup> FW) = [6.45 × (A532 − A600) − (0.56 × A450)] × V<sup>−</sup>1W, where V = volume (cm3); W = weight (g).

#### 2.2.7. Determination of Superoxide (O2 <sup>−</sup>) and Hydrogen Peroxide (H2O2)

Sweet pepper leaves were vacuum infiltrated with 10 mM potassium salicylate buffer containing 0.1 *w*/*v*% nitro blue tetrazolium (NBT) or 0.1 *w*/*v*% 3,3-diaminobenzidine (DAB). The leaves were incubated in the light for 140 min and two hours, respectively. The samples were cleared with trichloroacetic acid in ethanol: chloroform 4:1 *v*/*v* for 1 day, the samples were washed and placed in 50% glycerol. O2 <sup>−</sup> and H2O2 were determined as nmol·g−<sup>1</sup> FW according to Huckelhoven et al. [62] using a ChemiImager 4000 digital imaging system (Alpha Innotech Corp., San Leandro, CA, USA).

### 2.2.8. Assay of Enzymes Activity

For the determination of enzymes, 0.5 g fresh leaves were homogenized in 3 cm<sup>3</sup> of 50 mM Tris buffer at 0–4 ◦C, containing 1 mM EDTA-Na2 and 7.5% polyvinyl pyrrolidone. The samples were centrifuged 12,000× *g* for 20 min at 4 ◦C and the total soluble enzyme activities were measured using spectrophotometer in the supernatant [63]. Catalase activity (CAT) was determined through the decomposition of H2O2 by catalase results in the decrease of the ultraviolet absorption of H2O2 at 240 nm, catalase activity can be calculated from this decrease. The reaction mixture contained 2.15 cm3, 2 cm<sup>3</sup> 0.1 M Na-phosphate buffer, 100 μL H2O2, and 50 μL leaves extract. The solution is mixed, and the absorptions were recorded at 240 nm according to Aebi [64]. Peroxidase activity (POX) was calculated according to Hammerschmidt et al. [65]. The reaction mixture consisted of 2.9 cm<sup>3</sup> of a 100 mM sodium phosphate buffer containing 0.25% (*v*/*v*) guaiacol and 100 mM H2O2. The reaction was done with adding 100 μL of crude enzyme extract, the changes in absorbance were recorded every 30 s intervals for 3 min at 470 nm, the activity was determined for min−1·g−<sup>1</sup> fresh weight. Activity of superoxide dismutase (SOD) was measured according to Mishra et al. [66]. Then, we add 290 μL of a mixture containing 100 mM potassium phosphate buffer, 0.1 mM EDTA, 11 mm3 xanthine, cytochrome-c, and 0.002 units of xanthine oxidase to 20 μg of protein extracts was prepared. Xanthine oxidase regulation produced an increase in the absorbance due to the reduction of cytochrome-c (0.025 ± 0.005 min<sup>−</sup>1). Activity of SOD was stated by McCord and Fridovich [67]. According to Goldberg and Spooner [68], GR activity was measured, approximately 0.05 cm3 enzyme extract was mixed with 1 cm<sup>3</sup> phosphate buffer combined with EDTA, 0.1 cm<sup>3</sup> glutathione, and 0.1 cm<sup>3</sup> NADPH, the absorbance was determined at 340 nm.

#### 2.2.9. Fruit Yield

The harvest date starts at 120 days from transplanting to determine number of fruit plant<sup>−</sup>1, fruit fresh weight plant−<sup>1</sup> (g), and total fruit yield (ton hectare<sup>−</sup>1).

#### *2.3. Statistical Analysis*

Statistical analysis was done using analysis of variance (ANOVA) procedures according to the method of Gomez and Gomez [69] using the MSTAT-C statistical software package. The means between treatments were compared by Duncan [70] when the difference was significant (P ≤ 0.05). The correlation analysis was done using XLSTAT 2014.5.03 statistical software.

#### **3. Results**
